WO2015021741A1

WO2015021741A1 - Use of doxycycline

Info

Publication number: WO2015021741A1
Application number: PCT/CN2014/000721
Authority: WO
Inventors: 饶子和; 孙涛; 杨诚; 周红刚; 郭宇; 刘慧娟; 刘艳荣; 王静; 张博; 李珊; 翟佳黛
Original assignee: 天津国际生物医药联合研究院; 天津国际生物医药联合研究院有限公司; 旭和（天津）医药科技有限公司
Priority date: 2013-08-13
Filing date: 2014-07-29
Publication date: 2015-02-19
Also published as: CN104367584A; WO2015021704A1

Abstract

Provided is a use of doxycycline in the preparation of a drug for treating a highly metastatic malignant tumor. Doxycycline can inhibit growth, metastasis and invasion of tumor cells.

Description

Application of doxycycline

Technical field

The present invention relates to the field of medicinal chemistry and, in particular, to the use of doxycycline. Background technique

Doxycycline "Doxycycline" (also known as doxycycline), a tetracycline antibiotic, is a well-known and widely used antibiotic. Tetracyclines mainly act on the 30S subunit of the bacterial ribosome, interfering with the amino acid tRNA binding to the site of action on the 30S subunit, blocking the binding of the aminoacyl tRNA to the ribosome-mRNA complex, inhibiting protein synthesis, and also altering The permeability of the cell membrane exposes the nucleotides of important substances in the cell, inhibits DNA synthesis, and thus achieves an antibacterial effect. Doxycycline is a potent and widely used type of tetracycline (Adimora AA., 2002; Kovacova E, et al., 2002). Doxycycline is currently used clinically for the treatment of upper respiratory tract infections, biliary tract infections, urinary tract infections, chronic bronchitis, acute and chronic bronchitis, pneumonia, bronchitis, cellulitis and other symptoms.

Doxycycline on human invasive choroidal melanoma, human malignant melanoma, human breast cancer, human chronic myeloid leukemia, promyelocytic leukemia, human large cell lung cancer, human small cell lung cancer, human high metastatic liver cancer, human metastatic pancreas The role of several highly metastatic malignant tumors such as adenocarcinoma, cervical cancer, human poorly differentiated colon cancer, and human high metastatic gastric cancer has not been reported. Since the above-mentioned malignant tumors generally have the ability to metastasize and invade, once the above diseases occur, they are difficult to cure and cause great harm to human health. However, the current anti-tumor drugs against the above malignant tumors are not only low in anti-tumor activity, but also expensive, and patients are often unbearable, and even some patients give up treatment opportunities because they cannot afford expensive prices. Therefore, there is an urgent need to develop new drugs that are resistant to tumor growth, metastasis or invasion. Summary of the invention

In view of the defects in the prior art that high-metastatic malignant tumor treatment lacks an effective therapeutic drug, the object of the present invention is to provide a drug for preparing a drug for treating high metastatic malignant tumor. Wherein, the molecular structural formula of the doxycycline is

The high metastatic malignant tumor includes human invasive choroidal melanoma, human malignant melanoma, human breast cancer, human chronic myeloid leukemia, promyelocytic leukemia, human large cell lung cancer, human small cell lung cancer, human high metastatic liver cancer. Human metastatic pancreatic adenocarcinoma, cervical cancer, human high metastatic colon cancer, and human high metastatic gastric cancer.

In the above application, the use of the medicament for treating a highly metastatic malignant tumor includes use as a drug for inhibiting tumor growth, application as a drug for inhibiting tumor metastasis, and application for inhibiting tumor invasion.

In the above application, the medicament for treating a highly metastatic malignant tumor comprises doxycycline, doxycycline in a pharmaceutically acceptable salt, an ester, a hydrate or a combination thereof and an adjuvant.

In the above application, the dosage form of the medicament for treating a highly metastatic malignant tumor is selected from the group consisting of a tablet, a capsule, a pill, a suppository, an aerosol, a liquid preparation, a granule, a powder, an injection, a syrup, and a wine. , tinctures, lotions, films or combinations thereof.

In the above application, the administration of the drug for treating a highly metastatic malignant tumor includes oral administration, injection, implantation, external use, spraying, inhalation or a combination thereof.

The anti-tumor drug provided by the invention has the advantages that: the drug patient is not only easy to accept, but also low in price, widely available, easy to obtain, and easy to understand the patient's reaction to the drug. In addition, doxycycline will change the market pattern of existing cancer chemotherapy drugs, becoming a clinical drug that can be taken for a long time and effectively inhibits tumor metastasis, invasion and recurrence.

The doxycycline used in the present invention is doxycycline hydrochloride, yellow powder, manufactured by Kaifeng Pharmaceutical Co., Ltd.

(Group) Co., Ltd. provides, batch number: 201301011, purity: 91.0% (in accordance with the Chinese Pharmacopoeia 2010 edition two). The chemical structural formula of doxycycline hydrochloride is: • HCI

DRAWINGS

Figure 1A is a dose-response curve of the inhibitory effect of doxycycline on Mum-2C cells (pre-experimental results);

Figure 1B is a dose-response curve of the inhibition of Mum-2C cells by doxorubicin hydrochloride (pre-test results);

Figure 2A is a dose-response curve of doxycycline inhibition on Mum-2C cells (formal experimental results);

Figure 2B is a dose-response curve of doxorubicin hydrochloride inhibition on Mum-2C cells (official results);

Figure 3A is a dose-response curve of doxycycline inhibition on Mum-2B cells (pre-experimental results);

Figure 3B is a dose-response curve of doxorubicin hydrochloride inhibition on Mum-2B cells (pre-experimental results);

Figure 4A is a dose-response curve of doxycycline inhibition on Mum-2B cells (formal experimental results);

Figure 4B is a dose-response curve of doxorubicin hydrochloride inhibition on Mum-2B cells (official results);

Figure 5A is a dose-response curve of doxycycline inhibition on A875 cells (pre-experimental results);

Figure 5B is a dose-response curve of the inhibition of A875 cells by doxorubicin hydrochloride (pre-experimental results);

Figure 6A is a dose-response curve of doxycycline inhibition on A875 cells (formal experimental results);

Figure 6B is a dose-response curve of inhibition of A875 cells by doxorubicin hydrochloride (formal experiment) Result);

Figure 7A is a dose-response curve of doxycycline inhibition on A375 cells (pre-experimental results);

Figure 7B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on A375 cells (pre-experimental results);

Figure 8A is a dose-response curve of doxycycline inhibition on A375 cells (formal experimental results);

Figure 8B is a dose-response curve of the inhibition of A375 cells by doxorubicin hydrochloride (formal experimental results);

Figure 9A is the effect of doxycycline on the growth curve of Mum-2C cells (first test results);

(Where, the number of cells in each group on the sixth day was statistically analyzed, F=323.909, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.3975 μΜ, Δ and more西<0.01 in the 0.795 μΜ group of cisplatin compared with 1.5<0.01 in the 1.59 μΜ group of doxycycline compared with 多<0.01, ## compared with the 3.10 μΜ group of doxycycline Ρ<0.01, ** and doxycycline 6.36 μΜ group compared to Ρ<0.01) Figure 9Β is the effect of doxycycline on the growth curve of Mum-2C cells (the second test result);

(Where, the number of cells in each group on the sixth day was statistically analyzed, F=906.888, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.3975 μΜ, Δ and more西<0.01, + 0.01<0.01, + compared with doxycycline 1.59 μΜ group, Ρ<0.01, ## compared with doxycycline 3.18 μΜ group Ρ<0.01, ** and Dorsey环<0.01 for cyclin 6.36 μΜ group Figure 9C is the effect of doxycycline on the growth morphology of Mum-2C cells;

(wherein, A in Fig. 9C shows the cell morphology of the control group observed under an inverted microscope; and B in Fig. 9C shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope)

Figure 10A is the effect of doxycycline on the growth curve of Mum-2B cells (first test result);

(Where, the number of cells in each group on the sixth day was statistically analyzed, F=783.853, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.674 μΜ, Δ and Dorsey环<0.01, 环<0.01, + 多<0.01 compared with doxycycline 2.7 μΜ group, #<0.01 compared with doxycycline 5.4 μΜ group, ** and doxycycline 10.8 μΜ group compared to Ρ<0.01) Figure 10B is the effect of doxycycline on the growth curve of Mum-2B cells (second test result);

(Where, the number of cells in each group on the sixth day was statistically analyzed, F=724.774, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.674 μΜ, Δ and Dorsey环<0.01, 环<0.01, + 多<0.01 compared with doxycycline 2.7 μΜ group, #<0.01 compared with doxycycline 5.4 μΜ group, ** and doxycycline 10.8 μΜ group compared to Ρ<0.01)

Figure 10C is the effect of doxycycline on the growth morphology of Mum-2B cells;

(Where, A shows the cell morphology of the control group observed under an inverted microscope; and Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope)

Figure 11A is the effect of doxycycline on the growth curve of A875 cells (first test result);

(Where, the number of cells in each group on the sixth day was statistically analyzed, F=18599.225, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.602 μΜ, Δ and Dorsey环<0.01, 环<0.01, + 多<0.01 compared with the doxycycline 2.408 μΜ group, ## compared with the doxycycline 4.816 μΜ group Ρ<0.01, ** and doxycycline 9.632 Figure 11B is the effect of doxycycline on the growth curve of Α875 cells (the second test result);

(Where, the number of cells in each group on the sixth day was statistically analyzed, F=74919.505, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.602 μΜ, Δ and Dorsey环<0.01, 环<0.01, + 多<0.01 compared with the doxycycline 2.408 μΜ group, ## compared with the doxycycline 4.816 μΜ group Ρ<0.01, ** and doxycycline 9.632 Fig. 11C is the effect of doxycycline on the growth morphology of Α875 cells;

(Fig. Α shows the cell morphology of the control group observed under an inverted microscope; Fig. B shows the cell morphology of the doxycycline (Doxy) administration group observed under an inverted microscope).

Figure 12A is the effect of doxycycline on the growth curve of A375 cells (first test results); wherein, the number of cells in each group on the sixth day was statistically analyzed, F = 56641.287, * compared with the control group P < 0.01, # compared with doxycycline 1.03875 μΜ group Ρ<0.01, Δ compared with doxycycline 2.0775 μΜ group P<0.01, + compared with doxycycline 4.155 μΜ group P<0.01, ##与P < 0.01 for the doxycycline 8.31 μΜ group, P < 0.01 compared with the doxycycline 16.62 μΜ group. Figure 12B is the effect of doxycycline on the growth curve of A375 cells (second test result) (Where, statistical analysis was performed on the number of cells in each group on the sixth day, F=59812.053, * Compared with the control group, P<0.01, # 多<0.01 compared with the doxycycline 1.03875 μΜ group, Δ<0.01 compared with the doxycycline 2.0775 μΜ group, + compared with the doxycycline 4.155 μΜ group. Ρ<0.01, ## Ρ<0.01 compared with the doxycycline 8.31 μΜ group, Ρ<0.01 compared with the doxycycline 16.62 μΜ group. Figure 12C is the effect of doxycycline on the growth morphology of Α375 cells. ;

(Fig. Α shows the cell morphology of the control group observed under an inverted microscope; Fig. B shows the cell morphology of the doxycycline (Doxy) administration group observed under an inverted microscope)

Figure 13A shows the results of microscopic observation under the microscope of microscopic staining of doxanthin and doxorubicin for inhibiting the invasive ability of Mum-2C cells for 24 h;

Figure 13B is a statistical result of inhibition of invasion of Mum-2C cells by doxycycline and doxorubicin hydrochloride for 24 h. (Note: The concentration of doxycycline in Figure 13A and Figure 13B is: 1 μΜ, the concentration of doxorubicin hydrochloride is: 0.781 μΜ, **: compared with the control group Ρ < 0.01 ( η = 3 ) .

Figure 14A shows the results of microscopic observation under the microscope of microscopic staining of doxycycline and doxorubicin for inhibiting the invasion of human melanoma cells Mum-2B for 24 h;

Figure 14B is a statistical result of the inhibitory effect of doxycycline and doxorubicin on the invasion of human melanoma cells Mum-2B for 24 h;

(Note: The concentration of doxycycline in Figure 14A and Figure 14B is: 1 μΜ, the concentration of doxorubicin hydrochloride is: 0.5 μΜ, #: compared with the monthly group t匕Ρ>0.05, **: compared with the menstrual period Ρ < 0.01 ( η = 3 ). Figure 15A shows the results of microscopic observation under the microscope of microscopic staining of doxycycline and doxorubicin for inhibiting the invasive ability of Α875 cells for 24 h;

Figure 15B shows the statistical results of the inhibitory effect of doxycycline and doxorubicin on the invasive ability of A875 cells for 24 h;

(Note: The concentration of doxycycline in Figure 15A and Figure 15B is: 0.5 μΜ, the concentration of doxorubicin hydrochloride is: 0.4 μΜ, * Ρ < 0.05 compared with the control group, ** compared with the control group Ρ < 0.01 ( η = 3). Fig. 16A is the result of inhibition of the invasive ability of doxycycline and doxorubicin hydrochloride on human melanoma cell Α375 after 24 h;

Figure 16B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasive ability of human melanoma cell A375 after 24 h;

(Note: The concentration of doxycycline in Figure 16A and Figure 16B is: 0.2 μΜ, the concentration of doxorubicin hydrochloride is: 1 μΜ, ** compared with the control group Ρ < 0.01 ( η = 3 ). Figure 17A is a dose-response curve of the inhibitory effect of doxycycline on MCF-7 cells (pre-experimental results);

17 B is the dose-response curve of doxorubicin hydrochloride on MCF-7 cells (pre-experimental results);

Figure 18A is a dose-response curve of doxycycline inhibition on MCF-7 cells (formal experimental results);

Figure 18B is a dose-response curve of doxorubicin hydrochloride on MCF-7 cells (official experimental results);

Figure 19A is a graph showing the dose response curve of doxycycline on MDA-MB-231 cells (pre-experimental results);

Figure 19B is a dose-response curve of inhibition of MDA-MB-231 cells by doxorubicin hydrochloride

(pre-experimental results);

Figure 20A is a graph showing the dose-response curve of doxycycline on MDA-MB-231 cells (positive experiment results);

Figure 20B is a dose-response curve of inhibition of MDA-MB-231 cells by doxorubicin hydrochloride

(formal experimental results)

Figure 21A shows the effect of doxycycline on the growth curve of MCF-7 cells (first test results); (Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F = 57950.806, * with the control group Compared with P<0.01, # compared with the doxycycline 0.65625 μΜ group, Ρ<0.01, Δ compared with the doxycycline 1.3125 μΜ group, P<0.01, +P<0.01 compared with the doxycycline 2.625 μΜ group. ## Ρ<0.01 compared with the doxycycline 5.25 μΜ group, Ρ<0.01 compared with the doxycycline 10.5 μΜ group. Figure 21B is the effect of doxycycline on the growth curve of MCF-7 cells ( The second test result); (Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F=49792.408, *P<0.01 compared with the control group, # compared with the group of doxycycline 0.65625 μΜ <0.01, Δ was compared with doxycycline 1.3125 μΜ group P<0.01, +P <0.01 compared with doxycycline 2.625 μΜ group, ## compared with doxycycline 5.25 μΜ group Ρ<0.01, * * Ρ < 0.01 compared with the doxycycline 10.5 μΜ group. Figure 21C is the effect of doxycycline on the morphology of MCF-7 cells;

(Note: Figure Α shows the cell shape of the control group observed under an inverted microscope. B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope. Figure 22A is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasive ability of human breast cancer cell MCF-7 after 24 h;

Figure 22B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasive ability of human breast cancer cell MCF-7.

(Note: The concentration of doxycycline in Figure 22A and Figure 22B is 0.2 μΜ: the concentration of doxorubicin hydrochloride is: 0.2 μΜ, ** compared with the control group Ρ < 0.01 ( η = 3 ).

Figure 23 is the inhibition effect of doxycycline and doxorubicin on the invasive ability of human breast cancer cell MDA-MB-231. The results of microscopic observation under the microscope after 24 h;

Figure 23B shows the inhibitory effect of doxycycline and doxorubicin on the invasive ability of human breast cancer cell MDA-MB-231 24 h later;

(Note: The concentration of doxycycline in Figure 23A and Figure 23B is: 0.1 μΜ, the concentration of doxorubicin hydrochloride is: 0.1 μΜ, * compared with the control group Ρ < 0.05 ( η = 3 ).

Figure 24 is the dose-response curve of doxycycline inhibition on Κ562 cells (pre-experimental results);

Figure 24 is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on Κ562 cells (pre-experimental results);

Figure 25 is a dose-response curve of doxycycline inhibition on Κ562 cells (formal experimental results);

Figure 25 is a dose-response curve of doxorubicin hydrochloride on Κ562 cells (formal test results);

Figure 26 is the dose-response curve of doxycycline inhibition on HL60 cells (pre-experimental results);

Figure 26 is a dose-response curve of the inhibition of HL60 cells by doxorubicin hydrochloride (pre-experimental results);

Figure 27 Α is the dose-response curve of doxycycline inhibition on HL60 cells (formal experimental results);

Figure 27B is a dose-response curve of the inhibition of HL60 cells by doxorubicin hydrochloride (formal test results);

Figure 28A is the effect of doxycycline on the growth curve of K562 cells (first test results); (Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F=78.812, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.735 μΜ, Δ and doxycycline Compared with 多<0.01 in the 1.476 μΜ group, Ρ<0.01 compared with the 2.94 μΜ group in the doxycycline group, ## compared with the doxycycline 5.88 μΜ group Ρ<0.01, ** and doxycycline 11.76 μΜ The group is Ρ<0.01.)

Figure 28 is the effect of doxycycline on the growth curve of Κ562 cells (the second test result);

(Note: F=335.275, *P<0.01 compared with the control group, #Ρ0.01 compared with the doxycycline 0.735μΜ group, Δ<0.01 compared with the doxycycline 1.476 μΜ group, + and more Compared with 多<0.01, ##, compared with oxime 5.88 μΜ group, 西<0.01, ** compared with doxycycline 11.76 μΜ group Ρ<0.01;

Figure 28C is the effect of doxycycline on the morphology of human leukemia cells Κ562;

(Note: Figure Α shows the cell morphology of the control group observed under an inverted microscope; Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope)

Figure 29A is the effect of doxycycline on the growth curve of human leukemia cell line HL60 (first test result);

(Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F=29234.436, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.304 μΜ, Δ and Dorsey环<0.01, 环<0.01, + 多<0.01 compared with the doxycycline 1.214 μΜ group, #<0.01, ** and doxycycline 4.856 compared with the doxycycline 2.428 μΜ group. Μ<0.01 for the μΜ group. Figure 29Β is the effect of doxycycline on the growth curve of human leukemia cell line HL60 (the second test result);

(Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F=22546.666, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.304 μΜ, Δ and Dorsey环<0.01, 环<0.01, + 多<0.01 compared with the doxycycline 1.214 μΜ group, #<0.01, ** and doxycycline 4.856 compared with the doxycycline 2.428 μΜ group. Μ<0.01 for the μΜ group. Figure 29C is the effect of doxycycline on the morphology of human leukemia cell HL60;

Figure 30A is a dose-response curve of the inhibitory effect of doxycycline on NCI-H460 cells (pre-experimental results); Figure 30B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on NCI-H460 cells (pre-experimental results);

Figure 31A is a dose-response curve of doxycycline inhibition on NCI-H460 cells (formal experimental results);

Figure 31B is a dose-response curve of doxorubicin hydrochloride on NCI-H460 cells (positive experiment results);

Figure 32A is a dose response curve of doxycycline on NCI-H446 cells (pre-test results);

Figure 32B is a dose-response curve of the inhibition of doxorubicin hydrochloride on NCI-H446 cells (pre-experimental results);

Figure 33A is a dose response curve of doxycycline on NCI-H446 cells (official experimental results);

Figure 33B is a dose-response curve of doxorubicin hydrochloride on NCI-H446 cells (positive experiment results);

Figure 34A is the effect of doxycycline on the growth curve of NCI-H460 cells (first test result);

(Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F=1224.258, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.5 μΜ, Δ and more The oxime 1.0 μΜ group was Ρ<0.01, + compared with the doxycycline 2.0 μΜ group Ρ<0.01, ## compared with the doxycycline 4.0 μΜ group Ρ<0.01, ** and doxycycline 8.0 μΜ compared to Ρ<0.01.)

Figure 34 is the effect of doxycycline on the growth curve of human lung cancer cell line NCI-H460 (the second test result);

(Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F=655.857, *P<0.01 compared with the control group, #Ρ0.01 compared with the doxycycline 0.5 μΜ group, Δ and more The oxime 1.0 μΜ group was Ρ<0.01, + compared with the doxycycline 2.0 μΜ group Ρ<0.01, ## compared with the doxycycline 4.0 μΜ group Ρ<0.01, ** and doxycycline 8.0 μΜ compared to Ρ<0.01.

Figure 34C is the effect of doxycycline on the morphology of human lung cancer cell line NCI-H460;

(Note: Figure Α shows the cell morphology of the control group observed under an inverted microscope; Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope) Figure 35A is the effect of doxycycline on the growth curve of NCI-H446 cells (first test results);

(Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F=413.410, *P<0.01 compared with the control group, #Ρ0.01 compared with the doxycycline 0.25 μΜ group, Δ and more Compared with oxime <0.01, 西<0.01, + compared with doxycycline 1 μΜ group, Ρ<0.01, ## compared with doxycycline 2μΜ group Ρ<0.01, ** and doxycycline 4<0.01 for the 4 μΜ group.

Figure 35 is the effect of doxycycline on the growth curve of NCI-H446 cells (second test result);

(Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F = 525.848, * P < 0.01 compared with the control group, # 多 < 0.01, Δ with the doxycycline 0.25 μΜ group多<0.01, compared with 多<0.01, + 多<0.01, compared with doxycycline 1 μΜ group, #<0.01, ** and more than doxycycline 2 μΜ group The cyclin 4 μΜ group was Ρ<0.01.

Figure 35C is the effect of doxycycline on the morphology of human lung cancer cell line NCI-H446;

(Note: Figure Α shows the cell morphology of the control group observed under an inverted microscope; Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope.)

Figure 36A shows the inhibitory effect of doxycycline and doxorubicin on the invasion of NCI-H460 cells. The results of microscopic observation under a microscope after 24 h;

Figure 36B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasion of NCI-H460 cells after 24 h;

(Note: The concentration of doxycycline in Figure 36A and Figure 36B is: 0.08 μΜ, the concentration of doxorubicin hydrochloride is: 4 μΜ, ** compared with the control group Ρ < 0.01 ( η = 3 ).

Figure 37: Inhibition of invasion of human lung cancer cell line NCI-H446 by doxycycline and doxorubicin hydrochloride. Results observed under microscope microscopy after 24 h;

Figure 37B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasion of human lung cancer cell line NCI-H446.

(Note: The concentration of doxycycline in Figure 37A and Figure 37B is: 0.781 μΜ, the concentration of doxorubicin hydrochloride is: 0.1 μΜ, ** compared with the control group Ρ < 0.01 ( η = 3 ).

