CN110935012A

CN110935012A - A pharmaceutical composition for treating tumor

Info

Publication number: CN110935012A
Application number: CN201911315648.7A
Authority: CN
Inventors: 程海荣; 周义发; 陈磊; 台桂花
Original assignee: Northeast Normal University
Current assignee: Northeastern University China; Northeast Normal University
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2020-03-31

Abstract

The invention discloses a pharmaceutical composition for treating tumors. The invention discloses a medicine composition for treating tumor, which consists of DHCP and TRAIL. Experiments prove that the combination of the DHCP and the TRAIL can inhibit the growth of tumor cells, promote the apoptosis of the tumor cells and further inhibit the growth of tumors, and the effects are remarkably higher than those of the DHCP and the TRAIL which are used independently, so that the DHCP and the TRAIL can be used for treating tumors and have wide application prospects.

Description

A pharmaceutical composition for treating tumor

Technical Field

The invention relates to a pharmaceutical composition for treating tumors, belonging to the field of biomedicine.

Background

Colon cancer is a highly lethal malignancy worldwide. At present, the treatment means for colorectal cancer mainly comprises surgical resection, radiotherapy, chemotherapy, and targeted treatment means adopting angiogenesis inhibitor and epidermal growth factor receptor inhibitor. However, colon cancer differs in molecular characteristics, response to radiation and chemotherapy, clinical presentation, and prognosis of the disease. Therefore, there is a great need to develop effective therapeutic means.

Human TRAIL (TNF Related Apoptosis inducing ligand) is one of the members of the tumor necrosis factor superfamily. TRAIL can be combined with death receptors on the surface of cell membranes to transmit apoptosis signals into cells, so that tumor cells are specifically killed. TRAIL is a promising drug for treating tumors due to its selective killing effect on tumor cells, but the drug resistance of many tumor cells to TRAIL limits its application in tumor treatment. Therefore, the search for drugs capable of restoring the sensitivity of tumor cells to TRAIL becomes a new strategy for tumor therapy.

At present, a plurality of chemotherapeutic drugs or natural drugs and TRAIL are used in combination for treating tumors in clinical trials, and the results show that some chemotherapeutic drugs used in clinical trials can enhance the sensitivity of TRAIL-resistant cells to TRAIL. However, chemotherapeutic drugs not only kill tumor cells, but also are toxic to normal tissue cells of the body. Therefore, the search of natural products with small toxic and side effects and the combination of TRAIL for treating the tumor is of great significance.

Pectin is a plant polysaccharide with a complex structure and has various biological activities, including an anti-tumor effect. The pectin can reduce the polymerization degree and esterification after being subjected to chemical treatment, high-temperature and high-pressure treatment, radiation treatment or enzymolysis treatment, which is beneficial to enhancing the antitumor activity of the pectin, such as inhibiting tumor growth, inducing tumor cell apoptosis, inhibiting migration, regulating immune response and the like. DHCP is pectin modification derivative separated from lemon pectin after high temperature and high pressure treatment, and has killing effect on various tumor cells.

Disclosure of Invention

The technical problem to be solved by the invention is how to treat tumors, especially colon cancer.

In order to solve the above technical problems, the present invention provides a kit comprising DHCP ((Trans)4,5-dihydroxy-2-cyclopentene-l-one) and TRAIL (human tumor necrosis factor-related apoptosis-inducing ligand).

The kit may have any of the following uses:

a1, preparing a product for inhibiting the growth of tumor cells;

a2, preparing products for promoting apoptosis of tumor cells;

a3, preparing a product for inhibiting the growth of tumors;

a4, inhibiting the growth of tumor cells;

a5, promoting the apoptosis of tumor cells;

a6, inhibiting tumor growth.

DHCP is shown in equation 1:

the invention also provides a medicament, and the active ingredients of the medicament are DHCP and TRAIL.

The medicament may have any one of the following uses:

a1, preparing a product for inhibiting the growth of tumor cells;

a2, preparing products for promoting apoptosis of tumor cells;

a3, preparing a product for inhibiting the growth of tumors;

a4, inhibiting the growth of tumor cells;

a5, promoting the apoptosis of tumor cells;

a6, inhibiting tumor growth.

