CN114177189A

CN114177189A - Application of thebaine glucoside as antitumor drug

Info

Publication number: CN114177189A
Application number: CN202111394931.0A
Authority: CN
Inventors: 朱彦; 刘海鑫; 姜苗苗; 贺爽; 王跃飞; 高秀梅
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2019-03-27
Filing date: 2019-03-27
Publication date: 2022-03-15
Also published as: CN114129583A; CN109908160B; CN109908160A; CN114129582A; CN114099527A

Abstract

The application is divisional application of patent application with application number 201910236799.7, application date of 2019, 03 and 27, and invention name of application of fibraurea B glucoside as antitumor drug. The invention belongs to the technical field of natural medicines, and particularly relates to application of vincetoposide as an anti-tumor medicine. The tumors include osteosarcoma, breast cancer, cervical cancer, ovarian cancer, neuroblastoma and liver cancer. The research of the invention finds that the thebaine has broad-spectrum anticancer effect, has the functions of inhibiting proliferation and directly killing various tumor cells, and has no obvious influence on normal endothelial cells and myocardial cells. The anti-tumor mechanism mainly comprises the inhibition of tumor cell proliferation, the promotion of tumor apoptosis, cell cycle retardation, the inhibition of tumor angiogenesis, tumor immunity and the like.

Description

Application of thebaine glucoside as antitumor drug

The application is divisional application of patent application with application number 201910236799.7, application date of 2019, 03 and 27, and invention name of application of fibraurea B glucoside as antitumor drug.

Technical Field

The invention belongs to the technical field of natural medicines, and particularly relates to application of vincetoposide as an anti-tumor medicine.

Background

Tumors are new organisms formed by clonal abnormal hyperplasia of cells of local tissues which lose normal regulation and control on the growth of the cells under the action of various carcinogenic factors of organisms, and are divided into benign tumors and malignant tumors, wherein the malignant tumors have greater threat to human life health. At present, malignant tumors are treated by a plurality of methods, wherein early tumors are mainly treated by operation, and middle and late tumors are mainly treated by radiotherapy and chemotherapy. Malignant tumors are easy to metastasize in the early stage, which brings great difficulty to surgical excision; when the radiotherapy and the chemotherapy kill tumor cells, patients are affected by toxic and side effects, and the prognosis is poor. And the first-line chemotherapy medicaments in the prior art have the problems of easy medicament resistance, easy relapse, large side effect and the like.

Among the various malignancies, Osteosarcoma (OS) is the most common primary malignant bone tumor in long bones, with particular risks for children and adolescents, with a childhood incidence of 0.2-3 cases/10 million and adolescents of 0.8-11/10 million per year. Metastasis is a major cause of OS-related death. Although effective chemotherapeutic drugs, as well as neoadjuvant chemotherapy, increase the 5-year survival of patients to 66-82%, only 30% of metastatic OS patients reach a 5-year event-free survival. Those patients with detectable metastasis have a 5-year survival rate as low as 19%. Furthermore, even in patients without primary metastasis, 40% will continue to develop secondary metastasis; in one study, the survival rates of patients with subsequent metastases from non-metastatic high-grade osteosarcoma were 5% and 23%, respectively, and lung and bone metastases were 4 years old, respectively. Meanwhile, the traditional chemotherapeutic drugs are accompanied by large side effects, and the poor tolerance of the osteosarcoma patients to the traditional chemotherapeutic drugs is another important reason for the poor survival rate of the osteosarcoma patients.

Disclosure of Invention

Aiming at the problems of easy drug resistance, easy relapse, large side effect and the like of first-line chemotherapy drugs in the prior art, the invention provides the application of the vincetoposide as an anti-tumor drug.

Also, the present invention provides a method for inhibiting proliferation of proliferating cells of osteosarcoma in vitro.

The invention also provides a broad-spectrum anti-tumor pharmaceutical composition.

In order to achieve the above purpose, the embodiment of the invention provides the application of vincetoposide as an anti-tumor drug.

Compared with the prior art, theilerioside is a natural product of cardiac glycosides, and is derived from the extract of oleander malabaricum (Thevetia peruviana (Pers.) K.Schum.) of Apocynaceae. The invention discovers the broad-spectrum anticancer effect of the thebaine glucoside in research, the antitumor mechanism of the invention comprises inhibiting the proliferation of tumor cells, promoting apoptosis, cycle retardation, inhibiting migration, inhibiting angiogenesis, tumor immunity and the like, and the invention has the functions of inhibiting proliferation and directly killing various tumor cells. However, the thebaine has no obvious influence on normal endothelial cells and myocardial cells, as shown in fig. 24 and 15, the thebaine has no obvious influence on the body weight, organ index and electrocardiogram of the mice, and the selectivity and safety of the thebaine to the tumor are proved. .

Preferably, the tumor includes osteosarcoma, breast cancer, cervical cancer, ovarian cancer, neuroblastoma, and liver cancer. The thebaine glucoside has proliferation inhibiting or direct killing effect on the tumor cells of the above tumors.

Preferably, the anti-tumor drug is an anti-osteosarcoma drug.