Figure 38: Α is the dose-response curve of doxycycline inhibition on HCCLM3 cells (pre-experimental results); Figure 38 B is a dose-response curve of inhibition of HCCLM3 cells by doxorubicin hydrochloride (pre-experimental results);

Figure 39 A is the dose-response curve of doxycycline inhibition on HCCLM3 cells (official experimental results);

Figure 39 B is the dose-response curve of doxorubicin hydrochloride on HCCLM3 cells (normal experimental results);

Figure 40A is the effect of doxycycline on the growth curve of HCCLM3 (human hepatoma cells) (first test result);

(Note: The number of cells in each group on the sixth day was statistically scored, F=163.863, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.7805 μΜ, Δ and more西<0.01 in the 1.561 μΜ group of cyclin compared with 3.1<0.01 in the 3.122 μΜ group of doxycycline compared with 6.2<0.01, ## and doxycycline 6.214 μΜ group compared with 0.01<0.01, ** and doxycycline 12.488 μΜ group compared to Ρ<0.01.)

Figure 40 is the effect of doxycycline on the growth curve of HCCLM3 (human hepatoma cells) (second test result)

(Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F=52.628, *P<0.01 compared with the control group, #Ρ0.01 compared with the group of doxycycline 0.7805 μΜ, Δ and Dorsey环<0.01, 环<0.01, + 多<0.01 compared with doxycycline 3.122 μΜ group, #<0.01, ** and doxycycline 12.488 compared with doxycycline 6.244 μΜ group The μΜ group was Ρ<0.01.

Figure 40C is the effect of doxycycline on the morphology of HCCLM3 cells;

Figure 41A is a dose response curve of doxycycline on pancreatic cancer ASPC-1 cells (pre-experimental results);

Figure 41B is a dose-response curve of inhibition of doxorubicin hydrochloride on pancreatic cancer ASPC-1 cells (pre-experimental results);

Figure 42A is a dose response curve of doxycycline on pancreatic cancer ASPC-1 cells (normal experimental results);

Figure 42B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on pancreatic cancer ASPC-1 cells (formal experimental results); Figure 43A is the effect of doxycycline on the growth curve of ASPC-1 cells (first test results) (Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F = 200.330, * compared with the control group P<0.01, #Ρ0.01 compared with the doxycycline 0.977 μΜ group, Ρ<0.01 compared with the doxycycline 1.954 μΜ group, Ρ<0.01, compared with the doxycycline 3.909 μΜ group. #Ρ<0.01 compared with the doxycycline 7.818 μΜ group, Ρ<0.01 compared with the doxycycline 15.636 μΜ group.

Figure 43 is the effect of doxycycline on the growth curve of ASPC-1 cells (second test results) (Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F = 225.451, * compared with the control group P<0.01,# Ρ<0.01 compared with the doxycycline 0.977μΜ group, Ρ<0.01 compared with the doxycycline 1.954μΜ group, Ρ<0.01, compared with the doxycycline 3.909μΜ group. #Ρ<0.01 compared with the doxycycline 7.818μΜ group, Ρ<0.01 compared with the doxycycline 15.636μΜ group.

Figure 43C is the effect of doxycycline on the growth morphology of human pancreatic cancer cell line ASPC-1;

Figure 44A shows the inhibitory effect of doxycycline and doxorubicin on the invasion of pancreatic cancer cell ASPC-1. The results of microscopic observation under the microscope after 24 h;

Figure 44B is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasion of pancreatic cancer cell ASPC-1.

(Note: The concentration of doxycycline in Figure 44A and Figure 44B is 3 μΜ, the concentration of doxorubicin hydrochloride is 0.2 μΜ, and #: compared with the control group, Ρ>0.05, (η=3).)

Figure 45 is a dose-response curve of inhibition of doxycycline on cervical cancer cell line Hela (pre-experimental results);

Figure 45B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on cervical cancer cell line Hela (pre-experimental results);

Figure 46A is a dose-response curve of inhibition of doxycycline on cervical cancer cell line Hela (normal experimental results);

Figure 46B is a dose-response curve of the inhibitory effect of doxorubicin hydrochloride on cervical cancer cell line Hela (normal experimental results);

Figure 47A is the effect of doxycycline on the growth curve of Hela cells (first test results) (Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F = 101.211, * and control Compared with group P<0.01, # compared with doxycycline 0.3325 μΜ group Ρ<0.01, Δ compared with doxycycline 0.665 μΜ group Ρ<0.01, + compared with doxycycline 1.33 μΜ group Ρ<0.01, ## Ρ<0.01 compared with the doxycycline 2.66 μΜ group, Ρ<0.01 compared with the doxycycline 5.32 μΜ group. )

Figure 47 is the effect of doxycycline on the growth curve of Hela cells (second test results) (Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F = 61.892, * compared with the control group P < 0.01, # compared with the doxycycline 0.3325 μΜ group Ρ<0.01, Δ compared with the doxycycline 0.665 μΜ group Ρ<0.01, + compared with the doxycycline 1.33 μΜ group Ρ<0.01, # #Ρ<0.01 compared with the doxycycline 2.66 μΜ group, Ρ<0.01 compared with the doxycycline 5.32 μΜ group.

Figure 47C is the effect of doxycycline on the growth morphology of Hela cells;

(Note: Figure A. Cell morphology of the control group observed under an inverted microscope; Figure B. Cell morphology of the doxycycline (Doxy)-administered group observed under inverted microscopy)

Figure 48A shows the inhibitory effect of doxycycline and doxorubicin on human cervical cancer Hela invasion 24 h after crystal violet staining under microscope;

Figure 48B shows the inhibitory effect of doxycycline and doxorubicin on human cervical cancer Hela invasion 24 h later;

(Note: The concentration of doxycycline in Figure 48A and Figure 48B is 0.391 μΜ, the concentration of doxorubicin hydrochloride is 0.2 μΜ, **: Ρ<0.01, η=3 compared with the control group)

Figure 49 Α is the dose-response curve of doxycycline inhibition on colon cancer cell line S W620 (pre-experimental results);

Figure 49B is a dose-response curve of inhibition of colon cancer cell line SW620 by doxorubicin hydrochloride

(pre-experimental results);

Figure 50A is a dose-response curve of doxycycline inhibiting colon cancer cell line SW620 (normal experimental results);

Figure 50B is a dose-response curve of inhibition of colon cancer cell line SW620 by doxorubicin hydrochloride (formal experimental results);

Figure 51A shows the inhibitory effect of doxycycline and doxorubicin on the invasion of colon cancer cell line SW620.

Figure 5 1B is the statistical effect of doxycycline and doxorubicin on the invasion of colon cancer cell line SW620 for 24 h; (Note: Docetaxel concentration is 0.05 μΜ in Figure 48A and Figure 48B, and doxorubicin hydrochloride concentration is 0.1 μΜ; *: Ρ<0.05, (η=3) compared with the control group)

Figure 52Α is the dose-response curve of doxycycline on gastric cancer cell ΜΚΝ28 cells (pre-experimental results);

Figure 52 is a dose-response curve of doxorubicin hydrochloride on gastric cancer cell ΜΚΝ28 cells (pre-experimental results);

Figure 53 is a dose-response curve of doxycycline on gastric cancer cell ΜΚΝ28 cells (official experimental results);

Figure 53 is a dose-response curve of doxorubicin hydrochloride on gastric cancer cell ΜΚΝ28 cells (formal experimental results);

Figure 54 is the effect of doxycycline on the growth curve of ΜΚΝ28 cells (first test results) (Note: Statistical analysis of the number of cells in each group on the sixth day, F=5386.224, *Compared with the control group P <0.01, #Ρ<0.01 compared with the doxycycline 0.692 μΜ group, P<0.01 compared with the doxycycline 1.384 μΜ group, and P<0.01 compared with the doxycycline 2.678 μΜ group. P<0.01 compared with the doxycycline 5.536 μΜ group, **P<0.01 compared with the doxycycline 11.072 μΜ group. Figure 54B is the effect of doxycycline on the growth curve of MKN28 cells (second test) RESULTS) (Note: Statistical analysis was performed on the number of cells in each group on the sixth day, F = 8534.305, * P < 0.01 compared with the control group, # 0.01 < 0.01, Δ compared with the group of doxycycline 0.692 μΜ P<0.01 compared with doxycycline 1.384 μΜ group, P<0.01 compared with doxycycline 2.728 μΜ group, P<0.01 compared with doxycycline 5.536 μΜ group, ** and Dorsey P<0.01 for the cyclin 11.072 μΜ group. Figure 54C is the effect of doxycycline on the growth morphology of MKN28 cells;

(Note: Figure A shows the cell morphology of the control group observed under an inverted microscope; Figure B shows the cell morphology of the doxycycline (Doxy)-administered group observed under an inverted microscope)

Fig. 55A is a graph showing the inhibitory effect of doxycycline and doxorubicin on the invasion of gastric cancer cell MKN28 after 24 h;

Fig. 55B is a statistical result of inhibition of invasion of gastric cancer cell MKN28 by doxycycline and doxorubicin hydrochloride for 24 hours;

(Note: In Figure 55A and Figure 55B, doxycycline concentration was 1 μΜ, doxorubicin hydrochloride concentration was 1 μΜ, and #: compared with the control group, Ρ>0.05, (η=3). Figure 56 is a dose-response curve of inhibition of NIH-3T3 cells by doxycycline and doxorubicin hydrochloride (first experimental results);

Figure 57 is a dose-response curve of inhibition of NIH-3T3 cells by doxycycline and doxorubicin hydrochloride (second experiment results);

Figure 58 is a dose-response curve of inhibition of HaCaT cells by doxycycline and doxorubicin hydrochloride (first experimental results);

Figure 59 is a dose-response curve of inhibition of HaCaT cells by doxycycline and doxorubicin hydrochloride (second experimental results);

Figure 60 is a dose-response curve of the inhibitory effect of doxycycline (Doxy) and doxorubicin hydrochloride (ADR) on HEK 293 cells and HEK 293T cells (first experimental results);

Figure 61 is a dose-response curve of the inhibitory effect of doxycycline (Doxy) and doxorubicin hydrochloride (ADR) on HEK 293 cells and HEK 293T cells (the results of the second experiment);

Figure 62 is the effect of doxycycline on the growth of mouse B16 melanocytes xenografts (first trial); Figure 63 is the effect of doxycycline on the gross morphology of melanoma xenografts;

Figure 64 is the effect of doxycycline on body weight of B16 melanoma xenograft mice (first trial);

(Note: * P < 0.05 compared with the control group; ^# P < 0.05 compared with the cyclophosphamide group) Figure 65 is the effect of doxycycline on the growth of mouse B16 melanoma xenografts (repeated test); 66 is the effect of doxycycline on the body weight of B16 melanoma xenograft mice (repeated test); (Note: * P < 0.05 compared with the control group; ^# compared with the cyclophosphamide group, P < 0.05) Figure 67 Is the effect of doxycycline on the growth of mouse Lewis lung cancer xenografts (first trial); Figure 68 is the effect of doxycycline on the body weight of Lewis lung cancer xenografts (first trial); (Note: * with the control group In comparison, P <0.05;^# compared with the cyclophosphamide group, P < 0.05) Figure 69 is the effect of doxycycline on the growth of mouse Lewis lung cancer xenografts (repeated test); Figure 70 is the doxycycline pair Lewis lung tumor bearing mice body weight (replications); (Note: * compared with the control group, P <0.05;^# group compared with cyclophosphamide, P <0.05) FIG. 71 is a pair of doxycycline The effect of rat MCF-7 breast cancer xenografts (first trial); Figure 72 is the effect of doxycycline on the body weight of transplanted MCF-7 breast cancer mice (first Secondary test);

Figure 73 is the effect of doxycycline on the growth of transplanted mouse MCF-7 breast cancer (repeated test); FIG 74 is a bearing for doxycycline breast cancer MCF-7 xenografted mice body weight trees (replications); (Note: compared with control group * P <0.05;^# compared with cyclophosphamide group P <0.05 Figure 75 is the effect of doxycycline on the growth of transplanted mouse H446 small cell lung cancer (first trial);

Figure 76 is the effect of doxycycline on the body weight of transplanted H446 small cell lung cancer xenografts (first test);

Figure 77 is the effect of doxycycline on the growth of transplanted mouse H446 small cell lung cancer (repeated test);

Figure 78 is the effect of doxycycline on the body weight of transplanted H446 small cell lung cancer xenografts (repeated test);

Figure 79 is the effect of doxycycline on the survival of tumor-bearing mice (first trial);

Figure 80 is the effect of doxycycline on the survival of tumor-bearing mice (repeated trial);

Figure 81 shows the effect of doxycycline on migration of MCF-7 cells;

Figure 82 shows the effect of doxycycline on migration of HCCLM3 cells;

Figure 83 shows the effect of doxycycline on B16 cell migration;

Figure 84 shows the effect of doxycycline on the migration of human small cell lung cancer cell line NCI-H446; Figure 85 shows the effect of doxycycline on the migration of pancreatic cancer cell ASPC-1;

Figure 86 shows the effect of doxycycline on migration of human colon cancer cells SW620. detailed description

The technical solutions in the embodiments of the present invention are clearly and completely described in the following with reference to the accompanying drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of the present invention.

The inventors have confirmed the anti-tumor activity of doxycycline having a tetracycline structure after extensive experimental studies. The specific embodiment is as follows.

In Examples 1-10, the test materials used and their sources include:

(1) various cell lines

Human melanoma cell line: Mum-2C (human invasive choroidal melanoma cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

Mum-2B (human invasive choroidal melanoma cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

A875 (human malignant melanoma): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; A375 (human malignant melanoma): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; human breast cancer cell line:

MCF-7 (human breast cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

MDA-MB-231 (human breast cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

Human leukemia cell line:

K562 (human chronic myeloid leukemia cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

HL60 (human promyelocytic leukemia cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

Human lung cancer fine-moon package:

NCI-H460 (human large cell lung cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

NCI-H446 (human small cell lung cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

Human liver cancer cell line:

HCCLM3 (human high metastatic liver cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

Human pancreatic cancer cell line:

ASPC-1 (human metastatic pancreatic adenocarcinoma cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

Human cervical cancer cell line:

Hela (human cervical cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; human colon cancer cell line: SW620 (human high metastatic colon cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

Human gastric cancer cell line:

MKN28 (human gastric cancer high metastatic cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

Normal cell line:

NIH-3T3 (mouse embryo fibroblast cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

HaCaT (human normal skin cells): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; HEK 293 (human embryonic kidney cells): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

HEK 293T (human embryonic kidney cell derivative): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.;

(2) for the test

Doxycycline hydrochloride: yellow powder, supplied by Kaifeng Pharmaceutical (Group) Co., Ltd., batch number: 201301011, purity: 91.0% (in accordance with the Chinese Pharmacopoeia 2010 edition two).

(3) Positive control drugs

Doxorubicin hydrochloride: Orange-red powder, purchased from Beijing Enoch Technology Co., Ltd., manufacturer: Admas-beta, article number: 41701A, batch number: P11099, purity 98%+.

(4) Other reagents

Tetramethyl azozolium salt (MTT) : Shanghai Shenggong Bioengineering Co., Ltd.

TB0799-lg, lot number: XP1008B3012J, grade: analytical grade, size 1 g.

Dimercaptosulfoxide (DMSO) Cell culture grade: purchased from Beijing Suo Laibao Technology Co., Ltd., Item No.: D8370-500, Lot No.: 2P005970, Grade: Cell culture grade, Specification 500 mL, for cell cryopreservation, and drugs Formulated.

Dimercaptosulfoxide (DMSO): purchased from Shanghai Shenggong Bioengineering Co., Ltd., article number:

DN3039A, Lot No.: SJ0731S4013J, Grade: Analytical grade, size 500 mL, for dissolution of purple crystals by MTT method.

RPMI 1640 medium: purchased from Thermo Fisher Scientific, Hyclone brand, article number: SH30809.01B, lot number: NYG0920, specification: 500 mL. Trypsin: purchased from Shanghai Shenggong Bioengineering Co., Ltd., Item No.: T0458-10, Lot No.: 0301C314, Level: USP, Specification: 10 g.

Trypan Blue: Purchased from Biyuntian Biotechnology Research Institute, item number ST798-lg.

Crystal Violet: Purchased from Shanghai Shenggong Bioengineering Co., Ltd., Item No.: CB0331-25g, Lot No.: MC0807B4012J _o

Matrigel Matrigel: Manufacturer BD Medical Devices, Inc. No. 356234, Lot No.: 2229979.

(5) Instrument consumables

Biosafety Rejection: Telstar, Model: Bio II A.

C0 ₂ incubator: Shidukai Instrument Equipment (Shanghai) Co., Ltd., Model: STIK IL-161HI. Automated enzyme-linked immunosorbent assay: Thermo Fisher Scientific, model Multiscan FC. Inverted phase contrast microscope: Manufacturer: Olympus, Model: CKX41.

Inverted fluorescence microscope: Nikon, model: Nikon ECLIPSE Ti.

96-well plate: purchased from Thermo Fisher Scientific, Inc., Thermo Nunc brand, article number 167008.

24-well plate: purchased from Thermo Fisher Scientific, Inc., Thermo Nunc brand, article number 167008.

Transwell Chamber (BD-Falcon): Manufacturer BD Medical Devices, Catalog No. 353097, Lot No.: 3042760).

24 cell culture plate: Manufacturer Corning-Costar brand, Corn No.:

3524, Lot number: 34312004.

(6) The preparation methods of the drugs and reagents used include:

a) Preparation of doxycycline hydrochloride solution: Weigh 0.01 g of doxycycline hydrochloride and dissolve it in 1 mL of PBS solution to prepare a 10 mg/mL solution. After it is fully dissolved, filter it with a 0.22 μM filter. It is used after sterilization, and it is used now when it is used. Dilute to the desired concentration with cell culture medium during use. The formulation and use of the solution should be performed in a sterile biosafety refusal.

b) Preparation of doxorubicin hydrochloride solution: Weigh 0.005 g of doxorubicin hydrochloride and dissolve it in 0.5 mL of DMSO to prepare a 10 mg/mL solution, which is ready for use every time. Dilute to the desired concentration with cell culture medium at the time of use. The formulation and use of the solution should be performed in a sterile biosafety refusal. c) Preparation of MTT stock solution: Dissolve 1 g MTT in 200 mL PBS solution (concentration: 5 mg/mL), mix MTT completely, filter and sterilize with 0.22 μ πι filter, dispense, avoid The light was stored in a refrigerator at -20 °C. The formulation and use of the solution should be performed in a sterile biosafety refusal.

d) Preparation of trypan blue stock solution: Weigh 400 g of trypan blue powder in lOO mL of PBS and prepare 4% stock solution. After it is fully dissolved, filter it. Store in 4 ° (packed in the refrigerator). Dilute to 0.4% when used.

e) Preparation of crystal violet solution: 0.5 g of crystal violet powder was weighed and dissolved in 100 mL of PBS to prepare a 0.5% storage night. After it was sufficiently dissolved, it was filtered through a filter. Store in a 4°C refrigerator after dispensing and dilute to 0.1% when used. Example 1 The effect of doxycycline on human melanoma cells Mum-2C, Mum-2B, A875, A375 cells in vitro

1. MTT assay for the inhibitory effect of doxycycline on the growth of human melanoma cells Mum-2C, Mum-2B, A875, A375 and mouse melanoma B-16

The experimental method is MTT colorimetry: the detection principle is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT to water-insoluble blue-violet crystal sputum (Formazan) and deposit in cells, while dead cells have no this function. Dimercaptosulfoxide (DMSO) is capable of lysing sputum in cells, and its absorbance is measured at 490 nm by an enzyme-linked immunosorbent assay, which indirectly reflects the number of viable cells. Within a certain number of cells, the amount of strontium crystal formation is proportional to the number of cells. This method has been widely used for the detection of activity of some biologically active factors, large-scale anti-tumor drug screening, cytotoxicity tests, and tumor radiosensitivity assays.

Method steps:

(1) Cell recovery and culture

The frozen melanoma cells were taken out from the liquid nitrogen and immediately placed in a 37 ° C water bath to melt the cells. Biosafety Rejection The cell suspension was pipetted into a centrifuge tube containing appropriate medium and centrifuged at 800 rpm/min for 5 minutes. The supernatant was discarded, and the cells were suspended in 1 mL of medium, and the cells were filled with appropriate medium. In the culture dish, the cells were cultured under the conditions of 37 ° C, 5% CO ₂ , and saturated humidity. Passage when cells reach 80%-90% contact confluence, 0.2% trypsinization into single cell suspension, centrifugation at 800 rpm/min for 5 minutes; discard the supernatant, add 1-2 mL medium to suspend the cells, and fine The cells were transferred to 2-3 petri dishes containing appropriate medium and culture was continued.

(2) MTT assay for the inhibition of cell growth

The adherent cells in the logarithmic growth phase are trypsinized, dispersed into individual cells, and suspended in the corresponding cell culture medium. The cells were seeded on 96-well plates at 100 cell suspensions per well, 3000-4000 cells/well. The 96-well culture plate was placed in a carbon dioxide (5%) incubator for 24 hours at 37 ° C to allow the cells to adhere.

After the cells were completely adhered for 24 hours, the culture solution was discarded. The test group was added with cell culture medium containing different concentrations of doxycycline. The positive control group was added with different concentrations of doxorubicin hydrochloride cell culture solution, and no drug was added. Control wells with only the corresponding drug solvent (doxcycline solvent is PBS, doxorubicin hydrochloride solvent is DMSO), and only the medium-free zero-free wells are added. Set up 3-5 parallel holes in each group, then place the plate at 37 ° C for 48 hours in a carbon dioxide (5%) incubator.

After 48 hours, 20 μL of MTT (5 mg/mL) was added to each well and incubation continued for 4 h. Then, the culture solution was gently aspirated, 150 L DMSO was added to each well as a solvent to dissolve, and the absorbance at 570 nm was measured by a microplate reader after dissolution (experimental method reference: Wei Wei, Wu Ximei et al., Pharmacological Experimental Methodology, No. Fourth Edition, Beijing: People's Medical Publishing House, 2010: 1568-1569).

Each cell is tested twice, the first is a pre-experiment. In order to better study the dose-response relationship between the drug and the cell, the concentration of the drug used in the second formal test will be based on the first test. optimization.

(3) Data analysis

Calculate the survival rate of the cells and the inhibition was calculated drugs on cell half inhibition concentration IC _50. Survival rate % = (experimental group OD value - zero hole OD value) / (negative control group OD value - zero hole OD value) xlOO %; inhibition rate % = (1 - survival rate) xlOO %. IC _{5 was} performed using Graphpad Prism 5 software. The calculation of the value.

The dose-response curve of the drug to the cells was plotted using Excel software, and each index was expressed by mean ± standard deviation (X soil S ).

Experimental results:

(1) MTT assay for the inhibitory effect of doxycycline on the growth of Mum-2C cells

As shown in Table 1 and Figure 1 and Table 2 and Figure 2, doxycycline grows on Mum-2C cells. At low doses, it showed significant inhibition. As the concentration of doxycycline increased, the inhibitory effect on Mum-2C cells increased gradually, showing a dose-dependent manner.

The preliminary results showed that the half-inhibitory concentration (IC ₅ ) of doxycycline and the positive drug doxorubicin hydrochloride on Mum-2C were 1.2167+0.1409 μΜ and 0.3838+0.0319 μΜ, respectively. The results of formal experiments showed that doxycycline and the positive drug doxorubicin hydrochloride on the IC ₅ of Mum-2C. The values are 1.4007 ± 0.1089 μΜ and 0.6517 ± 0.0587 μΜ, respectively.

Table 1 Inhibition of Mum-2C cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR)

(2) MTT assay for the inhibitory effect of doxycycline on the growth of human melanoma cells Mum-2B as shown in Table 3 and Figure 3 and Table 4 and Figure 4, the growth of doxycycline on Mum-2B cells was low. At the dose, it showed obvious inhibition. With the increase of doxycycline concentration, the inhibitory effect on Mum-2B cells increased gradually, showing a dose-dependent manner.