The application of the kit or the medicament in preparing the product for inhibiting the growth of the tumor cells also belongs to the protection scope of the invention.

The application of the kit or the medicament in inhibiting the growth of tumor cells also belongs to the protection scope of the invention.

The application of the reagent set or the medicine in preparing the product for promoting the apoptosis of tumor cells also belongs to the protection scope of the invention.

The application of the kit or the medicament in promoting the apoptosis of tumor cells also belongs to the protection scope of the invention.

The application of the kit or the medicament in preparing a product for inhibiting the growth of the tumor also belongs to the protection scope of the invention.

The application of the kit or the medicament in inhibiting the growth of the tumor also belongs to the protection scope of the invention.

In the present invention, the tumor cell may be a solid tumor cell. The solid tumor cell can be a colon cancer cell.

The tumor may be a solid tumor. The solid tumor may be colon cancer.

In one embodiment of the invention, the tumor cells are human colon cancer cells HCT116 and HT-29, and the solid tumor is colon cancer caused by human colon cancer cells HCT116 or HT-29.

In the invention, DHCP can be prepared according to the method comprising the following steps: dissolving galacturonic acid in water to obtain a galacturonic acid solution; and (4) treating the galacturonic acid solution at high temperature, and drying to obtain the DHCP.

In the above method, the high temperature treatment may be carried out at 121 ℃ under 17.2 to 21.7psi (0.12 MPa). The high-temperature treatment can be carried out for 4 times, and each time lasts for 1 h.

The drying may be performed by lyophilization.

Experiments prove that the combination of the DHCP and the TRAIL can inhibit the growth of tumor cells, promote the apoptosis of the tumor cells and further inhibit the growth of tumors, and the effects are remarkably higher than those of the DHCP and the TRAIL which are used independently, so that the DHCP and the TRAIL can be used for treating tumors and have wide application prospects.

Drawings

Fig. 1 shows the detection result of DHCP.

Figure 2 shows the effect of combined DHCP and TRAIL treatment on HCT116 (left panel) and HT-29 (right panel) cell survival (.; P < 0.01). The ordinate represents cell viability (%).

FIG. 3 shows the effect of combined DHCP and TRAIL treatment on apoptosis of HCT116 (top panel) and HT-29 (bottom panel).

FIG. 4 is a graph of the effect of DHCP and TRAIL administration on mouse body weight.

FIG. 5 shows the effect of combined administration of DHCP and TRAIL on the growth of transplanted tumors.

FIG. 6 is the tumor-bearing nude mice and the ex vivo transplanted tumor tissue pictures.

FIG. 7 shows that immunohistochemistry tests detected Ki67 and clear-caspase-3 expression in transplanted tumor tissues (scale: 200. mu.M). H & E can observe the tissue structure, and provide basis and reference for detecting the expression of Ki-67 and clear-caspase-3. (Black dots in the figure indicate the amount of Ki67 and clear-caspase-3 expressed in the tissues).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 combination of DHCP and TRAIL inhibits colon cancer cells and colon cancer growth

First, experimental material

The human colon cancer cells HCT116 and HT-29 are ATCC products.

BALB/cA-nu Mice is a product of Beijing Huafukang Biotechnology GmbH.

TRAIL: is an abcam product, cat # ab 109360.

The specific DHCP preparation method reference (Akimoto-Tomiyama et al, Production of 5' -phosphor by Catharanthus roseus cells produced by fat-degraded products from urea acid, J Biosci Bioeng.2002; 94(2):154-9) was carried out as follows:

preparation of DHCP

Weighing 4g galacturonic acid (Sigma Aldrich, cat # 92478), adding 800mL distilled water, stirring to dissolve, placing the obtained sample solution in a sterilizing pot, treating at 121 deg.C and 17.2-21.7psi (0.12MPa) for 4 times at high temperature for 1h each time, cooling to room temperature, filtering, and lyophilizing to obtain DHCP.