Preferably, the anti-tumor drug is an anti-breast cancer drug. In vivo experiments show that thevinoside can inhibit the growth of breast cancer in mice. Currently, activation of the immune system has been shown to be able to treat cancer by using targeted immune checkpoint regulatory proteins (e.g., PD-1) in the clinic. The thevinoside can remarkably activate tumor immunity and down-regulate the expression of PD-1 through various ways, thereby inhibiting the growth of 4T1 breast cancer. The inventor finds that the thevinoside has no effect of inhibiting the proliferation of 4T1 breast cancer cells in vitro, but can obviously inhibit the growth of 4T1 breast cancer cells in vivo.

Preferably, the anti-tumor drug is a drug that activates tumor immunity. The thebaine glucoside can obviously activate tumor immunity and reduce the expression of PD-1, thereby achieving the anti-tumor effect.

Preferably, the anti-tumor drug is a drug for inhibiting tumor angiogenesis. Neovascularization (angiogenesis) is the basis of tumor growth. Due to hypoxia, tumor tissues produce and release angiogenic growth factors, such as Vascular Endothelial Growth Factor (VEGF), acidic and basic fibroblast growth factors (aFGF and bFGF), and platelet-derived endothelial growth factor (PD-ECGF), to form new blood vessels. The thebaine glucoside can obviously inhibit the angiogenesis of endothelial cells and has the activity of inhibiting the angiogenesis of tumor.

The embodiment of the invention also provides a method for inhibiting proliferation of in vitro osteosarcoma proliferating cells, which comprises the step of adding the vincetoxicoside into a culture solution of tumor cells, wherein the final concentration of the vincetoxicoside in the culture solution is 10-100 nM. In the concentration range of 10-100 nM, thebaine can inhibit the angiogenesis of endothelial cells and the migration of human osteosarcoma cells.

The embodiment of the invention also provides a broad-spectrum antitumor pharmaceutical composition, which is characterized by containing the vincetoxicoside.

Preferably, the broad-spectrum anti-tumor pharmaceutical composition is prepared into tablets, capsules, granules, oral liquid, sustained release preparations, controlled release preparations, nano preparations or injections for convenient clinical use.

Drawings

FIG. 1 is a diagram of different cells screened by the high content method of example 1IC for drug thebaine glucoside₅₀A curve;

FIG. 2 is the IC of the drug vincetoposide against different cells screened by the CCK-8 method in example 1₅₀A curve;

FIG. 3 is a graph showing the effect of U2OS cell cycle on high content fluorescence image analysis in example 2;

FIG. 4 is a quantification plot of the U2OS cell cycle for high content analysis in example 2;

FIG. 5 is a graph showing the effect of the flow cytometer in example 2 on the U2OS cell cycle;

FIG. 6 is a quantification of the U2OS cell cycle measured by the flow cytometer in example 2;

FIG. 7 is a graph showing the results of flow cytometry detection of apoptosis in U2OS in example 3;

FIG. 8 is a quantification chart of the flow cytometry detection of apoptosis in U2OS in example 3;

FIG. 9 is a graph showing the effect of Western blot analysis on the expression of the apoptosis-related proteins Bax, Bcl-2 and Caspepase-3 of U2OS in example 3;

FIG. 10 shows the results of in vitro scratch assay of U2OS cells in example 4;

FIG. 11 is a line graph of the mobility of endothelial cells at different time points in the U2OS cell scratch test of example 4;

FIG. 12 is a photograph of tumors of three groups of mice on day 20 after inoculation of tumor cells in example 5;

FIG. 13 is a wet weight plot of the groups of tumors from example 5;

FIG. 14 is a graph showing the size of the tumor and the body weight of the mouse in example 5 with time;

FIG. 15 is the index of each organ after the administration of three groups of mice on the 20 th day after the inoculation of tumor cells in example 5;

FIG. 16 is the results of electrocardiographic monitoring of the three groups of mice administered after the 19 th day after the tumor cells were inoculated in example 5;

FIG. 17 is a CT image of tumor sites of the solvent control group and the high dose group in example 6;

FIG. 18 is a graph showing the quantitative determination of the number of blood vessels formed in the treatment of each of the treatment groups consisting of thevinoside A, the blank control group and the positive control group in example 6;

fig. 19 shows the results of in vitro scratch assay of ea.hy926 cells of example 6;

fig. 20 is a line graph of the mobility of endothelial cells at different time points in the ea.hy926 cell scratch experiment of example 6;

FIG. 21 is a photograph of a tumor from a 4T1 tumor model mouse from a micro-CT scan of example 6;

FIG. 22 is a blood vessel distribution map of ImageJ extraction in example 6;

FIG. 23 is a histogram of the number of branched blood vessels in example 6;

FIG. 24 is a volcanic plot of the global distribution (fold change 2 and p value 0.05) of Differentially Expressed Genes (DEG) of the tumor model blank control group (C) and the tumor model administered group (H) in Experimental example 7;

FIG. 25 is a graph showing the general profiles of transcripts significantly and differentially expressed in samples of blank control group and administered group by hierarchical clustering in Experimental example 7;

FIG. 26 shows the first 10 signal paths of the tumor model blank control group and the tumor model administered group in Experimental example 7;

FIG. 27 shows the first 10 signal pathways of the upstream regulatory gene of the differential genes in the tumor model blank control group and the tumor model administration group in Experimental example 7;

