Application of cucurbitacine I in preparation of cisplatin sensitizing drug for treating ovarian cancer

Abstract

The invention discloses application of cucurbitacine I in preparation of cisplatin sensitization drugs for treating ovarian cancer, and belongs to the technical field of sensitization drugs for tumor chemotherapy. CuI and CDDP have a good synergistic tumor inhibition effect on ovarian cancer, CuI may directly act on EGFR and can be combined with CDDP to significantly inhibit phosphorylation of EGFR and downstream STAT3 protein thereof, induce DNA damage and ROS accumulation, amplify a pyroptosis effect, increase release of TAAs and DAMPs, promote DC maturation and promote infiltration of CD8 + T cells and memory T cells in TIME in vivo, so that the tumor inhibition effect on ovarian cancer is improved. The expression of an inhibitory costimulatory molecule PD-1 in CD8 + T cells is reduced, the whole body immune effect is activated, and the TIME of the ovarian cancer is remarkably improved. The combined treatment strategy of CuI-CDDP controls the progress of ovarian cancer from a dual mechanism of directly inhibiting tumor cells and promoting anti-tumor immune activation.

Description

Technical Field

[0001] This invention belongs to the field of sensitizing drugs for tumor chemotherapy, and specifically relates to the application of cucurbitacin I in the preparation of cisplatin sensitizing drugs for the treatment of ovarian cancer. Background Technology

[0002] Epithelial ovarian cancer (EOC) is the leading cause of death among malignant tumors of the female reproductive system. Currently, the basic clinical treatment for EOC is cytoreductive surgery combined with platinum-based chemotherapy. Cisplatin (CDDP) effectively inhibits the growth of surgically unresectable or distantly metastatic tumors, playing a crucial role in improving the prognosis of EOC patients. Over 70% of EOC patients are sensitive to initial platinum-based chemotherapy, achieving partial or even complete remission. However, the vast majority of patients relapse within two years, and after multiple rounds of platinum-based chemotherapy, most develop platinum resistance, severely impacting the 5-year survival rate of EOC patients. Furthermore, 20% of EOC patients exhibit primary resistance. Both primary and secondary resistance significantly affect the efficacy of platinum-based chemotherapy in EOC patients. Therefore, exploring platinum-sensitizing drug targets and synergistic treatment strategies is of great significance for improving the clinical outcomes of EOC treatment.

[0003] In recent years, the ability of CDDP to induce immunogenic cell death (ICD) in tumor cells has received considerable attention. ICD can trigger an effective anti-tumor immune response, enhancing the anti-tumor effect. A key molecular characteristic of ICD is the release of DAMPs, including CALR membrane eversion, ATP secretion, and extracellular release of HMGB1. DAMPs further activate the body's immune system, prompting immune cells to recognize and attack dying tumor cells. Studies have reported that CDDP can induce pyroptosis in tumor cells through the caspase-1 / GSDMD and caspase-3 / GSDME signaling pathways.

[0004] To address cisplatin resistance, most recent research has focused on combination therapies. Studies are also exploring ways to enhance cisplatin's ability to induce ICD by optimizing treatment regimens or combining it with other immunotherapies. For example, some studies are exploring the combined use of cisplatin and immune checkpoint inhibitors, aiming to achieve better therapeutic outcomes by simultaneously activating the body's immune system and directly killing cancer cells. Summary of the Invention

[0005] This invention provides the application of cucurbitacin I in the preparation of cisplatin-sensitizing agents for the treatment of ovarian cancer. Specifically, cucurbitacin I is used in combination with cisplatin in the preparation of agents that inhibit the proliferation of mouse ovarian epithelial carcinoma cells or human ovarian cancer cells, agents that induce pyroptosis in mouse ovarian epithelial carcinoma cells or human ovarian cancer cells, or agents that activate the body's anti-ovarian cancer immune response.