Pre-experimental results indicate that doxycycline and the positive drug doxorubicin hydrochloride on Mum-2B cells The half-inhibitory concentration (IC ₅₀ value) was 2.7543 + 0.5462 μΜ and 0.2658 + 0.0137 μΜ, respectively. The results of formal experiments showed that the IC ₅₀ values of doxycycline and the positive drug doxorubicin hydrochloride for Mum-2B were 3.3057±0.7184 μΜ and 0.1588±0.0440 μΜ, respectively.

Table 3 Inhibition of Mum-2B cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-experimental results)

Doxy-i x÷s survival rate AD -1 x÷s survival rate m

3⁄4 3⁄4( Μ;' ( OD57C, urn > '( 3⁄4 .) ί%) Concentration μΜ) (00;170 nm 3⁄4) (3⁄4>

0 i.23.24士 0.0786 100 inch 0 0 0.6293+0,0476 100 0

Ο.Γ953 1.1854^0.0146 96.19 3.81 0.0391 0.5745±0.a0 5 91 0 8.70

0.3906 i, 166 士 0,0249 94,61 539 0,0781 0.621±0.1022 98.68 1.32

0.7S125 0.9702±0.0543 78.73 21.27 0.15625 0.5750±0.0149 91.37 8,63

1.5625 .7293±0.0441 59.17 40.83 0. 125 0.4443+0.029S 70.60 29.40

0.5943^0.0632 4S.22 5178 0.625 45.01 54.99

6.25 0.4646^0.0321 37.70 62.30 L25 0.2009^0.0072 3L 2 6S.08

12.5 0J86 0.0G72 15.10 84.90 2.5 0.2248 into 0198 3 .72 64.2S

25 0.0624+0.0032 5.06 94.94 5 0.2i97±0.0016 34.91 65,09

50 0.0552士 0.0073 4.4S 552 iO 0.2566±0.0246 40.7 S

d

a

Table 4 Doxy and doxorubicin hydrochloride (ADR) w vs. inhibition of Mum-2B cells p

Role (formal experimental results)

Bu 1

Doxy-2 X±8 Survival rate AD -2 x±s Survival rate mm degree (μΜ.> ^{ ODSJO nm:》 {: (% ') Concentration (μ.Μ) ( OD570 nm } (%) (3⁄4)

0 0.S704士 0-0U3 100 0 (ϊ 0.9365=0.0553 100 0

0.0488 O.S26Si0.0679 94.99 5.01 0.03906 0.7472 ± 0.1272 79.7S 20.22

0.0976 0.6987±0.1455 80.27 19.73 0.0781 74.8 25. Π

0.1 53 .S09±0.0217 7,06 0.15625 0,5263÷0.0125 56.20 43,80

0.3906 0.7417i .1009 85.21 14.79 0.3125 0—5592 soil 0.0946 59.7 ί 40.29

0.6SS9士士 0.0570 79,14 20,86 0.625 29,33 70.67

0.44 6i0.0I59 5165 4S.35 1.25 0.3204i0.0656

0378 soil (.0104 43.43 56.57 2.5 0.2855±i) 0094 30.4 69.51

6.25 0.2S5±0.0153 67.25 5 0.29 士 0.00S4 30.97 69.03

12.5 0.0 15i0J) 010 10.51 9A9 10 0.2656=0.0521 28,36 71.64

(3) MTT assay for the inhibitory effect of doxycycline on the growth of A875 cells

As shown in Table 5 and Figure 5 and Table 6 and Figure 6, doxycycline significantly inhibited the growth of A875 cells at low doses, as the concentration of doxycycline increased, on A875 cells. The inhibitory effect is gradually increased, showing a significant dose dependency.

The preliminary results showed that the half-inhibitory concentration (IC ₅₀ value) of doxycycline and the positive drug doxorubicin hydrochloride on A875 cells were 3.0987±0.3297 μΜ and 0.5440±0.0399 μΜ, respectively. The results of the formal experiment showed that doxycycline and the positive drug doxorubicin hydrochloride had an IC ₅ on Α875. Values are 2.5683+0.0946 μΜ and 0.1271±0.0070 μΜ.

Table 5 Inhibition of A875 cells by Doxy and Doxorubicin hydrochloride (ADR) (pre-experimental r-fruit)

Doxy-1 士s survival rate inhibition rate AD -Ϊ survival rate inhibition rate concentration μΜ) ( OD o imi) (.%) degree ( μΜ:) (OE1⁄2o (3⁄4.> o

0 ϊ 6479 ΐ). Please o 9 Ϊ00 0 0 2.1, 3Si soil 0.0277 100 ϋ

0.3906 1.3264±0.0S9S 80.49 19.51 0.7S125 ϊ 0657 ±0.0089 49.84 50.1

0.7S125 1.088±0.Ϊ41Ι 66.02 33. S 1.5625 0.8426:±0.0Ι22 39.41 60.59

Ξ

1.5625 0.957 ]±0.0908 58.08 41.92 3.125 0.4183 ±0.0393 19.56 S .44 Bu

1125 09091 soil 0.0444 5517 44,83 6.25 0.143S± 0.0068 ■6,73 93,27

6.25 0.31; 36 00574 22.06 77.94 2.5 0.0362 soil 0.0043 Ϊ.6 9S31

12.5 0.0331±0.0020 2. 1 97.99 25 00457 soil 0.0093 2.14 97.86

25 ( 0292 ± 0,0011 1 98.23 50 0.0397:4:0.0282 1.S6 9SJ4

50 0031 ±0.0102 1.94 98.06 100 0.0825 soil 0.0143 3.86 96.14

100 0.054 ± 0,0015 3.329 96.671 200 0.2374 soil 0.0 i 73 11.10 8S.90 Table 6 Inhibition of A875 cells by Doxy and Doxorubicin hydrochloride (ADR) (Formal experimental results)

Doxy-2 x±s survival rate inhibition rate ADR-2 a x s ir ---r- inhibition rate

(OD570 (%) (%) concentration ( Μ; (OD570 mn.) ( )

0 1.1664 soil 0.02% 100 0 0 1.410Si0.0193 100 0

0.0391 1.0361 ±0.0983 88. S2 II. IS 0.04S 1.0461±0.0019 25.85

0.0781 -8.7 0.0976 0.9387=0.0245 66.53

0.15625 94.76 5.24 0.1953 0.761 iiO.01 0 ~ 46.05

0.3125 1,095 ±0.0268 93. S8 6.12 0.3906 0.5249 ± 0.0209 37.21 62,79

0.625 0.7948 ± 0.0977 68.14 3L86 17.94 S2.06

1.25 35.64 1.5625 0.3Q§9iO.Oi50 7§.I1

2.5 0.4845 0.0154 4153 58.47 22.30 877Q

5 17.64 82.36 6.25 0.2956 土 0.0090 20.95 79.05

10 3.49 96,51 12.5 0.2892 ± 0.011S 79.50

(4) MTT assay for the inhibitory effect of doxycycline on the growth of A375 cells

As shown in Table 7 and Figure 7 and Table 8 and Figure 8, doxycycline significantly inhibited the growth of A375 cells at low doses, and increased the concentration of doxycycline on A375 cells. The inhibitory effect is gradually increased, showing a significant dose dependency.

The pre-experimental measurement showed that the half-inhibitory concentration (IC ₅₀ value) of doxycycline and the positive drug doxorubicin hydrochloride on A375 cells were 2.4153±0.1415 μΜ and 0.1287±0.0073 μΜ, respectively. In formal experiments, the IC50 values of doxycycline and the positive drug doxorubicin hydrochloride for Α375 were 5.2740±0.3189 μΜ and 2.1843±0.1382 μΜ, respectively.

Table 7 inhibition of A375 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) Use (pre-experimental results)

Doxy-1 ±.s survival rate ADR-Ϊ x.±s survival rate inhibition rate

3⁄4 degrees < μΜ) (OD570匪) (3⁄4) Concentration JLM) OD570 nm) (3⁄4) 3⁄4

0 1.3201士 0.0327 100 0 0 0

0.1953 0,7747士 0.0455 5S.6S 41,32 £ 039063 0.S30S ±0.0232 100,09 -0.09

0.3906 0.6592^0. 139 49.93 50.07 0.078125 90.20 9.80

0.7S125 0,6425 ± 0.0064 4S.67 0.15625 (}, 6254 soil 0,0 23 75.35 24.65

0.5255 土谨 02 3 .S 60.2 0,625 0.2405 ±0.0376 28.9S

0.3647士 0.0289 27.63 72.37 125 86.43

6.25 0.1538±0.0045 11.65 2.5 0.1565 ±0.0590 81.15

12.5 .0359±0. 02 272 97.28 0.3068:1: 0.006? 36.96 63.04

25 0.0372士 0.0012 2.82 97.18 10 0J3SS soil 0.0150 16.72 83.28

50 0.0518±0.0021 3.92 96.08 Table 8 Inhibition of A375 cells by Doxy and Doxorubicin hydrochloride (ADR) (Formal experimental results)

x ± s ^ survival rate inhibition rate ADR-2 x ± s 1 ten ^in: -> ^¾ concentration inhibitory rate _{(.Μ) (OD 570 nm "} (¾ C% concentration (μΜ) O 50 nm) ( %>%)

0 1.82 newspaper 0.0225 100 0 0 O.8092iO.0O I 100 0 Bu

0.04SS i.7321±0.0145 94.94 5.06 03906 0.5 inch 017÷0.0327 62.00 3S.00

0.0976 ί.7742士 0.0209 97.25 2.75 0.7SI25 0.4676÷0.0099 57.79 42,21

0.1953 ί ό765± . 155 1.S9 S.ll 1.5625 0.36S ±0.0264 45.59 54.41

0.3906 1.5657±0.04ό7 S5.82 14.18 3.125 0.27 S±0.0034 33.47 6603

0.78125 1.22M士 0.0590 67.15 32.S5 ■6.25 0.174>±0,0115 21.61 78.39

1.5625 1.1503±0.0262 63.05 36.95 12,5 0.0497i0.0051 6.14 93.86

3,125 I.0399±0.00S4 57.00 43.00 25 0.0429i0.0014 5.30 9470

6.25 0.4 34±0.0029 27.05 72.95 5 0.0519±0.0064 6.42J - 93.5S

125 00907±0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Cyclulin has a good inhibitory effect on the growth of the above four cells and shows a significant dose-dependent, and IC ₅ . Values are below 10 μΜ (approx. 5 g/mL).

2. Growth curve assay for the inhibitory effect of doxycycline on the growth of human melanoma cells Mum-2C, Mum-2B, A875 and A375

Method steps:

(1) Cell recovery and culture

The frozen cells were removed from the liquid nitrogen and immediately placed in a 37 ° (water bath to melt the cells. Biosafety Rejection) The cell suspension was pipetted into a centrifuge tube containing the appropriate amount of medium and centrifuged at 800 rpm for 5 minutes. Discard the supernatant, suspend the cells in 1 mL medium, and pipette the cells in the appropriate amount of medium. In the dish, the cells were cultured at 37 ° C, 5% CO ₂ , and saturated humidity. Passage when cells reach 80%-90% contact confluence, 0.2% trypsinization into single cell suspension, centrifugation at 800 rpm/min for 5 minutes; discard the supernatant, add 1-2 mL medium to suspend the cells, transfer the cells Continue to culture in 2-3 petri dishes containing the appropriate amount of medium.

(2) Growth curve method to detect the inhibitory effect of drugs on cell growth

The adherent cells in the logarithmic growth phase are trypsinized, dispersed into individual cells, and suspended in the corresponding cell culture medium. The cells were seeded on a 24-well culture plate, 500 cells per well, 5000 cells/well, and 8 plates were inoculated. The above 24-well culture plates were placed in a 37 ° C carbon dioxide (5%) incubator for 24 hours to allow the cells to adhere.

After 24 hours, discard the culture solution, 6 plates were added with cell culture medium containing different concentrations of doxycycline, and the negative control wells were added without solvent and only solvent. The same concentration of doxorubicin hydrochloride was added to the plate. The IC ₅ concentration of doxycycline on each cell detected by MTT method. Four parallel holes were set in each group, and then the 24-well plates were placed in a carbon dioxide (5%) incubator at 37 °C.

Four parallel wells of doxycycline plus drug plate and doxorubicin hydrochloride were taken every day. The effect of the drug on cell morphology was first observed with an inverted microscope. After resuspending the cells in each well, the cell suspension was mixed with 0.4% trypan blue 9:1, counted within three minutes, and the number of viable cells was counted for a total of six days. Each cell was tested twice. (Experimental Methods Reference: Wei Wei, Wu Ximei, et al., Pharmacological Experimental Methodology, Fourth Edition, Beijing: People's Medical Publishing House, 2010: 1570-1571).

Statistical analysis: All statistical data were analyzed with SPSS 17.0 software and plotted with Excel software. Each index was expressed as mean ± standard deviation ( soil s ).

Experimental results:

(1) Growth curve assay for the inhibition of growth of Mum-2C cells by doxycycline. As shown in the growth curves of Figure 9A and Figure 9B, doxycycline has a certain inhibitory effect on the growth of Mum-2C cells. The results showed that the effect of doxycycline on the growth of Mum-2C cells was dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

According to the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated. The doxycycline significantly prolonged the doubling time of Mum-2C cells at a concentration of 0.3975 μΜ, 0.795 μΜ, 1.59 μΜ, When 3.18 μΜ and 6.36 μΜ of doxycycline, the doubling times of Mum-2C cells were 21.63, 22.37, 22.72, 24.04 and 35.56 hours, respectively, compared with the control group. Compared with 17.93 hours, they were extended by 20.67%, 24.75%, 26.73%, 34.12% and 98.32% respectively. Mum-2C is a clone of choroidal melanoma liver metastasis. Its cell characteristics are long fusiform, multi-pseudopod, infiltrating growth, and bidirectional differentiation. After six days of treatment with doxycycline (6.36 μΜ), the cells were spindle-shaped, with filopodia reduced, and the cells arranged like fibroblasts, suggesting that the cells had differentiated into the mesenchymal cells. At the same time, it can be seen that after the drug treatment, the number of cells is significantly reduced, and some cells are fragmented and aged to form flat disc cells (see Fig. 9C).

(2) Growth curve assay for the inhibition of growth of Mum-2B cells by doxycycline. As shown in the growth curves of Figure 10A and Figure 10B, doxycycline has a certain inhibitory effect on the growth of Mum-2B cells. The results showed that the effect of doxycycline on the growth of Mum-2B cells was dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on the doubling time of Mum-2B cells, and the experimental concentrations are 0.674 μΜ, 1.35 μΜ, 2.7 μΜ, After 5.4 μΜ and 10.8 μΜ of doxycycline, the cell doubling time of Mum-2B cells was 14.37, 16.09, 21.78, 26.34 and 29.87 hours, respectively, which was 16.30% and 30.23%, respectively, compared with 12.35 hours in the control group. 76.29%, 113.25% and 141.74%.

Mum-2B cells are primordial choroidal melanoma clones with epithelial differentiation characteristics, polygonal, paving stone, and clone-like growth. After administration of doxycycline (2.7 μΜ) for two days, apoptosis was directly induced, and the cells lost adhesion, fragmentation, and the boundary was unclear, and the number of cells was significantly reduced (see Fig. 10C).

(3) Growth curve method for the effect of doxycycline on the growth of Α875 cells

As shown in the growth curves of Fig. 11A and Fig. 11B, doxycycline inhibited the growth of Α875 cells, and the statistical results showed that the effect of doxycycline on the growth of Α875 cells was dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

According to the cell count results, the doubling time of the cells in the control group and each drug-added group was calculated. The doxycycline had a significant prolongation effect on the doubling time of Α875 cells. The experimental concentrations were 0.602 μΜ, 1.204 μΜ, 2.408 μΜ多西环The doubling time of Α875 cells was 37.85, 40.07, and 44.28 hours, respectively, which was extended by 23.05%, 30.26%, and 43.95% (4.86 μM and 9.632 μΜ, respectively, compared with 30.76 hours in the control group). The inhibition of Α875 cells is too strong, Causes the inability to calculate cell doubling time).

A875 is a human skin melanoma cell line. Under normal conditions, the cells have polygonal cells with slightly pseudopods. They tend to grow like clones, have clear cell contours, have good diopter in the nuclear region, and contain small amounts of particles in the cells. After administration of doxycycline (4.816 μΜ) for 2 days, the number of cells decreased, some cells shrunk, some cells showed a flat aging morphology, and the number of suspended cells increased, and a large number of particles were observed in the cells (see Fig. 11C).

(4) Growth curve method for the effect of doxycycline on the growth of Α375 cells

As shown in the growth curves of Fig. 12A and Fig. 12B, doxycycline inhibited the growth of Α375 cells, and the statistical results showed that the effect of doxycycline on the growth of Α375 cells was dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

According to the results of cell counting, the doubling time of the cells in the control group and each drug-added group was calculated. The doxycycline significantly prolonged the doubling time of A 375 cells. The concentration of the drug in the experiment was 1.03875 μΜ, 2.0775 μΜ, 4.155 μΜ. 8.31 μΜ and 16.62 μΜ doxycycline, the doubling time of Α375 cells was 25.89, 26.92, 30.12, 34.49 and 48.67 hours, respectively, compared with 24.18 hours of the control group, respectively, extended by 7.07%, 11.30%, 24.55%. , 42.64% and 101.26%.

Α375 cells are classical human skin melanoma cell lines, polygonal, filopodia, with more melanin particles in the cells. Two days after docetaxel (16.62 μΜ), the cells shrunk, the outline was unclear, the nuclear diopter disappeared, and the cells were fragmented and suspended (see Figure 12C).

It can be seen that the test results of in vitro growth curves show that doxycycline has a certain inhibitory effect on the growth of four human melanoma cells, Mum-2C, Mum-2B, A875 and A375. The statistical results show that doxycycline The doubling time of the above four melanoma cells can be significantly increased, and the effect of doxycycline on the above four cell doubling times is dose-dependent.

3. Effect of doxycycline on the invasive ability of human melanoma cells Mum-2C, Mum-2B, A875 and A375

Method steps:

(1) Cell recovery and culture

The frozen cells were removed from the liquid nitrogen and immediately placed in a 37 ° (water bath to melt the cells. Biosafety Rejection) The cell suspension was pipetted into a centrifuge tube containing the appropriate amount of medium and centrifuged at 800 rpm for 5 minutes. Discard the supernatant, suspend the cells in 1 mL medium, and pipette the cells in the appropriate amount of medium. In the dish, the cells were cultured at 37 ° C, 5% CO ₂ , and saturated humidity. Passage when the cells reach 80%-90% contact confluence, 0.2% trypsinization into a single cell suspension, centrifugation at 800 rpm/min for 5 minutes; discard the supernatant, add 1-2 mL medium to suspend the cells, and place the cells Transfer to 2-3 petri dishes containing appropriate medium and continue the culture.

(2) Trans well chamber preparation

Coating the basement membrane: Matrigel was diluted 1:1 with serum-free medium for the upper chamber of the bottom membrane of the Transwell chamber coated with a 24-well plate. Add 50 of the above culture solution per well and incubate in a 37 °C carbon dioxide incubator. 30 min-1 h.

Hydration basement membrane: Aspirate the residual liquid in the culture plate, add 50 serum-free medium containing 10 mg/mL BSA to each well, incubate at 37 °C for 30 min.

(3) Preparation of cell suspension

The cells in the logarithmic growth phase were trypsinized, the digestion was terminated, the culture was discarded by centrifugation, washed 1-2 times with PBS, and resuspended in serum-free medium containing BSA. Adjust the cell density to 5 X 10 ⁵ .

(4) Inoculated cells

Take 200 μL of cell suspension into Transwell chamber; 24 well plate lower chamber, add 500

10% FBS medium; no added drug in the control group, only the corresponding amount of solvent was added, the experimental group was added with the corresponding concentration of doxycycline (the highest drug concentration without obvious toxicity to the cells within 48 h), 37 °C Incubate for 24 h in a carbon dioxide incubator. Three replicates were set for each sample. The positive drug amylophylline hydrochloride was carried out in the same manner to determine the stability of the experiment.

(5) fine count

The matrigel and the cells in the upper chamber were wiped off with a cotton swab; the migrated cells were stained with 0.1% crystal violet, and the cells were counted under a microscope, and the results of the experiment were statistically calculated to calculate the migration inhibition rate (experimental method reference: Wei Wei, Wu Ximei, etc. The Pharmacological Experimental Methodology, Fourth Edition, Beijing: People's Medical Publishing House, 2010: 1627-1628).

Real horse results:

(1) Inhibition of invasive ability of Mum-2C cells by doxycycline

The inhibitory effect of doxycycline on Mum-2C cell invasion is shown in Fig. 13A and Fig. 13B: Fig. 13A shows the results of microscopic observation under the microscope of microscopy after treatment with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. , can be seen that compared with the control group, doxycycline attack on Mum-2C cells Ability has a strong inhibitory effect. The statistical results (Fig. 13B) showed that the inhibitory rate of doxycycline on Mum-2C cell migration at a concentration of 1 μΜ reached 45.12% (P<0.01, which was significantly different from the control group). When the doxorubicin hydrochloride was 0.781 μΜ, the inhibition rate of Mum-2C cell migration was 38.09% (P<0.01, which was significantly different from the control group).

(2) Inhibition of invasive ability of Mum-2B cells by doxycycline

The inhibitory effect of doxycycline on the invasive ability of Mum-2B cells is shown in Figures 14A and 14B: Figure 14A shows the results of microscopic observation under microscopic microscope after treatment of cells with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. It can be seen that the inhibitory effect of doxycycline on the invasive ability of Mum-2B cells is weak compared with the control group. The statistical results (Fig. 14B) showed no significant inhibition of the invasive ability of doxycycline on Mum-2B cells at 1 μΜ. (P>0.05, there was no significant difference compared with the control group). When the concentration of doxorubicin hydrochloride was 0.5 μΜ, the inhibition rate of Mum-2B cell migration was almost 100% (P < 0.01, which was significantly different from the control group).

(3) Inhibition of the invasion ability of A875 cells by doxycycline

The inhibitory effect of doxycycline on the invasive ability of A875 cells is shown in Figures 15A and 15B. Figure 14A shows the results of crystal violet staining under the microscope after treatment with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. It was found that doxycycline had a strong inhibitory effect on the invasive ability of A875 cells compared with the control group. The statistical results (Fig. 14B) showed that the inhibition rate of doxycycline on migration of Α875 cells at 0.5 μΜ reached 65.69% (Ρ<0.01, which was significantly different from the control group). The inhibition rate of doxorubicin hydrochloride on migration of Α875 cells reached 38.31% (Ρ<0.05, which was significantly different from the control group).

(4) Inhibition of invasive ability of Α375 cells by doxycycline

The inhibitory effect of doxycycline on the invasive ability of Α375 cells is shown in Figures 16A and 16B: Figure 16A shows the results of crystal violet staining under the microscope after treatment with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. It was found that doxycycline had a strong inhibitory effect on the invasive ability of A375 cells compared with the control group. The statistical results (Fig. 16B) showed that the inhibitory effect of doxycycline on the migration of Α375 cells at 0.2 μΜ reached 35.43% (Ρ < 0.01, which was significantly different from the control group). The inhibition rate of Α375 cells migration by doxorubicin hydrochloride at 1 μΜ reached 28.81% (Ρ < 0.01, which was significantly different from the control group).