Determination of DHCP

A C18 chromatographic column (4.6X 250mm, column temperature 35 ℃) and a Shimadzu LC-10AT VP high performance liquid chromatograph system are provided with an SPD-10AVD ultraviolet detector to detect the DHCP in the sample. The mobile phase was an aqueous trifluoroacetic acid-methanol solution (i.e., 0.05% and 5% by volume trifluoroacetic acid and methanol, respectively, obtained by adding trifluoroacetic acid and methanol to water), the flow rate was 0.6mL/min, and the detection wavelength was 235 nm. The specific operation is as follows: weighing 5mg of sample to be detected, adding 500 mu L of trifluoroacetic acid-methanol aqueous solution, filtering with a 0.22 mu m organic filter membrane, taking 10 mu L of sample, and calculating the DHCP content in the sample according to the peak area of the absorption peak of the sample to be detected at 235 nm. The results are shown in FIG. 1.DHCP is shown in equation 1:

second, cell survival experiment for detecting DHCP-enhanced TRAIL's effect in inhibiting colon cancer

The cells to be tested: human colon cancer cells HCT116, HT-29.

Cells were as defined by 1X 10⁴Perwell Density plated in 96-well cell culture plates at 37 ℃ with 5% CO₂And (3) carrying out overnight adherent culture under the constant temperature and humidity condition. Each cell was assigned to DHCP treatment group, DHCP and TRAIL co-treatment group, and DHCP, TRAIL and Z-VAD-fmk co-treatment group. The medium was discarded and treated as follows:

DHCP processing group: adding complete culture medium containing different DHCP concentrations, wherein the concentration of DHCP in the culture system is set to be 0 μ M, 100 μ M, 200 μ M and 300 μ M;

DHCP and TRAIL co-treatment group (DHCP + TRAIL): adding complete culture medium containing DHCP and TRAIL with different concentrations, wherein the concentration of DHCP in the culture system is set to be 0 μ M, 100 μ M, 200 μ M and 300 μ M, the concentration of TRAIL in HCT116 cells in the culture system is 25ng/mL, and the concentration of TRAIL in HT-29 cells in the culture system is 100 ng/mL;

DHCP, TRAIL and Z-VAD-fmk co-treatment groups: (DHCP + TRAIL + Z-VAD-fmk): Z-VAD-fmk (Beyotime, cat # C1202, an irreversible broad-spectrum caspase inhibitor that inhibits apoptosis caused by caspase activation) was added to the culture system on the basis of the group treated with DHCP and TRAIL at a concentration of 20. mu.M in the culture system.

The volume of the added liquid in each group is equal, and the culture is continued for 24 hours after the addition is finished. Discard the supernatant, add 100. mu.L of MTT working solution to each well, and place at 37 ℃ in 5% CO₂Incubating for 4-6 hr in cell incubator, adding 50 μ L of 20% HCl-SDS (20 g of SDS is dissolved in 60ml of distilled water, 3.3ml of concentrated hydrochloric acid is added, and the volume is adjusted to 100ml) per well, standing at 37 deg.C and 5% CO₂And (3) incubating in a cell incubator for 12h, detecting the absorbance value at the wavelength of 570nm by using an enzyme-labeling instrument, analyzing the result, and calculating the cell survival rate (cell viability,%), wherein the cell survival rate is the absorbance value at the wavelength of 570nm of the experimental group/the absorbance value is multiplied by 100% only by the wavelength of 570nm of the complete culture medium.

The results (fig. 2) show that DHCP significantly enhances the inhibitory effect of TRAIL on proliferation of colon cancer cells HCT116 and HT-29: after HCT116/HT-29 cells were treated with 0. mu.M, 100. mu.M, 200. mu.M and 300. mu.M DHCP and corresponding concentrations of TRAIL, respectively or together, for 24h, DHCP and TRAIL treatment significantly inhibited proliferation of HCT116 and HT-29 cells compared to DHCP and TRAIL treated groups alone. Among them, 200. mu.M DHCP was most effective in enhancing TRAIL inhibition of cell proliferation.

Flow cytometry detection of DHCP-enhanced TRAIL inhibition of colon cancer

The cells to be tested: human colon cancer cells HCT116, HT-29.