[0006] Among them, mouse ovarian epithelial cancer cells were ID8, and human ovarian cancer cells were A2780.

[0007] Preferably, for ID8: CuI (0.5 μmol / L) + CDDP (3 μmol / L); for A2780: CuI (1 μmol / L) + CDDP (6 μmol / L). Of course, other dosages can also be used.

[0008] Among them, the applications in the preparation of formulations that induce pyroptosis in mouse ovarian epithelial cancer cells or human ovarian cancer cells include: for mouse ovarian epithelial cancer cells or human ovarian cancer cells, in the preparation of formulations that upregulate the Annexin V-FITC+ / PI+ cell population ratio; for mouse ovarian epithelial cancer cells or human ovarian cancer cells, in the preparation of formulations that upregulate cellular LDH release; and for human ovarian cancer cells, in the preparation of formulations that upregulate GSDME-N protein expression.

[0009] Among them, the application in the preparation of agents that inhibit the proliferation of mouse ovarian epithelial cancer cells or human ovarian cancer cells includes: the application in the preparation of agents that inhibit cell viability for mouse ovarian epithelial cancer cells or human ovarian cancer cells; and the application in the preparation of agents that inhibit the growth of subcutaneous tumors in an animal model (C57BL / 6 mouse ID8-luc subcutaneous tumor model).

[0010] Among their applications in the preparation of agents that activate the body's immune response against ovarian cancer are: in animal models, in the preparation of agents that promote CD3+CD8+ T cell infiltration; in animal models, in the preparation of agents that downregulate the proportion of PD-1+CD8+ T cells in TIME; in animal models, in the preparation of agents that stimulate antigen presentation by DCs; in animal models, in the preparation of agents that promote CD8+ T cell infiltration in TIME; and in animal models, in the preparation of agents that downregulate the expression of the immune checkpoint PD-1 on the surface of CD8+ T cells.

[0011] Cucurbitacin I has direct targets including the epidermal growth factor receptor / STAT3 / Janus kinase signaling pathway.

[0012] Cucurbitacin I, when used in combination with cisplatin, has been applied in the preparation of formulations that inhibit the phosphorylation of EGFR and its downstream STAT3 protein, in the preparation of formulations that induce DNA damage accumulation, and in the preparation of formulations that upregulate ROS. Attached Figure Description

[0013] Figure 1 This study explores the combined effects of CuI and cisplatin in ovarian cancer cells A2780 and ID8 by using CompuSyn software to calculate CI values. In the figure, (A) shows the concentration settings of CDDP and CuI in A2780 cells, (B) shows the CI values ​​and combined effects scatter plot in A2780 cells, (C) shows the dose-normalized equivalent effect plot of the two drugs in A2780 cells, (E) shows the concentration settings of CDDP and CuI in ID8 cells, (F) shows the CI values ​​and combined effects scatter plot in ID8 cells, and (G) shows the dose-normalized equivalent effect plot of the two drugs in ID8 cells.

[0014] Figure 2 This study explores the synergistic effect of CuI and cisplatin in inducing an increase in the ratio of Annexin V+ / PI+ cells in the ovarian cancer cell line. In the diagram, (A) is a representative image from flow cytometry analysis of each drug combination; (B) is a bar chart of quantitative analysis of the Annexin V+ / PI+ cell population ratio detected by flow cytometry under the drug combination; (C) is a graph of LDH release from cell culture supernatant detected by chemiluminescence under the drug combination; (D) is a bar chart of quantitative analysis of intracellular ATP content under the drug combination; and (E) is a graph of protein expression levels of total pyroptosis executive protein GSDME and its splice variant GSDME-N detected by Western blotting under the drug combination. Con: Control, Com: Combination (combination group); *** indicates p < 0.00.