It can be seen that doxycycline is at the low concentration in vitro for three human melanoma cells Α875, Α375, The invasive ability of Mum 00-2C has a significant inhibitory effect. Low concentration of doxycycline on Mum-2B fine 4''

The invasive ability of the cells has no significant inhibitory effect. Example 2 Study on the effect of doxycycline on human breast cancer cells MCF-7 and MDA-MB-231 in vitro

1 MTT assay for the inhibitory effect of doxycycline on the growth of human breast cancer cells MCF-7 and MDA-MB-231

Method steps:

The method described under the corresponding MTT assay in Example 1 is the same except that the cell lines used in this example are human breast cancer cells MCF-7 and MDA-MB-231.

Experimental results:

(1) MTT assay for the inhibitory effect of doxycycline on the growth of MCF-7 cells

As shown in Table 9 and Figure 17 and Table 10 and Figure 18, the growth of MCF-7 cells by doxycycline showed a significant inhibitory effect at low doses. As the concentration of doxycycline increased, The inhibitory effect of MCF-7 cells gradually increased in a dose-dependent manner.

The preliminary results showed that the half-inhibitory concentration of doxycycline on MCF-7 cells (IC ₅ value)

,

It is 2.087±0.0268 μΜ. The official experimental results show that the IC _{5 ()} value of doxycycline on MCF-7 is

2.3103+0.1507 μΜ. The experimental results showed that the positive drug, doxorubicin hydrochloride, had an IC ₅₀ value of more than 50 μM for MCF-7 cells, and the inhibitory effect on the growth of MCF-7 cells was weak. Table 9 Inhibition of MCF-7 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-experimental results)

! Doxy-1 inhibition ratio ADR- survival rate survival sharing inhibition concentration _{': μΜ) COD ¾ 7o mil} ) (¾) (%) conc ¾ iM) (%) (: %)

0 Ϊ 045 0.0843 100 0 0 0.5755^0.0213 100 0

0.08 0.6537±0.0524 63.03 36,97 0.3906 0.4303^0.0024 74.78 25,22

0.2 0.536Η0.Ϊ043 4S.7 0.7S125 0Λ426 )Μ56 23.09

0.69 0.4969 0.1991 47 55 1.5625 0.390S±O.O097

2,06 0.4347i0.012S 41—60 5S.4 3.125 0.3661+0.0069 63.61 36.39

6.17 12.79 87.21 6.25 0.3589+0.0035 37.<53

0.0693 0.0274 6.63 93;37 12.5 0.37 3±0.0096 64.86 35.14

5556 0.0617^0.0059 5.91 94,09 25 0.3275±{}.Di46 56.90 43J0

O. I412iO.{)601 13 52 S6.4S 50 0.270 ί ±0,0044 46.93 53.07

100 0.1S66±OA>274 Table 10 Inhibition of MCF-7 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (official experimental results)

Doxy-2 x±s retention ' 'strain inhibition rate ADR-2 x±s retention rate

(%:} (3⁄4) Concentration μΜ) (ο _7ΰ ( _3⁄4 :'

0 1.0286H - 0,0255 100 α 0 0,765^0.0475 100 0

03906 0.5697i0.01S6 55.38 44.62 0.0488 84.71 15.29

0.7S125 0.5563i0.0383 54Λ")8 45.92 0.0976 0.6326^0.0013 82.6 17.3 1

1.5625 0.4336 0.0167 42, 16 0.6395=^0.0047 !6.41

:2i 2±0fine 4 3 1.23 0.6 0 0259 S0 16 19.84

6.25 0.15 1+0.0040 15.47 S4.53 .5305±0.016S 30.66

12 5 0.052 3⁄4±0.0028 5.08 94.92 1.5625 0.5416±0.0223 70 80 29.20

2 4 01 95.99 3.125 0.4965^0.0209 64.90 35Λ0

50 0,0404 0.0036 3.92 96,08 6,25 0.555 ii0.0 i36 72.57 27.43 a~

100 0.04S6i0.008S 4.73 ? o.7 12.5 0.466Si0. 1?2 61.02 38.98 ό

(2) MTT assay for the inhibitory effect of doxycycline on the growth of MD pA-MB-231 cells

·

As shown in Table 11 and Figure 19 and Table 12 and Figure 20, the effect of doxycycline on MDA-MB-231 cells

■

It has obvious inhibitory effect when it is grown at low dose. With the increase of doxycycline concentration,

Fl ■

The inhibitory effect of MDA-MB-231 cells gradually increased, showing a dose-dependent effect of i.

g

0·

The preliminary results showed that the half-inhibitory concentration (IC ₅₀ value) of doxycycline and the positive drug doxorubicin hydrochloride on MDA-MB-231 were 1.0436 ± 0.0845 μΜ and 0.7248 ± 0.2459 μΜ, respectively.

0

The results of formal experiments showed that the IC ₅₀ values of doxycycline and the positive drug doxorubicin hydrochloride for MDA-MB-231 were 1.5567 ± 0.0100 μΜ and 0.6660 ± 0.0938 μΜ, respectively.

Table 11 Inhibition of MDA-MB-231 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-experimental results)

Doxy-1 x±s Survival ίΦ rate AD -! Survival rate » System concentration (μΜ:》 ^{ : OD 7o inn ) (% ') ( 3⁄4■) Concentration (μΜ) i OD 70 wi) ( 3⁄4 ) (3⁄4

0 1 ,2455±0.0599 100 0 0 0 _Γ 9697÷ΐ} 0421 100 0

0.1953 0,708 i±0.0I93 56.S5 43.15 0.5641=0.0109 58.17 41. S3

0.3906 0.636±0.0537 51.1 48,9 0.3906 0.568i0. 273 5:8.57 41.43

0.7S125 0.5333±0.0466 57.19 0.78125 0.4055 0.0715 58.18

0.3 I38±0.0177 74.81 1.5625 33.03 66,97

0.223 1 ±0.0046 1 7.91 0.3168÷ 00 i6 32.67

6.25 0. Ϊ 1 7+0.0040 9.39 90.61 6,25 0.2856=0 0139 29.45 70.55 2.5 0,0353±0.0009 2.S3 97.17 12.5 24.10 75.90

25 D.03S3±0.0016 3.08 25 Ο. ί 993=0.0043⁄4 20.56 ?9.44

50 D.052()±O.OO31 4.17 95 83 50 Oi 907:0.0097 19.67 Table 12 Inhibition of MDA-MB-231 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (official results) ) D x -2 x±s AD -2 x±&

d Survival rate survival rate inhibition; rate concentration (μΜ) OD ₅ 7 mii) i%) (%, > degree ( μΜ } C3⁄4 ') ( )

0 I.5944±0.01S3 100 0 0 I.6239i0.0206 100 0

L2S3i±0.03"34 80.48 0.1953 I.i035i0.0416 32.05

0.3906 1.2579 0.0474 21.10 0.3906 1.0479^0.0093 64.53 35.47

0.7S125 .II45±0,0375 69.90 30,10 0.-357S chase 52.82 47.18

1.5625 .6i6i .OOS2 38-63 61.37 1.5625 0.6555i0.G52S 40.37

0,4923±0.0I65 30.88 69.12 0.6053 0.0483 37.28

6.25 0.3256±0.0144 20.42 79.58 6.25 0.5857i().0498 36.06 63.94

12.5 2.57 97.43 12.5 0.554 ±0.0471 34.17 65. S3

25 0.0567 0.0033 1.55 96.45 25 0.4957iO.G09i 30.53

50 0,0486^0.0050 3.05 96.95 50 0.399^0.009 24.57 75.43 o

It can be seen that the growth of MCF-7 and MDA-MB-231 cells by doxycycline in vitro by MTT assay,

Inhibition, it was found that doxycycline inhibited the growth of both breast cancer cells in a dose-dependent manner, IC ₅ . The values are all below 10 μΜ (about 5 g/mL).

2. Growth curve assay for the inhibitory effect of doxycycline on the growth of MCF-7 cells

Method steps:

d Only the cell line used in this example is human breast cancer cell MCF-7

Experimental results:

(1) Growth curve assay for the inhibitory effect of doxycycline on the growth of MCF-7 cells

As shown in the growth curves of Fig. 21A and Fig. 21B, doxycycline has a certain inhibitory effect on the growth of MCF-7 cells. Statistical results show that doxycycline has a dose-dependent effect on the growth of MCF-7 cells. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

According to the results of cell counting, the doubling time of the cells in the control group and each drug-added group was calculated. The doxorubicin had a significant prolongation effect on the doubling time of MCF-7 cells. The concentration of the drug in the experiment was 0.65625 μΜ 1.3125 μΜ 2.625 μΜ 5.25. When μΜ and 10.5 μΜ of doxycycline, the doubling time of MCF-7 cells was 31.43 35.87 44.89 54.73 and 87.75 hours, respectively, which was extended by 6.54%, 21.59%, 52.17% and 85.52, respectively, compared with 29.50 hours of the control group. % and 197.41%

MCF-7 is a highly metastatic breast cancer cell line with clonal growth of polygonal epithelial cells with intercellular bridges. After administration of doxycycline (10.5 μΜ) for three days, a large number of vacuoles appeared in the cells, the bridge disappeared, the contours were blurred, the cells were shrunk and suspended, and the number of cells was significantly reduced (see Figure 21C). The cell growth curve test was performed in vitro. It was found that doxycycline is fine for human breast cancer. The growth of MCF-7 has a certain inhibitory effect, which can significantly prolong the doubling time of cells. The statistical results show that the effect of doxycycline on the doubling time of MCF-7 cells is dose-dependent.

3. Effect of doxycycline on the invasive ability of human breast cancer cells MCF-7 and MDA-MB-231 Method steps:

The method described under the corresponding invasiveness test in Example 1 was the same except that the cell lines used in the present example were human breast cancer cells MCF-7 and MDA-MB-231.

Experimental results:

(1) Inhibition of invasive ability of human breast cancer cell line MCF-7 by doxycycline

The inhibitory effect of doxycycline on the invasive ability of human breast cancer cell line MCF-7 is shown in Figure 22A and Figure 22B: Figure 22A shows crystal violet staining after treatment of cells with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. The results observed under the microscope showed that doxycycline had a strong inhibitory effect on the invasion ability of MCF-7 cells compared with the control group. The statistical results (Fig. 22B) showed that the inhibition rate of doxycycline on migration of MCF-7 cells at 0.2 μΜ reached 40.43% (Ρ < 0.01, which was significantly different from the control group), and the concentration of doxorubicin hydrochloride at 0.2 μΜ The inhibition rate of migration to MCF-7 cells reached 52.34% (Ρ < 0.01, which was significantly different from the control group).

(2) Inhibition of invasive ability of human breast cancer cell line MDA-MB-231 by doxycycline The inhibitory effect of doxycycline on the invasive ability of human breast cancer cell line MDA-MB-231 is shown in Figs. 23A and 23B. Fig. 23A shows the results of 24 hours of crystal violet staining after treatment of cells with doxycycline and the positive drug doxorubicin hydrochloride. It can be seen that doxycycline to MDA-MB-231 compared with the control group. The invasive ability of cells has a strong inhibitory effect. The statistical results (Fig. 23B) showed that the inhibition rate of doxycycline on migration of MDA-MB-231 cells was 0.15% at 0.1 μΜ (P < 0.05, which was significantly different from the control group). The inhibition rate of doxorubicin hydrochloride on migration of MCF-7 cells at 0.1 μΜ was 20.4% (Ρ < 0.05, which was significantly different from the control group).

It can be seen that the effect of doxycycline on cell invasion ability was examined by Transwell method. The results showed that doxycycline can significantly inhibit the invasion of human breast cancer cell lines MCF-7 and MDA-MB-231 at low dose in vitro. ability. Example 3 Effect of doxycycline on human leukemia cells K562 and HL60 in vitro

1. MTT assay for the inhibitory effect of doxycycline on the growth of human leukemia cells K562 and HL60

Method steps:

The method described in the corresponding MTT assay in Example 1 was the same except that the cell lines used in the present example were human leukemia cells K562 and HL60. At the same time, K562 and HL60 cells are suspension cells, which do not need to be digested with trypsin before passage and plating.

Experimental results:

(1) MTT assay for the inhibitory effect of doxycycline on the growth of K562 cells

As shown in Table 13 and Figure 24 and Table 14 and Figure 25, doxycycline significantly inhibited the growth of K562 cells at low doses, and increased the concentration of doxycycline on K562 cells. The inhibitory effect is gradually increased, showing a significant dose dependency.

The preliminary results showed that the half-inhibitory concentration (IC ₅ ) of doxycycline on K562 cells was 1.4483+0.0996 μΜ. The results of formal experiments indicate that doxycycline affects IC ₅ in Κ562 cells. The value is 2.9403 + 0.2778 μΜ. The experimental results showed that doxorubicin hydrochloride inhibited the growth of Κ562 cells in vitro (1( ₅ . value greater than 50^1).

Table 13 Inhibition of K562 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-experimental results)

Doxy-l ±s Survival rate ADR-1 x±3⁄4 Survival rate of concentration ί' μΜ.) (OD. _17a ian) (%) Concentration ί μΜ ) ''' ODj g nmi ( % ) (% }

0 3.7504±0.0512 100 0 0 1.6S52 times 776 100 0

0.3906 2.4783± .02S1 66.08 33.92 0.0977 1.238±0.0273 73.46 26.54

0.7S I25 2.6304±0.19I 1 70.14 29.86 0.1953 1.4392i0.0537 85.40 14.60

1.5625 1.9913±0.0868 53.10 46.90 0.3906 i .3353±0.07S8 79.24 20.76

3.125 1.15 S±0.035S 30.69 69-31 0.78125 1.2814i0.1091 76.03 23.97

6.25 0.65S3±0.046 17.55 82.45 1.5625 1.263±0.0?57 74.94 25.06

12.5 0.3O8±O.OO37 8.21 9L7 3.125 1.2477±0.1063 74.04 25.96

25 0.3033 soil 0.0143 8.09 91.91 6.25 1.2743±0.0801 75.61 24.39

50 0.424±0.0039 1 1.30 88.70 12.5 1.1525 ± 0.07] 8 68.39 31.61 i OO 0:7220 ± 0.0124 19.25 80-75 25 1.287 Gentleman 0.127S 76.38 23.62 Table 14 Inhibition of K562 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (official experimental results)

Doxy-2 X s storage '牟A rate of ADR- 2 xs survival rate»rate concentration μΜ) o (OD ί OD _57t} iim) ! %)

0 1- 3Ϊ4 0.16 4 100 0 0 100 0

0.0977 0.7252±0 o.02S9 0.1953 L 724^0.013 98.34 1.66

0.1953 0.654 ±O.OS2 tο·5 33.91 66.09 03906 1.S92S 0.0947 94. 7 5.63

0.3906 32.32 67,68 0.7S125 79.96 20.04

0.78125 0.5S4± .Oi72 30.24 1.5625 77.S3 2217

1.5625 0 9 0.0707 22,31 77.69 3.125 1.567Si0. 85 21.84

625 1, 55 i 9 0,0546 22,63

625 772 12.5 78.53

12,5 0.043 0.0022 Q ii7 r 77 25 1.5716^0.0633 7S.3 21.64

25 GDI 7g .0034 0.92 99. OS 50 2.2282^0.0496 11109 -1L09

(2) MTT assay for the inhibitory effect of doxycycline on the growth of HL60 cells

See Table 25 and Figure 26 and Table 16 and Figure 27. The growth of HL60 cells with more than 1 cyclin showed a significant inhibitory effect at low doses. As the concentration of doxycycline increased, the inhibitory effect on HL60 cells increased gradually, showing a dose-dependent effect.

Pre-experimental results indicate doxycycline and the positive drug doxorubicin hydrochloride. The half-inhibitory concentration (IC ₅₀ value) for HL60 was 1.4813 ± 0.2241 μΜ and 0.6532 ± 0.0660 μΜ, respectively. The results of formal experiments indicate the IC _{5 of} doxycycline and the positive drug doxorubicin hydrochloride on HL60. The value is j

0.9774 ± 0.0799 μΜ and 0.3560 ± 0.0121 μΜ

Table 15 Inhibition of HL60 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-experimental results)

Dox -2 control rate ADR-2 survival rate mm concentration (μΜ (%.) concentration ίμΜ) OD ₅₇ dish) (3⁄4)

0 1.6711^0.0703 100 0 0 1.1027i0.0212 100 0

0.1953 1.5201±0.09ό5 90.96 9.04 0.1953

Π4.57 -14.57

0.3906 L2849^0.0562 76. S 23.11 03906 1.4087+01)684 127.75 -27.75

0.78125 1.069 M).0236 64.02 35.9S 0.7SI25 0.9906+0.0221 S9.S3 10.17

1,5625 0.977^0. 181 58.47 4L53 1.5625 0.4926^0.0154 44.68 55.32

0.5769i0.0014 34.52 65.4S 3.125 0.4928^0.0016 44..69 55.31

6.25 O.3578i .ai09 21.41 78.59 6.25 0,4379 0,0136 39.71 60.29

12.5 0,2169 0.0017 1 . S S7.02 Ϊ2-5 33.81 66.19

25 0.2047±0.0042 12.25 87.75 25 0.3175=^)0172 28.80 71.20

50 0,1804^0.0089 1 .S S9.2 50 0.2985+01)231 27.07 72.93 Table 16 Inhibition of HL60 cells by Doxy and doxorubicin hydrochloride (ADR) (official experimental results)

Dox -2 x±s survival rate castration rate AD -2 x±s survival rate inhibition rate

(OD _57. Leg) ( ) (.%) Concentration (μΜ) OD5 nm) (%) i3⁄4)

0 1.93S5i0. 707 Ϊ00 0 0 I.3W>-C>143 100 0

1.6105i0. 1 3 S3. OS 16.92 .Ϊ953 117.58

0.3906 i.3 7÷0.1023 32.5S 0.3906 1.3071=0.0293 99.78 0.22

0.78 Ϊ25 1150S±{}.0735 59.37 40,63 0.8985=0.0150 68.59 31.41

0.7293+0.0126 1.5625 O, 5S03÷O.025S 44.3 55.7,

0.5504÷0. 199 28,.39 7i.61 3,125 0 736 0.0088 36.15 63,85

6.25 0.2824^0.0011 14.57 85.43 6.25 0.4046=0.0160 30.89 69.il

12.5 O.ni9i0. 043 5,77 12.5 0.3421=0.0035 73. S8

25 0.14S9±0.0033 7.6S 25 0.3326^0.0024 2,5.39 74.61

50 01521 Discussion 12 7.S5 92 J 5 50 0—2724 0057 20.79 ?9.21 It can be seen that the inhibition of the growth of K562 and HL60 cells by in vitro detection of doxycycline by MTT method

The effect of doxycycline on the growth of these two leukemia cells was found to be in a dose-dependent manner, IC ₅ . Values are below 10 μΜ (approx. 5 g/mL).

H ■

2. Growth curve method to detect the inhibitory effect of doxycycline on the growth of human leukemia cells K562 and HL60

Method steps: Only the cell lines used in this example were human leukemia cells K562 and HL60. At the same time, r-K' 562 and HL60 cells were suspended cells, which were not digested with trypsin before passage and plating.

Experimental results:

(1) Growth curve assay for the inhibitory effect of doxycycline on the growth of K562 cells

As shown in the growth curves of Fig. 28A and Fig. 28B, doxycycline has a certain inhibitory effect on the growth of K562 cells. The statistical results show that the effect of doxycycline on the growth of K562 cells is dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: when 1.476 μΜ, 2.94 μΜ, 5.88 μΜ, and 11.76 μΜ of doxycycline, the doubling time of the Κ562 cells was 14.79, 15.00, respectively. 16.50, 17.74, and 19.72 hours, compared with 13.56 hours in the control group, were extended by 9.08%, 10.62%, 21.64%, 30.85%, and 45.40%, respectively. K562 is a chronic myeloid leukemia cell line in which cells are suspended and have a distinct concentric lens contour. After administration of doxycycline (11.76 μΜ), the cells shrunk, shattered, and the refractive index increased, the concentric contour disappeared, and the cell volume decreased and the number decreased (see Figure 28C).

(2) Growth curve assay for the inhibitory effect of doxycycline on the growth of HL60 cells

As shown in the growth curves of Fig. 29Α and Fig. 29Β, doxycycline has a certain inhibitory effect on the growth of HL60 cells. Statistical results show that doxycycline has a dose-dependent effect on the growth of HL60 cells. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on the doubling time of HL60 cells, and the experimental concentrations are 0.304 μΜ, 0.607 μΜ, 1.214 μΜ, and 2.428. When μΜdoxcycline was used, the doubling time of HL60 cells was 32.42, 42.52, 42.83 and 52.82 hours, respectively, which was 8.14%, 41.82%, 42.86% and 76.19%, respectively, compared with 29.98 hours in the control group. μ Μ doxycycline inhibited HL60 cells too much, which made it impossible to calculate the doubling time).

HL60 is a promyelocytic leukemia cell line. The cells are suspended and have a distinct concentric circular membrane contour, and some cells tend to adhere to a cluster. After doxycycline ( 1.214 μΜ ), the cells shrunk, shattered, concentric contours disappeared, cell agglomeration was evident, cell volume decreased, and the number decreased (see Figure 29C).

It can be seen that the in vitro cell growth curve test found that doxycycline has a certain inhibitory effect on the growth of human leukemia cells Κ562 and HL60, and can significantly increase the doubling time of cells. The statistical results show that doxycycline on Κ562 cells and The effect of HL60 cell doubling time was dose dependent. Example 4 Study on the effect of doxycycline on human lung cancer cells NCI-H460 and NCI-H446 in vitro

1. Inhibition of doxanthin on the growth of human lung cancer cells NCI-H460 and NCI-H446 by sputum method

Method steps:

The method described in the corresponding sputum detection in Example 1 was the same except that the cell lines used in the present example were human lung cancer cells NCI-H460 and NCI-H446.

Experimental results: (1) MTT assay for the inhibition of the growth of NCI-H460 cells by doxycycline as shown in Table 17 and Figure 30 and Table 18 and Figure 31. The growth of doxycycline on NCI-H460 cells at low doses Significant inhibition was observed. As the concentration of doxycycline increased, the inhibitory effect on NCI-H460 cells increased gradually, showing a dose-dependent manner.

The preliminary results showed that the half-inhibitory concentration (IC ₅₍₎ value) of doxycycline on NCI-H460 cells was 1.0638±0.1266 μΜ. The results of formal experiments indicate the IC _{5 of} doxycycline on NCI-H460. The value is 1.9340 ± 0.0286 μΜ. The experimental results showed that doxorubicin hydrochloride did not significantly inhibit the growth of NCI-H460 cells.

