Preparation method of medicine for treating ovarian cancer and endometrial carcinoma as well as medicine and application

Abstract

The invention discloses a preparation method of a medicine for treating ovarian cancer and endometrial carcinoma and the medicine for treating the ovarian cancer and the endometrial carcinoma. The invention belongs to the technical field of medicine preparation, and aims to solve the problem of preparation of medicines for treating ovarian cancer and endometrial carcinoma, polyoxyethylene hydrogenated castor oil 40, 1, 2-propylene glycol and ethyl oleate are adopted to construct a self-emulsifying medicine delivery system to prepare polyphyllin-I nanoparticles, and then the medicines are prepared, so that the bioavailability and targeting property of the medicines are improved. Aiming at the problems of poor curative effect and large side effect when the existing medicine is used for treating ovarian cancer and endometrial carcinoma, the medicine can be prepared by combining polyphyllin-I nanoparticles and rapamycin.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology. More specifically, the present invention relates to a preparation method, a drug and an application of a drug for treating ovarian cancer and endometrioid carcinoma. Background Art

[0002] Ovarian cancer and endometrioid carcinoma are one of the common malignant tumors in women, seriously threatening the life and health of women. At present, chemotherapy, radiotherapy and surgery are mainly used for treatment clinically, but the curative effect is not ideal and there are relatively large side effects. Therefore, it is of great significance to develop a drug with significant curative effect and small side effects for treating ovarian cancer and endometrioid carcinoma.

[0003] However, there are many problems and disadvantages in the existing anti-cancer drugs when treating ovarian cancer and endometrioid carcinoma. For example, the bioavailability of the drug is low, resulting in difficulty in reaching an effective therapeutic concentration in the body; the targeting of the drug is poor, and it is easy to damage normal tissues; the side effects of the drug are large, bringing great pain to patients. Therefore, how to solve these problems and improve the curative effect and safety of the drug is an urgent technical problem to be solved in the current field of pharmaceutical technology. Summary of the Invention

[0004] One object of the present invention is to solve at least the above problems and provide at least the advantages described hereinafter.

[0005] In order to achieve these objects and other advantages according to the present invention, there is provided a preparation method of a drug for treating ovarian cancer and endometrioid carcinoma, comprising the following steps:

[0006] Using polyoxyl 40 hydrogenated castor oil, 1,2-propanediol and ethyl oleate to construct a self-emulsifying drug delivery system, and preparing paris saponin-I nanoparticles by using the self-emulsifying drug delivery system.

[0007] Preferably, the mass percentages of polyoxyl 40 hydrogenated castor oil, 1,2-propanediol and ethyl oleate are 47.38%, 36.73% and 15.89% respectively.

[0008] Preferably, it includes:

[0009] Step 1, weighing an appropriate amount of paris saponin-I and adding it to ethyl oleate, stirring to dissolve it fully to form an oil-phase solution;

[0010] Step 2, measuring an appropriate amount of polyoxyl 40 hydrogenated castor oil and 1,2-propanediol, stirring evenly to form a mixed solvent phase;

[0011] Step 3: Pour the oil phase solution into the mixed solvent phase while stirring. Control the stirring speed at 200 - 500 revolutions per minute until fully mixed to form a preliminary mixture.

[0012] Step 4: Ultrasonically treat the preliminary mixture until nanoparticles are preliminarily formed.

[0013] Step 5: Subject the solution after ultrasonic treatment to high-pressure homogenization to make the distribution uniform, obtaining Paris saponin-I nanoparticles.

[0014] Provide a drug for treating ovarian cancer and endometrioid carcinoma, including the prepared Paris saponin-I nanoparticles, or including the prepared Paris saponin-I nanoparticles and rapamycin, or including Paris saponin-I, or including Paris saponin-I and rapamycin.

[0015] Provide an application of a drug in the preparation of a drug for treating ovarian cancer.

[0016] Provide an application of a drug in the preparation of a drug for treating endometrioid carcinoma.

[0017] The present invention has at least the following beneficial effects:

[0018] First, it is discovered for the first time that PPI blocks autophagic flux by disrupting the assembly of the SNARE complex, specifically by promoting chaperone-mediated autophagy (CMA), resulting in the degradation of YKT6p, VAMP8, and STX17, and ultimately causing autophagic accumulation and cell death. This provides a new perspective for understanding the anti-tumor effect of PPI and also provides a key theoretical basis for developing gynecological cancer treatment strategies based on autophagy regulation.

[0019] Second, the present invention studies the effects of PPI on various gynecological cancer cells (such as ovarian cancer cells and endometrioid carcinoma cells), and confirms that PPI can inhibit tumor cell proliferation, induce cell death, and arrest the cell cycle at the G2 / M phase. It is also found that PPI-induced cell death is partly due to the interruption of autophagic flux, which is different from the effects and mechanisms of PPI on other cancer cells in previous studies, providing direct experimental evidence for the application of PPI in the treatment of gynecological cancers.

[0020] Third, the present invention observes an enhanced autophagic flux during the metastasis of ovarian cancer, and clarifies the key role of autophagy in the formation of ovarian cancer spheroids, that is, inhibiting autophagy can significantly inhibit the formation of ovarian cancer spheroids. This reveals the close connection between ovarian cancer metastasis and autophagy, providing new potential targets and theoretical support for intervening in ovarian cancer metastasis.

[0021] Fourth, the present invention proposes a treatment strategy of combining PPI with rapamycin. In vitro and in vivo experiments have verified that this combination therapy has an obvious synergistic effect on inhibiting the proliferation and peritoneal metastasis of ovarian cancer, and does not affect the body weight of mice, with good safety. Previously, the anti-cancer effect of the mTOR inhibitor rapamycin used alone did not meet expectations, while the combination therapy of the present invention provides a more effective new scheme for the treatment of ovarian cancer.