FIG. 28 is the ratio of the differential genes in tumor model blank control group and tumor model administration group in tumor and immune related targets in Experimental example 7;

FIG. 29 is the results of the experimental validation of the differential genes associated with tumor immunity in the Dendritic Cell (DCs) maturation pathway in example 7.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

The embodiment of the invention provides the influence of the eticoside serving as a broad-spectrum anti-tumor medicament on the in-vitro proliferation activity of various tumor cells, which specifically comprises the following steps:

1. experimental methods

1.1 Experimental cell lines

Hy926 (human umbilical vein cell fusion cell line) from the national key laboratory of modern Chinese medicine of Tianjin Chinese medicine university;

MYO (primary myocardial cell line in suckling mice) from the national focus laboratory of modern Chinese medicine of Tianjin Chinese medicine university;

u2OS (human osteosarcoma cell strain) from the national focus laboratory of modern Chinese medicine of Tianjin Chinese medicine university;

a2780 (human cervical cancer cell strain) from national key laboratory of modern Chinese medicine of Tianjin Chinese medicine university;

hela (human ovarian cancer cell line) from the national key laboratory of modern Chinese medicine of Tianjin Chinese medicine university;

MCF7 (human Sanyang breast cancer cell line) from the modern Chinese medicine national key laboratory of Tianjin Chinese medicine university;

SK-N-SH (neuroblastoma cell) from the central laboratory of modern Chinese medicine of Tianjin Chinese medicine university;

MDA-MB-231 (human triple negative breast cancer cell line) is from the national key laboratory of modern Chinese medicine of Tianjin Chinese medicine university;

4T1-luc (mouse breast cancer cell line with luciferase) from the modern Chinese medicine national focus laboratory of Tianjin Chinese medicine university.

1.2 cell culture method

DMEM high-sugar medium containing 10% FBS at 37 deg.C and 5% CO₂And performing conventional culture on the cells in an incubator, selecting 3 rd-9 th generation cells, digesting for about 1min by using 0.25% pancreatin when the cells are fused to 80% -90%, and using for subsequent experiments.

1.3 sample preparation

Mixing thebaine with DMSO to obtain a mixture of 10^-1mol/L、10^-2mol/L、10^-3The solution was divided into 10. mu.L/tube and stored at-20 ℃. Get 10^-2M, adding a mother solution of the organic solvent,diluting to 10% with phenol-free red medium containing 1% CS-FBS^-3M、10^-4M、10^- ⁵M、10^-6M、10^-7M、10^-8M、10^-9M in a sample solution. 1.4 high content cell imaging System for detecting the Effect of Dietyoxyfen on the Activity of different cells

Cells in the 1.2 are inoculated in a 96-well cell culture plate by using 1% FBS, 8000 cells are cultured in an adherent manner for 24h in each well, 1.3 yellow-clamp-glucoside samples (10mmol/L, 1mmol/L, 100nmol/L, 10nmol/L and 1nmol/L) with different concentrations are added for incubation for 24h, then the medicine is sucked out, cell nuclei are stained by using Hoechst 33342(1 mu g/L), incubated for 30min at 37 ℃, and washed for 3 times by using 1 XPBS. And (3) selecting 5 middle visual fields in each hole under a high content analyzer, photographing by using a 10-time objective lens, and calculating the number of cells in each hole. The data were analyzed for one-way anova (n-3) using SPSS16.0 statistical software, with the least significant difference level set to P<0.05，**P<0.01, and using Graph Pad Prism 6 as a statistical chart, as shown in figure 1, and respectively showing the IC of MCF-7, Hela, U2OS, A2780, SN-N-SH, hy926, MYO, 4T1 and MDA-MB-231 on the drug xanthophyllin₅₀(concentration of inhibitor half inhibited) curve. IC of each cell₅₀The values are shown in Table 1.

TABLE 1 IC of Dioscoreaside on different cells₅₀Value of

Cells	IC₅₀(mol/L)
		MCF7	5.452×10^-8
Hela	1.708×10^-8
		U2OS	1.742×10^-8
A2780	2.687×10^-8
		SK-N-SH	5.076×10^-8
EA.hy926	——
		MYO	——
4T1	——
		MDA-MB-231	——

As can be seen from FIG. 1 and Table 1, the vincetoposide has significant proliferation inhibiting effect on five cells, namely A2780, Hela, MCF7, SK-N-SH and U2OS, has no significant effect on normal cells (EA.hy926 and MYO), and has no in vitro inhibiting effect on 4T1 and MDA-MB-231. IC (integrated circuit)₅₀The values are shown in table 1, which suggests that the thebaine glycoside has selectivity in inhibiting cell proliferation, may have a significantly stronger killing effect on human tumor cells than normal cells, and may be a relatively safe antitumor drug.