[0015] Figure 3 This study explores the combined use of CuI and cisplatin to inhibit tumor growth in mice bearing subcutaneous ovarian cancer tumors. In the figures, (A) is a tumor growth curve of subcutaneous tumors in mice under the action of each drug combination; (B) is a gross image of tumor tissue in mice under the action of each drug combination; (C) is a bar chart of quantitative weight analysis of tumor tissue dissected by mice at the observation endpoint under the action of each drug combination; (D) is a mean growth curve of subcutaneous tumors in mice under the action of each drug combination; (E) is a line graph of weight change of tumor-bearing mice in each group after treatment under the action of each drug combination; and (F) is a representative image of Ki-67 protein expression in subcutaneous tumor tissue of mice under the action of each drug combination. Each group consisted of 6 mice. * indicates P < 0.05, and ** indicates P < 0.01.

[0016] Figure 4 This is an exploration of the combined application of CuI and cisplatin in the infiltration of immune cells in subcutaneous tumor tissue; In the figure, (A) shows representative images of the proportion of cells in each component detected and analyzed by flow cytometry under the action of each drug combination; (B) is a bar chart showing the quantitative analysis of the proportion of CD45+ immune cells infiltrating subcutaneous tumors among living cells (leftmost), lymphocytes (leftmost), CD3+CD8+ T cells (leftmost), and PD-1+ T cells among CD8+ T cells (leftmost) under the action of each drug combination. Six mice were included in each group. * indicates P<0.05, ** indicates P<0.01.

[0017] Figure 5 This study explores the combined use of CuI and cisplatin to activate a systemic immune response in ovarian cancer-bearing mice. Among them, (A) is a representative image of the proportion of cells in each component detected and analyzed by flow cytometry in the spleen; (B) is a representative image of the proportion of cells in each component detected and analyzed by flow cytometry in the bilateral inguinal lymph nodes; (C) is a bar chart (left) showing the quantitative analysis of the proportion of CD3+CD8+ T cells infiltrating the spleen under the action of drugs in each combination in the proportion of CD45+ T cells; (second from left) is a bar chart (right) showing the quantitative analysis of the proportion of CD80+CD86+ DCs infiltrating the bilateral inguinal lymph nodes in each group in the proportion of CD11c+MHCII+ DCs.

[0018] Figure 6 The AutoDock molecular docking experiment was used to analyze the binding modes of CuI with EGFR, JAK2 and STAT3 proteins.

[0019] Figure 7 This study explores the interaction between CuI and EGFR, JAK2, and STAT3 proteins through molecular dynamics simulation experiments.

[0020] Among them, (A) is the root mean square deviation (RMSD) curve of the CuI complex interacting with EGFR, JAK2, and STAT3 proteins over time; (B) is the root mean square fluctuation (RMSF) curve of the CuI complex interacting with EGFR, JAK2, and STAT3 proteins calculated based on molecular dynamics simulations; (C) is the curve showing the change in the number of hydrogen bonds (Hbonds) between CuI and the protein. Hydrogen bonds are one of the strongest non-covalent binding interactions; a higher number indicates a stronger binding force; and (D) is the binding energy diagram of CuI and the protein calculated using the MM-GBSA method. Negative values ​​indicate that the two molecules have a binding affinity to the target protein, and lower values ​​indicate a stronger binding. Where ΔEvdW: van der Waals energy, ΔEelec: electrostatic energy, ΔGGB: electrostatic contribution to solvation, ΔGSA: non-polar contribution to solvation, and ΔGbind: binding free energy.

[0021] Figure 8 The results are from surface plasmon resonance (SPR) measurements of the interaction between CuI and EGFR protein.

[0022] Figure 9 This study explores the synergistic effect of CuI and cisplatin in inducing intracellular ROS generation and DNA damage accumulation in ovarian cancer cell lines.

[0023] In this diagram, (A) shows representative images of ROS levels in A2780 and ID8 cells detected by flow cytometry; (B) is a bar chart of quantitative analysis of ROS levels in ID8 (top) and A2780 cells (bottom), (n=3); and (C) shows the expression levels of DNA damage marker proteins p-γH2AX and PARP splice variants detected by Western blotting. Con: Control, * indicates P<0.05, ** indicates P<0.01. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0025] I. CuI and CDDP can synergistically inhibit the proliferation of ovarian cancer cells.