Table 17 Inhibition of NCI-H460 cells by Doxy and Doxorubicin hydrochloride (ADR) (pre-experimental results)

Doxy-1 X s Survival rate ADR-I Survival inhibition rate concentration ''μΜ) (OD;, 7oiim) % (%; 'concentration ί μΜ ) ( OD _{: :7fJ} nm (%■}

0 75±()Mi2 100 0 0 2.0906±0.i240 WO 0

0.3906 0.76 2±0.02S4 86.67 13.33 0.7S125 2.0672±0.07i3 986 ί.4

0.5674±.03Ϊ9 63.94 36.06 1.5625 1.9 84±0.115S 93.41 6.59

1.5625 D.3793±0.01S6 4273 57.27 3.125 l.SO71^0.1854 6A9 13,81

3.125 0.3125±0.01S7 35.21 64.79 6.25 2.14S0i0.03 7 102.44 -2.44

6.25 0.1945±0.0075 21.9! 78,09 125 2.(}743i(},1374 98.93 1.067

12.5 0,069S±0.0022 7.S7 92,13 25 2.1733i0lS36 103.66 -3.66

25 0.0322 chase 0071 3.63 96.37 50 2. 16S±0.Q502 139.12 -39.12

50 0.0252^0.0072 2M 97.16 100 3.1343+O.0S6O 149.49 -49.49 Table 18 Doxy and doxorubicin hydrochloride (ADR) for NCI-H460 cells

'Production (official experiment)

Doxy-2 activity Φ 'jfc ij rate AD -2 survival rate (μΐνυ 0 31%) concentration (μΜ.) ( ()D ₅ 7,[j Ι1Ϊ11 ) 3⁄4) (%)

0 3.3365^0.0074 100 0 0 3.39S±0,007i iOO 0

0.1953 I.627S± .0223 48.79 51.21 0.Ϊ953 3.31S+ .0209 97.65 2.35

0.3906 L450S± 0 1C) 43.48 56.52 0.390ό 3.3069±0.0S0S 97-32 2.68

0.78125 1.27S ± .0S98 38.33 61.67 0.78125 3.3033±O.O793 t722 2.78

1.5625 1.0309^0.0006 30.9 69.1 [3625 3, 2743 0.0 i 01 96.36 3.64

3.125 0.5563 0.0066 16,61 83.33 3.125 3 HO, 1149 93.28 6.72

625 0.4S17i0.0 57 14.44 S5.56 6.25 2.9858^0.2425 87. S7 12.13

12.5 0.2741 0.0024 S.2I 91.79 12.5 3.2729 0.0520 96.32 3.68

25 0.0711 ±0.0005 2.13 7.S7 25 3.2863 O.i 100 96.71 3.29

50 .05iS± .0002 1.55 9845 50 3.3778±0.0452 99-41 0.59 ( 2) MTT assay for the inhibitory effect of doxycycline on the growth of NCI-H446 cells

As shown in Table 19 and Figure 32 and Table 20 and Figure 33, the growth of NCI-H446 cells by doxycycline showed a significant inhibitory effect at low doses. As the concentration of doxycycline increased, The inhibitory effect of NCI-H446 cells gradually increased in a dose-dependent manner. The preliminary results showed that the half-inhibitory concentration (IC ₅ o value) of doxycycline and the positive drug doxorubicin hydrochloride on NCI-H446 were 0.3871±0.1203 μΜ and 3.2700±0.0644 μΜ, respectively. The results of formal experiments showed that the IC ₅₍₎ values of doxycycline and the positive drug doxorubicin hydrochloride for NCI-H446 were 1.7043±0.1241 μΜ and 4.3313±0.3911 μΜ, respectively.

Table 19 Inhibition of NCI-H446 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-experimental results)

It can be seen that the inhibitory effect of doxycycline on the growth of NCI-H460 cells and NCI-H446 cells was detected by MTT assay in vitro. It was found that doxycycline inhibited the growth of these two lung cancer cells in a dose-dependent manner. . IC ₅ . Values are below 10 μΜ (approx. 5 g/mL).

2. Growth curve method for the detection of doxycycline on human lung cancer cells NCI-H460 and NCI-H446 Long inhibition

Method steps: Only the cell lines used in this example were human lung cancer cells NCI-H460 and NCI-H446.

Real horse results:

(1) Growth curve assay for the inhibitory effect of doxycycline on the growth of NCI-H460 cells. As shown in the growth curves of 34A and 34B, doxycycline inhibited the growth of NCI-H460 cells. The results showed that the effect of doxycycline on the growth of NCI-H460 cells was dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on the doubling time of NCI-H460 cells at a concentration of 0.5 μΜ, 1.0 μΜ, 2.0 μΜ, The doubling time of NCI-H460 cells after 4.0 μΜ and 8.0 μΜ of doxycycline was 48.56, 58.60, 81.13, 116.13 and 208.47 hours, respectively, which was 17.97%, 42.34% and 97.09%, respectively, compared with 41.17 hours in the control group. , 182.09% and 406.43%.

NCI-H460 cells are human large cell lung cancer cell lines, characterized by epithelial cells, clone-like growth, polygonal shape, and very few cells are fusiform. After administration of doxycycline ( 7.724 μΜ ) for 3 days, the cell outline disappeared, the refractive index decreased, the intercellular bridge disappeared, and most of the cells were fragmented or condensed (see Figure 34C).

(2) Growth curve assay for the inhibitory effect of doxycycline on the growth of NCI-H446 cells. As shown in the growth curves of 35A and 35B, doxycycline inhibited the growth of NCI-H446 cells. The results showed that the effect of doxycycline on the growth of NCI-H446 cells was dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on cell doubling time at doses of 0.25 μΜ, 0.5 μΜ, 1 μΜ, and 2 μΜ In the case of cyclin, the doubling time of NCI-H446 cells was 16.32, 20.03, 23.56 and 25.81 hours, respectively, which was 18.18%, 45.07%, 70.59% and 86.90%, respectively, compared with 13.81 hours in the control group.

NCI-H446 is a small cell lung cancer of neuroectodermal origin. The cells are small, clone-like, some cells are suspended, and a small number of cells have pseudopods. After administration of doxycycline (3.9404 μΜ) for 3 days, the cell volume became larger, the refractive index disappeared, the pseudopod decreased, the intracellular particles increased, and the suspended cells increased. Most, most cells have aging performance (see Figure 35C).

It can be seen that the in vitro cell growth curve test found that doxycycline has a certain inhibitory effect on the growth of NCI-H460 cells and NCI-H446 cells. The statistical results show that doxycycline on NCI-H460 and NCI-H446 cells. The effect of doubling time is dose dependent.

3. Effect of doxycycline on the invasive ability of human lung cancer cells NCI-H460 and NCI-H446 Method steps:

The method described in the corresponding invasiveness test in Example 1 was the same except that the cell lines used in the present example were human lung cancer cells NCI-H460 and NCI-H446.

Experimental results:

(1) Inhibition of invasive ability of NCI-H460 cells by doxycycline

The inhibitory effect of doxycycline on the invasive ability of NCI-H460 cells is shown in Figures 36A and 36B: Figure 36A shows the crystal violet staining fluoroscopy after 24 h treatment with doxycycline and the positive drug doxorubicin hydrochloride. As a result of the observation, it can be seen that compared with the control group, doxycycline has a strong inhibitory effect on the invasion of NCI-H460 cells. The statistical results (Fig. 36B) showed that the inhibitory effect of doxycycline on NCI-H460 cell migration at 0.08 μΜ reached 50.5 % (Ρ < 0.01 was significantly different from the control group). The inhibition rate of doxorubicin hydrochloride on migration of NCI-H460 cells at 4 μΜ was 49.5%.

(2) Inhibition of invasive ability of NCI-H446 cells by doxycycline

The inhibitory effect of doxycycline on the invasive ability of NCI-H446 is shown in Fig. 37Α and 37Β: Fig. 37ΑAnalysis of cells treated with doxycycline and the positive drug doxorubicin hydrochloride after 24 h, crystal violet staining As a result, it can be seen that compared with the control group, doxycycline has a strong inhibitory effect on the invasion ability of NCI-H446 cells. The statistical results (Fig. 37B) showed that the inhibition rate of doxycycline on the migration of NCI-H446 cells at a concentration of 0.781 μΜ reached 51.84% (Ρ<0.01, which was significantly different from the control group), and the doxorubicin hydrochloride was at 0.1. The inhibition rate of NCI-H446 cell migration was 50.61% (Ρ<0.01, which was significantly different from the control group).

It can be seen that the effect of doxycycline on cell invasion ability was examined by Transwell method. The results showed that doxycycline can significantly inhibit NCI-H460 (human lung adenocarcinoma cell line) and NCI-H446 (human small cell lung cancer) in vitro. Cell line) The ability of cells to invade. Example 5 Study on the effect of doxycycline on human hepatoma cell line HCCLM3 in vitro

1. MTT assay for the inhibitory effect of doxycycline on the growth of HCCLM3 cells

Method steps:

The method described in the corresponding MTT assay in Example 1 is the same except that the cell line used in this example is human hepatoma cell HCCLM3.

Experimental results:

(1) MTT assay for the inhibitory effect of doxycycline on the growth of HCCLM3 cells

As shown in Table 21 and Figure 38 and Table 22 and Figure 39, doxycycline significantly inhibited the growth of HCCLM3 cells at low doses. As the concentration of doxycycline increased,

The inhibitory effect of HCCLM3 cells gradually increased in a dose-dependent manner.

The results show that pre, doxycycline IC ₅ HCCLM3 pair of cells. The value is 2.2367 ± 0.1528 μΜ. The results of formal experiments showed that the IC _{5 ()} value of HCCLM3 cells was 2.2770 ± 0.1576 μΜ. The inhibition of growth of HCCLM3 cells by doxorubicin hydrochloride was weak in vitro.

Table 21 inhibition of HCCLM3 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR)

Q,

Table 22 Inhibition of HCCLM3 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (official experimental results)

Doxy-2 survivor inhibition rate ADR-2 x s rate inhibition rate

(OD _: - _ι7ϋ 1Ίί!1.>':■%) Concentration ' μΜ) ( % )

0 1.196 0.0093 100 0 0 1.3532=0.0934 100

0.7S125 O.S935±O, O570 74.71 25.29 0.7S125 1.2551 0.0723 7,24

1.5625 0.8547i0.0256 71-46 28.54 1 ,5625 1.2033^0.0664 11.07

3.125 0.5335±0.0125 44.6 55.4 3.125 LI 13± .0403 SS.04 11.96

6.25 0.4183±0.0213 34-97 65.03 6.25 1.1616i0.04 0 8 . S4 14.16

12.5 0.0665 0.0037 5.56 94.44 12.5 1.2069^0,0140 89.19 10.81

25 0Λ319± .0041 .66 97.34 8S.84 11.16

50 0.0327 into 0004 2.73 97.27 50 1.2432^0,0152 91.87 8.13

100 0. 543÷0.00i3 4.54 95.46 100 1.0917÷ .0553 80.67 1933

200 0.1533=0.0035 12. S2 87.18 200 0.8981=0.0300 66.37 33,63 It can be seen that the inhibitory effect of doxycycline on the growth of HCCLM3 cells in vitro by MTT assay showed that doxycycline inhibited the growth of HCCLM3 cells in a dose-dependent manner, IC ₅ . The value is below 10 μΜ (about 5 g/mL).

2. Growth curve method for detecting the inhibitory effect of doxycycline on the growth of hepatoma cells HCCLM3. Method steps: Only the cell line used in the present example was human hepatoma cell line HCCLM3.

Experimental results:

(1) Growth curve method for the inhibition of the growth of HCCLM3 by doxycycline

As shown in the growth curves of Fig. 40A and Fig. 40B, doxycycline has a certain inhibitory effect on the growth of HCCLM3 cells. The statistical results show that the effect of doxycycline on the growth of HCCLM3 cells is dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on cell doubling time at doses of 0.7805 μΜ, 1.561 μΜ, 3.122 μΜ, 6.244 μΜ, and 12.488. When μΜdoxcycline was used, the doubling time of HCCLM3 cells was 22.72, 24.12, 25.91, 27.27, and 35.18 hours, respectively, which was 6.52%, 13.07%, 21.46%, and 27.82%, respectively, compared with 21.33 hours in the control group. And 64.92%.

HCCLM3 cells are human metastatic liver cancer, clone-like growth, and clones are often aggregated. After administration of doxycycline (12,488 μΜ) for 3 days, it was observed that the cells formed small clones, or the clones were poorly formed, and there were a large number of cell suspensions, and the fusion ability between the clones was weakened, and the total number of cells was reduced (see Fig. 40C).

It can be seen that the in vitro cell growth curve test found that doxycycline has a certain inhibitory effect on the growth of human hepatoma cell line HCCLM3. The statistical results show that the effect of doxycycline on the doubling time of HCCLM3 cells is dose dependent. Example 6 Study on the effect of doxycycline on human pancreatic cancer cell line ASPC-1 in vitro

1. Detection of the inhibitory effect of doxycycline on the growth of human pancreatic cancer cell line ASPC-1 by method: The method is the same as that described in the corresponding MTT assay in Example 1, except that the cell line used in the present example is human pancreatic cancer cell ASPC-1.

Experimental results:

(1) MTT assay for the inhibitory effect of doxycycline on the growth of human pancreatic cancer cell line ASPC-1 as shown in Table 23 and Figure 41 and Table 24 and Figure 42, the growth of doxycycline on ASPC-1 cells was low. At the dose, it showed obvious inhibition. With the increase of doxycycline concentration, the inhibitory effect on ASPC-1 cells increased gradually, showing a dose-dependent manner.

The pre-experimental results showed that the half-inhibitory concentration (IC ₅ ) of doxycycline and the positive drug doxorubicin against ASPC-1 were 2.2337+0.1284 μΜ and 0.6884+0.0341 μΜ, respectively. Official results showed that doxycycline and positive drug doxorubicin hydrochloride IC ₅ Dui ASPC-1's. Value f

2.3490±0.1285 μΜ and 2.4783±0.3667 μΜ

Table 23 Inhibition of ASPC-1 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-experimental results)

Doxy-1 throw rate AD -1 '±i Survival rate Inhibition rate Concentration (μΜ) (OD57 is rich 3⁄4 μΜ ( ODf 1101 > ,>

0 0.429SiO.O035 100 0 0 1.4018^0.1345 100 0 i.04 4-23 0 \25 1.3447^0.0456 95.93 4 7

2.0S 0.3575+0.0023 S3. IS 1.5625 1.123 iiO.0406 SO.12 19.SS

4,165 0.22S ±0.0059 53.26 46,74 3Λ25 39.45 60.55

0

33 0.18 4i0. 062 4407 6.25 0.305 ±0.00a2 21.S2 78.18

16.66 0.144ϋ0.0026 66.47 12.5 0.1986^0.0209 14,17 85.83 f-

33/3 0.0536iO.O023 12.47 87.53 25 O.i23S±O.0O35 S. 3 91.17

66.65 0.034ό±0.001δ S.04 91.96 50 0.i3 SiO.012S 9.33 90.6665

133.33 ΟΛ)577±0.006 "100 0.15 5i0.0024 11.38 88.62

266.625 0.344SiO.O0SS 34,48 65.52 200 0, 015÷0,0 66 21.51 78.49 Table 24 Detonation of ASPC-1 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR)杲)

Doxy-2 storage rate AD -2 Survival inhibition rate concentration (μ Μ · (OD _{: r} 3⁄4i i.) %) (concentration ' Μ) ( OD50 inn .)

0 l.3⁄4!57±0.0976 100 0 0 1J 1S±0J421 !00 0

0.7S125 i.7103±0-0626 94.19 5.81 0.1953 0.5003± .0207 43.43 56.57

1.5625 1.436^0.0369 79.09 20.91 03906 0.6316^0.1455 54.84 45.16

3,125 08765 00300 4S.2S 51.72 0,5263 0.0190 45.69 54,31

6-25 0.7929± .0164 43.67 56.33 1.5625 0.4429± .00 9 38.45 61.55

12.5 0.59H0.0034 67.45 0.314 ±0.0296

25 0.09^0,0081 4.96 95.04 6.25 0.2569=0.0290 77.70

50 0.0644± .0016 3.55 96.45 12.5 0.195 Ii .0262 Ϊ .94 83,06

100 0.1133±( 0027 6.24 2 0.Π76 0.0063 15.42 84.58

200 0.2917± .0152 16.067 S3933 50 0.i80S±0.00 1 15.70 84.30 It can be seen that the inhibitory effect of doxycycline on ASPC-1 in human pancreatic cancer cells was detected by MTT assay in vitro. It was found that doxycycline had a good inhibitory effect on the growth of ASPC-1 cells in a dose-dependent manner, IC ₅ . The value is below ΙΟ μΜ (approx. 5 g/mL).

2. Growth curve assay for inhibition of growth of human pancreatic cancer cell ASPC-1 by doxycycline Method step: Only the cell line used in this example is human pancreatic cancer cell ASPC-1.

Experimental results:

(1) Growth curve assay for the effect of doxycycline on the growth of ASPC-1 cells

As shown in the growth curves of Fig. 43A and Fig. 43B, doxycycline inhibited the growth of ASPC-1 cells in a dose-dependent manner. The statistical results showed that the effect of doxycycline on the doubling time of ASPC-1 cells was dose-dependent. . At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on cell doubling time. When administered at concentrations of 0.977 μΜ, 1.954 μΜ, 3.909 μΜ, 7.818 μΜ and 15.636 μΜ of doxycycline, the doubling times of ASPC-1 cells were 26.51, 27.70, 29.44, 29.83 and 42.94 hours, respectively, compared with 20.89 hours of the control group. In comparison, they were extended by 26.95%, 32.64%, 40.97%, 42.85% and 105.61%, respectively.

The effect of doxycycline on the growth morphology of pancreatic cancer ASPC-1 cells can be seen: ASPC-1 is a human metastatic pancreatic cancer cell line with bidirectional differentiation potential, and the cells are mostly clonal epithelial-like growth, and some cells have extraneural nerves. The germ layer features a filamentous pseudopod. After administration of doxycycline (15.636 μΜ), the cells were suspended, and most of them were epithelial-like, with suspension and apoptosis of filopodia. Epithelial-like cells age and the cell outline is unclear (see Figure 43C).

It can be seen that the in vitro cell growth curve test found that doxycycline has a certain inhibitory effect on the growth of human pancreatic cancer cell line ASPC-1. The statistical results show that the effect of doxycycline on the doubling time of ASPC-1 cells is dosed. Dependence.

3. Effect of doxycycline on the invasion ability of human pancreatic cancer cell line ASPC-1

Method steps:

The same procedure as described in the corresponding invasiveness test in Example 1 was carried out except that the cell line used in the present example was human pancreatic cancer cell ASPC-1. Experimental results:

(1) Inhibition of invasive ability of doxcycline on human pancreatic cancer cell line ASPC-1

As can be seen from Figures 44A and 44B, the effect of doxycycline on the invasive ability of ASPC-1 cells in vitro was not significant. The statistical results (Fig. 44B) showed that there was no significant difference between the doxycycline-dosing group and the azithromycin hydrochloride-administered group and the control group (P>0.05).

It can be seen that the effect of doxycycline on cell invasion ability was examined by Transwell method in vitro. The results showed that doxycycline had no significant effect on the invasion ability of human pancreatic cancer cell ASPC-1 in vitro. Example 7 Study on the effect of doxycycline on human cervical cancer Hela in vitro

1. MTT assay for the inhibitory effect of doxycycline on the growth of human cervical cancer Hela cells

Method steps:

The method described in the corresponding MTT assay in Example 1 was the same except that the cell line used in the present example was human cervical cancer cell Hela.

Real horse results:

(1) MTT assay for the inhibitory effect of doxycycline on Hela cell growth

As shown in Table 25 and Figure 45 and Table 26 and Figure 46, the growth of HeLa cells by doxycycline showed a significant inhibitory effect at low doses. As the concentration of doxycycline increased, the Hela cells were increased. The inhibitory effect is gradually increased, showing a significant dose dependency.

The preliminary results showed that the half-inhibitory concentration (IC ₅ ) of doxycycline and the positive drug doxorubicin hydrochloride on Hela cells were 1.3343±0.1346 μΜ and 2.4560±0.1736 μΜ, respectively. Official results showed that doxycycline and positive drug doxorubicin hydrochloride IC ₅ of Hela's. The values are 4.0750 + 0.1907 μΜ and 2.8520 + 0.2959 μΜ, respectively. 3⁄4 - Table 25 Inhibition of Hela cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-1 experimental results)

Doxy-1 : s survival rate AD x s survival

Concentration (μΜ.) ( ) (3⁄4.) Concentration (, μΜ) OD mil ) 3⁄4) (3⁄4)

0 0-7247 0.0150 100 0 0 100 0

0.3906 00006 64.54 35.46 0.1 53 0.9672=0.0363 73.35 26.65

0.7S13 0.4020 0. 7 55.47 44.53 0,3906 0.S 92 ^0.0316 67.71

1.5625 0.3081±0.0088 42,52 0.78125 0.79§5÷0.0140 60.55 39.45

3.125 0.2054±0.0il4 28. 3⁄44 71.66 0.76S3i0. 020 5S.26 41.74

6.2500 0.1183± . 1Ι9 16.33 83.67 3.125 0.5414÷0.065 41.06 58.94

12.5 00S32 i0.0020 11.49 8S.51 6.25 0.339= 0.0202 25.71 74.29

25 0.0662± . 06ό 9.14 90.86 12.5 0.3372÷0.0i2 17.99

50 0.0555±0, 0045 7.66 92.34 25 0.238i>i0.0040 IS.12

100 0.0538±0. 031 7 A3 92.57 50 0.2272 0.0062 17.23 82.77

f

Table 26 inhibition of Hela cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) o

Use (formal experimental results)

Doxy-2 x±s survival rate, God's rate, ADR-2, rate, rate, amortization rate

· 'OD (3⁄4) (%. concentration (uM.) (OD %) (3⁄4:'

1.4S4 0.0622 Ϊ00 0 0 2Λ416± 39\5 100 0

0.1953 I,22S1±0.0082 S2,7i 17.29 0.1953 1,0834 0306 44.15

0.3906 1.1475±0.i344

0.3906 1.060 iJn■ 0.0296 43.2 56.8

0.7SI25 0.9475± .0096 36.19 0.904S±0.0202 3 . S7 63.13

1.5625 Ο.8597±0.0119 57.9 42.1 1.5625 O.S5Si0.0466 34.96 65.04

0.7718±0Λ)298 5L98 JiS6÷ 0S48 29.2S 70.72

6.25 0.45S1±0.0426 30.85 69.15 6.25 0.3123÷00294 S7.27

12,5 O. S05=b0.0055 5.42 94.58 25 .3 89 0J97S 13. SI 86.Ϊ

25 0I) 57±O. O03 3.S4 96.16 12.5 9.59 90.41

50 0.0542 0.0020 3.65 96,35 50 0.1684^0.0111 6.S6 Inhibition of human cervical cancer Hela by doxycycline in vitro by MTT assay o, it was found that doxycycline has a good inhibitory effect on the growth of Hela cells and Showing a significant dose-dependent, IC ₅ . The value is below 10 μΜ (approx. 5 g/mL).

2. Growth curve method for detecting the inhibitory effect of doxycycline on the growth of human cervical cancer Hela cells. Method steps: Only the cell line used in this example is human cervical cancer cell line Hela

Experimental results:

(1) Growth curve method for the effect of doxycycline on the growth of Hela cells

As shown in the growth curves of Fig. 47A and Fig. 47B, doxycycline has a certain inhibitory effect on the growth of Hela cells. The statistical results show that the effect of doxycycline on the growth of Hela cells is dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment. Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on the doubling time of Hela cells at doses of 0.3325 μΜ, 0.665 μΜ, 1.33 μΜ, 2.66 μM. When compared with 5.32 μΜ of doxycycline, the doubling time of Hela cells was 21.31, 22.52, 23.56, 24.42, and 27.11 hours, respectively, which was 3.21%, 9.07%, and 14.13%, respectively, compared with 20.64 hours of the control group. , 18.28% and 31.34%.

Hela cells are cervical cancer epithelial cell lines and are classical epithelial tumor cell lines with epithelial-like growth. After administration of doxycycline (5.32 μΜ), the cell outline disappeared, most cells aged, cytoplasmic particles increased, and some cells shattered (see Figure 47C).

It can be seen that the in vitro cell growth curve test found that doxycycline has a certain inhibitory effect on the growth of human cervical cancer Hela. The statistical results show that doxycycline can significantly increase the doubling time of Hela cells, doxycycline. The effect on Hela cell doubling time was dose dependent.