1.5CCK-8 kit for detecting the influence of thebaistin on the activity of different cells

Inoculating the cells in 1.2 in 96-well cell culture plate with 1% FBS, 8000 cells per well, performing adherent culture for 24h, adding different concentrations of xanthophyll samples (10mmol/L, 1 mmol/L) in 1.3Per L, 100nmol/L, 10nmol/L, 1nmol/L) for 24 h. According to the specification of a CCK-8 kit, 10 percent of CCK-8 solution is added into each hole, and the mixture is incubated for 2 hours in an incubator at 37 ℃, and the application is realized

And 3, detecting the absorbance value of each hole with the wavelength of 450nm by using the multifunctional plate reading machine. The results are shown in FIG. 2 and Table 2, in which FIG. 2 shows the EC of MCF7, MDA-MB-231, Hela, A2780, SK-N-SH, U2OS, hy926, MYO and 4T1 against the drug xanthophyll₅₀Curve line. The result obtained by the CCK-8 kit detection is consistent with the result of high content cell imaging analysis, the thebainoside has obvious proliferation inhibition effect on A2780, Hela, MCF7, SK-K-SH and U2OS, and has no in vitro inhibition effect on 4T 1.

TABLE 2 IC of Dioscoreaside on different cells₅₀Value of

Cells	IC₅₀(mol/L)
		MCF7	5.806×10^-7
MDA-MB-231	1.589×10^-7
		Hela	9.179×10^-8
A2780	1.837×10^-8
		SK-N-SH	7.059×10^-8
U2OS	1.735×10^-8
		EA.hy926	2.290×10^-8
MYO	——
		4T1	——

Example 2

The embodiment of the invention provides the influence of the eticoside serving as an anti-osteosarcoma drug on the cell cycle of osteosarcoma.

1. High-content cell imaging system for detecting influence of eticoside on osteosarcoma cell cycle

The cells in the 1.2 are inoculated in a 96-well cell culture plate by using 1% FBS, 8000 cells are cultured in an adherent manner for 24h in each well, DMEM is fed to a control group, and DMEM with the concentrations of the samples containing the theiletin of 100nmol/L, 10nmol/L and 1nmol/L is fed to an administration group. 3 multiple wells per group, cells at 37 ℃ in 5% CO₂Culturing under saturated humidity condition, and after 24 hours of action, performing immunofluorescence staining: labeling EDU with the fluorescent dye Alexa488 (green), identifying S-phase cells; labeling (red) with phosphorylated histone PHH3 and its fluorescent dye Alexa647, identifying M phase cells; all nuclei are identified by a fluorescent dye Hochest33342 (blue), and cell thresholds of G1 stage and G2 stage are determined (chromosomes of cells in G1 stage are in diploid stage, and G2 stage is in mitotic stage, and two complete diploid chromosomes are contained in the cells, so that the fluorescence expression quantity of cells in G2 stage is twice that of cells in G1 stage, and the threshold range is set, so that G1 and G2 stages are distinguished. In the experimental setting of a high content analytical instrument, 13 middle visual fields are selected in each hole and are all used by 10 timesPhotographing by an objective lens, selecting three channels (Alexa647, Alexa488 and Hoechst33324), respectively setting exposure time to 3000ms, 1000ms and 200ms, calculating the number of cell nuclei marked by different fluorescent dyes, calculating the number of cell nuclei marked by different antibodies or dyes and the fluorescence intensity of cell nuclei marked by EdU by using high content analysis software, representing by mean +/-standard deviation, performing one-factor variance analysis on each group of data by using SPSS16.0 statistical software, and setting the minimum significant difference level as P<0.05，**P<0.01，***P<0.001. And Graph Pad Prism 6 is used as a statistical chart, and the results are shown in FIGS. 3-4.

The ratio of the number of cells of U2OS cells in different cell cycles to the number of cells in the whole cell cycle is shown in fig. 3 and 4: at the G0/G1 phase of the cell cycle, compared with a control group, the proportion of the administration group cells containing 100nM and 10nM thebaioside is obviously reduced (P <0.01), and the proportion of the administration group cells containing 10nM and 1nM thebaioside is not statistically different; at the S phase of the cell cycle, compared with a control group, the proportion of the cells of the administration group containing 100nM theiletin is obviously increased (P <0.01), and the proportion of the cells of the theiletin of 10nM and 1nM is not statistically different; at the G2 and M phases of the cell cycle, the proportion of cells in each administration group is not statistically different compared with the control group. Namely, 100nM thebaine can induce the cell cycle S phase block of U2OS cell (P < 0.01). This result is in concert with the result that philliptin at a concentration of 100nM significantly inhibited cell proliferation.

2. Flow cytometry method for detecting influence of etilenin on cell cycle of osteosarcoma cells

Selecting 3-9 generation U2OS cells, adding 10% FBS-containing DMEM high-sugar medium, and culturing at 37 deg.C under 5% CO₂Performing conventional culture in an incubator, changing 1% FBS for 24h when fusion reaches about 80%, feeding DMEM to a control group, feeding DMEM to an administration group, and incubating for 24h, wherein the concentrations of the samples containing the vincetoxicoside are 100nmol/L, 10nmol/L and 1nmol/L respectively; collecting cells, the number of which is about 1X 10⁶～5×10⁶Centrifuging at 1000r/min for 3min per mL, and discarding the culture solution; washing with PBS 1 time, centrifuging to remove PBS, adding ice-cold 70% v/v ethanol, and fixing at 4 deg.COver 12 hours; centrifuging to remove the stationary liquid, adding 3mL of PBS for re-suspension for 5min, centrifuging at 500-1000 r/min for 5min, and removing the PBS; staining with 1mL PI staining solution and protecting from light at 4 ℃ for 30 min.