[0026] Different concentration combinations (concentration settings such as...) Figure 1After 24 hours of treatment with CuI and / or CDDP (as shown in Figure A), ID8 and A2780 cells were analyzed using the CCK8 assay to detect cell viability and calculate the inhibition rate. The drug concentration and cell inhibition rate data were then imported into CompuSyn software to calculate the CI values. As shown in Figures 1B and 1C, in the A2780 cell line, six concentration combinations had CI values ​​less than 0.9, and the corresponding concentration points all fell to the lower left of the oblique line on the isodynamic plot. The CI values ​​were particularly low for the concentration combinations CuI (1 μmol / L) + CDDP (6 μmol / L) and CuI (1 μmol / L) + CDDP (12 μmol / L). In the ID8 cell line, seven concentration combinations (concentration settings as shown in Figure A) were analyzed. Figure 1 The CI values ​​(shown in E) were less than 0.9, and the points were all located in the lower left of the iso-effect curve. The CI values ​​were particularly low for the concentration combinations CuI (0.5 μmol / L) + CDDP (6 μmol / L) and CuI (0.5 μmol / L) + CDDP (12 μmol / L), suggesting that CuI can enhance the sensitivity of ovarian cancer cell lines A2780 and ID8 to CDDP, and the two drug molecules exhibit a strong synergistic inhibitory effect on proliferation. Considering the cytotoxicity issues associated with higher doses of CDDP, we selected the combination regimens of ID8: CuI (0.5 μmol / L) + CDDP (3 μmol / L) and A2780: CuI (1 μmol / L) + CDDP (6 μmol / L) for further synergistic effect evaluation in subsequent studies.

[0027] II. CuI and CDDP can synergistically induce pyroptosis in ovarian cancer cells. To investigate whether CuI combined with CDDP can synergistically induce pyroptosis in ovarian cancer cells, this patent investigated the effects of CuI and / or CDDP on A2780 and ID8 cells for 24 hours, followed by flow cytometry analysis to detect Annexin-V FITC / PI staining. (See also...) Figure 2 A, for ID8: CuI monotherapy group (CuI (1 μmol / L), CDDP monotherapy group (CDDP (6 μmol / L)), combination therapy group (CuI (0.5 μmol / L), CDDP (3 μmol / L)). For A2780, see [link to relevant documentation]. Figure 2 A down.

[0028] In the ID8 cell line, the combined treatment group had the highest proportion of Annexin V-FITC+ / PI+ cell population (p<0.001), at (39.23±0.96%), which was 11.65 times, 1.76 times, and 4.74 times that of the control group, CuI monotherapy group, and CDDP monotherapy group, respectively. In the A2780 cell line, the combined treatment group also had the highest proportion of Annexin V-FITC+ / PI+ cell population, at (21.13±1.36%), which was 10.36 times, 2.94 times, and 3.10 times that of the control group, CuI monotherapy group, and CDDP monotherapy group, respectively. Figure 2 (A and 2B).

[0029] Further analysis of LDH release levels and intracellular ATP content revealed that, compared to the single-drug group, the combination therapy group exhibited the strongest LDH release capacity in ovarian cancer cells. Figure 2 C, and its intracellular ATP content was the lowest among all groups. Figure 2 D). Meanwhile, Western blot results showed that both CuI and CDDP monotherapy groups induced GSDME protein cleavage in the A2780 cell line, while the combined treatment group showed a significant increase in GSDME-N protein expression in both ovarian cancer cell lines. In the ID8 cell line, CuI monotherapy induced GSDME cleavage, while CDDP monotherapy at a dose of 3 μmol / L induced an increase in the Annexin V-FITC+ / PI+ cell population ratio accompanied by increased LDH release. However, Western blot did not show significant GSDME-N protein expression, possibly because ID8 cells at 3 μmol / L CDDP did not produce a band detectable by Western blot. Figure 2 E). The results of the combined use of the two drugs mentioned above suggest that CuI has a more stable and stronger ability to induce pyroptosis in ovarian cancer cells than CDDP. The combined use of CuI and CDDP can significantly induce a synergistic effect and significantly enhance the pyroptosis-inducing effect in ovarian cancer cells.