3. Effect of doxycycline on the invasive ability of human cervical cancer Hela

Method steps:

The method described in the corresponding invasiveness test in Example 1 was the same except that the cell line used in the present example was human cervical cancer cell Hela.

Experimental results:

(1) Inhibition of invasive ability of dolacycline on Hela (cervical cancer cell line)

The inhibitory effect of doxycycline on the invasive ability of Hela (cervical cancer cell line) is shown in Figures 48A and 48B: Figure 48A shows the crystal violet staining after 24 h treatment with doxycycline and the positive drug doxorubicin hydrochloride. The results observed under the microscope showed that doxycycline had a strong inhibitory effect on the invasion ability of Hela cells compared with the control group. The statistical results (Fig. 48B) showed that the inhibitory effect of doxycycline on Hela cell migration at a concentration of 0.391 μΜ reached 34.1% (P<0.01, which was significantly different from the control group), and the doxorubicin hydrochloride concentration was 0.2 μΜ. The inhibition rate of Hela cell migration was 65.8% (P<0.01, which was significantly different from the control group).

It can be seen that the effect of doxycycline on the invasion ability of Hela cells was detected by Transwell method in vitro. The results showed that doxycycline inhibited the invasion ability of Hela cells at low dose in vitro. Example 8 Study on the effect of doxycycline on human colon cancer cell line SW620 in vitro

1 MTT assay for the inhibition of doxycycline on the growth of human colon cancer cell line SW620 Method steps:

The method described in the corresponding MTT assay in Example 1 is the same except that the cell line used in this example is human colon cancer cell S W620.

Experimental results:

(1) MTT assay for the inhibitory effect of doxecycline on the growth of colon cancer cell line SW620 is shown in Table 27 and Figure 49 and Table 28 and Figure 50. The growth of SW620 on SW620 is evident at low doses. Inhibition, and exhibit a dose response. As the concentration of doxycycline increased, the inhibitory effect on SW620 cells increased gradually, showing a dose-dependent manner.

The preliminary results showed that the half-inhibitory concentration (IC ₅₀ value) of doxycycline and the positive drug doxorubicin hydrochloride on S W620 were 0.9116±0.1485 μΜ and 0.2763±0.0152 μΜ, respectively. Official results showed that doxycycline and positive drug doxorubicin hydrochloride IC ₅ Dui SW620 is. The values are 0.8013+0.0766 μΜ and 0.3563±0.0674 μΜ, respectively.

Table 27 Inhibition of SW620 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (pre-experimental results)

- Survival rate Survival rate Inhibition rate Concentration ( 〈 , concentration

Shishi

Earth

Table 28 Inhibition of SW620 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (official experimental results)

, ^ viability inhibition rate concentration ^: degree;

〗 It can be seen that the inhibitory effect of doxycycline on the growth of SW620 cells was examined by in vitro MTT assay. The results showed that doxycycline significantly inhibited the growth of SW620 cells in a dose-dependent manner. IC ₅ . The value is below 10 μΜ (about 5 g/mL).

2. Effect of doxycycline on the invasion ability of human colon cancer cell line SW620

Method steps:

The method described under the corresponding invasiveness test in Example 1 was the same except that the cell line used in the present example was human colon cancer cell S W 620.

Experimental results:

(1) Inhibition of invasive ability of colon cancer cell SW620 by doxycycline

The inhibitory effect of doxycycline on the invasion ability of colon cancer cell line SW620 is shown in Fig. 51A and 51B. Fig. 51A shows the results of microscopic observation under the microscope of microscopic staining of cells treated with doxycycline and the positive drug doxorubicin hydrochloride for 24 h. It can be seen that compared with the control group, doxycycline has a strong inhibitory effect on the invasion ability of SW620 cells. The statistical results (Fig. 51B) showed that the inhibition rate of doxycycline on SW620 cell migration at a concentration of 0.05 μΜ reached 36.14% (Ρ<0.05, which was significantly different from the control group), and doxorubicin hydrochloride at 0.1 μΜ The inhibition rate of SW620 cell migration was 14.45% (Ρ<0.05, which was significantly different from the control group).

It can be seen that the effect of doxycycline on cell invasion ability was examined by Transwell method in vitro. The results showed that doxycycline significantly inhibited the invasion ability of SW620 cells at low dose in vitro. Example 9 Study on the effect of doxycycline on human gastric cancer cell line MKN28 in vitro

1. MTT assay for the inhibitory effect of doxycycline on the growth of gastric cancer cell line MKN28

Method steps:

The method described in the corresponding MTT assay in Example 1 was the same except that the cell line used in the present example was human gastric cancer cell MKN28.

Experimental results:

(1) MTT assay for the inhibitory effect of doxycycline on the growth of gastric cancer cell line MKN28 As shown in Table 29 and Figure 52 and Table 30 and Figure 53, doxycycline significantly inhibited MKN28 cells at low doses. , as the concentration of doxycycline increases, it is fine for MKN28 The inhibition of o is gradually increased in a dose-dependent manner.

The half-inhibitory concentration (IC ₅ ) of doxycycline on MKN28 was 2.4517 ± 0.25 o 91 μΜ as measured in the pre-experiment. In the formal experiment, the IC ₅₀ value of doxycycline for ΜΚΝ28 was 1.6640+0.1853 μH -Μ. The experimental results show that doxorubicin hydrochloride almost grows on ΜΚΝ28 cells.

H- has no inhibition.

Table 29 Inhibition of Μ ΚΝ 28 cells by doxycycline and doxorubicin hydrochloride (pre-experimental results)

Dox -I x soil s survival j rate w rate ADR-1 - rate of concentration (u ( OD ₅₇ o诵) (%) i%) concentration (μΜ) (OD _i7tl nm) ( > ί3⁄4)

0 3.2iI9i0.i260 100 0 0 3.5292^0.01 0 100 0

0,3906 ί-344ό 0Λ474 4LS6 o 5S.14 0.3906 95,59 4.41

1.237 Machinery 0156 38.53 61.4, 7 0.78125 9534 4.66

1.5625 0.9SS7±0.022S 30.73⁄4 3.2485i0. 2 4 92.05 7.95 J25 22.00 7S.0O 3.2219^0.03 6 1.29 S.71

625 0.451 Zhuang 0.0712 14,07 S5.93 6,25 93.03 6.97

12-5 01864 0.014S 5.80 94.20 12.5 3.26iS÷0.047S 92.42 7.5S

25 00737 0 i 2.30 25 3.2286^0.0198 9L4S S.52

50 0.0708 土谨 75 2.20: 50 97.12 2M

100 0.0946i0. 015 2.94 97.06 100 3.5 g2''2ii}l>470 99.80 0.20

[,

n ■

Table 30 The inhibition of doxycycline and doxorubicin hydrochloride on ΜΚΝ28 cells.

Doxy-2 xs ίΦ copper ratio ADR-2 ν ¹ -·"',

Xs ,: Shen Ning concentration ( μ, Μ) (.0 _7ΰ mil ) (%) (. ) Concentration ( μΜ) (Ο ₇₀ ηηι) (%.) (%.)

t J

0 1 0 0 0 2.0112^0.0660 100 0

0.1953 0.432Si0.0425 21.53 7S.47 0.1953 l.SS01i0.126 6.52

0.3906 0.3S02i0.0136 IS.91 81.09 0.3906 1.793Si0.0779 89.19 1 .S1

0.7S125 0.3143 0.0Π6 1.7361i0.0961 13.68

11.06 SS. 4 1.5625 94.5 5.5

3.125 0.1495 ± 0.0120 744 3, 125 1.7S41 0. 1I2 SS.71 11.29

6.25 O.I483i0. 174 7.38 92.62 6.25 92.89 7.11

12.5 0.S 99.14 12.5 L834S 0.1461 S.77

*"? 0.003 0.0006 0.16 99.84 25 1.7954 soil 0.0997 89.27 10.73

50 .0066±0,004S 0.33 99.67 50 93.61 6.39 It can be seen that the inhibitory effect of doxycycline on the growth of human gastric cancer cell line MKN28 was detected by MTT assay. It was found that doxycycline inhibited MKN28 cells in vitro and presented Dose dependent. The IC ₅ Q value is below 10 μΜ (about 5 g/mL).

2. Growth curve method for detecting the inhibitory effect of doxycycline on the growth of human gastric cancer cell MKN28. Method steps: Only the cell line used in the present example is human gastric cancer cell MKN28. Experimental results:

(1) Growth curve assay for the effect of doxycycline on the growth of gastric cancer cell line MKN28 As shown in the growth curves of 54A and 54B, doxycycline has a certain inhibitory effect on the growth of MKN28 cells. The effect of cyclulin on the doubling time of MKN28 cells was dose dependent. At the same time, doxorubicin hydrochloride was used as a positive control in the experiment.

Based on the cell count results, the doubling time of the cells in the control group and each of the drug-added groups was calculated: Doxycycline has a significant prolongation effect on cell doubling time at doses of 0.692 μΜ, 1.384 μΜ, 2.768 μΜ, 5.536 μΜ, and 11.072. After μΜdoxcycline, the doubling time of ΜΚΝ28 cells was 31.48, 35.79, 45.93, 56.52 hours and 87.50 hours, respectively, which was 0.37%, 14.09%, 46.44%, 80.23%, and 31.36 hours, respectively. 178.96%.

ΜΚΝ28 is a human metastatic gastric cancer cell line with epithelial-like features and a common divisional phase. After administration of doxycycline (11.07 μΜ) for 3 days, the cells shrunk into spheres, most of which were suspended, showing anoikis, and few adherent cells remained (see Figure 54C).

It can be seen that the in vitro cell growth curve test found that doxycycline has a certain inhibitory effect on the growth of ΜΚΝ28. The statistical results show that the effect of doxycycline on the doubling time of ΜΚΝ28 cells is dose-dependent.

3. Effect of doxycycline on the invasion ability of human gastric cancer cells ΜΚΝ28

Method steps:

The method described under the corresponding invasiveness test in Example 1 was the same except that the cell line used in the present example was human gastric cancer cell ΜΚΝ28.

Experimental results:

(1) Inhibition of invasive ability of ΜΚΝ28 cells by doxycycline

The inhibitory effect of doxycycline on the invasion of ΜΚΝ28 cells is shown in Fig. 55Α and 55Β: Fig. 55Α The results of observation under the microscope of crystal violet staining after treatment of cells with doxycycline and the positive drug doxorubicin hydrochloride for 24 h, can be seen Compared with the control group, the inhibitory effect of doxycycline on MKN28 cells was not obvious at low concentrations. The statistical results (Fig. 55B) showed that doxycycline had no significant effect on the invasion of ΜΚΝ28 cells at 1 μΜ concentration (Ρ>0.05, no significant difference compared with the control group), and the apoptosis of ΜΚΝ28 cells by doxorubicin hydrochloride was also observed. There was no significant effect (Ρ>0.05, no significant difference compared with the control group). It can be seen that doxycycline has no obvious inhibitory effect on the invasive ability of MKN28 cells at low concentrations. Example 10 Study on the effect of doxycycline on normal cells in vitro

1. MTT assay for the inhibitory effect of doxycycline on the growth of NIH-3T3, HEK 293, HEK 293T, HaCaT cells

■ ■

Method steps: i bo

The method described in the corresponding MTT assay in Example 1 was the same except that the cell lines used in this example were NIH-3T3, HEK 293, HEK 293T, HaCaT cells.

Experimental results:

(1) MTT assay for the inhibitory effect of doxycycline on the growth of NIH-3T3 cells

0 d

As shown in Table 31 and Figure 56 and Table 32 and Figure 57, the effect of doxetine on the growth of NIH-3T3 cells was weak, and the inhibition rate of cells at 50 μΜ was only about 0 to 35%. Adriamycin hydrochloride has a strong inhibitory effect on the growth of ΝΙΗ-3Τ3, and the inhibition rate of cell growth at 1.5625 μΜ is about 80%. The results showed that the inhibitory effect of doxycycline on the growth of ΝΙΗ-3Τ3 cells was weaker than that of the positive drug doxorubicin hydrochloride, indicating that doxycycline was less toxic to ΝΙΗ-3Τ3 cells.

Table 31 Inhibition of NIH-3T3 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (Results of the first experiment)

Doxy-l xis rate of ownership rate ADR-1

Survival inhibition rate concentration (μΜ) (OD ₅₇₀ mn ) concentration 'μΜ) COD57M dish) (3⁄4 3⁄4)

0 0.8584 soil 0.0i69 100 0 0 1,6906 ±0.06 II 100 0

0.7 19 ;!;0.0124 92.25 7.75 0.1953 38.38 61.62

0.3906 0.7292 ±0.0214 S4. 4 15.06 0 6±0.0210 60.93

0.78125 .7309±0.014 i 85.14 14.86 0.7S125 0.3741 : ί:ϋ.002"1 22.13 77.87

L5625 0.7576± .016I Π.75 1.5625 0.1469±0. 036 S.69 91.31

3.125 0.6520:!: 0.0106 75.96 3.125 0.2470 soil 0.0034 14.61 85.39

625 0.6903 ±0.0077 S0.42 19.58 6.25 0.3276 ±0.0127 Ϊ938 S0.62

12-5 0.6422 soil 0.0i26 25.19 12.5 0.2S48 士. Photo 3 16.S4 83.16

2 0,5813 ±0.1333

25 0.2094 soil 0,0004 12.3S 87.62

50 ί 5820:!: 0.01 &5 67.80 50 10.18 9.S2 ο

.1-'-Table 32 Doxy and doxorubicin hydrochloride (ADR) inhibit NIH-3T3 cells y (second experiment result)

Doxy-2 x survival rate x÷s survival rate neural rate

d

Concentration (μΜ) (OD ₅ 7omn:) <,3⁄4) OD570 <%)

0 0.9 4S± 0.0451 100 0 0 1.0115 ±00097 100 0

0 i953 96.97 3.03 0.1953 0.5480

d d soil 0.0113 54AS 45. S2

0.3 06 0.9198 ± „01S7 95.34 4.66 0.3906 0.4938 ±0.0111 48.82 5i.iS

0.8962 ±0.0346 92.89 7.11 0.7S125 0.3752 soil 0.0313 37.10 62.90

1.5625 08423: ±Ο 038 S7.30 12.70 1.5625 0.2047 ±0.0071 20,24 79.76

3.125,7666±Ο.Ϊ312 79.46 20.54 3.125 0.2485 ±0,0264 24.57 75,43

6.25 O.S443± 0.0041 87.5 i 12.49 625 0.3495 ±0.0035 34.55 65.45

12.5 SI.75 18.25 12.5 0.2S59 ± 0.0205 71.74

25 0,8239 ± 0.0148 S5.40 14.60 25 0.1923 ±0,0108 19.02 80.9S

50 .62S ± 0.0301 65.09 34.91 50 0.1 SOS ±0.0036 17.SS 82.12

(2) MTT assay for the inhibitory effect of doxycycline on the growth of HaCaT cells

As shown in Table 33 and Figure 58 and Table 34 and Figure 59, doxycycline inhibited the growth of HaCaT cells very weakly, and the concentration of doxycycline at 50 μΜ had almost no inhibitory effect on cell growth. Doxorubicin hydrochloride inhibited cells, and the inhibition of cells at 0.1953 μΜ reached 90.77%. The results showed that the inhibitory effect of doxycycline on the growth of HaCaT cells was weaker than that of the positive drug doxorubicin hydrochloride, indicating that doxycycline was less toxic to HaCaT cells.

Table 33 Inhibition of HaCaT cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (Results of the first experiment)

D xy- - deposit _; tongue rate ADR- Ϊ 'survival rate suppressed car concentration fi I > OD ₅₇ oi 3⁄4 ) ) concentration uNi OD »; ) ( «

0 0.2773 ά: 0. 62 1 oo 0 0 .3402 ; ;0. 42 1GO 0

0,1 53 0.3027 ;!; 0.0331 109.13 -9.13 0.1 53 0.0314 ± 0.0012 S>.23 90.77

0.3906 0.3221 ± 001 S9 I 16 14 -16.14 0.39O6 0055Sv -O.Oi 3S i .40 S 3

0.7SI25 0.332SJ 0.0078 Ϊ20.04 -2O.04 0.7S125 0.0179 ± 0.0077 5.26 94.74

1.5 25 0.2637.1·. . 171 95.10 4.90 1.5625 O.012 . 0. G73 96.22

0.31674:0.0086 114.21 -14.21 3Λ25 .(X)54; ;0.O 31 5S 98.42 0.3Ι71±:0.0186 114.33 -14.33 6.25 0.CQ21土0.0041 3. 5 96.45 ί2.5 0.3 ΟΟ8± 00096 I OS 45 -S 45 12.5 004 2 -OO 34 S701 25 0.351 ; ί; 0. 02Ο 126.00 -26.60 25 θ 0677 0.0118 so 50 0.3400.1-. .O062 122.58 -22.58 50 0.1091.1.0.0207 67.94 Table 34 Doxycycline (Doxy) And the inhibitory effect of doxorubicin hydrochloride (ADR) on HaCaT cells (the second experimental result)

Doxy-2 ±s Survival Rate Suppression Rate ADR-2 Preservation Rate Concentration μΜ (OD _i7 Dish (. 3⁄4 ); Farming) ( OD (%

0 0.2838 ±0.0259 100 0 0 0.3167±0.0178 Ϊ00 0

0.1953 0.2609 ±0.02S6 1.94 S.06 0.1953 0.049i soil 0.0062 15.49 S4.51

0.3906 0.2711 ±0.0190 95.52 4.4S 0.3906 0.0234± 0.0043 7.38 92.62

0.2254;};.0.0396 79.43 20.57 0.78125 00144vh-0.00aS 4.56 95.44

1.5625 0,2105 ±0.0476 74.1 25, SI 1.5625 O.0 S ± 0.0074 2.52 97.48

3.丄 25 0.3I63±0.0150 111.48 -HAS 3.125 0.0121 ±0.0074 3.S2 96. IS

6.25 02770;i;. .0*;t72 97.63 2.37 625 0.0226 vh" 0.0032 7.15 92.

12.5 0.3094 ±0.0024 109.02 -9.02 12.5 O.03OO±0.OO52 9.46 90.54

25 0,2770 ±0.0074 97.62 2 25 0.035S±0.007 11 30 S8.70

50 0.2743 ±0,0126 96.66 3.34 50 0.1075 ±0.0094 33.94 66.06 (3) MTT assay for the inhibitory effect of doxycycline on the growth of HEK 293 and HEK 293T cells

From Table 35, Table 37, Figure 60 and Figure 61: Doxycycline has a certain inhibitory effect on the growth of HEK 293 cells. When the concentration is 25 μΜ, the inhibition rate is about 60%. Doxorubicin hydrochloride achieved a 70% inhibition rate of 293 293 cells at a concentration of 0.78125 μM. The results showed that the inhibitory effect of doxycycline on the growth of ΗΕΚ293 cells was weaker than that of the positive drug doxorubicin hydrochloride. This indicates that doxycycline is less toxic to ΗΕΚ 293 cells.

From Table 36, Table 38, Figure 60 and Figure 61, doxycycline has a strong inhibitory effect on the growth of HEK 293 cells. At a concentration of 3.125 μΜ, the inhibition rate is 71.05%. The inhibitory rate of azin hydrochloride to ΗΕΚ 293 cells at a concentration of 0.3906 μΜ reached 71.96%.

The inhibitory effect of doxycycline on HEK 293 cells was stronger than that on 293 cells. This is mainly because the HEK 293 Τ cell line is a high transfection efficiency derivative produced by the ΗΕΚ 293 cell line inserted into the temperature-sensitive gene of SV40 Τ-antigen, and the transduction of S V40 Τ-antigen induces malignant transformation of cells. This indicates that doxycycline has a strong inhibitory effect on the growth of malignant transformed cells, and that doxycycline has a certain selectivity for the inhibition of normal cells and malignant transformed cells.

Table 35 Inhibition of HEK 293 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (Results of the first experiment)

Doxy-i P"^ rate cotton rate ADR-i activity inhibition concentration (μ > OD-- ₇₀ ιιΐϊ ; ': .; concentration · ^: : ΝΓ ) OD- _;7 .;;, imi) <: )

0 1.03 1.±0.0594 1 0 0 0 1.1264 soil 〖) 0205 100 0

0.1953 0.8004-1:0.0 4 77 10 22.90 0.1953 0.4496 0.0099 3992 60.08

0.3906 0.6S 5±0.02S4 66.14 33. S6 0.3906 Ο.35ΟΟ·±Ο.Ο310 31.07 6S. 3

0.7S125 0.7349 soil O.ftZSi 70. SO 29.20 .7S125 0.32iO soil 0.0252 2S.50 71.50

1.5625 0.6δ41 ± 00163 63.97 36.03 1.5625 02926.†.· 0.0426 25.97 7403

3.125 0.6S86; i.0.O574 66.33 33.67 3Λ25 0.20 SO .±0.0070 IS.47 S i .53

6.25 0.531S 0 :)5 1.23 48.77 6.25 0.3666士— 0—0585 32. 5 67.45

12.5 0.4S 1± 0.0275 47.12 52.8S 12.5 G..3S 0±0. (i042 34.59 65. 1

25 0.46SS±0.0S17 45.16 54.84 25 0.2917d:C 0196 25. S 9 74. ί 1 Table 36 Doxy and doxorubicin hydrochloride (ADR) for the production of HEK293T cells (first experiment Result)

D x -1 s s rate inhibition rate AD -1 survival rate spur rate

< OO _J urn ί' 3⁄4 .> (concentration (μΜ) ⁽ OD _i70 urn ) (°b >

0 0.6666 ; 0.0iS9 丄 00 0 0 0,6399土 0-0087 100 0 ϋ.1 3 0.6051 ±0.0294 90.7S 9.22 0.1 53 0,2607土 0.0193 40.74 59.26

0.3906 0.5662 ±0.0054 84.94 1 ,06 0.3906 0.1794 ±0.0252 28.04 71.96

0.7S125 0.447S±0.008S 67. IS 32..S2 0.7S125 0.1745±0.0006 727 72..73

1.5625 0.3367 0.0024 50.52 49.48 1.5625 0.1458 ±0.0137 227S 7

3.125 0.1930± 0.0197 28.95 71.05 3.125 Q.1699 ±00116 26.56 73.44

6.25 0.2123 ±0.0016 1.S5 6S.I5 6.25 0.16S2±0.0060 26.29 73.71

125 0. ί 758 ±00280 26.37 73.63 12.5 0.18 3± .Ο145 2S 17 71.S3

0.20 6± 0.0050 31.29 68.71 25 0.1594±0-0121 24,90 75.10 Table 37 Inhibition of HEK 293 cells by doxycycline (Doxy) and doxorubicin hydrochloride (ADR) (Results of the second experiment)

Doxy-2 survival rate ADR-2 ^ viability rate of concentration ίμΜ) (OD mil ) , ■%) (%) ( ) (%)

0: 1,0963 ±0.0822 100 0 0 i.13 SS± 0,0063 100 0

70.00 30.00 0.1953 0.3SI5±0.0122 33.50 66.50

0.3906 0:7432士 0.0495 67.S0 0.3906 0.5967 ±0.0108 52.40

0,78125 0.6S95士 0 116 0.78125 0.3όΙ3±0.0036 31.73 6S.27

1.5625 0.6202±0.0358 56.57 43.43 1.5625 0.3342±0.0I64 29.34 70.66

3,125 0.6312:1:00178 51.5 f,1 42.43 0.2592: :0.0224 22.76 77.24 o

6,25 0.579? Earth 00094 47.12 6:25 0.4512 soil 0.0119 39.62 60.3S

12.5 0 97 ±0.017 45.42 54.5 S 12.5 0.3555 ±0.0670

25 0.4396 :!:0.0383 40.10 59,9'0 25 0.3670 ±0.0233

67.78 o

Table 38 Inhibition of HEK293T cells by Doxy and Doxorubicin hydrochloride (ADR) (Results of the second experiment)

Doxy-2 χ s activity inhibition rate ADR-2 x s 3⁄4-live inhibition rate

( OD J mi ) (3⁄4 J (%) concentration (μΜ) ^ (%)

0 0.6927±0.0009 ΐΰθ 0 0 0.5160±0.020i 100 0

O o

0J953 0.566 i:i 0.0254 81.7Ϊ 18.29 0.1953 0.21 Q66 ±0.001? 41.9S

0.3906 0.53I5±0.012 0.3906 0.1922 soil 0.0036

0.78125 0.4395 ±0.0092 36.55 .7SI 5 0.1 S63 ±0.0092 36J0 63.90

0.3697 ±0.01 S 46.64 1.5625 0.1443 ±0.0053 27,97

3,125 0—iS20 soil 0.0121 26.28 3.125 0.1643 soil 0.0139 68.15

6-25 0.164 Ϊ ±0.0155 6.25 0.1425 ±δ„0 30 t

J -

12.5 0. Ϊ 600 : ; 0.0067 23.09 76.91 12.5 0.1 94 ±0.0052 30.SS 69.12 It can be seen that the inhibition of normal cell growth by doxycycline shows that it is more than the positive drug doxorubicin hydrochloride. The inhibitory effect of cyclin on normal cell growth is generally weak, indicating that doxycycline is less toxic to normal cells. Doxycycline has a certain selectivity for the inhibition of normal cells and tumor cell growth.