[0022] Fifth, in view of the problems of low solubility of PPI in water and high cytotoxicity, self-emulsifying drug delivery systems (SMEDDS) are used to prepare PPI nanoparticles. Experiments have shown that nanoparticle delivery can enhance the bioavailability of PPI, improve its tumor targeting ability, and reduce cytotoxicity, providing a more feasible dosage form option for the clinical application of PPI.

[0023] Other advantages, objectives, and features of the present invention will be partially reflected by the following description, and partially will also be understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0024] Figure 1 It is a schematic diagram of the chemical structure of Paris vietnamensis plants and Polyphyllin-I;

[0025] Figure 2 It is the inhibitory effect of PPI on the cell viability of SKOV3, A2780, A1847, and COV362I detected by the CCK-8 method for 24 h;

[0026] Figure 3 It is the inhibitory effect of PPI on the cell viability of SNU251, Kuramochi, ID8, and Ishikawa detected by the CCK-8 method for 24 h;

[0027] Figure 4 It is the inhibitory effect of PPI on the cell viability of KLE, Case1#, and IOSE80 detected by the CCK-8 method for 24 h;

[0028] Figure 5 It is the cell cycle phase distribution diagram of Ishikawa and SKOV3 after being treated with PPI for 24 h and stained with PI;

[0029] Figure 6 It is the result of the cell clone formation experiment (7 - 10 days) of Ishikawa, SKOV3, and ID8 treated with PPI (inhibitory effect result);

[0030] Figure 7Results of the apoptosis assay of PPI-treated Ishikawa cells for 12 h analyzed by AnnexinV-FITC / PI (EA: early apoptosis, LA: late apoptosis, Necr: necrosis);

[0031] Figure 8 Results of the apoptosis assay of PPI-treated SKOV3 cells for 12 h analyzed by AnnexinV-FITC / PI;

[0032] Figure 9 Western blot analysis of the protein levels of LC3Ⅰ, LC3Ⅱ, β-actin, and p62 in SNU251 cells, Kuramochi cells, A2780 cells, and A1847 cells treated with different concentrations of PPI;

[0033] Figure 10 SKOV3 and Ishikawa cells were transfected with LC3-GFP plasmid, and after 24 h of PPI treatment, LC3 punctate structures were observed by confocal microscopy and quantitatively analyzed. Views of LC3II puncta in Ishikawa cells and SKOV3 cells;

[0034] Figure 11 Number of LC3II puncta in Ishikawa cells and SKOV3 cells treated with PPI;

[0035] Figure 12 Western blot analysis of the effects of different concentrations of PPI treatment for 24 h on the protein levels of autophagy markers LC3B and p62 in tumor cells (Ishikawa cells and SKOV3 cells);

[0036] Figure 13 Western blot analysis of the protein levels of p62 and LC3B in tumor cells (SNU251 cells, Kuramochi cells, A2780 cells, A1847 cells) treated with different concentrations of PPI for 24 h;

[0037] Figure 14 Western blot analysis of the effects of PPI treatment on LC3B and p62 in tumor cells (shikawa cells, SKOV3 cells, ID8 cells) at different time points;

[0038] Figure 15 For Figure 14 Bar chart;

[0039] Figure 16Results of ULK1 (S555) phosphorylation and p-beclin1 (S14) levels in Ishikawa cells, SKOV3 cells, and A1847 cells treated with PPI; Western blot detection of ULK1 and p-ULK1 (S555) protein levels in tumor cells (Ishikawa cells, SKOV3 cells, A1847 cells) after treatment with different concentrations of PPI for 24 h; and Western blot detection of p-Beclin1 (S14) levels;

[0040] Figure 17 Effect of PPI treatment of tumor cells (Ishikawa cells and SKOV3 cells) on autophagy receptors Tax1bp1, Calcoco2, and NBR1 at different time courses detected by Western blot;

[0041] Figure 18 Western blot detection of LC3Ⅱ and p62 protein levels in SKOV3 cells and Ishikawa cells after treatment with HCQ and PPI or without treatment for 24 h;

[0042] Figure 19 Quantitative diagram of LC3Ⅱ and p62 protein levels in SKOV3 cells and Ishikawa cells after treatment with HCQ and PPI or without treatment for 24 h detected by Western blot;

[0043] Figure 20 Detection of autophagosomes in SKOV3 cells and Ishikawa cells after treatment with HCQ and PPI or without treatment for 24 h by transmission electron microscopy;

[0044] Figure 21 Detection of the number of autophagosomes in SKOV3 cells and Ishikawa cells after treatment with HCQ and PPI or without treatment for 24 h by transmission electron microscopy;

[0045] Figure 22 After transfection of phosphorylated GFP-LC3-RFP into SKOV3 cells and treatment with PPI or HCQ for 24 h, confocal microscopy was used to observe the changes in LC3 punctate fluorescence;

[0046] Figure 23 Cell mortality of SKOV3 cells and Ishikawa cells after treatment with PPI combined or not combined with 3-MA or HCQ for 24 h;

[0047] Figure 24 Colony formation ability of SKOV3 cells and Ishikawa cells after treatment with PPI combined or not combined with 3-MA or HCQ for 24 h;

[0048] Figure 25Changes in cell viability after treating SKOV3 cells and Ishikawa cells with PPI with or without 3-MA or HCQ for 24 hours;

[0049] Figure 26 Effect of the combination of MRT68921 (2.5 μM) and PPI (0.5 μM) in SKOV3 cells in the colony formation assay;

[0050] Figure 27 Schematic diagram of the SNAP29 complex mediating the fusion of autophagosomes and lysosomes. After treating with different concentrations of PPI for 24 h, the protein levels of SNAP29, VAMP8, STX17, and YKT6p, which are components of the SNAP29 protein complex, were detected by Western blot. After transfecting SKOV3 cells with SNAP29-GFP, the cells were treated with or without PPI for 24 h, and the levels of SNARE complex component proteins YKT6p, STX17, and VAMP8 were detected by immunoprecipitation;