[0030] III. Synergistic Tumor Suppressive Effect of CuI and CDD Combined in Vivo Based on the above findings, CuI and CDDP can synergistically inhibit the proliferation of ovarian cancer cell lines and induce pyroptosis. To further evaluate the synergistic antitumor effects of CuI and CDDP in vivo, a C57BL / 6 mouse ID8-luc subcutaneous tumor model was constructed. When the tumor volume in the mice reached 50-100 mm, the tumor was detected. 3 At approximately 10:00 AM, mice were randomly divided into four groups: a PBS control group, a CuI monotherapy group, a CDDP monotherapy group, and a CuI and CDDP combination group. The drugs were administered via intraperitoneal injection: CuI (1 mg / kg, every other day) and CDDP (2 mg / kg, three times a week). Figure 3 As shown, compared with the control group, although the ID8-luc subcutaneous tumor tissues of mice in the three treatment groups also showed continuous growth, the growth rate was significantly reduced, especially in the combination treatment group. Compared with the two single-drug groups, the CuI and CDDP combination treatment group showed a significantly enhanced inhibitory effect on ID8-luc subcutaneous tumors in mice. After subcutaneous tumor dissection and weighing at the end of the observation period, the tumor weight in the combination treatment group was significantly reduced, and the difference was statistically significant. At the same time, immunohistochemical staining showed a significant reduction in the expression of Ki-67 protein, a cell proliferation indicator, in the tumor tissues of mice in the combination treatment group. Throughout the treatment, compared with the untreated normal control group, the CuI single-drug treatment group did not show a significant decrease in body weight; however, the CDDP single-drug treatment group showed a sustained and significant decrease in body weight, but all mice remained above 15g; while the combination treatment group showed the same degree of weight loss as the CDDP single-drug group, without further significant reduction, suggesting that CuI in combination treatment does not enhance the toxic side effects of CDDP in vivo, thus preventing further weight loss. The results in summary indicate that CuI combined with CDDP can produce an effective synergistic tumor-suppressing effect in vivo. IV. Combined use of CuI and CDDP to activate the body's immune response against ovarian cancer Because the above experiments showed that CuI and CDDP synergistically enhance the induction of pyroptosis in ovarian cancer cells, the effects of the combined use of CuI and CDDP on tumor immune response in vivo were further explored. The animal model construction, administration method, and dosage were the same as in Experiment 3 (synergistic tumor-suppressive effect of CuI and CDDP combined in vivo). Figure 4 As shown, in all groups of ID8-luc tumor-bearing mice subcutaneous tumor tissues, the CuI and CDDP combination treatment group had the highest proportions of CD45+ lymphocytes and CD3+CD8+ T cells. Compared with the untreated tumor-bearing control group and the CuI and CDDP monotherapy groups, the combination treatment group showed a significant increase, suggesting that the combination treatment can synergistically promote the infiltration of CD3+CD8+ T cells in mouse ovarian cancer subcutaneous tumor tissues. The proportion of PD-1+CD8+ T cells in the TIME of mouse subcutaneous tumors was significantly lower in the CuI and CDDP combination treatment group than in the control group and the monotherapy group. Compared with the untreated tumor-bearing control group, the proportions of CD45+ lymphocytes and CD45+CD3+CD8+ T cells in the TIME of ID8-luc mouse subcutaneous tumors were significantly upregulated in the CuI monotherapy group, while these two indicators in the CDDP administration group were not significantly different from those in the control group.

