Application of matrine combined with medroxyprogesterone acetate in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to application of matrine combined with medroxyprogesterone acetate in preparation of antitumor drugs. According to the invention, matrine and medroxyprogesterone are combined for use, and the matrine and medroxyprogesterone are used for developing products such as drugs, preparations, kits and the like for treating endometrial cancer. Experimental results prove that after sophocarpidine and medroxyprogesterone acetate are combined for medication, the composition shows relatively strong activity of inhibiting endometrial cancer and relatively good synergistic inhibition effect, so that the composition has relatively good practical application value. Experimental results prove that the dosages of the two can be remarkably reduced after drug combination, and the anti-tumor effect is remarkably improved. Animal experiments show that in-vivo anti-tumor effects of sophocarpidine and medroxyprogesterone acetate can be improved by combined medication compared with that of single medication. In conclusion, the combined medication of the matrine and the medroxyprogesterone can become a new method for treating endometrial cancer patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to the application of matrine combined with medroxyprogesterone acetate in the preparation of anti-tumor drugs. Background Art

[0002] The uterus is an important part of the female reproductive system and is the place where the embryo grows and develops. Endometrial cancer (EC) is one of the three major tumors of the female reproductive system and ranks first among female reproductive system cancers in developed countries. In China, endometrial cancer is the second most threatening reproductive system tumor to women's health after cervical cancer. Affected by factors such as lifestyle changes and obesity, the incidence and mortality rates of endometrial cancer patients are both on the rise and tend to be younger. Existing data show that some endometrial cancer patients have not achieved their fertility wishes at the time of diagnosis, and the issue of fertility preservation treatment for endometrial cancer patients has become increasingly prominent. Currently, the treatment options for fertility preservation include high-dose oral progesterone and non-oral progesterone regimens based on GnRH-a. Among them, the most widely used progesterone is medroxyprogesterone acetate. When patients receive high-dose oral progesterone regimens, they will develop resistance to other hormone drug treatments. At the same time, some patients who initially respond may experience recurrence during hormone treatment, and long-term use will form blood clots, cause abnormal glucose and lipid metabolism, and damage liver function, etc.

[0003] Medroxyprogesterone acetate (MPA) is a synthetic progestin drug that can play a role in regulating menstruation by inhibiting the release of luteinizing hormone from the anterior pituitary gland and inhibiting the ovulation process of the ovary. It is mainly used to treat irregular menstruation, functional uterine bleeding, endometriosis, atypical endometrial hyperplasia, and endometrial cancer, etc. Medroxyprogesterone acetate is commonly used in the conservative treatment of young endometrial cancer patients, especially those who hope to preserve their fertility. In the treatment of early endometrial cancer patients characterized by germline mutations in the mismatch repair system (MMRd) gene, the complete remission rate of MPA is 61.8%, but the late recurrence rate is as high as 41.2%. In advanced patients, the complete remission rate of medroxyprogesterone acetate is less than 20%. Medroxyprogesterone acetate used alone often causes side effects such as nausea, weight gain, blood clots, and headache. Clinically, metformin combined with medroxyprogesterone acetate is used to treat early endometrial cancer patients to improve their fertility. Patients receiving combined medication show obvious side effects such as gastrointestinal reactions, headache, and insomnia.

[0004] Matrine (MA) is an alkaloid extracted from the dried roots of Sophora flavescens, and is one of the main active components of Sophora flavescens. Matrine can inhibit the growth and metastasis of tumors such as liver cancer, colorectal cancer, lung cancer, and pancreatic cancer; Matrine induces apoptosis of tumor cells and inhibits the migration and invasion of tumor cells by regulating the expression of proteins such as Bcl-2, Bax, p-Akt, EGFR, E-cadherin, and MMP. Matrine can also induce autophagic cell death of multiple myeloma cells by activating the ROS / AMPK / mTOR signaling pathway. In addition, Matrine exerts an anti-tumor effect by combining with a variety of clinical drugs such as all-trans retinoic acid, sorafenib, 5-fluorouracil, cisplatin, and doxorubicin, and enhances the sensitivity of tumors to chemotherapeutic drugs. In female reproductive system tumors, Matrine downregulates the phosphorylation levels of multiple proteins and inhibits the progression of ovarian cancer; Matrine induces ferroptosis of cervical cancer cells by activating the Piezo1 channel.

[0005] At present, Matrine has not been applied to the treatment of endometrial cancer, and the effect of combined use of Matrine (MA) and medroxyprogesterone acetate (MPA) in the treatment of tumors has not been reported. Clinical studies have shown that Sophora flavescens, the main source of Matrine, has multiple functions such as inhibiting tumors and regulating tumor immunity. The treatment effect of medroxyprogesterone acetate on some endometrial cancer patients is not ideal. Therefore, exploring more effective and safe treatment regimens helps to improve the survival and quality of life of patients. Summary of the Invention

[0006] In view of the problems existing in the prior art, through long-term practical exploration, the inventor has proposed an application of Matrine (MA) combined with medroxyprogesterone acetate (MPA) in the preparation of products for the treatment of endometrial cancer. The present invention combines Matrine and medroxyprogesterone acetate for use in developing products such as drugs, preparations, and reagent kits for the treatment of endometrial cancer. Experimental results have proved that after combining Matrine and medroxyprogesterone acetate, it shows strong activity in inhibiting endometrial cancer and good synergistic inhibitory effect, and thus has good practical application value.