Flow cytometry detection: the PI uses argon ions to excite fluorescence, the wavelength of laser light is 488nm, the wavelength of emitted light is more than 630nm, and a histogram of red fluorescence analysis PI fluorescence intensity is generated;

when analyzing the histogram of PI fluorescence, the gating technology is firstly used to eliminate cells which are doubled or aggregated and cell fragments which emit weak fluorescence, and on the histogram of PI fluorescence, apoptotic cells show a hypodiploid peak before the G1/G0 phase. If the fluorescence intensity of the G1/G0 phase position is 1.0, the fluorescence intensity of a hypodiploid peak of a typical apoptotic cell sample is 0.45, and the PI fluorescence intensities of red blood cells of chicken and salmon are taken as reference standards, which are respectively 0.35 and 0.7, so that cells which are not cell fragments but are intact can be ensured between the two. The results are shown in FIGS. 5 and 6.

As shown in fig. 5 and 6: the ratio of the number of cells of U2OS cells in different cell cycles to the number of cells in the whole cell cycle: at the G0/G1 phase of the cell cycle, compared with a control group, the proportion of the administration group cells containing 10nM and 100nM thebaioside is obviously reduced (P <0.01), and the proportion of the administration group cells containing 1nM thebaioside is not statistically different; at the S phase of the cell cycle, compared with a control group, the proportion of the cells of the administration group containing 100nM theiletin is obviously increased (P <0.01), and the proportion of the cells of the theiletin of 10nM and 1nM is not statistically different; at the G2 and M phases of the cell cycle, the proportion of cells in each administration group is not statistically different compared with the control group. Namely, 100nM thebaine can induce the cell cycle S phase block of U2OS cell (P < 0.01). This result is in concert with the result that philliptin at a concentration of 100nM significantly inhibited cell proliferation.

Example 3

The embodiment of the invention provides the influence of the eticoside serving as an anti-osteosarcoma drug on the apoptosis of osteosarcoma cells.

1. Flow cytometry for detecting influence of thebaine glucoside on apoptosis of U2OS cells

Selecting 3-9 generation U2OS cells, adding 10% FBS-containing DMEM high-sugar medium, and culturing at 37 deg.C under 5% CO₂Performing conventional culture in an incubator, changing 1% FBS for 24h when fusion reaches about 80%, feeding DMEM to a control group, feeding DMEM to an administration group, and incubating for 24h, wherein the concentrations of the samples containing theiletin are 100nmol/L, 10nmol/L and 1nmol/L respectively; and (3) sucking the culture medium supernatant, adding a proper amount of PBS (phosphate buffer solution) for rinsing, slightly shaking the cell culture vessel to enable the PBS to cover the whole vessel surface, and then sucking the PBS as clean as possible.

Adding a proper amount of pancreatin, incubating at 37 ℃, taking out every 1 minute or so, slightly shaking the cell culture vessel or slightly beating the side wall of the cell culture vessel, and observing whether the cells float up smoothly. When the cells were able to float smoothly, 3 volumes of the medium (containing 10% FBS) supplemented with pancreatin was added to terminate the digestion reaction. Centrifuge at 1000rpm for 5 minutes and remove the supernatant as clean as possible.

The cell sediment is re-suspended by using binding buffer and is placed in hot water at 50-55 ℃ to be used as the osteosarcoma cell to be detected.

Blank Control (Control): untreated, unstained cells were taken for initial voltage adjustment.

Annexin V single-stained tube: and taking the osteosarcoma cell to be detected, and singly staining the osteosarcoma cell with Annexin V.

Nucleic acid single staining tube: and taking the osteosarcoma cells to be detected, and singly dyeing the osteosarcoma cells with PI dye.

Negative Control (Negative Control): cell suspensions without vincetoposide were prepared as negative control samples as described above, double stained with Annexin V and PI, and used to draw a portal.

Positive Control (Positive Control): taking the osteosarcoma cell to be detected, Annexin V and nucleic acid dye for double dyeing.

Cells were resuspended in 100. mu.L binding buffer (cell number typically 10)⁵Respectively), adding proper amount of reagent according to the instruction.

Incubate for 15 minutes at room temperature in the dark.

After adding 400. mu.L binding buffer to the stained sample, it was placed on ice and examined within 1 day using an Attune NxT flow cytometer.

The results of flow cytometry to detect apoptosis of U2OS are shown in FIGS. 7 and 8, and the 10nM and 100nM of whether or not the thebaine glycoside could induce apoptosis of U2 OS.

2. Western blot analysis is used for researching the influence of the avicularin on the expression of human osteosarcoma cell U2 OS-related apoptosis protein

The results are shown in fig. 9, with Actin (β -Actin) as an internal reference, compared with a blank control group, the expression of the avidinide apoptosis-promoting protein Bax protein of 10nM and 100nM is significantly increased (P <0.05), and the avidinide of 1nM has no statistical difference; the expression of the apoptosis inhibiting protein Bcl-2 is obviously reduced (P is less than 0.05, P is less than 0.01), and the 1nM theiletin has no statistical difference; caspase-3 has a significant increase in the ratio of activated to unactivated (P <0.01), and there was no statistical difference in 1nM thebainoside.