In Examples 11-15, the test materials used and their sources include:

(1) Various cell lines

B16-F10 mouse melanoma cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd. Lewis lung cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; human breast cancer cell line MCF-7: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; Human small cell lung cancer cell line H446: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; B16-BL6 mouse melanoma cell line: purchased from Nanjing Kaiji Biotechnology Development Co., Ltd. (2) Laboratory animals

The animal strains used in Examples 11 and 12 were C57 mice, SPF grade. A total of 120 C57 mice aged 4-6 weeks and 16-18 g (60 for the first trial and repeated trials) were introduced in this test. Females and animals were introduced in strict accordance with the Standard Operating Procedures for the Introduction of Test Animals (SOPA). -001-V00 ) and recorded in the "Test Animal Introduction Record Form" (BG-015-V00), provided by the Experimental Animal Center of the Chinese Academy of Military Medical Sciences and Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. : 0001631 (first test), 11400700018994 (repeated test).

After the animal arrives, the animal is received by the special person in the double-corridor barrier environment mouse breeding room 2, and the "Test Animal Reception Record Form" (BG-017-V00) is filled in. The general condition of the animal is observed at the time of receiving, and the animal is randomly selected. Weigh to ensure that the test animals are basically in line with the introduction criteria. Laboratory Animal Use License No.: SYXK (Tian) 2012-0003.

Example 13 The animal strain was selected as BABc/nu mice, and the SPF grade was used. In this trial, a total of 78 C57 mice with a mouse age of 5-6 weeks and 18-20 g were introduced (39 for the first trial and repeated trials respectively). Females and animals were introduced in strict accordance with the Standard Operating Procedures for the Introduction of Test Animals (SOPA-001). -V00 ) and recorded in the "Introduction Record of Test Animals" (BG-015-V00), provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the People's Liberation Army, with the certificate number: 0000804 (first test), 0022032 (repeated test).

Example 14 The animal strain was selected as BABc/nu mouse, SPF grade. In this trial, a total of 78 C57 mice with a mouse age of 5-6 weeks and 18-20 g were introduced (39 for the first trial and repeated trials respectively). Females and animals were introduced in strict accordance with the Standard Operating Procedures for the Introduction of Test Animals (SOPA-001). -V00 ) and recorded in the "Introduction Record of Test Animals" (BG-015-V00), provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the People's Liberation Army, with the certificate number: 0000804 (first test), 0022032 (repeated test).

After the animal arrives, the person receives the animal in the double-corridor barrier environment mouse breeding room 2, fill in "Test Animal Reception Record Form" (BG-017-V00), the general situation of the animals was observed at the time of reception, and the animals were randomly selected for weighing to ensure that the test animals were basically in agreement with the introduction standards. Laboratory animal use license number: SYXK (Tian) 2012-0003.

Example 15: Animal strain was selected as C57 mouse, and SPF grade was introduced in this experiment. A total of 120 C57 mice of 4-6 weeks and 16-18 g were injected (60 for the first trial and repeated trials), female, The introduction of animals is strictly in accordance with the "Standard Operating Procedures for the Introduction of Test Animals" (SOPA-001-V00) and recorded in the "Introduction Record of Test Animals" (BG-015-V00), by the Experimental Animal Center of the Chinese Academy of Military Medical Sciences and Beijing. Provided by Vitallihua Laboratory Animal Technology Co., Ltd., certificate number: 000804 (first test), 11400700020415 (repeated test).

After the animal arrives, the person receives the animal in the double-corridor barrier environment mouse breeding room 2, fill in

The Test Animal Receiving Record Form (BG-017-V00) was used to observe the general condition of the animals at the time of receiving, and the animals were randomly selected for weighing to ensure that the test animals were basically in agreement with the introduction standards. Laboratory Animal Use License No.: SYXK (Tian) 2012-0003.

(3) Test samples and positive control drugs

Test sample: Doxycycline hydrochloride: Yellow powder, supplied by Kaifeng Pharmaceutical (Group) Co., Ltd., batch number: 201301011, Purity: 91.0% (in accordance with the Chinese Pharmacopoeia 2010 edition 2).

Positive control drug: Cyclophosphamide: white granules, Alfa-Aesa, article number: L11508, lot number: K17X007, purity: 97+%.

Solvent: 0.9% normal saline.

Test sample and positive control drug preservation: 4 ° C

(4) Configuration of drugs and reagents

a: Preparation of doxycycline hydrochloride solution: Weigh 720 mg of doxycycline and dissolve it in 60 mL of 0.9% physiological saline solution to prepare 12 mg/mL doxycycline hydrochloride solution. After that, it was suction filtered through a 0.22 μM filter. The diluted 0.9% physiological saline was separately diluted to 6 mg/ml and 3 mg/ml in a clean bench.

b: Configuration of cyclophosphamide solution: 120 mg of cyclophosphamide was weighed and dissolved in 30 mL of physiological saline to prepare a 4 mg/ml cyclophosphamide solution. After it was sufficiently dissolved, it was filtered with a 0.22 μΜ filter. Example 11 Effect of doxycycline on C57BL/6 mouse B16-F10 melanoma xenografts

Method steps:

(1) Test grouping and dose setting

The test consisted of a negative control group, a positive control group, and a treatment group. The negative control group consisted of 20 rats in each group, and the positive control group, the high dose group, the middle dose group and the low dose group each had 10 rats. The treatment group was set in three dose groups: high, medium and low. The dose is set at 4:2:1. According to the clinical maximum dose, this experiment will prepare three different concentrations (high, medium and low) of doxycycline solution at concentrations of 12mg/ml, 6mg/ml and 3mg/ml, respectively, at a dose of 60mg/kg. , 30mg/kg and 15mg/kg. The positive control group was treated with cyclophosphamide (4 mg/ml) at a dose of 20 mg/kg. The negative control group used physiological saline.

(2) Cell culture

The frozen cells were removed from the liquid nitrogen and immediately placed in a 37 ° (water bath to melt the cells. Biosafety Rejection) The cell suspension was pipetted into a centrifuge tube containing the appropriate amount of medium and centrifuged at 800 rpm for 5 minutes. The supernatant was discarded and 10 ml of RPMI1640 containing 10% calf serum was added. The cells were cultured at 37 ° C, 5% CO ₂ , saturated humidity to 80% contact confluence, and 0.2% trypsin was digested into single cell suspension. The cells were counted under a light microscope using a counting plate, and the number of cells was adjusted to lx 10 ⁷ /ml with sterile physiological saline.

(3) In vivo test

Biosafety refused to disinfect for 30 min. Ignite the alcohol lamp (the device is sterilized during the inoculation). The operator wears sterile gloves, a hat, and a mask. Single-handed mice, 75% alcohol cotton balls were used to disinfect the right groin, each subcutaneously injected with 0.1 ml of B 16 malignant melanoma cell suspension. After the injection, the small tweezers pinch the pinhole for a while to prevent the liquid from overflowing. The vital signs and tumor growth of the mice were observed daily. C57BL/6 mice with melanoma B 16-F10 with strong tumor growth and no ulceration were selected and sacrificed by cervical dislocation. In a sterile clean bench, the skin of the animal was disinfected with alcohol, the skin was cut, and the tumor was removed. The tumor tissue was cut into 1.5 mm ^{3 to} prepare B 16 melanoma cell suspension, and then sterile saline was added in a certain proportion and inoculated subcutaneously into the mouse murine. The number of tumor cells per mouse was 1 X 10 . ⁶ , each 0.1ml. Animals were randomized the next day after tumor inoculation. The intragastric administration was started when the tumor grew to 40 mm ³ . The negative control group received 0.1 ml of normal saline per day, and the positive control group received 0.1 ml of cyclophosphamide (4 mg/ml) per day. The high dose group, the middle dose group and the low dose group were respectively 0.1 ml of different concentrations (12 mg/ml, 6 mg/ml and 3 mg/ml) of doxycycline were administered. The diameter of the transplanted tumor was measured daily with a vernier caliper.

(4) Evaluation method (Example 11 - Example 14)

Tumor volume was calculated using the method of measuring the tumor diameter. The tumor volume (TV) is calculated as: V = l/2 xaxb ² . Where a and b represent the length and width, respectively. According to the measurement results, the relative tumor volume (RTV) was calculated, and RTV = Vt / V0. Where V0 is the measured tumor volume at the start of dosing (ie, dO) and Vt is the tumor volume at each measurement. The anti-tumor activity evaluation index is the relative tumor inhibition rate (%) = (1-T/C) X 100%. T/C ( % ) = TRTV/CRTV x 100%. TRTV: treatment group RTV; CRTV: negative control group RTV. Efficacy evaluation criteria: Relative tumor inhibition rate (%) > 60%, and statistically treated P < 0.05 is effective; relative tumor inhibition rate (%) 60% is invalid. All data were analyzed by ANOVA ANOVA with SPSS 17.0 statistical software. The difference was statistically significant at P < 0.05.

Experimental results:

1, the first test r

The mouse B16 melanoma xenograft model established in this experiment was the first after tumor cell inoculation.

A nodular mass of about 40 mm ³ was reached subcutaneously in the mouse sac. As can be seen from the tumor growth curve (see Table 39 and Figure 62), on the 11th day of tumor growth, the tumor volume of the cyclophosphamide group and the doxycycline high-dose group were smaller than the control group, and the difference was statistically significant ( P<0.05). On the 13th day of tumor growth, the tumor volume of the cyclophosphamide group and the doxycycline high, medium and low dose groups were smaller than the control group, the difference was statistically significant (P<0.05). On days 14 and 15 of tumor growth, the tumor volume in the middle and low dose groups of doxycycline was smaller than that in the control group, and the tumor volume in the low dose group of doxycycline on day 14 was smaller than that in the cyclophosphamide group. Significance (P<0.05).

In addition, compared with the control group, the transplanted tumor after administration of doxycycline showed a decrease in blood supply vessels and easy exfoliation of the tumor when the tumor was dissected, suggesting that doxycycline inhibits tumor angiogenesis and infiltration of surrounding normal tissues. The role (see Figure 63).

Compared with the control group, the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 36%; high dose group: 49%; medium dose group: 65%; low dose group: 63% (see Table 39).

In addition, compared with the control group, the doxycycline high and middle dose group weight was greater than the control group, the difference was statistically significant (P<0.05); compared with the cyclophosphamide group, the doxycycline high, middle dose group Body The weight was greater than that of the cyclophosphamide group, and the difference was statistically significant (P<0.05) (see Figure 64). First test of the horse)

Tumor inhibition

Control cycloal acid saddle in the middle of the low dose group

Day

夭夭

Day

Tumor inhibition rate Note: * P < 0.05 compared with the control group; # P < 0.05 compared with the cyclophosphamide group. 2, repeat the test

The mouse B 16 melanin xenograft model established in this experiment can also reach about 40 mm ³ of nodular mass in the mouse sac on the 8th day after tumor cell inoculation, which is consistent with the first test results. The results of this trial showed (Table 40 and Figure 65) that on the 9th day of melanoma xenograft growth, the tumor volume in the high-dose and middle-dose groups was significantly smaller than that in the control group, about 1/2 of the control group; On the 10th day of growth, the tumor volume of the cyclophosphamide group and the doxycycline high and middle dose groups were smaller than the control group; on the 11th day of tumor growth, only the doxycycline high dose group had a smaller tumor volume than the control group. On days 12, 16, 17, and 18 of tumor growth, the tumor volume of the cyclophosphamide group, doxycycline high, and low dose groups was smaller than that of the control group; at 13, 14, 15 and 19, 20 days of tumor growth At 5 time points, the tumor volume of the cyclophosphamide group and the doxycycline high, medium and low dose groups were smaller than those of the control group. In addition, on the 19th day of tumor growth, the tumor volume of the high dose group of doxycycline was smaller than that of the middle dose group; the above results were statistically significant (P < 0.05).

Compared with the control group, the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 59%; high dose group: 73%; medium dose group: 28%; low dose group: 56% (see Table 40).

In addition, compared with the control group, the doxycycline middle dose group was larger than the control group, while the cyclophosphamide group weight was lower than the control group, the difference was statistically significant (P<0.05); compared with the cyclophosphamide group, more The body weight of the high, middle and low dose groups of the cyclin was higher than that of the cyclophosphamide group, and the difference was statistically significant. (P<0.05) (see Figure 66)

Table 40 Tumor volume and tumor inhibition rate of mouse B16 melanoma xenografts (mm ³ ± s complex test) Time / inhibition ί

Group t ring 3⁄4 acid amine quotient group middle dose group low dose group

Ε天. 76,δ52±5ϊ.94!> 55.512^42.569 59-08 · 30.994 Flat

9 days 9"; 7.826^86-940 64.210i36.553' 77.495^45,940'

10夭 I93.43⁄4t±105.200 !10.72Sig8.177'

P;"

Day 1 3.1 2±IQ9 52 "1 1. 3±i07.2S? ^v 67.617±3S 948 ί65.045±141.427

12 days 436ΛΜ±Ϊ5Η 626 195.261=^125.944* 3 4.296±i75285

13 ¾ 540.23S ± 336.406 162.148 ± 5..207 "269. 77 ± 26S.09 240.S4iil.35.245 '¾

14 days 676.230±354.S23 241.04§±154..67?'

15 3⁄4 S04.68S=b472.4SS i55.279±91.S9S* i 2.752±I 2.49f 231.22S±153.697 ^ft

16 days 10 1.752± S.5S2 50S.394±300. 55" " 486.15Si376.295*

17 days! IS5.13 ± 9S.S2 569.384x348.1 "13""

Si2.2SS 255.035 542.262+407J7T Day "i374..68S±i025.061 621.924=t396.563- 4 6,S06=1S3732" 94 .724^ 92.405 5S0.944i416.I66"

:19 days 436.032±562.364 ^t÷ 566.521+529.430"

20 days 25§5.¾0 ± 1208.517 101T.204 ± 415.67t 505.9ST = 302.629 * 1217.340 ± g60,63g ¾ god disease rate 59%, 2S% 56%

s

i i-- Note: * P<0.05 compared with the control group; + P<0 i j.05 compared with the middle dose group; This part of the experiment established and analyzed the C16 mouse B16 melanoma xenograft model. Effect of cyclulin on the growth of mouse B16 melanoma xenografts. The results showed that: Doxe H-inhibition significantly inhibited the growth of mouse B16 melanoma xenografts, with the highest tumor inhibition rate of 73%; doxycycline can increase the body weight of mice bearing B16 melanoma xenografts, improve the load The survival status of tumor mice indicates that doxycycline has good antitumor activity against melanoma and has low toxicity and side effects. Example 12 Effect of doxycycline on Lewis lung cancer xenografts in C57BL/6 mice

Method steps:

(1) Test grouping and dose setting

The same test grouping and dose setting as in the corresponding item in Example 11

(2) Cell culture

The operation method is the same as that under the corresponding cell culture in Example 11, except that The cultured cells were mouse Lewis lung cancer cells.

(3) In vivo test

Biosafety refused to disinfect for 30 min. Ignite the alcohol lamp (the device is sterilized during the inoculation). Operator with sterile gloves, hat, mask. Single-handed mice, 75% alcohol cotton balls were used to disinfect the right rat's ankle, and each was subcutaneously injected with 0.1 ml of Lewis lung cancer cell suspension. After the injection, the small tweezers pinch the pinhole for a while to prevent the liquid from overflowing. The vital signs and tumor growth of the mice were observed daily.

C57BL/6 mice bearing Lewis lung cancer with strong tumor growth and no ulceration were selected and sacrificed by cervical dislocation. In a sterile clean bench, the skin of the animal was disinfected with alcohol, the skin was cut, and the tumor was peeled off. The tumor tissue was cut into 1.5 mm ^{3 to} prepare B 16 melanoma cell suspension, then sterile saline was added in a certain proportion, and inoculated into the back of the mouse. The number of tumor cells per mouse was 1 10 ⁶ . Each 0.1ml. Animals were randomized the next day after tumor inoculation. The intragastric administration was started when the tumor grew to 40 mm ³ . The mice in the negative control group were given 0.1 ml of normal saline per day, and the mice in the positive control group were given 0.1 ml of cyclophosphamide (4 mg/ml) per day. The high-dose group, the middle-dose group and the low-dose group were given 0.1 ml of different concentrations each day (12 mg). /ml, 6mg/ml and 3mg/ml) of doxycycline. The diameter of the transplanted tumor was measured daily with a vernier caliper.

(4) Evaluation method

Same as the evaluation method under the corresponding item in Example 11.

Experimental results:

1, the first test r

The mouse Lewis lung cancer xenograft model constructed in this experiment can reach about 40 mm ³ tumor subcutaneously about 11 days after tumor cell inoculation. The tumor growth curve obtained after measuring the tumor size can be seen ( See Table 41, Figure 67), when the tumor grew to the 15th day, the tumor volume of the cyclophosphamide group and the doxycycline high, medium and low dose groups were smaller than the control group; subsequently, when the tumor grew to the 16th day, The tumor volume of the middle and low dose groups of doxycycline was smaller than that of the control group; on day 17, the tumor volume of the high, medium and low dose groups of doxycycline was smaller than that of the control group; on the 18th day, the doxycycline was low. The tumor volume of the dose group was smaller than that of the control group and the cyclophosphamide group; on the 19th day, the tumor volume of the high and middle dose groups of doxycycline was smaller than that of the control group; the above results were statistically significant (P<0.05).

Compared with the control group, the inhibition rate of each group at the terminal time point was: cyclophosphamide group: 41%; High dose group: 68%; medium dose group: 68%; low dose group: 27% (see Table 41). In addition, compared with the control group, the weight of the high, middle and low dose groups was greater than that of the control group, the difference was statistically significant (P<0.05); the doxycycline high, medium and low dose compared with the cyclophosphamide group. The body weight of the group was greater than that of the cyclophosphamide group, and the difference was statistically significant (P<0.05) (see Figure 68).

Table 41 Tumor volume and tumor inhibition rate of mice with Lewis lung cancer xenografts (mm ³ , ± _s , first test)

Time/tumor inhibition rate control group ring 3⁄43⁄4 amine commercial dose group Zhongjing group side dose s

11 days. 41.i7tH16 41 ^14. 2 40.57 ±12.124

Day 43.445 .902

13夭

14 days

15 days 30.iS9 35.41S* 40.256±32.474 ^A

16夭

Haotian

IS day 87,773 ± 6i.226 7S = 44 42351 ± 32.S3S ¾ *

,

1 day

Tumor inhibition rate Note: * P < 0.05 compared with the control group; ^# P < 0.05 compared with the cyclophosphamide group.

2, repeat the test

The mouse Lewis lung cancer xenograft model constructed in this experiment can reach about 40 mm ³ of tumor under the skin about 8 days after tumor cell inoculation, and measure the tumor size.

The tumor growth curves obtained after i r- are shown in Fig. 69 and Table 42. As can be seen from the graph, on the 12th day of tumor growth, the tumor volume of the cyclophosphamide group, the doxycycline high, medium and low dose groups was smaller than that of the control group; on the 13th day of tumor growth, doxycycline was high, The tumor volume of the middle dose group was smaller than that of the control group; on the 14th day, the tumor volume of the cyclophosphamide group, the doxycycline high and low dose groups was smaller than that of the control group; on the 16th and 17th days, cyclophosphamide and doxycycline The tumor volume of the high-dose group was smaller than that of the control group; on the 20th day, the tumor volume of the high dose group of doxycycline was smaller than that of the control group. The above results were statistically significant (P < 0.05).

Compared with the control group, the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 30%; high dose group: 61%; medium dose group: 53%; low dose group: 43% (see Table 42).

In addition, compared with the control group, the weight of the cyclophosphamide group was lower than that of the control group, and the difference was statistically significant (P<0.05). Compared with the cyclophosphamide group, the body weight of the high, medium and low dose groups of doxycycline was The difference was statistically significant (P<0.05) than the cyclophosphamide group (see Figure 70). Table 42 Tumor volume and tumor inhibition rate of mouse Lewis lung cancer xenografts (mm ³ ±s repeated test)

Time 阆 / 撺绉 rate control group 3⁄4aamine 3⁄4 dose group medium dose 俎 low dose group

S Day 42.4«2±19.621 42.S05±40536 56..768±3S.777 64.519±30.560 40.01 ±lS.56i ί j

9 days 6 ..10 ±i?. 77 67.33^17.363

10 days 9397Q±51A 0 I 4.4&3÷36.614 92.S53⁄4S.407 0.73 ±34.346

11 days 137.6 5±^^ 300 i44396±47.S26 Ϊ0ό.577±2 ^9ί 117.1⁄23=63.726

12 days. 272.25S-136. 68 17i.077 ± 61..55S ¾ 126336 ± 73.1½ Λ 159.307 ± 75.ΰ'52 Α

13 days 247.225^78.735 Π0..8 7ϋ21;741 226.257±S5.049.