[0007] Another object of the present invention is to provide an anti-tumor drug composition containing Matrine and medroxyprogesterone acetate.

[0008] To achieve the above object, the technical solution of the present invention is realized as follows:

[0009] An application of Matrine (MA) combined with medroxyprogesterone acetate (MPA) in the preparation of an anti-tumor drug.

[0010] Specifically, the structural formula of the Matrine is as follows:

[0011]

[0012] Specifically, the structural formula of the medroxyprogesterone acetate is as follows:

[0013]

[0014] Specifically, the tumor includes but is not limited to breast cancer, endometrial cancer, cervical cancer or ovarian cancer.

[0015] Specifically, when matrine and medroxyprogesterone acetate are used in combination, matrine and medroxyprogesterone acetate can be used in a mixed manner or separately.

[0016] Specifically, when matrine and medroxyprogesterone acetate are used in combination, matrine and medroxyprogesterone acetate are added once or added continuously for multiple times.

[0017] Specifically, when matrine and medroxyprogesterone acetate are added once, the dosage ratio of matrine and medroxyprogesterone acetate is (12-400) g: (1-160) mol, and the treatment time is 12-72 hours (preferably 12-24 hours); when matrine and medroxyprogesterone acetate are added continuously for multiple times, the mass ratio of matrine and medroxyprogesterone acetate added each time is (0.25-1): 1, and the continuous addition is 7-15 times in total.

[0018] Preferably, when matrine and medroxyprogesterone acetate are added once, the dosage ratio of matrine and medroxyprogesterone acetate is 2.5g:1mol, and the treatment time is 24-72 hours (preferably 24 hours); when matrine and medroxyprogesterone acetate are added continuously for multiple times, the mass ratio of matrine and medroxyprogesterone acetate added each time is 3:4, and a total of 15 times are added continuously.

[0019] Specifically, when matrine and medroxyprogesterone acetate are added at one time, the molar ratio of matrine to medroxyprogesterone acetate is (0.24-8.2):(5-80), and the treatment time is 12-72 hours (preferably 12-24 hours).

[0020] Preferably, when matrine and medroxyprogesterone acetate are added at one time, the molar ratio of matrine to medroxyprogesterone acetate is (0.003-1.64):1, and the treatment time is 24-72 hours (preferably 24 hours).

[0021] More preferably, when matrine and medroxyprogesterone acetate are added at one time, the molar ratio of matrine to medroxyprogesterone acetate is (0.1-0.102):1, and the treatment time is 24-72 hours (preferably 24 hours).

[0022] Furthermore, the present invention also provides the use of matrine (MA) combined with medroxyprogesterone acetate (MPA) in the preparation of a drug for inhibiting the proliferation or promoting the apoptosis of endometrial cancer cells, wherein the endometrial cancer cells are AN3CA or Ishikawa cells.

[0023] Specifically, when used alone, matrine can inhibit the proliferation and / or the number or size of colony formation of endometrial cancer cells, or can induce apoptosis of endometrial cancer cells, at a concentration of 0.06 μg / μL - 2 μg / μL and a treatment time of 24 - 72 h.

[0024] Preferably, when used alone, matrine can inhibit the proliferation and / or the number or size of colony formation of endometrial cancer cells, or can induce apoptosis of endometrial cancer cells, at a concentration of 1 or 2 μg / μL and a treatment time of 48 h or 72 h.

[0025] Specifically, when used in combination, matrine at a concentration of 0.06 μg / μL - 2 μg / μL and a treatment time of 24 - 72 h, and medroxyprogesterone acetate at a concentration of 5 - 80 μM and a treatment time of 24 - 72 h, can inhibit the proliferation and / or the number or size of colony formation of endometrial cancer cells, or can induce apoptosis of endometrial cancer cells.

[0026] Preferably, when used in combination, matrine at a concentration of 0.125 μg / μL, 0.25 μg / μL, 0.5 μg / μL, 1 μg / μL or 2 μg / μL and a treatment time of 24 h, 48 h or 72 h; and medroxyprogesterone acetate at a concentration of 5 μM, 10 μM, 20 μM, 40 μM, 60 μM or 80 μM and a treatment time of 24 h, 48 h or 72 h, can inhibit the proliferation and / or the number or size of colony formation of endometrial cancer cells, or can induce apoptosis of endometrial cancer cells.

[0027] More preferably, when used in combination, matrine at a concentration of 1 μg / μL or 2 μg / μL and a treatment time of 24 h or 48 h, and medroxyprogesterone acetate at a concentration of 40 μM or 80 μM and a treatment time of 24 h or 48 h, can inhibit the proliferation and / or the number or size of colony formation of endometrial cancer cells, or can induce apoptosis of endometrial cancer cells.

[0028] Furthermore, the present invention also provides the use of matrine (MA) combined with medroxyprogesterone acetate (MPA) in the preparation of a drug for inhibiting the growth of human or murine xenograft tumor models of endometrial cancer.

[0029] Specifically, when the dosage of matrine is 50 - 100 mg / kg / day, the dosage of medroxyprogesterone acetate is 10 - 100 mg / kg / day, and the treatment time is 1 - 15 days, it can inhibit the growth of human or murine tumors of endometrial cancer.