[0031] like Figure 5As shown, further examination of immune cell infiltration in the spleen and TDLNs of tumor-bearing mice in each group revealed that, compared with the untreated tumor-bearing control group and the CuI and CDDP monotherapy group, the proportion of CD3+CD8+ T cells in the spleen and CD80+CD86+ DCs in the TDLNs of the CuI and CDDP combined treatment group was significantly increased.

[0032] The results suggest that the combined application of CuI and CDDP can further stimulate antigen presentation of DCs, promote the infiltration of CD8+ T cells in TIME, and at the same time significantly reduce the expression of the inhibitory immune checkpoint PD-1 on the surface of CD8+ T cells, thereby significantly improving the inhibitory TIME in ovarian cancer.

[0033] V. Direct Targets of the Synergistic Effect of CuI and Cisplatin Connectivity Map analysis revealed that CuI treatment significantly altered the expression profile of EOC cell lines. Further analysis extracted transcriptional expression characteristics after CuI treatment and used the CMap data platform to evaluate the correlation scores between CuI and the characteristic expression profiles of cell lines treated with small molecule drugs in the library, aiming to explore the potential molecular targets, pathways, and mechanisms of CuI inhibition of EOC cells. Results showed a significant association between CuI and inhibitors of the epidermal growth factor receptor (EGFR) / STAT3 / Janus kinase (JAK) signaling pathway, suggesting that this pathway may be a target of CuI. The high correlation was concentrated in treatment arrays of EGFR inhibitors (10), DNA inhibitors (9), STAT inhibitors (6), and JAK2 / STAT3 inhibitors, as detailed in Table 1 (Representative results of CMap analysis of differentially expressed gene profiles of ovarian cancer cells after 24 hours of treatment with 0.5 μmol / L CuI).

[0034] The EGFR pathway plays an important role in tumor development and the development of platinum resistance. JAK2 / STAT3 is an important downstream pathway. Given that the three are significantly correlated with CuI-treated EOC cells in CMap analysis, this embodiment selects these three candidate target molecules for further analysis and exploration, such as molecular docking.

[0035] Table 1

[0036]

[0037] Where: Pert_iname: drug name; Cell_iname: cell line name; Per_idose: drug concentration during treatment; Pert_itime: drug treatment time; MoA: CMap mode of action analysis; CS: Connectivity score.

[0038] VI. Molecular docking and molecular dynamics simulation. Combined with pattern analysis, such as Figure 6 As shown, the interaction between small molecule CuI and EGFR, STAT3, and JAK2 proteins, from Figure 6 As shown above, CuI forms hydrogen bonds with LYS-745, ASN-842, ARG-803, THR-854, ARG-841, and ASP-800 on the EGFR protein. These hydrogen bonds strengthen the bond between the protein and smaller molecules. CuI also forms hydrophobic interactions with LEU-799, ARG-841, LEU-844, and VAL-726, providing strong van der Waals forces for the molecules. Figure 6 The diagram shows the interaction between CuI and the JAK2 protein. As can be seen, CuI forms hydrogen bonds with THR-636, LYS-630, LYS-581, ASN-678, and SER-633 on the JAK2 protein. These hydrogen bonds strengthen the bond between the protein and smaller molecules. CuI also forms hydrophobic interactions with LEU-579, LEU-551, ILE-559, and TYR-637, providing strong van der Waals forces for the molecule. Figure 6 The diagram shows the interaction between CuI and the STAT3 protein. It reveals hydrogen bonds formed between CuI and SER-636, LYS-658, and TYR-640 on the STAT3 protein. These hydrogen bonds strengthen the bond between the protein and smaller molecules. CuI also forms hydrophobic interactions with TYR-657 and TRP-623, providing strong van der Waals forces. The root mean square (RMSD) of molecular dynamics simulations reflects the complex's motion; a larger RMSD and more pronounced fluctuations indicate vigorous motion, while a smaller RMSD indicates more stable motion.