Western blot results prove that, compared with a blank control group, the 10nM and 100nM penethamine can promote the expression of the apoptosis-promoting protein Bax, inhibit the expression of the apoptosis-inhibiting protein Bcl-2 and activate the apoptosis-executing protein Caspepase-3.

Example 4

The embodiment of the invention provides the influence of the eticoside serving as an anti-osteosarcoma drug on the migration capacity of osteosarcoma cells.

1. Study of whether thebaine glycoside can inhibit migration of osteosarcoma cells (U2OS)

U2OS cells were subjected to in vitro scratch experiments. As shown in fig. 10, 100nM and 10nM of vincetoposide significantly inhibited U2OS cell migration.

Mobility line graph 11 for U2OS cells at different time points shows that, at 12h time point, 100nM and 10nM of vincetoposide significantly inhibited U2OS cell migration compared to the control group (P < 0.05); 1nM theiletin inhibits U2OS cell migration without statistical difference; at the 24h time point, 100nM and 10nM of vincetoposide significantly inhibited U2OS cell migration (P <0.01) compared to the control group, and 1nM of vincetoposide inhibited U2OS cell migration with no statistical difference.

Example 5

The embodiment of the invention provides an effect of vincetoposide serving as an anti-breast cancer medicament for inhibiting growth of breast cancer of a mouse.

ICR mice, 18, 4-5 weeks old and female, were purchased from the institute for laboratory animal resources, SPF grade, China institute for food and drug testing. Raising at room temperature, alternately illuminating for 12h, and illuminating for 8:30a.m-8:30 p.m. The use of the animals is approved by animal ethics committee of Tianjin International biomedical Union research institute, and meets the ethical examination requirements.

Before the experiment, 500mg/mL mother liquor of the etidinin solid powder is prepared by DMSO, and the mother liquor is subpackaged into 4mLEP tubes, 5.5 mu L/tube and stored at the temperature of minus 20 ℃.

Mouse breast cancer cells grown in log phase were diluted to 5X 10 with PBS⁷Cell suspension in 5X 10/mL⁶Inoculating one seed/one seed under the axilla of nude mouse, and allowing the seed to grow to 300mm³(sixth day after inoculation of tumor cells), mice were randomly divided into three groups according to the size of tumor volume, i.e., high-dose group of vincetoposide (1mg/kg), low-dose group of vincetoposide (0.5mg/kg) and blank control group (physiological saline containing 0.2% DMSO), 6 mice each were intraperitoneally administered 1 time per day, and the state of the mice was observed. The administration period was 15 days, and the body weight and tumor volume of the mice were measured every 2 days throughout the treatment period, and the tumor volume was calculated using the following formula: tumor volume is the square of length by width/2.

Since the thebaine glycoside is a cardiac glycoside compound, on the 19 th day after inoculation, the tribromoethanol is anesthetized, an electrocardiograph is used for detecting the cardiac function of the mouse, and the safety of the thebaine glycoside is detected. On day 20, blood was collected from the eyeball, the mouse was sacrificed after removing the cervical vertebrae, and the liver, heart, spleen, lung, kidney and thymus were weighed as damp weight. And taking the tumor, weighing the tumor by wet weight, taking a picture, quickly freezing the tumor in liquid nitrogen for 2 hours, and then transferring the tumor to a refrigerator at the temperature of-80 ℃ for storage for subsequent experimental investigation.

As can be seen from FIGS. 12-14, the high-dose xanthophyll group significantly inhibited tumor growth (P <0.01) compared to the blank control group, but the weights of the three groups of 4T1 tumor model mice did not statistically differ with time. The ratio of the wet weight of heart, liver, spleen, lung, kidney, thymus and brain to the body weight of the mice in the three groups is shown in fig. 15, compared with the control group, the ratio of the wet weight of heart, liver, spleen, lung, kidney, thymus and brain to the body weight of the mice in the high-dose and low-dose administration groups is not statistically different, which indicates the selectivity and safety of the thebaine B to the tumor.

The electrocardiogram of three groups of mice detected by the small animal electrocardiograph is shown in figure 16, the heart work index is shown in table 3, and compared with the control group, the heart work indexes have no statistical difference.

Example 6

The embodiment of the invention provides the influence of the vincetoxicoside serving as an anti-breast cancer drug on the tumor angiogenesis capacity.

1. Study on inhibition of vascular endothelial cell tubule formation ability by vincetoxicoside

Selecting EA.hy926 (human umbilical vein cell fusion cell, purchased from cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences), incubating with DMEM high-sugar medium containing 10% FBS, standing at 37 deg.C and 5% CO₂The incubator is used for conventional culture. And (3) selecting 3 rd-6 th generation cells, digesting the cells for about 1min by using 0.25% of pancreatin when the cells are fused to 80% -90%, and using the cells for subsequent experiments.