[0051] Figure 28 To evaluate the binding potential of PPI with YKT6p and VAMP8 by molecular and protein docking and show the potential binding sites. After treating with MG132 (1 μM) and PPI together for 24 hours, the SNAP29 complex-related proteins were detected by Western blotting;

[0052] Figure 29 SKOV3 cells were treated with Bafilomycin and HCQ respectively in combination with PPI for 24 h, and the expression of SNARE complex-related proteins was detected by Western blot;

[0053] Figure 30 The HSP70 inhibitor VER-155008 (0.5 μM) rescued the degradation of SNARE proteins STX17, VAMP8, and YKT6p induced by PPI after co-treatment for 24 hours;

[0054] Figure 31 Confocal immunofluorescence showed that the level of YKT6p was significantly decreased after PPI treatment, and YKT6p co-localized with HSP70 (red arrows indicate the co-localization of YKT6p and HSP70);

[0055] Figure 32 In the SKOV3 cell line, Co-IP was used to detect the interaction between YKT6p and HSP70 after treating with or without PPI for 24 hours;

[0056] Figure 33 Schematic diagram of the mechanism of PPI-mediated YKT6p degradation;

[0057] Figure 34 It is a schematic diagram of the autophagy process of cells;

[0058] Figure 35 Ovarian cancer spheres were collected at different time points, and the dynamic changes of autophagy markers LC3B, P62 / SQSTM1, ULK1 and p-ULK (S555) were detected by Western blot;

[0059] Figure 36 Ovarian cancer spheres were collected at different time points, and the dynamic change amounts of autophagy markers LC3B, P62 / SQSTM1, ULK1 and p-ULK (S555) were detected by Western blot;

[0060] Figure 37 Autophagy markers of ovarian cancer cells (A2780) were detected by Western blot under hypoxic and normoxic conditions;

[0061] Figure 38 Autophagy markers of ovarian cancer cells (SKOV3) were detected by Western blot under hypoxic and normoxic conditions;

[0062] Figure 39 Ovarian cancer cells were treated with autophagy inhibitors HCQ and 3-MA for 7 - 10 days to evaluate the sphere formation ability after blocking autophagy of ovarian cancer cells;

[0063] Figure 40 Ovarian cancer cells were treated with autophagy inhibitors MRT6892 and SAR405 for 7 - 10 days to evaluate the sphere formation ability after blocking autophagy of ovarian cancer cells;

[0064] Figure 41 It is a flow chart for the establishment of an ovarian cancer mouse model and drug treatment;

[0065] Figure 42 It is the tumor weight growth curve after subcutaneous injection of PPI and injection of PPI alone, and the tumor weights of the PPI group and PBS group of mouse tumors were excised;

[0066] Figure 43 It is the change in the body weight of mice after 3 weeks of PPI administration;

[0067] Figure 44 It is the image of tumors in the abdominal cavity of mice taken by a live imaging system and the ascites collected at the end of the experiment;

[0068] Figure 45 It is the change in the body weight of mice treated with PPI under the protection of nanoparticles;

[0069] Figure 46To evaluate the inhibitory effect of the combination of PPI and rapamycin on the viability of ovarian cancer cells using the CCK-8 method, the CompuSyn software was used to calculate the Cl (Cl: combination index, a value less than 1 indicates a synergistic effect on average), and to evaluate the synergistic inhibitory effect of the combination of PPI and rapamycin on the spheroid formation ability of ovarian cancer cells using the spheroid formation assay;

[0070] Figure 47 PPI (ig 20 mpk) and rapamycin (ip 2 mpk) were injected into mice every two days (n = 5). After continuous administration for 3 weeks, the in vivo tumor growth and illuminance intensity in the abdominal cavity of the mice were captured using an in vivo animal imaging system, and the ascites collected at the end of the experiment;

[0071] Figure 48 For the body weight curve of mice treated with the combination of rapamycin and PPI.

[0072] English Explanation of Drawings in the Specification: Rhizoma Paridis, Polyphyllin I, Cell Viability (%control), Cell cycle distribution (%), colony numbers, Cell proportion (%), Control, LC3B puncta / cell, Tubulin, LC3Ⅱ / LC3Ⅰ represents the LC3Ⅱ / LC3Ⅰ ratio, p62 protein level, CTR, Relative protein expression, CTL, LC3Ⅱ protein level, Autophagosome / field, LC3 puncta / cells, Ctr, Fusion of AL, lysosome, input, IP:GFP, Autophagy initiation, Autophagosome formation, Degradation, Days, Days of drug treatment, Monolayer, spheroid, Hypoxia, Ctrl, Modeling, Drug administration, Q.O.D, weeks, Sacrificed, Tumor Volume, Days after drug treatment, Tumor weight (g), Body weight (g), Ascites volume (mL), Weight (g), Relative growth (%), Combination, Luminous intensity. Detailed implementation mode

[0073] The present invention will be further described in detail below with reference to the accompanying drawings (1 to 48) so that those skilled in the art can implement it with reference to the text of the specification.

[0074] It should be noted that the experimental methods described in the following implementation examples are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified; in the description of the present invention, the orientation or positional relationship indicated by the terms is based on the orientation or positional relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0075] 1. Test materials

[0076] Table 1 shows the main reagents, antibodies and plasmids, and Table 2 shows the main cells.

[0077] Table 1 Reagents, antibodies, plasmids

[0078]

[0079] Table 2 Cells

[0080] Cell line name Source Human umbilical vein endothelial cell line HUVEC The University of Hong Kong Human ovarian cancer cell line OVCA429 The University of Hong Kong Human ovarian cancer cell SKOV3 The University of Hong Kong Human ovarian cancer cell line A2780 The University of Hong Kong Immortalized normal ovarian cell IOSE80 The University of Hong Kong Human ovarian cancer cell A1847 Kebai Biotechnology Human ovarian cancer cell line COV362 Kebai Biotechnology Human ovarian endometrioid carcinoma cell SUN251 Kebai Biotechnology Human ovarian cancer cell Kuramochi Kebai Biotechnology Human endometrial adenocarcinoma cell line Ishikawa Shanghai Institute of Cell Biology Human endometrial adenocarcinoma cell line KLE Shanghai Institute of Cell Biology Primary endometrial carcinoma sample Case1# Patient Case1#

[0081] 2. Test methods

[0082] 2.1 Cell culture

[0083] The OVCA429, SKOV3, A2780, IOSE80, HUVEC, Ishikawa and KLE cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin; the A1847, COV362, SUN251 and Kuramochi cell lines were grown in RPMI1640 medium containing 10% FBS and penicillin / streptomycin. All cells were cultured in a cell incubator at 37 °C and 5% CO2.