Cells were aligned at 5X 10⁵Density per well plated in 6 well cell culture plates at 37 ℃ with 5% CO₂And (3) carrying out overnight adherent culture under the constant temperature and humidity condition. The treatment groups are divided into a control group, a DHCP treatment group, a TRAIL treatment group, a DHCP + TRAIL treatment group, a Z-VAD-fmk treatment group and a DHCP + TRAIL + Z-VAD-fmk treatment group. The medium was discarded and treated as follows:

control group: adding a complete culture medium;

DHCP processing group: adding complete culture medium containing different DHCP concentrations, wherein the concentration of DHCP in the culture system is set to be 200 μ M;

TRAIL treatment group: adding a complete medium containing TRAIL, wherein the concentration of TRAIL in the HCT116 cell in the culture system is 25ng/mL, and the concentration of TRAIL in the HT-29 cell in the culture system is 100 ng/mL;

DHCP + TRAIL treatment group: adding complete culture medium containing DHCP and TRAIL, wherein the concentration of DHCP in the culture system is set to be 200 μ M, the concentration of TRAIL in HCT116 cells in the culture system is 25ng/mL, and the concentration of TRAIL in HT-29 cells in the culture system is 100 ng/mL;

Z-VAD-fmk treatment group: adding complete medium containing Z-VAD-fmk, wherein the concentration of Z-VAD-fmk in the culture system is 20 mu M;

DHCP + TRAIL + Z-VAD-fmk treatment group: Z-VAD-fmk was added to the culture system on the basis of the DHCP + TRAIL-treated group, and the concentration of Z-VAD-fmk in the culture system was 20. mu.M.

The volume of the added liquid in each group is equal, and the culture is continued for 24 hours after the addition is finished. The medium was collected separately from each well, the wells were rinsed with PBS and the rinse was collected, and after digesting the cells for 2min with pancreatin (Beyotime, cat # C0201), 2mL of complete cell medium was added to terminate the pancreatin digestion reaction, and the cells were gently blown apart and cell suspensions were collected separately. Centrifuge at 1000rpm for 5min, add PBS (Beyotime, cat # ST476) and wash the cells once. The supernatant was discarded, 500. mu.L of PBS was added to resuspend the cells, 5mL of 75% ethanol aqueous solution pre-cooled at-20 ℃ was added, mixed well and fixed on ice for 30min or overnight at-20 ℃. Centrifuging at 2000rpm for 5min, discarding the supernatant, washing the cells once with PBS, adding 200 μ L PI staining solution (meilunbio, cat # MA0137) and staining in dark for 30min, detecting with flow cytometry, and analyzing the experimental result with ModFit 4.0, wherein the percentage of Sub G1 stage cells is the apoptosis rate.

The results (FIG. 3) show that DHCP-enhanced TRAIL induces apoptosis in colon cancer cells HCT116 and HT-29: after 24h of treatment of HCT116/HT-29 cells with 200. mu.M DHCP and corresponding concentrations of TRAIL, respectively or together, DHCP and TRAIL treatment significantly increased apoptosis of HCT116 and HT-29 cells compared to DHCP and TRAIL alone. In HCT116 cells, the control group, DHCP-treated group, TRAIL-treated group, DHCP + TRAIL-treated group, Z-VAD-fmk-treated group, DHCP + TRAIL + Z-VAD-fmk-treated group had apoptosis rates of 1.18%, 21.12%, 18.69%, 90.07%, 6.28%, 7.35%, respectively, and in HT-29 cells, the control group, DHCP-treated group, TRAIL-treated group, DHCP + TRAIL-treated group, Z-VAD-fmk-treated group, DHCP + TRAIL + Z-VAD-fmk-treated group had apoptosis rates of 3.58%, 12.95%, 17.86%, 93.41%, 2.58%, 2.74%, respectively.

Fourthly, detecting the function of DHCP enhanced TRAIL in inhibiting colon cancer at living body level

1. Establishment and treatment of nude mouse subcutaneous transplantation tumor model

(1) Raising experimental animals

The nude Mice (BALB/cA-nu Mice) are bred in a clean animal breeding center, and the breeding and experimental processes of the nude Mice follow the relevant guidelines in the handbook for experimental animal breeding management and use. Animals were fed ad libitum and water, changing bedding 2 times per week.