[0030] Preferably, when the dosage of matrine is 75 mg / kg / day, the dosage of medroxyprogesterone acetate is 100 mg / kg / day, and the treatment time is 15 days, it can inhibit the growth of human or murine tumors of endometrial cancer.

[0031] Specifically, the murine source uses female NU / NU mice.

[0032] Furthermore, the present invention also provides an anti-tumor pharmaceutical composition containing matrine and medroxyprogesterone acetate, including matrine, its salts or its derivatives, and medroxyprogesterone acetate, its salts or its derivatives.

[0033] Specifically, in the anti-tumor pharmaceutical composition containing matrine and medroxyprogesterone acetate, the dosage ratio of matrine to medroxyprogesterone acetate is (12 - 400) g : (1 - 160) mol.

[0034] Preferably, in the anti-tumor pharmaceutical composition containing matrine and medroxyprogesterone acetate, the molar ratio of matrine to medroxyprogesterone acetate is (0.24 - 8.2) : (5 - 80).

[0035] Furthermore, the present invention further provides a preparation of the above-mentioned pharmaceutical composition, which is composed of the pharmaceutical composition and a pharmaceutically acceptable carrier.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] Experimental results prove that the matrine (MA) and medroxyprogesterone acetate (MPA) of the present invention have a certain anti-tumor effect when used alone, but the in vitro anti-tumor effect of low-concentration or low-dose matrine and low-concentration or low-dose medroxyprogesterone acetate is not good. After combined medication, the dosages of both can be significantly reduced, and the anti-tumor effect is significantly improved. Animal experiments show that compared with single drug use, combined medication can improve the in vivo anti-tumor effect of matrine and medroxyprogesterone acetate. In summary, the combined use of matrine and medroxyprogesterone acetate of the present invention can become a new method for the treatment of endometrial cancer patients. Description of the Drawings

[0038] Figure 1 Results of detecting cell proliferation inhibition by MTT assay 24 hours after matrine (MA) is used alone on endometrial cancer cells AN3CA and Ishikawa;

[0039] Figure 2The results of detecting cell proliferation inhibition by MTT assay after matrine (MA) was used alone to act on endometrial cancer cells AN3CA and Ishikawa for 48 hours;

[0040] Figure 3 The results of detecting cell proliferation inhibition by MTT assay after matrine (MA) was used alone to act on endometrial cancer cells AN3CA and Ishikawa for 72 hours;

[0041] Figure 4 The results of detecting cell growth inhibition by EDU staining assay after matrine (MA) was used alone to act on endometrial cancer cells AN3CA and Ishikawa;

[0042] Figure 5 The results of colony formation after matrine (MA) was used alone to act on endometrial cancer cells AN3CA and Ishikawa;

[0043] Figure 6 The results of detecting cell apoptosis by Annexin V-FITC / PI staining after matrine (MA) was used alone to act on endometrial cancer cells AN3CA and Ishikawa;

[0044] Figure 7 The results of cell cycle arrest after matrine (MA) was used alone to act on endometrial cancer cells AN3CA and Ishikawa;

[0045] Figure 8 The results of DNA damage in cells after matrine (MA) was used alone to act on endometrial cancer cells AN3CA and Ishikawa;

[0046] Figure 9 The results of detecting the weight of solid tumors in mice after matrine (MA) was used alone (100 mg / kg / day) to act on mice for 12 days in in vivo drug administration experiments;

[0047] Figure 10 The results of detecting the body weight of mice after matrine (MA) was used alone (100 mg / kg / day) to act on mice for 12 days in in vivo drug administration experiments;

[0048] Figure 11 The results of detecting the organ indices of mice after matrine (MA) was used alone (100 mg / kg / day) to act on mice for 12 days in in vivo drug administration experiments;

[0049] Figure 12In the in - vivo drug - administration experiment on mice, the results of hematoxylin - eosin staining of tissues such as the heart, liver, spleen, lung, and kidney of mice treated with matrine (MA) alone (100 mg / kg / day) for 12 days during growth;

[0050] Figure 13 The results of detecting cell proliferation inhibition by MTT assay when medroxyprogesterone acetate (MPA) was used alone on endometrial cancer cells AN3CA and Ishikawa;

[0051] Figure 14 The results of detecting cell proliferation inhibition by MTT assay 24 hours after co - administration of different concentrations of matrine (MA) and different concentrations of medroxyprogesterone acetate (MPA) on endometrial cancer cells AN3CA and Ishikawa;

[0052] Figure 15 The results of detecting cell proliferation inhibition by MTT assay 48 hours after co - administration of different concentrations of matrine (MA) and different concentrations of medroxyprogesterone acetate (MPA) on endometrial cancer cells AN3CA and Ishikawa;

[0053] Figure 16 The results of detecting cell growth by colony - formation assay when 1 μg / μL matrine (MA) and 40 μM medroxyprogesterone acetate (MPA) were co - administered on endometrial cancer cells AN3CA and Ishikawa;

[0054] Figure 17 In the in - vivo drug - administration experiment on mice, the results of inhibiting the in - vivo growth of endometrial cancer by co - administration of matrine (MA) and medroxyprogesterone acetate (MPA);

[0055] Figure 18 In the in - vivo drug - administration experiment on mice, the results of detecting the body weight of mice treated with co - administration of matrine (MA) and medroxyprogesterone acetate (MPA) for 15 days during growth. Detailed implementation mode

[0056] The present invention will be further described in detail below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to skilled personnel in the art. In addition, any methods and materials similar or equivalent to the recorded content can be applied to the method of the present invention.