[0084] Case1# represents a primary endometrial cancer sample isolated from a patient, which was cultured in DMEM medium supplemented with 10% FBS and penicillin / streptomycin. After 3 passages, most of the stromal cells were removed, and then the purified cancer cells were used in this study.

[0085] 2.2 CCK-8 cell viability assay

[0086] The target cells were seeded in 96-well plates at a density of 3000 cells / well. The next day, they were treated with different concentrations of Paris saponin-I (PPI) for 24 h, 10 μL of CCK8 solution was added to each well, and after incubation for 1 h, the absorbance of the substrate at OD 450 was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated using the following formula:

[0087] Cell survival rate = [(A实验组 -A 空白孔 ) / (A 对照组 -A 空白孔 )]×100%

[0088] 2.3, Immunoblotting (Western blotting)

[0089] Pre - inoculate cells into a 6 - well plate (3×10 5 cells per well), and treat with different concentrations of PPI for 24 h. Wash the cells twice with phosphate - buffered saline (PBS), and then lyse the cells on ice for 30 min with RIPA buffer containing protease inhibitor mixture (P1006) and protease phosphatase inhibitor mixture (P1045). After the samples are denatured for 10 min, load 20 - 30 μg of protein for gel separation and transfer to a polyvinylidene fluoride (PVDF) membrane. Block the membrane with 5% skim milk at room temperature for 1 h, incubate with the primary antibody at the indicated dilution, and incubate overnight at 4°C. Incubate the horseradish peroxidase (HRP) - conjugated secondary antibody (1:5000) with the membrane for 1 h at room temperature. Between the completion of membrane transfer and the antibody incubation steps, the membrane needs to be washed with TBST, and the HRP signal is detected with ECL substrate in an illuminance imaging system.

[0090] 2.4, AnnexinV - FITC / PI apoptosis analysis

[0091] Annexin V - FITC / PI apoptosis analysis: Inoculate cells into a 6 - well plate and treat with different concentrations of PPI for 12 h. After treatment, collect and count the cells. Use 1×10 5 cells for each sample, resuspend in staining buffer, and then stain with 5 μL of PI and 5 μL of Annexin V - FITC for 10 min. Perform compensation adjustment using single staining with PI or Annexin V - FITC. Analyze the samples by flow cytometry. By detecting the intensities of green fluorescence (Annexin V - FITC) and red fluorescence (PI), the cells are divided into different populations, such as normal cells (Annexin V - / PI - ), early apoptotic cells (Annexin V + / PI - ), late apoptotic cells (Annexin V + / PI + ), and necrotic cells (Annexin V - / PI + ). Among them, FITC is the green fluorescent dye fluorescein isothiocyanate.

[0092] 2.5, Cell colony formation assay

[0093] Cells were seeded in 6-well plates at a density of 300 cells / well. After 10 h, the medium was replaced with the medium containing the indicated PPI concentration, and fresh medium was changed every 5 days for 7 - 10 days. The cells were rinsed twice with PBS, then fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet solution for 10 min at room temperature. The cells were photographed and analyzed using Image J software.

[0094] 2.6. Confocal immunofluorescence observation

[0095] Tumor cells were pre-seeded on sterile cover slips in 12-well cell culture plates before cross-sectioning. The next day, the EGFP-LC3 plasmid (plasmid number: addgene#11546) was transfected into tumor cells using a cell transfection reagent (Lipofectamine 3000). For SUN251 cells, cells stably expressing pMRX-IP-GFP-LC3-RFP (plasmid number: addgene#84573) were seeded in 12-well cell culture plates containing sterilized cover slips. The above cells were treated with PPI for 24 h. After the treatment, the cells were fixed with 4% paraformaldehyde for 5 min. Then the samples were gently washed with PBS buffer three times to remove impurities and residual reagents on the sample surface. Subsequently, the samples were washed three times with DAPI solution to ensure thorough washing. After washing, an appropriate amount of mounting medium containing an anti-fluorescence quencher was used to mount the samples to prevent fluorescence signal quenching during observation and ensure the stability and persistence of the fluorescence signal. Finally, the mounted samples were placed under a confocal fluorescence microscope for observation. By adjusting the microscope parameters, the distribution and morphology of LC3 puncta were clearly presented for analysis and research.

[0096] 2.7. Transmission electron microscope (TEM) observation

[0097] After the cells were treated with PPI for 24 h, they were quickly transferred to a 2.5% glutaraldehyde solution containing 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C and fixed for 2 h to stably preserve the cell structure.

[0098] After fixation, the cells were successively placed in ethanol solutions of gradient concentrations for dehydration to remove the water in the cells and prepare for the subsequent embedding step. After dehydration, the cells were embedded so that the cells were completely wrapped by the embedding medium to form a stable solid structure for subsequent sectioning.

[0099] The embedded samples were cut into ultra-thin sections with a thickness of about 70 nm using a microtome, and these sections were carefully placed on nickel grids for subsequent staining and observation.

[0100] In the staining step, a specific stain is used to stain the sections on the nickel grid to enhance the contrast of the cell structure, enabling the fine structures of the cells to be clearly presented under the electron microscope. After staining, the sample is placed in a Jeol electron microscope (JEM-1400) at 120 kV and observed at an accelerating voltage of 80 kV. A Gatan-832 digital camera is used to photograph the observed cell ultrastructure, recording the detailed morphology and structural information inside the cells, providing intuitive image data for subsequent analysis and research.