14 days

1Π·457 248. 26±I75.3S3 217.78 ±97.5θΓ

15 days .5<0±36 .S 5 2 .3 2±15 j7.16S 25S.lls5±i2 .63S 2^a.069±197 SS 249..16 ±10I.7S7

16 days 541.294±313. 3⁄433 302.SI9=21S.254 ^V 379.467±210. 3i 36S.930±201.5S0

17 days. 3n.9l9±2G6.2"9^ 446.777±2Ϊ . 62

18 days. 407.191±I 74-811 524JS4±1S4.325

19 days 625.464±3 7.540 455.S52±1SS, 43 44d274±202.253 57ί.4Ιό±33⁄49-489 5.17.,020±I .053

20 days S4S.766=4S6.1S2 6G1.C 0±376.60S 447. ±223.031 603.G33±401. & 27 610 ^170.76 Tumor inhibition rate 30% 6l3⁄4 533⁄4 433⁄4 Note: *P<0.05 compared with the control group

In this part of the experiment, we established the C57 mouse Lewis lung cancer xenograft model, and observed the effect of doxycycline on the growth of Lewis lung cancer xenografts in mice. The results showed that t: doxycycline significantly inhibited the growth of Lewis lung cancer xenografts in mice, with the highest tumor inhibition rate of 68%; in addition, compared with the traditional cytotoxic drug cyclophosphamide, doxycycline can Increasing the body weight of Lewis lung cancer xenograft mice and improving the survival status of tumor-bearing mice showed that doxycycline has good anti-tumor activity against Lewis lung cancer, and the toxicity and side effects are low. Example 13 Effect of doxycycline on transplanted tumor of MCF-7 breast cancer in mice

Method steps:

(1) Test grouping and dose setting

The test consisted of a negative control group, a positive control group, and a treatment group. The negative control group consisted of 15 rats in each group, and the positive control group, the high dose group, the middle dose group and the low dose group each had 6 rats. The positive control group used cyclophosphamide (4 mg/ml). The treatment group was set in three dose groups: high, medium and low. The dose is set at 4:2:1. According to the clinical maximum dose, three different concentrations (high, medium and low) of doxycycline solution will be prepared in this experiment. The concentrations are: 12mg/ml 6mg/ml and 3mg/ml, respectively, the dose is 60mg/kg 30mg /kg and 15mg/kg. The positive control group was treated with cyclophosphamide (4 mg/ml) at a dose of 30 mg/kg. (2) Cell culture

The procedure was the same as in the corresponding cell culture in Example 11, except that the cells cultured were MCF-7 breast cancer cells.

(3) In vivo test

Biosafety refused to disinfect for 30 min. Ignite the alcohol lamp (the device is sterilized during the inoculation). The operator has sterile gloves, a hat, and a mask. Single-handed mice, 75% alcohol cotton balls were used to disinfect the right groin, and each human breast cancer MCF-7 cell suspension was 0.1 ml. After the injection, the small tweezers pinch the pinhole for a moment to prevent the liquid from overflowing. The vital signs and tumor growth of the mice were observed daily.

BALB/C mice bearing MCF-7 breast cancer with vigorous growth and no ulceration were selected and sacrificed by cervical dislocation. In the sterile clean bench, the skin of the animal was disinfected with alcohol, the skin was cut, and the tumor was removed. The tumor tissue was cut into 1.5 mm ^{3 to} prepare MCF-7 breast cancer cell suspension, then sterile saline was added in a certain proportion, and inoculated into the back of the mouse. The number of tumor cells per mouse was 1 10 ⁶ , each 0.1ml. Animals were randomized the next day after tumor inoculation. The intragastric administration was started after the tumor was grown to about 40 mm ³ . The mice in the negative control group were given 0.1 ml of normal saline per day, and the mice in the positive control group were given 0.1 ml of cyclophosphamide (4 mg/ml) per day. The high-dose group, the middle-dose group and the low-dose group were given 0.1 ml of different concentrations each day (12 mg). /ml, 6mg/ml and 3mg/ml) of doxycycline. The diameter of the transplanted tumor was measured every other day using a vernier caliper.

(4) Evaluation method

Same as the evaluation method under the corresponding item in Example 11.

Experimental results:

1, the first test r

The mouse MCF-7 breast cancer xenograft model constructed in this experiment can reach about 40 mm ³ of tumor nodules subcutaneously on the 16th day after tumor inoculation. As can be seen from the tumor growth curve (Fig. 71, data shown in Table 43), the tumor volumes in the high, medium and low dose groups of doxycycline were smaller than those in the control group on days 20, 24, 28 and 30 of tumor growth. On days 22 and 26 of tumor growth, the tumor volume of the high and middle dose groups of doxycycline was smaller than that of the control group; in addition, on days 20 and 22 of tumor growth, the doxycycline high, medium, and low dose groups The tumor volume was smaller than that of the cyclophosphamide group; on the 28th day of tumor growth, the tumor volume of the high and medium dose groups of doxycycline was smaller than that of the cyclophosphamide group. The above results were statistically significant (P<0.05) Compared with the control group, the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 45%; high dose group: 67%; medium dose group: 75%; low dose group: 72% (see Table 43).

In addition, there was no statistical difference in body weight between the groups in the MCF-7 breast cancer xenograft model.

''

Significance (P>0.05) (see Figure 72).

Table 43 Tumor volume and tumor inhibition rate of each group of MCF-7 breast cancer xenografts (mm ³ , ^ ± s , first test horse full)

Time' / tumor inhibition

Control group f3⁄43⁄4 volume group medium dose 3⁄4 & dose group rate

Day

"

Day

22 days

Day 150.342ϋ40.674 ^¾ days

28 days 1 & .377±Ι 17.3 ^# 229.765=194.065"

30 days

Tumor inhibition rate

Note: * ρ < 0.05 compared with the control group; ^# Ρ < 0.05 compared with the cyclophosphamide group.

2, repeat the test results

The rat model of MCF-7 breast cancer xenograft model established in this experiment can reach about 40 mm ³ of tumor nodules subcutaneously on the 12th day after tumor inoculation. As can be seen from the tumor growth curve (Fig. 73, data shown in Table 44), on the 20th to 34th day of tumor growth, the tumor volume of the high dose group of doxycycline was smaller than that of the control group; At 22 days, the tumor volume of doxycycline was smaller than that of the positive drug cyclophosphamide group, which was about 1/3 of the cyclophosphamide group; on the 28th and 30th day of tumor growth, the tumor volume of the high dose group of doxycycline Less than the low dose group; on days 32 and 34 of tumor growth, the tumor volume of the high dose group of doxycycline was smaller than that of the middle dose group. The above results were statistically significant (P < 0.05).

Compared with the control group, the tumor inhibition rates of the groups at the terminal time point were: cyclophosphamide group: 68%; high dose group: 98%; medium dose group: 44%; low dose group: 68% (see Table 44).

In addition, compared with the control group, the body weight of the cyclophosphamide group was lower than that of the control group, and the difference was statistically significant (P<0.05). Compared with the cyclophosphamide group, the body weight of the high, medium and low dose groups of doxycycline was higher than that of the cyclophosphamide group. The difference was statistically significant (P<0.05) than the cyclophosphamide group (see Figure 74). Table 44 Tumor volume and tumor inhibition rate of each group of murine MCF-7 breast cancer xenografts (mm ³ ± s repeat test)

Time / tumor rate control group ring phosphorus fiber: fatigue high dose group medium volume group low dose group

12夭 32.244±29.32 37.4όί±21.50 57.330=^47.100 50.234+44.780

1 day i. 9±33157 95.56 & ±46.777 64.644i40.i38 61. ?67^46.520

16 days 176,102±102.00S 154.996±S6 7 252.407±277.506

IS夭132.051^5.118 306,008^344.7S6 ¹ I84. 7±95 "1

20 days 407.I 4±214.22S 62ϊ.72(ϊ÷347..13⁄42 458.59ΰ±207. 31

22 days 7S5.S7C»±366.074 7 Ml ± 472.S42 5I7.520±i74.6S7 492(7fe4)S.340

24 days 1164.712±501.67S Sg5.0GI±527.1S3⁄4 S70.950i977.06

26 days 1577.31S±6.59,920 1019.927=769.538 190〗 6±li'5.0S 785.796 295.975 1036.197=925.594

28 days iS7 .6 1±76L936 Ι323.22 ±1θεθ.(17

I64.401÷1425.56i

30 3⁄4 2 S4.5g9i7S6.662 125S.969±I24I.990 154.249 275.6K ^{+ :} 3⁄4 ^;

34 days 3595.299=1249.372 13ίΰ, 149±1408.Ι93" 143.116±I65.4I2'" 3115375±S7S 03 220 2 29.646

r

Dog rate 68% 44% 0S% Note: compared with control group * P <0.05;^# cyclophosphamide group compared with P <0.05; + dose group compared with P <0.05; compared to low dose volumes P<0.05

In this part of the experiment, the effect of doxycycline on the growth of transplanted MCF-7 breast cancer was observed and analyzed by establishing a mouse model of MCF-7 breast cancer. The results showed that: doxycycline significantly inhibited the growth of transplanted MCF-7 breast cancer, the highest tumor inhibition rate was 98%; in addition, compared with the traditional cytotoxic drug cyclophosphamide, doxycycline It can increase the body weight of mice bearing MCF-7 breast cancer and improve the survival of tumor-bearing mice, indicating that doxycycline has good anti-tumor activity and toxicity and side effects on n MCF-7 breast cancer xenografts. Both are lower. Example 14 Study on the effect of doxycycline on transplanted tumor of H446 small cell lung cancer in nude mice

Method steps:

(2) Cell culture

The operation method was the same as that in the corresponding cell culture in Example 11, except that the cultured cells were H446 small cell lung cancer cells.

(3) In vivo test

Biosafety refused to disinfect for 30 minutes. Ignite the alcohol lamp (the device is sterilized during the inoculation). The operator wears sterile gloves, a hat, and a mask. Single-handed mice, 75% alcohol cotton balls were used to disinfect the right groin, and each cell suspension was injected with 0.1 ml. After the injection, the small tweezers pinch the pinhole for a while to prevent liquid Spillover. The vital signs and tumor growth of the mice were observed daily.

BALB/C mice bearing H446 small cell lung cancer with vigorous growth and no ulceration were selected and sacrificed by cervical dislocation. In the sterile clean bench, the skin of the animal was disinfected with alcohol, the skin was cut, and the tumor was removed. The tumor tissue was cut into 1.5 mm ^{3 to} prepare H446 small cell lung cancer cell suspension, then sterile saline was added in a certain proportion, and inoculated into the back of the mouse. The number of tumor cells per mouse was IX 106, each Only 0.1ml. Animals were randomized the next day after tumor inoculation. The intragastric administration was started after the tumor was grown to about 40 mm ³ . The mice in the negative control group were given 0.1 ml of normal saline per day, and the mice in the positive control group were given 0.1 ml of cyclophosphamide (4 mg/ml) per day. The high-dose group, the middle-dose group and the low-dose group were given 0.1 ml of different concentrations each day (12 mg). /ml, 6mg/ml and 3mg/ml) of doxycycline. The diameter of the transplanted tumor was measured every other day using a vernier caliper.

(4) Evaluation method

Same as the evaluation method under the corresponding item in Example 11.

Experimental results:

1, the first test r

The mouse H446 small cell lung cancer xenograft model established in this experiment can reach about 40 mm ³ of tumor nodules under the skin of the mouse on the 16th day after inoculation of the tumor cells. Tumor growth curves plotted after tumor size measurements (see Table 45 and Figure 75, showing that on day 20 of tumor growth, the tumor volume in the low dose group of doxycycline was smaller than in the control group; in the 22nd and At 24 days, the tumor volume of the cyclophosphamide group and the doxycycline medium and low dose groups were smaller than the control group; then on the 26th day of tumor growth, the tumor volume of the middle and low dose groups of doxycycline was smaller than that of the control group; On days 28 and 30, only the doxycycline mid-dose group had a smaller tumor volume than the control group. The above results were statistically significant (P < 0.05).

Compared with the control group, the tumor inhibition rates of the groups at the end time were: high dose group: 65%; medium dose group: 58%; low dose group: 55% (see Table 45). In the cyclophosphamide group, the tumor inhibition rate could not be calculated because all animals died at the terminal point.

In addition, there was no significant difference in body weight between the groups in the H446 small cell lung cancer xenograft model (P>0.05) (see Figure 76).

】

Will test)

Photo ff3⁄4 agent: amount a

Day

夭 - day m

Day suppression rate Note: * Compared with the control group, P<0.05

2, repeat the test results

The mouse H446 small cell lung cancer xenograft constructed in this experiment can reach about 40 mm ³ of tumor nodules in the skin of the mouse on the 16th day after tumor inoculation. Tumors were measured for long and short diameters and tumor volumes were calculated. The resulting tumor growth curves are shown in Table 46 and Figure 77. On the 22nd day of tumor growth, the tumor volume of the doxycycline middle dose group and the low dose group was smaller than that of the control group; on the 24th day, the tumor volume of the middle dose group was smaller than that of the control group, and the difference was statistically significant (P>0.05). )

Compared with the control group, the inhibition rate of each group at the terminal time point was: Compared with the control group, the inhibition rate of each group at the terminal time point was: cyclophosphamide group: 94%; High dose group: 66%; medium dose group: 81%; low dose group: 72% (see Table 46)

In addition, there was no statistical difference in body weight between the groups in the H446 small cell lung cancer xenograft model (P>0.05) (see Figure 78).

Table 46 Tumor volume and tumor inhibition rate of each group of mouse H446 small cell lung cancer xenografts (mm ³ , ± s, repeated test)

Control group high dose group fine group low dose group day

IS day

20夭

22夭

24夭 ;

Tumor inhibition rate Note: * P < 0.05 compared with the control group.

In this part of the experiment, the rat model of H446 small cell lung cancer xenograft was established and the effect of doxycycline on the growth of transplanted mouse H446 small cell lung cancer was observed and analyzed. The results showed that: doxycycline significantly inhibited the growth of transplanted H446 small cell lung cancer xenografts, the highest tumor inhibition rate was 81%; indicating that doxycycline has good antitumor activity against H446 small cell lung cancer xenografts. . Example 15 Effect of doxycycline on survival time of mouse B16-BL6 melanoma high metastatic model mice

Method steps:

(1) Test grouping and dose setting

(2) Cell culture

The water bath was preheated to 43 °C, and the cryopreserved B 16 malignant melanoma cells were rapidly warmed up in hot water at 20 ~ 30 °C, then centrifuged at 12000r/min for 10min, the supernatant was discarded, and 10ml was added to contain 10%. In RPMI1640 of calf serum, cultured to 80% contact confluence at 37 ° C, 5% CO ₂ , saturated humidity, 0.2% trypsin digestion into single cell suspension, cell counting with counting plate under light microscope Adjust the number of cells to lx 10 ⁷ /ml with sterile saline.

(3) In vivo test

Biosafety refused to disinfect for 30 min. Ignite the alcohol lamp (the device is sterilized during the inoculation). The operator has sterile gloves, a hat, and a mask. Single-handed mice, 75% alcohol cotton balls were used to disinfect the right groin, and each cell suspension was 0.1 ml. After the injection, the small tweezers pinch the pinhole for a while to prevent the liquid from overflowing. The vital signs and tumor growth of the mice were observed daily.

C57BL/6 mice with vigorously growing and unbroken melanoma B 16-BL6 were selected and sacrificed by cervical dislocation. In a sterile clean bench, the skin of the animal was disinfected with alcohol, the skin was cut, and the tumor was removed. The tumor tissue was cut into 1.5 mm ^{3 to} prepare B16 melanoma cell suspension, then sterile saline was added in a certain proportion, and tumor cells were inoculated into the tail vein of the mouse. The number of tumor cells inoculated into each mouse was lx l0 ⁶ , each 0.1ml, and administered intragastrically on the day after inoculation. The negative control group received 0.1 ml of normal saline per day. The positive control group received 0.1 ml of cyclophosphamide (4 mg/ml) per day. The high-dose group, the middle-dose group and the low-dose group were given 0.1 ml of different concentrations each day (12 mg). /ml, 6 mg/ml and 3 mg/ml) of doxycycline. After administration, the time of death of the animals was observed and the number of days of survival was calculated. Survival time was observed until the 60th day after modeling, and the remaining surviving mice were sacrificed by cervical dislocation.

(4) Evaluation method

The median survival time (MST) was used to evaluate the survival time of each group. The formula was: MST = (intermediate survival days -0.5) + intermediate survival days. The comparison between the treatment group and the control group is expressed by T/C (%), and the calculation formula is: T/C = T MST/ C MST X 100%. T MST: treatment group MST; C MST: negative control group MST. The evaluation criteria are bounded by 125%, when T/C% > 125%. It is considered valid, otherwise it is invalid. Test results:

1, the first test r

The median survival time of the negative control mice was 15.0 days. The median survival time of the cyclophosphamide group and the control group was 171%; the median survival time of the high, middle, and low dose groups of doxycycline and the control group were: 235%, 197%, and 184%, respectively. . It is indicated that the high, medium and low doses of positive drug and doxycycline can effectively prolong the survival of tumor-bearing mice. The survival curves of the mice in each group are shown in Figure 79. At the end of the 60-day observation period, 7 (7/37) mice remained healthy in the doxycycline group; all mice in the control group died.

2, repeat the test results

The median survival time of the negative control mice was 24.0 days; the median survival of the cyclophosphamide group was the same as that of the control group. The median survival time of the high, medium, and low dose groups of doxycycline and the control group were: 133%, 154%, and 104%, respectively. It indicated that the high and middle dose groups of doxycycline can effectively prolong the survival of tumor-bearing mice. The survival curves of the mice in each group are shown in Fig. 80. At the end of the 60-day observation period, 6 (6/45) mice in the doxycycline group survived healthyly; all mice in the control group died.

In this part of the experiment, the mouse B16-BL6 melanoma artificial lung metastasis model was established to observe and analyze the effect of doxycycline on the survival of lung metastasis mice. The results showed that doxycycline can significantly prolong the survival of tumor-bearing mice, improve the survival status of tumor-bearing mice, and increase their body weight, indicating that doxycycline has a good inhibitory effect on the hematogenous metastasis of melanoma cells. Example 16 Detection of the effect of doxycycline on cell migration in vitro at the cellular level

Cell line types and sources: B-16 cells (mouse melanoma): purchased from Beijing Jin Zijing Biomedical Technology Co., Ltd.; MCF-7 (human breast cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; HCCLM3 (human liver cancer cell line) : purchased from Beijing Jin Zijing Biomedical Technology Co., Ltd.); NCI-H446 (human small cell lung cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd.; ASPC-1 (pancreatic cancer cell line): purchased from Nanjing Kaiji Bio Technology Development Co., Ltd.; SW620 (human colon cancer cell line): purchased from Nanjing Kaiji Biotechnology Development Co., Ltd. Real horse full method:

After the above cells are trypsinized, they are dispersed into individual cells and suspended in the corresponding medium. The cells were seeded on 96-well plates at 4000 cells/well. The cells were cultured overnight in a 37 ° (carbon dioxide (5%) incubator. The next day, the culture solution was discarded and a medium containing a series of concentrations of the test substance (doxycycline) was added. Day, aspirate the culture solution, wash the cells twice with PBS, add Hochest 33342 (dissolved in phosphate buffer pH 7.4, final concentration 5 g / mL), staining solution 50 μΙ well, 37 ° (carbon dioxide (5%) Incubate for 20 min in the incubator. Aspirate the staining solution, wash twice with PBS, add the medium containing a certain concentration of the drug to be tested, and then put the effect of the 'J test drug on cell migration.

The effect of doxycycline on cells can be analyzed by the software that comes with the high content analysis platform. The effect of the drug on cell migration rate is primarily indicated by the slope of the curve in the resulting average distance-time plot. The higher the slope, the faster the cell migration rate, and the smaller the slope, the slower the cell migration rate.

Experimental results 1 inhibition of migration of MCF-7 cells by doxycycline

The inhibitory effect of doxycycline on MCF-7 cell migration is shown in Figure 81. In the figure, D7 is the control group, and D8 and D9 are the force. In the drug group, the drug concentration in the D8 group was 20 μΜ, and the drug concentration in the D9 group was 4 μΜ. As can be seen from the figure, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. It was proved that doxycycline significantly inhibited the migration of MCF-7 cells.

Experimental results 2. Inhibition of migration of HCCLM3 cells by doxycycline

The inhibitory effect of doxycycline on HCCLM3 cell migration is shown in Figure 82. In the figure, C6 is the control group, C7 and C8 are the doxycycline group, and the C7 and C8 groups are all 20 μΜ. As can be seen from the figure, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. Proof of Dorset It inhibits the migration of HCCLM3 cells.

Experimental results 3 Inhibition of B16 cell migration by doxycycline

The inhibitory effect of doxycycline on B16 cell migration is shown in Figure 83. In the figure, B6 is the control group, B7 and B8 are the dosing group, the drug concentration in the B7 group is 4 μΜ, and the drug concentration in the Β8 group is 20 μΜ. As can be seen, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. It was demonstrated that doxycycline significantly inhibited the migration of B16 cells.

Experimental results 4 inhibition of migration of NCI-H446 cells by doxycycline

The inhibitory effect of doxycycline on NCI-H446 cell migration is shown in Figure 84. In the figure, D11 is the control group, D9 and D10 are the dosing group, the D9 group has a drug concentration of 4 μΜ, and the D10 group has a drug concentration of 20 μΜ. As can be seen, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. It was proved that doxycycline significantly inhibited the migration of NCI-H446 cells.

Experimental results 5 inhibition of ASPC-1 cell migration by doxycycline

The inhibitory effect of doxycycline on ASPC-1 cell migration is shown in Figure 85. In the figure, C2 is the control group, C3, C4, and C5 are the dosing groups, and the drug concentration is 20 μΜ. As can be seen, the slope of the motion curve of the cells in the dosing group was smaller than that of the control group. It was proved that doxycycline has a certain inhibitory effect on the migration of ASPC-1 cells.

Experimental results 6 inhibition of migration of SW620 cells by doxycycline

The inhibitory effect of doxycycline on SW620 cell migration is shown in Figure 86. D2 was the control group, C9, CIO, and C11 were the dosing group. The C9 drug concentration was 20 μΜ, the C10 drug concentration was 10 μΜ, and the Cl l drug concentration was 4 μΜ. As can be seen, the slope of the motion curve of the cells in the dosing group was significantly smaller than that of the control group. It was proved that doxycycline has a certain inhibitory effect on the migration of SW620 cells.

Through the above experiments at the in vitro cell level and animal experiment level, doxycycline on human invasive choroidal melanoma, human malignant melanoma, human breast cancer, human chronic myeloid leukemia, promyelocytic leukemia, human large cell lung cancer , human small cell lung cancer, human metastatic liver cancer, human metastatic pancreatic adenocarcinoma, cervical cancer, human high metastatic colon cancer, human high metastatic gastric cancer and other high metastatic malignant tumor cells have different degrees of inhibition, such as growth, migration and invasion, At the same time, it can well inhibit the growth and metastasis of tumors in tumor-bearing mice, and can be used for the treatment of the above tumors.

Moreover, the doxycycline of the present invention has advantages unmatched by other drugs, and is easily accepted by patients and is easy to understand the patient's response to drugs. Doxycycline will alter existing cancer chemotherapy drugs The market pattern has become a clinical drug that can be taken for a long time and effectively inhibits tumor metastasis, invasion and recurrence.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalents, improvements, etc., which are included in the spirit and scope of the present invention, should be included in the present invention. Within the scope of protection.

Claims

Claim

The use of doxycycline for the preparation of a medicament for treating a highly metastatic malignant tumor, wherein the molecular structural formula of the doxycycline is:

2. The use according to claim 1, wherein the application of the medicament for treating a highly metastatic malignant tumor includes use as a drug for inhibiting tumor cell growth, application as a drug for inhibiting tumor cell metastasis, and inhibition of tumor cells. The application of invasive drugs.

3. The use according to claim 1, wherein the drug for treating a highly metastatic malignant tumor comprises doxycycline, doxycycline in a pharmaceutically acceptable salt, an ester, a hydrate or a combination thereof. And accessories.

The use according to any one of claims 1 to 3, wherein the dosage form of the medicament for treating a highly metastatic malignant tumor is selected from the group consisting of a tablet, a capsule, a pill, a suppository, an aerosol, and a mouth. A liquid preparation, granule, powder, injection, syrup, wine, tincture, lotion, film, or a combination thereof.

The use according to claim 4, wherein the administration of the medicament for treating a highly metastatic malignant tumor comprises oral administration, injection, implantation, external application, spraying, inhalation or a combination thereof.