(2) Establishment of nude mouse transplantation tumor model

HCT116 cell suspension was prepared by suspending HCT116 in complete cell culture medium at a density of 1X 10⁷Adding matrigel melted on ice with equal volume per mL, mixing uniformly to obtain HCT116 injection, and placing on ice for later use. The HCT116 injection is sucked by a 1mL syringe and injected into the lower right side and the lower back of a nude mouse under the aseptic condition, 0.2mL of cell suspension is injected into each nude mouse, and 32 nude mice are inoculated to obtain the HCT116 nude mouse.

HT-29 was prepared to a density of 2X 10 using complete cell culture medium⁷After one/mL cell suspension, the mice were inoculated subcutaneously on the left and lower sides of the back by the method of HCT116 inoculation, and 32 mice were inoculated to obtain HT-29 nude mice.

(3) Grouping and administration of tumor-bearing nude mice

The grouping method comprises the following steps: nude mice inoculated with cells were housed in cages of 4 mice each for a total of 8 cages. Subcutaneous neoplasia was observed daily and, after about 5 days of inoculation, subcutaneous transplants were grouped when they reached a diameter of about 0.2 cm. The animals were randomly divided into 4 groups, i.e., a control group, a DHCP-administered group, a TRAIL-administered group, and a DHCP + TRAIL-administered group, and 8 animals were kept in cages. Using a 1mL disposable sterile syringe, after dissolving DHCP and TRAIL in physiological saline respectively, dosing was started according to the corresponding protocol for 21d continuously, and tumor volumes were measured:

DHCP group for administration: the medicine is administrated by adopting an intraperitoneal injection mode according to the dose of 0.5 mg/kg/time;

TRAIL administration group: the medicine is administrated by adopting a mode of fundus intravenous injection according to the dosage of 100 mu g/patient/time;

DHCP + TRAIL administration group: the medicine is administrated according to the dosage of 0.5 mg/kg/time DHCP and 100 mu g/time TRAIL, wherein DHCP is injected in the abdominal cavity, and TRAIL is injected in the fundus vein.

Control group: injecting normal saline into abdominal cavity and fundus;

the same site in each group was dosed in equal volumes. Tumor volume calculation formula: tumor volume (mm)³) 0.52 × minor diameter²X major axis.

(4) Observation of tumor-bearing nude mice and measurement of size of transplanted tumor

After administration, general conditions (mental, activity, diet, etc.) of the nude mice were observed every day, the nude mice were weighed weekly, the length and the short diameter of the transplanted tumor were measured every two days, and the volume of the transplanted tumor was calculated.

(5) Isolation of transplantable tumors

Mice were sacrificed by cervical dislocation after anesthesia with high dose of anesthetic. Stripping fresh tumor tissue from mouse, placing into 50mL centrifuge tube containing 4% paraformaldehyde, standing at 4 deg.C overnight, changing the soaking solution into 70% ethanol water solution the next day, and storing at 4 deg.C for a long time or preparing into slices.

2. Immunohistochemistry

(1) Slicing and dewaxing

The slices were washed in xylene buffer for 15min × 2 times, then in 100% ethanol aqueous solution for 5min × 2 times, and then washed for 5min in the order of 90%, 85%, and 70% ethanol concentration. Finally, wash with PBS 3min × 3 times.

(2) Antigen retrieval

Placing the slices on a plastic slicing frame, immersing in 10mM sodium citrate buffer solution with pH6.0, placing in a microwave oven, heating for 15min, cooling for 15min at room temperature, adding 0.1% pancreatin dropwise, placing at 37 deg.C for reaction for 3min, and washing with PBS for 3min × 3 times.

(3) Antibody incubation

The method is carried out by adopting a KIT (product number KIT-9710) of Fuzhou Michi New Biotechnology development Co., Ltd, 50 mu L of peroxidase blocking solution (reagent in the KIT) is dripped into each section, the section is incubated for 10min at room temperature, and the section is washed for 3min multiplied by 3 times by PBS. PBS was removed and 50. mu.L of serum from normal non-immunized animals (reagent in kit) was added dropwise to each section and incubated at room temperature for 10 min. Serum was removed, 50. mu.L of primary antibody (reagent in kit) was added dropwise, incubated at room temperature for 1h, and washed 5min X3 times with PBST. PBST was removed, 50. mu.L biotin-labeled secondary antibody (reagent in kit) was added dropwise, incubated at room temperature for 10min, and washed with PBS 3min X3 times. PBS was removed, 50. mu.L of Streptomyces antibiotic-antioxidant enzyme solution (reagent in kit) was added dropwise, incubated at room temperature for 10min, and washed with PBST 5min X3 times.