[0101] 2.8, Co-Immunoprecipitation (Co-IP)

[0102] During the experiment, with the help of a lentiviral packaging system (DVPR and VSVG), lentiviruses expressing SNAP29-GFP or YKT6p genes were successfully generated in HEK293T cells. Subsequently, these lentiviruses were used to infect SKOV3 cells, and after puromycin screening, an ovarian cancer cell line with stable expression of the target gene was successfully established.

[0103] Next, the constructed ovarian cancer cell line was treated. An experimental group and a control group were set up. The experimental group was treated with PPI, and the control group was not treated. The treatment duration was 24 h for both groups. After the treatment, the cells in a 100 mm culture dish were collected, 500 μL of IP lysis buffer was added, and the cells were lysed on ice for 10 min to fully lyse the cells. After lysis, centrifugation was performed at a centrifugal force of 12,000 xg, and the supernatant was collected to obtain the cell lysate.

[0104] The collected supernatant was mixed with 10 μL of anti-GFP magnetic beads and incubated overnight at 4 °C to allow the anti-GFP magnetic beads to specifically bind to the target protein. After incubation, the magnetic beads were washed 3 times with PBS to remove unbound impurities. After washing, 100 μL of 1× SDS sample loading buffer was added to each sample, and denaturation treatment was carried out at 95 °C for 10 min to fully denature the protein. Subsequently, the samples were subjected to Western blot analysis to detect the expression and interaction of related proteins.

[0105] 2.9, Mouse Ovarian Cancer Model

[0106] Female C57BL / 6 mice at 5 weeks of age were then housed in a specific pathogen-free (SPF) animal facility.

[0107] Three days after lentiviral infection and puromycin screening, an ID8 cell line stably expressing luciferase was successfully established. 5×10 6 cells of this type were introduced into the mice by intraperitoneal injection or subcutaneous implantation.

[0108] The administration methods and dosages of specific compounds are as follows: PPI is administered by subcutaneous injection (sc) or intragastric administration (ig), with a dosage of 20 mg / kg; carboplatin is administered by intragastric administration, with a dosage of 5 mg / kg; rapamycin is administered by intraperitoneal injection (ip), with a dosage of 2 mg / kg. The above drugs are administered once every two days for 3 weeks continuously.

[0109] Considering the solubility problem of PPI, a self-emulsifying drug delivery system (SMEDDS) was used to prepare PPI nanoparticles. Its formulation is Cremophor RH40, 1,2-propanediol, and ethyl oleate, and the ratio of the three is 47.38%:36.73%:15.89%. Under these conditions, the drug loading of the prepared PPI nanoparticles can reach 4 mg / g.

[0110] To evaluate the tumor metastasis in the abdominal cavity of mice, mice were first anesthetized with pentobarbital, and then each mouse was injected with 3 mg of D-luciferin. The fluorescence signal was captured by an In-vivo Xtreme (in vivo imaging system) real-time imaging system to analyze and evaluate the tumor metastasis situation.

[0111] 2.10. Statistical analysis method

[0112] Statistical analysis of this invention was carried out using Prism software. Experimental data are all presented in the form of "mean ± standard error", and statistical analysis was performed by Student's t-test. All t-tests were two-tailed tests, and when P < 0.05, the difference was considered statistically significant.

[0113] 3. Summary of results:

[0114] 3.1. PPI inhibits tumor cell proliferation and induces cell death

[0115] Polyphyllin-I (PPI), as a natural compound extracted from the Rhizoma Paridis plant ( Figure 1 ), is regarded as a highly potential candidate anti-cancer drug. At the beginning of the study, the effect of PPI on inhibiting cell viability was tested by CCK-8 assay. First, ovarian cancer cells were detected, and the data clearly showed that PPI could significantly reduce the viability of ovarian cancer cells. Subsequently, the effects of PPI on endometrial cancer cells Ishikawa, KLE, and primary endometrial cancer cells taken from a patient (Case1#) were further explored. After treatment with PPI, the viability of these endometrial cancer cells was inhibited ( Figures 2 - 4 ). However, it is worth noting that PPI also inhibited the viability of immortalized ovarian epithelial cells IOSE80 ( Figures 2 - 4)。The above data fully demonstrate that the cytotoxicity induced by PPI is not only targeted at cancer cells.

[0116] Elucidating the mechanism of PPI-induced cell death is crucial for the future development and application of this compound. Based on this, the research team first analyzed the cell cycle, and the results confirmed that after treatment with PPI, the proportion of cells in the G2 / M phase increased significantly, while there was no obvious change in the S-phase cell population ( Figure 5 ). The colony formation assay further showed that PPI treatment reduced the tumorigenicity of cancer cells ( Figure 6 ). To further explore the specific way of PPI-induced cell death, a cell apoptosis assay based on FITC Annexin V / PI was used for research. The results showed that after 12 h of PPI treatment, the number of necrotic cells and late apoptotic cell populations increased significantly, with an increase rate exceeding that of the early apoptotic cell population ( Figures 7 - 9 ).

[0117] 3.2. PPI reduces autophagic flux by impairing the formation of autophagolysosomes

[0118] To further explore the mechanism of PPI-induced cell death, the hypothesis was proposed that PPI might induce cell death by disrupting the autophagy process. Based on this, LC3-GFP was transfected into cancer cells, and then they were treated with PPI. The experimental results showed that in Ishikawa cells and SKOV3 cells, PPI treatment significantly increased the number of LC3II punctate aggregates ( Figures 10 - 11 ). Further research found that after PPI treatment, the content of LC3II increased significantly, while the content of p62 did not decrease as in the case of classical autophagy induction conditions ( Figures 12 - 13 ). Moreover, with the prolongation of PPI treatment time, the accumulation of LC3II and p62 was further verified ( Figures 14 - 15 ).