[0057] Matrine (MA) and medroxyprogesterone acetate (MPA) used in the present invention were purchased from MedChemExpress (MCE) company.

[0058] The present invention mainly provides an application method of combined use of matrine (MA) and medroxyprogesterone acetate (MPA). Before use, prepare a matrine (MA) stock solution with physiological saline for standby; prepare a medroxyprogesterone acetate (MPA) stock solution with dimethyl sulfoxide (DMSO) for standby.

[0059] The cancer cell lines selected in the following experiments of the present invention include: human endometrial cancer cells AN3CA and Ishikawa (purchased from Shanghai Cell Bank, Chinese Academy of Sciences).

[0060] Example 1: In vitro biological activity evaluation of matrine (MA) alone

[0061] Experiment 1, inhibition test on cancer cell activity:

[0062] Respectively take the endometrial cancer cells AN3CA and Ishikawa in the logarithmic growth phase, digest the cancer cells with trypsin, collect the cells, centrifuge at a speed of 1000 r / min for 5 minutes, discard the supernatant, resuspend with complete medium (RPMI 1640 medium + 10% fetal bovine serum + double antibodies), count the number of cells with a hemocytometer, inoculate into a 96-well plate (5000 cells per well), and allow them to adhere and grow for 24 hours. Then add matrine (MA) to make the final concentrations 0, 0.125, 0.25, 0.5, 1, and 2 μg / μL respectively, and then incubate for 24 hours, 48 hours, and 72 hours respectively. After culturing for the specified time, add 50 μL of MTT solution (prepared by dissolving MTT in PBS buffer, where the MTT concentration is 2.5 mg / mL) to each well, remove the liquid in the 96-well plate after incubating for 4 hours, and add 100 μL of dimethyl sulfoxide solution, and gently shake on a shaker for 10 minutes to fully dissolve the purple crystals in the 96-well plate. Then, measure the absorbance value of each well at 570 nm with a multifunctional microplate reader, and calculate the cell activity inhibition rate; where the inhibition rate (%) = (OD 570 control group - OD 570 experimental group / OD 570 control group) × 100%.

[0063] The test results are as shown in Figure 1 、 Figure 2 and Figure 3 shown, Figure 1 、 Figure 2 and Figure 3 The results of show that with the increase of the incubation time, matrine (MA) can inhibit the growth of endometrial cancer cells.

[0064] Experiment 2, inhibition test on cancer cell proliferation:

[0065] Respectively take the endometrial cancer cells AN3CA and Ishikawa in the logarithmic growth phase, digest the cancer cells with trypsin, collect the cells, and inoculate the cells into a 6-well plate at a density of 3×10 5 cells per well, and place them in an incubator containing 5% CO2 at 37°C. After treating the cells with matrine (MA) at concentrations of 0 and 1 μg / μL for 24 hours respectively, stain them according to the experimental method in the paper by Yu Qifan, Han Feng, Yuan Zhangqin, Zhu Zhuang, Liu Changjiang, Tu Zhengdong, Guo Qianping, Zhao Runze, Zhang Weidong, Wang Huan, Mao Haijiao, Li Bin, Zhu Caihong. Fucoidan-loaded nanofibrous scaffolds promote annulus fibrosus repair by ameliorating the inflammatory and oxidative microenvironments in degenerative intervertebral discs. [J]. Acta Biomater. 2022, 148: 73-89. Specifically: Add an equal volume of pre-warmed 2x EDU working solution (20 μM) at 37°C to the 6-well plate to make the final concentration of EDU in the 6-well plate become 1x. Continue to incubate the cells in the incubator for 2 hours. After the EDU labeling of the cells is completed, remove the culture medium in the 6-well plate, and add 1 mL of 4% paraformaldehyde to fix at room temperature for 15 minutes. Remove the fixing solution, wash the cells in each well 3 times with 1 mL of washing solution, 3-5 minutes each time. Remove the washing solution, incubate each well with 1 mL of 0.3% Triton X-100 at room temperature for 10-15 minutes. Remove the permeabilizing solution, wash the cells in each well 1-2 times with 1 mL of PBS, 3-5 minutes each time. Remove the washing solution from the previous step, add 0.5 mL of Click reaction solution to each well, and gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample. Incubate in the dark at room temperature for 30 minutes, aspirate the Click reaction solution, and wash 3 times with the washing solution, 3-5 minutes each time. Then use 1x DAPI solution to stain the cell nuclei, and incubate in the dark at room temperature for 10 minutes. Aspirate the 1x DAPI solution, and wash 3 times with the washing solution, 3-5 minutes each time. Subsequently, fluorescence detection can be carried out. DAPI is blue fluorescence, with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm.

[0066] The test results are as Figure 4 shown, Figure 4The results showed that matrine (MA) could inhibit the proliferation of endometrial cancer cells.