[0039] like Figure 7Figure A shows the RMSD (Reactive Power Score) curves of the EGFR / CuI, JAK2 / CuI, and STAT3CuI systems during the simulation. The EGFR / CuI system gradually converged in the first 10 ns of the simulation, then maintained stable fluctuations, with the RMSD fluctuating around 0.2 nm. JAK2 / CuI and STAT3 / CuI showed dramatic fluctuations during the 100 ns molecular dynamics simulation, indicating that CuI can stably bind to EGFR, while its stability and effectiveness with JAK2 and STAT3 are weaker compared to EGFR. RMSD reflects the flexibility of proteins during molecular dynamics simulations. Typically, after a drug binds to a protein, the protein's flexibility decreases, thereby stabilizing the protein and allowing it to exert its enzymatic activity. Figure 7 As shown in Figure C, all proteins, except for their ends, exhibited low RMSF after binding different small molecules, indicating that the protein core structure possesses good rigidity. EGFR, JAK2, and STAT3 showed overall RMSF less than 0.2 nm, and these proteins exhibited even greater rigidity when bound to CuI, especially EGFR, indicating a close EGFR / CuI binding. Hydrogen bonds are one of the strongest non-covalent binding interactions; a higher number of hydrogen bonds indicates better binding. Figure 7 B shows that EGFR / CuI has the best number and density of hydrogen bonds among small molecules and proteins, with a stable number of 2-4 hydrogen bonds throughout the simulation. In contrast, JAK2 / CuI and STAT3 / CuI show greater changes in hydrogen bond count during the simulation, with fewer hydrogen bonds in the later stages. This indicates that hydrogen bonds make a significant contribution to EGFR / CuI, but a weaker contribution to JAK2 / CuI and STAT3 / CuI.

[0040] Based on molecular dynamics simulation trajectories, the MM-GBSA method was used to calculate binding energies, enabling a more accurate evaluation of the binding modes between small molecules and target proteins. For example... Figure 7 As shown in Figure D, the binding energies of the EGFR / CuI, JAK2 / CuI, and STAT3 / CuI complexes are -30.88±2.91, -7.60±3.67, and -9.25±3.42 kcal / mol, respectively. Negative values ​​indicate that these molecules have a binding affinity to the target protein; lower values ​​indicate stronger binding. The results show that CuI and the corresponding proteins all possess a certain binding affinity, with EGFR / CuI exhibiting the strongest binding energy, less than -30.0 kcal / mol.

[0041] VII. Surface Plasmon Resonance Experiment like Figure 8 As shown, surface plasmon resonance experiments indicate that the dissociation constant of CuI and EGFR protein is 3.18 nM, suggesting a strong binding force. These results indicate that EGFR is a direct target of CuI.

[0042] The affinity of CuI for the interaction with Human EGFR protein was determined using Biacore, employing a CM5 chip-coupled protein assay. KD: dissociation constant, reflecting the affinity of the analyte for the target; a smaller value indicates stronger affinity. Ka: association rate constant, representing the speed of intermolecular binding; a larger value indicates faster binding. Kd: dissociation rate constant, representing the speed of intermolecular dissociation; a larger value indicates faster dissociation.

[0043] VIII. The effect of combined CuI and CDDP on the EGFR signaling pathway in, Figure 9 The experimental procedure for A was as follows: A2780 and ID8 cells were treated with CuI and cisplatin alone or in combination for 1 hour, followed by flow cytometry to detect intracellular ROS levels in A2780 and ID8 cells. After treating A2780 and ID8 cells with CuI and / or CDDP for 24 hours, Western blotting was used to detect the expression levels of DNA damage marker proteins p-γH2AX and PARP splice variants. Figure 9 As shown, the expression of p-γH2AX was upregulated after treatment with CuI and CDDP alone compared to the control group, indicating a certain accumulation of DNA damage, which was significantly increased in the combination therapy group. Simultaneously, the ROS level was also significantly higher in the combination therapy group compared to the single-drug treatment group.