[0119] To exclude the possibility that PPI increases LC3II by initiating autophagy, relevant detections were carried out. The results showed that PPI treatment did not significantly increase the phosphorylation level of ULK1 (S555) and the level of p-beclin1 (S14), and the changes of these two are usually closely related to the initiation of autophagy in cells ( Figure 16 ). In addition to studying p62, other autophagy receptors, including NBR1, Calcoco2, and Tax1bp1, were also detected. The experimental results showed that in Ishikawa cells and SKOV3 cells, after PPI treatment, the content of these autophagy receptor proteins increased ( Figure 17 ), which strongly implies that PPI might hinder the degradation process of autophagic cargo.

[0120] To accurately verify the effect of PPI on autophagic flux, a new experiment was designed. The autophagosome and lysosome fusion inhibitor hydroxychloroquine (HCQ) was used, and treatment groups with and without PPI addition were set up respectively. The experimental results showed that treatment with PPI or HCQ alone could increase the content of LC3II, and at the same time, the level of p62 protein also increased. However, when HCQ and PPI were used in combination, the level of LC3II did not increase further( Figures 18 - 19 ). By observing the change in the number of autophagosomes, it was found that under PPI treatment, the number of autophagosomes increased significantly, but when co-treated with HCQ, the number of autophagosomes no longer increased( Figures 20 - 21 ). Considering the characteristic that the low pH value in lysosomes quenches GFP fluorescence, the mCherry-GFP-LC3 reporter gene was used to monitor the function of autolysosomes. The experimental results showed that the number of RFP / GFP dots increased, while the number of RFP dots did not change significantly( Figure 22 ). Combining the above results strongly indicates that PPI blocks autophagic flux rather than the initiation process of autophagy.

[0121] To further clarify whether autophagy induced by PPI leads to cell death, an in-depth study was carried out. The results showed that 3-MA could effectively rescue the inhibitory effects of PPI on cell viability, cell formation, and cell death induction, while HCQ could not restore the anti-tumor effect of PPI( Figures 23 - 26 ). These series of experimental results fully indicate that part of the reason for PPI-induced cell death is the interruption of autophagic flux.

[0122] 3.3. PPI impairs autophagosome-lysosome fusion by delaying the assembly of the SNARE complex

[0123] When exploring the root cause of PPI-induced autophagosome accumulation, it was speculated that PPI might disrupt the fusion process of autophagosomes and lysosomes. Research has shown that the SNARE complex plays a key role in autophagy, mainly mediating membrane fusion events during autophagosome formation and subsequent fusion with lysosomes. In this process, several SNARE proteins play different roles, among which YKT6p and STX17 are located on the autophagosome membrane, while STX7 and VAMP8 are located on the lysosome membrane( Figure 27 ). Research has pointed out that disrupting the function of components of the SNARE complex involved in autophagy impairs autophagosome-lysosome fusion, thereby leading to autophagy defects.

[0124] Based on the above background, relevant experimental studies were carried out. The results showed that in ovarian cancer cells, PPI treatment caused a certain degree of decrease in the protein levels of VAMP8 and STX17, and a significant decrease in the protein level of YKT6p, while the level of SNAP29 did not change significantly(Figure 27 ) To deeply explore whether PPI can disrupt the assembly of the SNARE complex, the SNAP29-GFP plasmid was transfected into the ovarian cancer cell line SKOV3, and then co-immunoprecipitation was performed using an anti-GFP antibody to isolate the SNARE complex. The experimental results confirmed that PPI treatment could disrupt the SNARE complex by weakening the interaction between YKT6p and STX17 with SNAP29( Figure 27 ).

[0125] To further clarify the binding characteristics of PPI with related SNARE proteins, the protein binding affinities of YKT6p and VAMP8 with PPI were analyzed by molecular-protein docking technology. The results showed that the minimum binding energies of PPI with STX17, VAMP8, and YKT6 were 4.70 kcal / mol, 6.20 kcal / mol, and -11.17 kcal / mol, respectively( Figure 28 ).

[0126] When studying the specific mechanism by which PPI reduces the SNARE proteins VAMP8 and YKT6p, it was found that the use of the proteasome inhibitor MG132 could not restore the protein levels of VAMP8 and YKT6p( Figures 28 - 29 ). However, bafilomycin and HCQ could achieve this effect. Combining these results, it was inferred that PPI might achieve the degradation of YKT6p by promoting chaperone-mediated autophagy.

[0127] 3.4. PPI promotes the degradation of dYKT6p through the CMA-mediated pathway

[0128] Chaperone-mediated autophagy (CMA) is an important protein degradation pathway, in which the chaperone protein HSP70 plays a key role. The KFERQ-like motif, as a conserved sequence for HSP70 to recognize substrates and guide their hydrolysis in lysosomes, is of great significance in the CMA process.

[0129] To explore the mechanism of the effect of PPI on related proteins, the amino acid sequences of VAMP8, YKT6p, and STX17 were analyzed using the KFERQ motif finder Profit. The results showed that, except for VAMP8, there was a typical KFERQ-like motif in both YKT6p and STX17 respectively.

[0130] Table 3 Recognition results of the KFERQ motif finder

[0131]

[0132] Cells were co-treated with the inhibitor VER-155008 of the molecular chaperone HSP70 and PPI. The experimental results showed that VER-155008 could effectively restore the protein levels of YKT6, STX17, and VAMP8 that were decreased by PPI treatment ( Figure 30 ). Given that the binding ability of YKT6p to PPI is the strongest among these proteins and its protein level decreased more significantly after PPI treatment, it is speculated that PPI may mainly inhibit the assembly of the SNARE complex by inducing CMA-mediated degradation of YKT6p.