[0067] Experiment 3. Inhibition test of cancer cell colony formation ability:

[0068] AN3CA and Ishikawa endometrial cancer cells in the logarithmic growth phase were separately taken, the cancer cells were digested with trypsin, the cells were collected, and the cells were inoculated into 6-well plates at a density of 3×10 5 cells per well, and placed in an incubator containing 5% CO2 at 37°C; then the cells were treated with matrine (MA) at concentrations of 0, 0.25, 0.5, 1, and 2 μg / μL for 48 hours, and then the cancer cells were digested with trypsin, the cells were collected, centrifuged, resuspended, counted, and plated; then cultured in an incubator at 37°C for 10 days. During this period, the culture medium was changed every 2 days. After 10 days, the original culture medium in the culture plate was discarded, the cells were washed 2-3 times with PBS, fixed with 4% (mass / volume) paraformaldehyde solution for 30 minutes, stained with 1% crystal violet for 5 minutes, then washed with distilled water, dried in an oven, photographed and imaged with a gel imager, and the number of cell colonies was counted.

[0069] The test results were as Figure 5 shown. The results showed that compared with the control group, matrine (MA) significantly inhibited the colony formation ability of endometrial cancer cells at concentrations of 0.5, 1, and 2 μg / μL.

[0070] Experiment 4. Inducing cancer cell apoptosis test:

[0071] AN3CA and Ishikawa endometrial cancer cells in the logarithmic growth phase were separately taken, the cancer cells were digested with trypsin, the cells were collected, and the cells were inoculated into each well at 3×10 5The cells were seeded into 6-well plates at a density and placed in an incubator with 5% CO2 at 37°C. Then, the cells were treated with matrine (MA) at concentrations of 0, 0.25, 0.5, and 1 μg / μL for 48 hours. The supernatant was discarded, and the cells were first washed with PBS and then digested with trypsin. The cells were collected and centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended with PBS and centrifuged at 1500 rpm for 10 minutes again. The supernatant was discarded again. According to the method in the paper by Ge Chaochao, Wang Yuxia, Feng Yongli, Wang Senzhen, Zhang Kemeng, Xu Xiaojuan, Zhang Zhiyang, Zhao Yuan, Wang Yanming, Gao Lei, Dai Fujun, Xie Songqiang, Wang Chaojie. Suppression of oxidative phosphorylation and IDH2 sensitizes colorectal cancer to a naphthalimide derivative and mitoxantrone. [J]. Cancer Letters. 2021, 519: 30-45., an Annexin V-FITC / PI apoptosis detection kit (BD Biosciences, USA) was used for testing. Specifically, 300 μL of binding buffer, 5 μL of AnnexinⅤ-FITC solution, and 5 μL of PI solution were added to each sample. After mixing evenly, the samples were incubated at 37°C in the dark for 20 minutes, and then centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, 700 μL of pre-cooled PBS was added, and the cell pellet was gently pipetted, filtered, and then the proportion of apoptotic cells was detected by flow cytometry.

[0072] The test results are as Figure 6 shown. The results showed that compared with the control group, matrine (MA) could induce apoptosis in human endometrial cancer cells AN3CA and Ishikawa.

[0073] Experiment Five: Inducing Cancer Cell Cycle Arrest Experiment:

[0074] Logarithmic growth phase endometrial cancer cells AN3CA and Ishikawa were separately taken, the cancer cells were digested with trypsin, the cells were collected, and the cells were seeded at 3×10 per well 5Cells were seeded into 6-well plates at a certain density and placed in an incubator with 5% CO2 at 37°C. Then, the cells were treated with matrine (MA) at concentrations of 0, 0.25, 0.5, and 1 μg / μL for 48 hours. The supernatant was discarded, and the cells were first washed with PBS and then digested with trypsin. The cells were collected and centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended with PBS and centrifuged at 1500 rpm for 10 minutes again. The supernatant was discarded again. According to the method in the paper by Ge Chaochao, Wang Yuxia, Feng Yongli, Wang Senzhen, Zhang Kemeng, Xu Xiaojuan, Zhang Zhiyang, Zhao Yuan, Wang Yanming, Gao Lei, Dai Fujun, Xie Songqiang, Wang Chaojie. Suppression of oxidative phosphorylation and IDH2 sensitizes colorectal cancer to a naphthalimide derivative and mitoxantrone. [J]. Cancer Letters. 2021, 519: 30 - 45., PI staining was used. Specifically, 1 mL of cell suspension was slowly added dropwise to 4 mL of pre-cooled 70% absolute ethanol. After thorough mixing, it was fixed overnight at 4°C. After fixation, the samples were taken out and centrifuged at 1000 rpm for 3 minutes in a 4°C centrifuge. The supernatant was discarded, and the cells were resuspended with 1 mL of pre-cooled PBS and centrifuged again. This was repeated once. The supernatant was discarded, and the precipitated cells were collected. 200 μL of Triton-100 working solution was added to each tube to resuspend the cells. Then, an appropriate amount of RNase A was added to each tube to make its final concentration 50 μg / mL, and it was incubated at 37°C for 5 minutes. After incubation, it was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The prepared PI staining solution was used to slowly and thoroughly resuspend the cells, and they were incubated in the dark at 37°C for 30 minutes. After incubation, they were centrifuged to collect the cells. 2 mL of PBS was used to gently mix them, and they were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 200 μL of PBS was added to the cell pellet and gently blown to mix evenly. They were filtered, and then the distribution ratio of the cell cycle was detected using a flow cytometer.