(4) DAB color development

Adding 1mL of distilled water and A, B, C three reagents into a DAB chromogenic kit (DAB-0031, product number DAB-New Biotechnology development Co., Ltd.) respectively one drop in 2mL of EP tube, mixing uniformly, dropwise adding into slices, developing at room temperature for 10min, and observing under a microscope. Washing with tap water, air drying, counterstaining with hematoxylin and eosin for 5min, washing with tap water, sequentially eluting with ethanol at concentrations of 70%, 85%, 90%, 100% and 100% for 5min, eluting with xylene for 15min × 2 times, air drying, adding neutral gum dropwise, covering with glass cover, and performing microscopic examination.

The results show that DHCP enhances the inhibition of TRAIL on the growth of subcutaneous transplanted tumors in nude mice:

the tumor major and minor diameters were measured at 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d and 21d of administration, respectively, and the tumor volume was calculated according to the formula. In the experimental process, the nude mice inoculated with the subcutaneous transplantation tumor have good growth state, normal diet and activity and no death of the nude mice. The measured data show that DHCP and TRAIL do not cause drug toxicity to tumor-bearing nude mice, and the body weight does not change obviously (FIG. 4).

The group co-administered with DHCP and TRAIL was able to significantly inhibit the growth of HCT116 and HT-29 cell transplantable tumors compared to the saline-injected group, DHCP-administered group and TRAIL-administered group (FIG. 5), and the tumor sizes of the control group, DHCP-administered group, TRAIL-administered group and DHCP + TRAIL-administered group were 1056 + -62, 798 + -86, 701 + -77 and 462 + -94 mm, respectively, in the HCT116 experiment at 21 days after administration³In HT-29 experiment, the tumor sizes of the control group, DHCP-administered group, TRAIL-administered group and DHCP + TRAIL-administered group were 983 + -54, 629 + -94,730±93、407±93mm³。

After administration of 21d, tumor-bearing nude mice and tumor tissues exfoliated from their bodies were photographed and recorded, thereby intuitively indicating that DHCP and TRAIL synergistically inhibit the growth of transplanted tumors (fig. 6).

Immunohistochemistry was used to detect Ki67 and clear-caspase-3 expression in transplanted tumor tissues from different treatment groups. The results show that: in HCT116 and HT-29 transplanted tumor tissues, the expression level of Ki67 was significantly reduced in the group co-treated with DHCP and TRAIL as compared to the other groups, and the expression level of clear-caspase-3 was significantly increased as compared to the other groups (FIG. 7). The results indicate that DHCP and TRAIL significantly inhibited the proliferation and promoted apoptosis of tumor cells in transplanted tumors in vivo.

Claims

1. The kit consists of DHCP and TRAIL.

2. A medicine contains DHCP and TRAIL as active ingredients.

3. Use of a kit according to claim 1 or a medicament according to claim 2 for the manufacture of a product for inhibiting the growth of tumor cells.

4. Use of the kit of claim 1 or the medicament of claim 2 for inhibiting the growth of tumor cells.

5. Use of a kit of parts according to claim 1 or a medicament according to claim 2 for the manufacture of a product for promoting apoptosis of tumor cells.

6. Use of the kit of claim 1 or the medicament of claim 2 for promoting apoptosis of tumor cells.

7. Use according to any one of claims 3 to 6, characterized in that: the tumor cell is a solid tumor cell.

8. Use of a kit according to claim 1 or a medicament according to claim 2 for the manufacture of a product for inhibiting the growth of a tumour.

9. Use of the kit of claim 1 or the medicament of claim 2 for inhibiting the growth of a tumor.

10. Use according to claim 8 or 9, characterized in that: the tumor is a solid tumor.