[0133] To further verify this speculation, confocal immunofluorescence technology was used to observe the co-localization of YKT6p and HSP70. The results showed that after PPI treatment, the level of YKT6p was significantly decreased, and obvious co-localization of YKT6p and HSP70 occurred ( Figure 31 ). In addition, through co-immunoprecipitation (co-IP) experiments, it was found that the interaction between YKT6p and HSP70 was significantly enhanced after PPI treatment ( Figure 32 ). These experimental results together revealed the mechanism by which PPI inhibits the assembly of the SNARE complex through CMA-mediated degradation of YKT6p ( Figure 33 ).

[0134] 3.5 Enhanced autophagic flux during ovarian cancer metastasis

[0135] Cancer metastasis is the main cause of death in cancer patients, and autophagy plays an important role in this process. Autophagy mainly includes three key steps: initiation, autophagosome formation, and autophagolysosome formation ( Figure 34 ). In the process of ovarian cancer metastasis, the formation of cell spheres is crucial for the survival of cancer cells. Based on this, an in-depth study was carried out on the state of autophagy during the formation of cell spheres.

[0136] During the research, the dynamic changes of LC3B and p62 / SQSTM1 during the formation of cell spheres were detected. The results showed that the phosphorylation level of ULK increased, and at the same time, the contents of LC3-II and p62 also increased. This phenomenon indicated that autophagy had been initiated, but the function of autophagolysosomes did not increase synchronously ( Figures 35 - 36 ). Considering the hypoxic conditions in the peritoneal microenvironment and inside the cell spheres, the effect of hypoxia on autophagy was further explored. The experimental results showed that hypoxia could not only increase autophagic flux in monolayer-cultured cells but also promote it under sphere growth conditions, specifically manifested as a significant increase in the content of LC3-II and a decrease in the content of p62 ( Figures 37 - 38 ).

[0137] To determine whether autophagy activation is beneficial for cancer cell survival and sphere formation, experiments were conducted using the autophagy inhibitors 3-MA (a VPS34 inhibitor), MRT68921 (an ULK1 inhibitor), and SAR405 (a VPS34 inhibitor). The experimental data clearly showed that autophagy inhibition significantly suppressed the formation of ovarian cancer cell spheres ( Figures 39 - 40 ).

[0138] 3.6 In vivo study: PPI inhibits ovarian cancer metastasis

[0139] To investigate the anti-tumor effect of PPI in vivo, mouse models of ovarian cancer with subcutaneous implantation and peritoneal dissemination were constructed ( Figure 41 ). The experimental results showed that subcutaneous injection of PPI into mice at a dose of 20 mg / kg (mpk) effectively inhibited tumor growth ( Figure 42 ). However, it is worth noting that after 3 weeks of continuous administration, the mice showed weight loss, indicating that PPI has certain cytotoxicity ( Figure 43 ).

[0140] In addition, our previous studies have shown that oral administration of 80 mg / kg of PPI has a moderate inhibitory effect on lung cancer in vivo. However, the original data of this experiment showed that oral administration of 20 mg / kg of PPI three times resulted in the death of all mice. Based on these results, it is speculated that PPI has problems of low bioavailability and organ toxicity.

[0141] To solve the above problems, PPI nanoparticles were prepared using a "self-emulsifying drug delivery system" (SMEDDS) to eliminate peritoneal dissemination of ovarian cancer. Given that the safety of SMEDDS has been tested previously and to reduce the number of experimental animals used, no nanoparticle control group was set in this experiment. The results of the animal experiment showed that under the protection of the nanoparticles, 20 mg / kg of PPI was comparable to 5 mg / kg of carboplatin in terms of controlling the volume of ascites and inhibiting intraperitoneal tumor dissemination ( Figure 44 ). Moreover, under the protection of the nanoparticles, PPI did not cause weight loss in mice ( Figure 45 ), indicating that this preparation reduced the toxic and side effects of PPI to a certain extent.

[0142] The AKT / mTOR pathway is an important signaling pathway that regulates cell proliferation and has a negative regulatory effect on autophagy. mTOR inhibitors can activate autophagy and thus inhibit cancer cell proliferation. However, it was found in clinical trials that the use of mTOR inhibitors alone did not achieve the expected anti-cancer effect. Based on this, a treatment strategy of rapamycin combined with PPI was proposed to induce excessive autophagic stress and promote tumor cell death by inhibiting autophagic flux.

[0143] The results of in vitro CCK-8 assay and tumor sphere formation assay showed that the combination of rapamycin and PPI had a significant synergistic effect on inhibiting the proliferation of ovarian cancer cells and tumor sphere formation ( Figure 46 ). In the mouse model experiment, this combination also demonstrated a strong synergistic effect, effectively inhibiting the peritoneal dissemination of ovarian cancer ( Figure 47 ), and having no adverse effect on the body weight of mice ( Figure 48 ), which fully indicated that this combination administration had good safety in vivo.

[0144] 4. Analysis and Discussion

[0145] During the past few decades, a large number of studies have shown that Polyphyllin-I (PPI) has the potential to inhibit the malignant characteristics of cancer cells as a chemotherapeutic drug. Its mechanism of action mainly achieves this by inducing cell cycle arrest and promoting apoptosis of different cancer cells. Although PPI is widely used in traditional Chinese medicine, however, there is still a lack of comprehensive evaluation and interpretation of its specific mechanism of action and potential side effects.

[0146] Previously, some studies have reported that PPI can regulate the autophagy process by evaluating autophagy markers, such as the ratio of LC3II / LC3I after treatment and the formation of puncta. Most of these studies believe that PPI can initiate autophagy by activating the AMPK / mTOR / ULK1 pathway or inhibiting the AKT / mTOR / ULK1 pathway. However, the research results of the present invention are different from the above findings. After PPI treatment, the level of LC3II increased and the accumulation of puncta increased, which was consistent with the previous reports. However, the phosphorylation of ULK1 at the S555 site and the phosphorylation of Beclin1 at the S12 site, the key events of autophagy initiation, were not observed. In addition, multiple cargo receptors, including p62, Calcoco2, NBR1, and Tax1bp1, were detected, and the results showed that the contents of these receptors all increased after PPI treatment. At the same time, through the use of autophagy inhibitors and transmission electron microscopy (TEM) analysis, it was confirmed that PPI led to an increase in LC3 puncta and an increase in the LC3-II / LC3-I ratio by weakening the function of autophagolysosomes.