[0075] The test results are as Figure 7 shown. The results showed that compared with the control group, matrine (MA) could induce the arrest of human endometrial cancer cells AN3CA and Ishikawa in the G1 phase.

[0076] Experiment 6, Inducing DNA Damage in Cancer Cells Experiment:

[0077] Take the endometrial cancer cell line AN3CA in logarithmic growth phase, digest the cancer cells with trypsin, collect the cells, and inoculate the cells into a 24-well plate with coverslips at a density of 8×10 4 cells per well, and place it in an incubator containing 5% CO2 at 37°C; then treat the cells with matrine (MA) at concentrations of 0 and 1 μg / μL for 24 hours respectively. After the drug treatment, aspirate the culture medium, add PBS to wash the cells 3 times, 5 minutes each time. According to the method in the paper by Wang Senzhen, Xu Xiaojuan, Che Delu, Fan Ronghui, Gao Mengke, Cao Yue, Ge Chaochao, Feng Yongli, Li Jinghua, Xie Songqiang, Wang Chaojie, Dai Fujun, Gao Lei, Wang Yuxia. Reactive Oxygen Species Mediate 6c-Induced Mitochondrial and Lysosomal Dysfunction, Autophagic Cell Death, and DNA Damage in Hepatocellular Carcinoma. [J]. Int J Mol Sci. 2021, 22(20):10987, perform immunofluorescence experiments. The specific method is as follows: Add 1 mL of 4% paraformaldehyde and fix at room temperature for 20 minutes. Aspirate the paraformaldehyde and wash the cells 3 times with PBS, 5 minutes each time. Add 0.5% Triton X-100 and permeabilize at room temperature for 20 minutes. Discard Triton X-100 and wash the cells 3 times with PBS, 5 minutes each time. Block with 0.5% BSA at room temperature for 2 hours, aspirate the blocking solution, add the antibody against γ-H2AX to each well, and incubate overnight at 4°C. Wash the cells 3 times with PBS, 5 minutes each time. Add the secondary antibody to each well and incubate at 37°C in the dark for 1 hour, wash the cells 3 times with PBS, 5 minutes each time. Aspirate the secondary antibody and wash the cells 3 times with PBS, 5 minutes each time. Add DAPI to stain the cell nuclei, incubate in the dark for 5 minutes, and wash the cells 3 times with PBS, 5 minutes each time. Observe the fluorescence in the cells through a fluorescence microscope.

[0078] The test results are as Figure 8 shown. The results show that compared with the control group, the fluorescence intensity in the human endometrial cancer cell line AN3CA treated with matrine (MA) is enhanced, indicating that matrine (MA) induces DNA damage in human endometrial cancer cells.

[0079] Example 2: Evaluation of the in vivo biological activity of matrine (MA) alone

[0080] Test VII:

[0081] Take AN3CA cells in the logarithmic growth phase, digest the cancer cells with trypsin, then centrifuge at 1000 rpm for 5 minutes, wash the cells twice with PBS, resuspend the cells with sterile pre-cooled physiological saline, and adjust the cell density to 6×10 7 cells / mL. Inject AN3CA cells into the armpits of mice (female NU / NU mice) (6×10 6 cells per mouse). When the tumor volume reaches 50-100 mm 3 , randomly divide the mice into two groups, namely the control group and the drug administration group, and inject normal saline (control group) and matrine (MA) into the mice by intraperitoneal injection. After continuous drug administration for 12 days, end the experiment, weigh the tumors, and perform statistical analysis.

[0082] The test results are as Figure 9 shown. The results show that compared with the control group, matrine (MA) can inhibit the in vivo growth of endometrial cancer, and the inhibition rate is 68.97%.

[0083] During the experiment, the body weight changes of the mice were statistically analyzed. The results are as Figure 10 shown. Compared with the control group, matrine (100 mg / kg / day) has little effect on the body weight of the mice.

[0084] After continuous drug administration for 12 days, end the experiment, dissect and take tissues such as the heart, liver, spleen, lung and kidney, weigh them, calculate the organ index and analyze. The results are as Figure 11 shown. Compared with the control group, matrine (100 mg / kg / day) has no obvious effect on the organ index of the mice.

[0085] According to the method in the paper "Suppression of oxidative phosphorylation and IDH2 sensitizes colorectal cancer to a naphthalimide derivative and mitoxantrone" by Ge Chaochao, Wang Yuxia, Feng Yongli, Wang Senzhen, Zhang Kemeng, Xu Xiaojuan, Zhang Zhiyang, Zhao Yuan, Wang Yanming, Gao Lei, Dai Fujun, Xie Songqiang, Wang Chaojie. [J]. Cancer Letters. 2021, 519: 30 - 45., tissues such as the heart, liver, spleen, lung, and kidney of the above - mentioned control group and matrine (100 mg / kg / day) group mice were stained with hematoxylin - eosin to detect the effect of the drug on the tissues. The results are as Figure 12 shown. The results showed that single - drug use of matrine (100 mg / kg / day) had no obvious effect on the morphology of the tissues.