Routinely inoculating digested EA.hy926 cells at 75cm²And (4) in a culture bottle, after 12 hours of adherent culture, starving for 24 hours by replacing a serum-free culture medium. The BD matrigel was thawed overnight at 4 ℃ in advance, and 200. mu.L of sterilized pipette tips were pre-cooled in a 96-well plate. Operating on ice box, rapidly adding matrigel into the middle position (45 μ L/hole) of the bottom of each hole, shaking vigorously to make matrigel spread over the whole bottom of the hole, placing in refrigerator at 4 deg.C, standing for 15min, centrifuging at room temperature 2500rpm for 10min, and standing at 37 deg.C with 5% CO₂Equilibrate in the incubator for 30 min. Cells were collected by trypsinization and counted. At a rate of 1.5X 10 per hole⁴The density of each cell is inoculated in a 96-well plate, a blank control group is a culture medium containing 0.1% FBS, 100nmol/L of thevetin is added into the cell culture medium of an intervention group, and a positive control group is added with 50ng/mL of Vascular Endothelial Growth Factor (VEGF) at 37 ℃ and 5% CO respectively₂CulturingCulturing in a box for 16 h. The middle 9 fields were selected per well under high content analysis instrumentation and photographed with a 10 x objective as shown in figure 17.

The number of vascular mesh structures in each well was counted and a one-way anova (n-3) was performed on each set of data using SPSS16.0 statistical software, with the minimum significant difference level set at P <0.05 or P < 0.01. As shown in FIG. 18, 100nM of vincetoposide significantly inhibited endothelial vascularization (P <0.01) and 50ng/mL of VEGF significantly promoted endothelial vascularization (P <0.01) compared to the blank control.

As can be seen from the results of FIGS. 17 and 18, 100nM of vincetoposide significantly inhibited endothelial cell angiogenesis, suggesting that vincetoposide may have activity in inhibiting tumor angiogenesis.

2. Effect of Dimethoside on migration Capacity of vascular endothelial cells

Hy926 cells were subjected to in vitro scratch experiments. As shown in fig. 19 and 20, 100nM of vincetoposide significantly inhibited endothelial cell migration.

With the control group of non-medicated 1% FBS as a negative control, the mobility line graph of endothelial cells at different time points is shown in FIG. 20, and at the time point of 12h, the 100nM thebaine significantly inhibits endothelial cell migration compared with the control group (P < 0.05); whether 10nM and 1nM of vincetoposide inhibit endothelial cell migration was not statistically different, and whether 100nM vincetoposide significantly inhibited endothelial cell migration (P <0.01) or 10nM and 1nM of vincetoposide inhibited endothelial cell migration at the 24h time point compared to the control group.

The results of the experiments in fig. 19 and 20 are consistent with those of angiogenesis, suggesting that it is possible that vincetoposide has the potential to inhibit tumor angiogenesis.

3. Study of whether Sphaerothiolane has the ability to inhibit tumor angiogenesis in mice

On day 16 of the group administration (see example 5 for details of modeling and administration process), tribromoethanol anesthesia, contrast agent administration to radial artery, and micro-CT to detect the distribution of blood vessels around tumor.

FIG. 21 is a micro-CT scanned tumor image of 4T1 tumor model mouse, the circled area is the subcutaneous tumor of the mouse, FIG. 22 is the blood vessel distribution map extracted by ImageJ, and the circled area is the blood vessel distribution on the subcutaneous tumor of the mouse. Fig. 23 is a histogram of the number of branch vessels. The blood vessel distribution situation around the tumor can be visually seen, the blood vessel distribution around the tumor of the high-dose xanthophyll group is obviously sparser than that of a solvent control group, and the number of branch blood vessels is obviously less than that of the solvent control group.

Experimental example 7

The experimental example provides the research on the anti-tumor immune action mechanism of the vincetoxicoside as an anti-breast cancer drug, the RNA extraction is carried out on the tumor preserved in the refrigerator in the example 5 by using a conventional RNA extraction method, the research on tumor genes is carried out on the extracted RNA, and the action mechanism of the vincetoxicoside for inhibiting the breast cancer of a mouse is detected.

1. Experimental methods

1.1 sample Collection and preparation

RNA degradation and contamination were monitored on a 1% agarose gel: the RNA purity was checked using a NanoPhotometer spectrophotometer (IMPLEN, CA, USA)

2.0 Fluorometer

RNA concentration was measured by the RNAAssay Kit (Life Technologies, CA, USA) and RNA integrity was assessed using the RNA Nano 6000Assay Kit from Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

1.2 library preparation for transcriptome sequencing

Each sample used 1 μ gRNA as input material for RNA sample preparation. Use of

Ultra^TMThe RNA library preparation kit (NEB, USA) generates a sequencing library and adds an index code to the attribute sequence of each sample. Finally, the PCR products were purified (AMPure XP system) and the libraries evaluated on the Agilent Bioanalyzer 2100 systemAnd (4) quality.

1.3 clustering and sequencing

Clustering of index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia). After cluster generation, library preparations were sequenced on the Illumina Hiseq platform and paired-end reads of 125bp/150bp were generated.

1.4 data analysis

a quality control

Raw data in fastq format (raw read) is first processed through an internal perl script. In this step, clean data (clean read) is obtained by deleting reads containing adapters, reads containing ploy-N and low quality reads read from the original data. Meanwhile, clean data were calculated for Q20, Q30, and GC content. All downstream analyses were based on high quality cleaning data.

b reads mapping to reference genome

An index of the reference genome was constructed using Hisat2 v2.0.4 and paired-end clean reads were aligned to the reference genome. Hisat2 is selected as the mapping tool.