[0147] Particularly importantly, a new molecular mechanism was discovered: PPI can block autophagic flux by disrupting the assembly of the SNARE complex, specifically by activating chaperone-mediated autophagy, which in turn leads to the degradation of YKT6p, VAMP8, and STX17. According to the data, the binding affinity of PPI to YKT6p is higher than the other two, which indicates that PPI may directly bind to YKT6p, resulting in the degradation of the SNARE complex, although this still needs to be further studied and verified.

[0148] Previous studies have reported that PPI can induce apoptosis and cell cycle arrest. In this study, it was further confirmed that PPI can induce cell cycle arrest at the G2 / M phase and trigger apoptosis. The data indicate that PPI causes a strong blockade of autophagic flux and cell death, while other autophagy inhibitors have relatively weak effects on cancer cell survival. Therefore, the study shows that PPI-induced tumor cell death is closely related to autophagic dysfunction.

[0149] Peritoneal metastasis is the main factor leading to the treatment failure and death of patients with ovarian cancer. Ovarian cancer has a tendency to metastasize via the peritoneal cavity, and the peritoneal environment is characterized by low oxygen content and limited energy supply. Autophagy plays a crucial role in cell survival, especially when cancer cells face metastatic stress and encounter reduced oxygen and energy supply.

[0150] In this study, a variety of inhibitors targeting different autophagy steps were used, and the experimental results showed that these inhibitors significantly reduced cell viability ( Figures 39 - 40 ). This result is consistent with previous related reports, further emphasizing the key role of autophagy in tumor survival. Although genetic models such as gene knockdown or gene knockout were not used in this study, a large number of powerful studies have shown that inhibiting autophagy can effectively slow down tumor progression.

[0151] Rapamycin, as an mTOR inhibitor and autophagy activator, is an immunosuppressive drug approved by the US Food and Drug Administration (FDA) and has currently been applied in the field of anti-aging, as well as the treatment of metastatic cancer and lymphoma. Based on this, a treatment strategy of combining rapamycin with PPI was proposed, hoping to induce excessive accumulation of autophagosomes through this combination of drugs, thereby leading to the death of tumor cells. This study was the first to verify the effectiveness and safety of this combined drug regimen in inhibiting the growth and peritoneal metastasis of ovarian cancer.

[0152] However, PPI has the problem of low solubility in water and has significant cytotoxic effects. The results of this study show that when mice were directly intraperitoneally (ip) injected with PPI at a dose of 20 mg / kg, the mice died after only three treatments; on the contrary, when administered intragastrically (ig) at a dose of 20 mg / kg, no anti-cancer effect was observed. However, another research team pointed out that using nanoparticles as a drug carrier for PPI can enhance its anti-cancer efficacy. In addition, using nanoparticles as a carrier does not cause weight loss in mice. Based on the above research results, nanoparticles were used to load PPI. The experimental results clearly show that the nanoparticle delivery system can not only enhance the bioavailability of PPI but also improve its tumor targeting ability.

[0153] This study provides strong evidence for the autophagy-related cell death induced by Polyphyllin-I (PPI) in tumor cells. We have confirmed through experiments that PPI can disrupt the assembly process of the SNARE complex by promoting the degradation of YKT6p, VAMP8, and STX17. This mechanism ultimately leads to the massive accumulation of autophagosomes in cells, thereby triggering cell death. In addition, we have also observed that autophagy is activated during the metastasis of ovarian cancer.

[0154] Based on the above important findings, a treatment strategy can be provided, that is, by combining PPI with rapamycin to overcome the problem of ovarian cancer metastasis. This combination therapy is expected to effectively block the metastasis of ovarian cancer and show great application potential in the field of ovarian cancer treatment.

[0155] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details and the examples shown and described here.

Claims

1. A method for preparing a drug for treating ovarian cancer and endometrioid carcinoma, characterized in that, Comprising the following steps: Using a self-emulsifying drug delivery system composed of polyoxyl 40 hydrogenated castor oil, 1,2-propanediol and ethyl oleate, and preparing paris saponin-I nanoparticles using the self-emulsifying drug delivery system.

2. The preparation method of the drug for treating ovarian cancer and endometrioid carcinoma according to claim 1, characterized in that, The mass percentages of polyoxyl 40 hydrogenated castor oil, 1,2-propanediol and ethyl oleate are 47.38%, 36.73% and 15.89% respectively.

3. The preparation method of the drug for treating ovarian cancer and endometrioid carcinoma according to claim 1, characterized in that, Including: Step 1: Weigh an appropriate amount of paris saponin-I and add it to ethyl oleate, and stir to dissolve it completely to form an oil-phase solution. Step 2: Measure appropriate amounts of polyoxyl 40 hydrogenated castor oil and 1,2-propanediol, and stir evenly to form a mixed solvent phase. Step 3: Pour the oil-phase solution into the mixed solvent phase, stirring while pouring, and controlling the stirring speed at 200 - 500 revolutions per minute until fully mixed to form a preliminary mixture. Step 4: Ultrasonically treat the preliminary mixture until nanoparticles are preliminarily formed. Step 5: Subject the solution after ultrasonic treatment to high-pressure homogenization treatment to make the distribution uniform and obtain paris saponin-I nanoparticles.

4. A drug for treating ovarian cancer and endometrioid carcinoma, characterized in that, Comprising the paris saponin-I nanoparticles prepared according to any one of claims 1 - 3, or comprising the paris saponin-I nanoparticles prepared according to any one of claims 1 - 3 and rapamycin, or comprising paris saponin-I, or comprising paris saponin-I and rapamycin.

5. Use of the drug according to claim 4 in the preparation of a drug for treating ovarian cancer.

6. Use of the drug according to claim 4 in the preparation of a drug for treating endometrioid carcinoma.

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