[0086] Example 3: In vitro biological activity evaluation of medroxyprogesterone acetate (MPA) used alone

[0087] Experiment VIII:

[0088] Endometrial cancer cells AN3CA and Ishikawa in the logarithmic growth phase were taken respectively. The cancer cells were digested with trypsin, and the cells were collected. They were centrifuged at a speed of 1000 r / min for 5 minutes, and the supernatant was discarded. The cells were resuspended with complete medium (RPMI 1640 medium + 10% fetal bovine serum + double antibodies). The number of cells was counted with a hemocytometer and inoculated into 96 - well plates (5000 cells per well) to allow them to adhere and grow for 24 hours. Then medroxyprogesterone acetate (MPA) was added to make its final concentration 0, 5, 10, 20, 40, and 80 μM. Then they were incubated for 24 hours and 48 hours respectively. After culturing for the specified time, 50 μL of MTT solution (prepared by dissolving MTT in PBS buffer, with MTT concentration of 2.5 mg / mL) was added to each well. After incubating for 4 hours, the liquid in the 96 - well plate was removed, and 100 μL of dimethyl sulfoxide solution was added. It was placed on a shaker and gently shaken for 10 minutes to fully dissolve the purple crystals in the 96 - well plate. Then, the absorbance value of each well at 570 nm was measured with a multifunctional microplate reader, and the cell activity inhibition rate was calculated; where the inhibition rate (%) = (OD 570 control group - OD 570 experimental group / OD 570 control group) × 100%.

[0089] The test results are as follows Figure 13 shown. As the drug concentration and time increase, medroxyprogesterone acetate (MPA) can inhibit the growth of endometrial cancer cells.

[0090] Example 4: In vitro mechanism of action of matrine (MA) and medroxyprogesterone acetate (MPA) in combination

[0091] Test IX, effect of combined drug use on cancer cell viability:

[0092] Respectively take the endometrial cancer cells AN3CA and Ishikawa in the logarithmic growth phase, digest the cancer cells with trypsin, collect the cells, centrifuge at a speed of 1000 r / min for 5 minutes, discard the supernatant, resuspend with complete medium (RPMI 1640 medium + 10% fetal bovine serum + double antibodies), count the number of cells with a hemocytometer, inoculate into a 96-well plate (5000 cells per well), and let it adhere and grow for 24 hours. Then add drug treatment. The final concentration of MA is 0, 0.06, 0.13, 0.25, 0.5, 1 μg / μL, the final concentration of MPA is 0, 5, 10, 20, 40, 80 μM, and the final concentration of MA combined with MPA is MA (0.06 μg / μL) + MPA (5 μM), MA (0.06 μg / μL) + MPA (10 μM), MA (0.06 μg / μL) + MPA (20 μM), MA (0.06 μg / μL) + MPA (40 μM), MA (0.06 μg / μL) + MPA (80 μM), MA (0.13 μg / μL) + MPA (5 μM), MA (0.13 μg / μL) + MPA (10 μM), MA (0.13 μg / μL) + MPA (20 μM), MA (0.13 μg / μL) + MPA (40 μM), MA (0.13 μg / μL) + MPA (80 μM), MA (0.25 μg / μL) + MPA (5 μM), MA (0.25 μg / μL) + MPA (10 μM), MA (0.25 μg / μL) + MPA (20 μM), MA (0.25 μg / μL) + MPA (40 μM), MA (0.25 μg / μL) + MPA (80 μM), MA (0.5 μg / μL) + MPA (5 μM), MA (0.5 μg / μL) + MPA (10 μM), MA (0.5 μg / μL) + MPA (20 μM), MA (0.5 μg / μL) + MPA (40 μM), MA (0.5 μg / μL) + MPA (80 μM), MA (1 μg / μL) + MPA (5 μM), MA (1 μg / μL) + MPA (10 μM), MA (1 μg / μL) + MPA (20 μM), MA (1 μg / μL) + MPA (40 μM), MA (1 μg / μL) + MPA (80 μM).

[0093] Place the 96-well plate in an incubator at 37 °C and 5% CO2 and continue to culture for 24 hours or 48 hours. Then, add 50 μL of MTT solution (prepared by dissolving MTT in PBS buffer, where the MTT concentration is 2.5 mg / mL) to each well. After incubating for 4 hours, remove the liquid in the 96-well plate, and add 100 μL of dimethyl sulfoxide solution. Place it on a shaker and gently shake for 10 minutes to fully dissolve the purple crystals in the 96-well plate. Then, measure the absorbance value of each well at 570 nm using a multifunctional microplate reader, and calculate the cell proliferation inhibition rate; where the inhibition rate (%) = (OD 570 control group - OD 570 experimental group / OD 570 control group) × 100%.

[0094] The test results are as shown in Figure 14 and Figure 15 It shows that the combined use of matrine (MA) and medroxyprogesterone acetate (MPA) significantly enhances the effect on cell viability, and determines that the final concentration of matrine (MA) is 1 μg / μL and the final concentration of medroxyprogesterone acetate (MPA) is 80 μM.

[0095] Experiment 10: Inhibitory test on the clone formation ability of cancer cells by combined drug use

[0096] Respectively take endometrial cancer cells AN3CA and Ishikawa in the logarithmic growth phase, digest the cancer cells with trypsin, collect the cells, inoculate the cells into 6-well plates at a density of 3×10 5 cells per well, and place them in an incubator containing 5% CO2 at 37 °C; then treat the cells with matrine (MA) at a concentration of 1 μg / μL and medroxyprogesterone acetate (MPA) at a concentration of 40 μM for 24 hours respectively. Digest the cancer cells with trypsin, collect the cells, centrifuge, resuspend, count, and plate; then culture in an incubator at 37 °C for 10 days. During this period, the culture medium is changed every 2 days. After 10 days, discard the original culture medium in the culture plate, wash the cells 2 - 3 times with PBS, add 4% (mass / volume ratio) paraformaldehyde solution to fix for 30 minutes, stain with 1% crystal violet for 5 minutes, then wash with distilled water, place in an oven for drying, take pictures with a gel imager, and count the number of cell clones.