Quantification of c Gene expression levels

Reads mapped to each gene were recorded with HTSeq v0.9.1, and then FPKM for each gene was calculated based on the length of the gene and read counts were mapped to the gene.

d differential expression analysis

Differential expression analysis of the two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). The resulting P value was adjusted using the methods of Benjamini and Hochberg to control the false discovery rate. Genes found by DESeq with an overall P value <0.05 were designated as differentially expressed.

e (for DEGSeq without biological replicates) prior to differential gene expression analysis, read counts were adjusted by the edgeR package by a scaling normalization factor for each sequencing library. Differential expression analysis of the two conditions was performed using the DEGSeq R package (1.20.0). The P value was adjusted using the Benjamini & Hochberg method. Corrected P values of 0.005 and log2 (fold change) were set as thresholds for significant differential expression.

f analysis of differentially expressed genes was performed by IPA (Induction Path analysis) software.

2. Results of the experiment

The tumor tissues were subjected to transcriptome analysis as shown in FIG. 24, where the X-axis represents the variation of gene expression in different samples, the Y-axis represents the statistical significance of the difference in gene expression, up-regulated genes in the upper right region and down-regulated genes in the upper left region. As can be seen in FIG. 24, the blank control group and the administration group differentially expressed 102 genes, 55 genes were up-regulated and 47 genes were down-regulated. Then, these 102 genes were analyzed by IPA analysis software, and as shown in FIG. 25, the degree of difference between the genes in the control group and the administration group was found, and the difference between the colors of the genes in the two groups was larger, indicating that the difference between the genes was larger.

In the ten pathways of fig. 26, the first is the Dendritic Cell (DCs) maturation pathway, the second is the adrenal medulla signaling pathway, the third is the natural killer cell signaling pathway, the fourth is the colorectal cancer metastasis signaling pathway, the fifth is the Ephrin a signaling pathway, the sixth is the eNOS signaling pathway, the seventh is the γ -chain cytokine signaling pathway acting through JAK1/JAK3, the eighth is the macrophage, fibroblast and endothelial cell acting pathway in rheumatoid arthritis, the ninth is the activating T-cell nuclear factor acting pathway in immune response, and the tenth is the leptin acting pathway in obesity. It can be seen from FIG. 26 that there are 4 pathways closely related to immunity. Among the ten pathways in FIG. 27, the first is the helper T cell differentiation pathway, the second is the kinase-mediated immune response pathway, the third is the glucocorticoid receptor signaling pathway, the fourth is the macrophage and T helper cell production-related cytokine signaling pathway by IL-17A and IL-17F, the fifth is the mode recognition receptor role in bacterial and viral recognition, the sixth is the high mobility group box B1(HMGB1) signaling pathway, the seventh is the asthma inflammation pathway, the eighth is the intestinal epithelial cell production-related cytokine pathway by IL-17A and IL-17F, the ninth is the interleukin 15(IL-15) signaling pathway, and the tenth is the activation of the T helper 1 and T helper 2 signaling pathways. As can be seen in fig. 27, 80% of the first ten pathways are closely associated with the immune pathway. The upstream regulatory gene of the differential expression gene of the blank control group and the thebaine glucoside group is analyzed to find that the channel with the highest P value is an auxiliary T cell differentiation channel, the auxiliary T cell differentiation is an intermediate process in the immune reaction, and further proves that the thebaine glucoside inhibits 4T1 breast cancer through activating the immune pathway. In addition, analysis of the differential genes recognized in Gene Card showed that the differential genes in the tumor model blank control group and the tumor model administered group accounted for 73% of the tumor and immune-related targets, as shown in FIG. 28.

The research analyzes differential expression genes of an air-white control group and a penethamine group to find that the most central pathway is a Dendritic Cell (DCs) maturation pathway, the dendritic cell is a professional antigen presenting cell with the strongest organism function, the dendritic cell can efficiently take, process and present antigens, immature DCs have stronger migration capacity, and mature DCs can effectively activate initial T cells and are in a central link of starting, regulating and maintaining immune response. DCs are closely related to the occurrence and development of tumors, and the number of DCs infiltrated in most solid tumors is large, so that the prognosis of patients is good. The results of this assay also indicate that vincetoposide inhibits 4T1 breast cancer by activating the immune pathway. The RT-PCR method is utilized to analyze and verify the differential genes related to tumor immunity in the DCs maturation pathway, and the result shows that the thevinoside can obviously up-regulate FCGR3A and HLA-DRB1 and down-regulate FGFR4, and as shown in figure 29, the result is consistent with the analysis result of a transcriptome. In addition, the transcriptional level of a marker gene PD-1 of tumor immunity is analyzed, so that the xanthophyll is found to obviously down-regulate the expression of PD-1, and further the fact that the xanthophyll inhibits the proliferation of 4T1 breast cancer cells in a mouse body through a tumor immunity way is proved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The application of the thebaine glucoside in preparing the anti-breast cancer medicament is characterized in that the anti-breast cancer medicament is a medicament for inhibiting tumor angiogenesis.