[0097] The test results are as shown in Figure 16 It shows that, compared with the control group and the single drug use group, the combined use of matrine (MA) and medroxyprogesterone acetate (MPA) significantly inhibits the clone formation ability of endometrial cancer cells.

[0098] Example 5: In vivo biological activity evaluation of the combined use of matrine (MA) and medroxyprogesterone acetate (MPA)

[0099] Experiment XI:

[0100] AN3CA cells in the logarithmic growth phase were taken, and the cancer cells were digested with trypsin. Then, they were centrifuged at 1000 rpm for 5 minutes, washed twice with PBS, resuspended with sterile pre-cooled normal saline, and the cell density was adjusted to 6×10 7 cells / mL. The AN3CA cells were injected into the axilla of mice (female NU / NU mice) (6×10 6 cells per mouse). When the tumor volume reached 50 - 100 mm 3 , the mice were randomly divided into four groups: control group, matrine (MA) (75 mg / kg / day) group, medroxyprogesterone acetate (MPA) (100 mg / kg / day) group, and combined drug group. After continuous drug administration for 15 days, the experiment was terminated, the tumors were weighed, and statistical analysis was performed.

[0101] The experimental results are as Figure 17 shown. Figure 17 The results show that, compared with the control group, the combined use of matrine (MA) and medroxyprogesterone acetate (MPA) can significantly inhibit the growth of endometrial cancer in mice. Statistical results show that, compared with the control group, the tumor inhibition rate of the matrine (MA) group is 41.2%, the tumor inhibition rate of the medroxyprogesterone acetate (MPA) group is 35.3%, and the tumor inhibition rate of the combined drug group is 78.4%. These results indicate that, compared with single drug use, the combined drug use can enhance the anti-tumor activity of single drug use.

[0102] The above experimental data show that, compared with the single drug group, the combined drug use can significantly inhibit the in vivo growth of endometrial cancer cells and significantly improve the anti-tumor activity of the single drug group.

[0103] During the experiment, the body weight changes of the mice were statistically analyzed. The results are as Figure 18 shown. It can be seen from Figure 18 that, compared with the control group and the single drug use group, the combined drug use has little effect on the body weight of the mice.

Claims

1. The use of matrine combined with medroxyprogesterone acetate in the preparation of anti-tumor drugs, characterized in that: The tumor includes breast cancer, endometrial cancer, cervical cancer or ovarian cancer.

2. The use according to claim 1, characterized in that: The structural formula of matrine is as follows: The structural formula of the medroxyprogesterone acetate is as follows:

3. The use according to claim 1, characterized in that: When matrine is used alone or when matrine and medroxyprogesterone acetate are used in combination, the proliferation and / or the number or size of clone formation of endometrial cancer cells can be inhibited; or, apoptosis of endometrial cancer cells can be induced.

4. The use according to claim 3, characterized in that: The endometrial cancer cells include AN3CA or Ishikawa cells.

5. The use according to any one of claims 1 to 4, characterized in that: When matrine and medroxyprogesterone acetate are used in combination, matrine and medroxyprogesterone acetate can be added all at once or added continuously in multiple times.

6. The use according to any one of claims 1 to 4, characterized in that: When matrine and medroxyprogesterone acetate are added at one time, the dosage ratio of matrine and medroxyprogesterone acetate is (12-400) g: (1-160) mol, and the treatment time is 12-72 hours; when matrine and medroxyprogesterone acetate are added continuously for multiple times, the mass ratio of matrine and medroxyprogesterone acetate added each time is (0.25-1): 1, and the continuous addition is 7-15 times in total.

7. The use according to any one of claims 1 to 4, characterized in that: When used in combination, matrine at a concentration of 0.06 μg / μL-2 μg / μL and a treatment time of 24-72 hours, and medroxyprogesterone acetate at a concentration of 5-80 μM and a treatment time of 24-72 hours can inhibit the proliferation and / or number or size of clones formed by endometrial cancer cells; or, can induce apoptosis of endometrial cancer cells.

8. The use according to any one of claims 1 to 4, characterized in that: Application of matrine combined with medroxyprogesterone acetate in the preparation of drugs for inhibiting tumor growth in human or mouse transplanted tumor models of endometrial cancer.

9. The use according to claim 8, characterized in that: When the dosage of matrine is 50-100 mg / kg / day and the dosage of medroxyprogesterone acetate is 10-100 mg / kg / day, and the treatment time is 1-15 days, the growth of human or mouse endometrial cancer tumors can be inhibited.

10. An anti-tumor pharmaceutical composition containing matrine and medroxyprogesterone acetate, characterized in that: Including matrine, its salt or derivatives thereof, and medroxyprogesterone acetate, its salt or derivatives thereof; In the anti-tumor pharmaceutical composition containing matrine and medroxyprogesterone acetate, the usage ratio of matrine to medroxyprogesterone acetate is (12-400) g: (1-160) mol.

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