[0044] IX. Conclusion In summary, both in vivo and in vitro experiments confirmed that CuI and CDDP have a good synergistic antitumor effect on ovarian cancer. CuI may directly act on EGFR, and when used in combination with CDDP, it can significantly inhibit the phosphorylation of EGFR and its downstream STAT3 protein, induce DNA damage and ROS accumulation, amplify the pyroptosis effect, increase the release of TAAs and DAMPs, promote DC maturation, promote the infiltration of CD8+ T cells and memory T cells in TIME (time-sensitive period) in vivo, reduce the expression of the inhibitory co-stimulatory molecule PD-1 in CD8+ T cells, activate systemic immune effects, and significantly improve the TIME of ovarian cancer. Therefore, the CuI-CDDP combined treatment strategy controls the progression of ovarian cancer through a dual mechanism of directly inhibiting tumor cells (promoting DNA damage, etc.) and promoting antitumor immune activation (improving TIME).

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of cucurbitacin I in the preparation of cisplatin sensitizers for the treatment of ovarian cancer.

2. The application according to claim 1, characterized in that, Used in combination with cisplatin in the preparation of formulations that inhibit the proliferation of mouse ovarian epithelial carcinoma cells or human ovarian carcinoma cells, induce pyroptosis in mouse ovarian epithelial carcinoma cells or human ovarian carcinoma cells, or activate the body's immune response against ovarian cancer.

3. The application according to claim 2, characterized in that, The mouse ovarian epithelial cancer cells were ID8, and the human ovarian cancer cells were A2780.

4. The application according to claim 3, characterized in that, For ID8: CuI (0.5 μmol / L) + CDDP (3 μmol / L); For A2780: CuI (1 μmol / L) + CDDP (6 μmol / L).

5. The application according to claim 2, characterized in that, Its applications in the preparation of formulations that induce pyroptosis in mouse ovarian epithelial carcinoma cells or human ovarian carcinoma cells include: Application of Annexin V-FITC+ / PI+ cell population ratio in the preparation of formulations for mouse ovarian epithelial cancer cells or human ovarian cancer cells; Application in the preparation of formulations that upregulate the release of LDH from mouse ovarian epithelial cancer cells or human ovarian cancer cells; Application in the preparation of formulations that upregulate GSDME-N protein expression in human ovarian cancer cells.

6. The application according to claim 3, characterized in that, Its applications in the preparation of agents that inhibit the proliferation of mouse ovarian epithelial cancer cells or human ovarian cancer cells include: Application in the preparation of agents that inhibit the activity of mouse ovarian epithelial cancer cells or human ovarian cancer cells; Its application in animal models for preparing agents that inhibit the growth of subcutaneous tumors.

7. The application according to claim 1, characterized in that, Its applications in the preparation of agents that activate the body's immune response against ovarian cancer include: Application in animal models in the preparation of formulations that promote CD3+CD8+ T cell infiltration; Application in animal models in the preparation of formulations that downregulate the proportion of PD-1+CD8+ T cells in TIME; Application in animal models in the preparation of formulations that stimulate antigen presentation in DCs; Application in animal models in the preparation of formulations that promote CD8+ T cell infiltration during TIME; Application in animal models for the preparation of formulations that downregulate the expression of the immune checkpoint PD-1 on the surface of CD8+ T cells.

8. The application according to claim 2, characterized in that, Direct targets include the epidermal growth factor receptor / STAT3 / Janus kinase signaling pathway.

9. The application according to claim 8, characterized in that, Applications include: preparation of formulations that inhibit the phosphorylation of EGFR and its downstream STAT3 protein; preparation of formulations that induce DNA damage accumulation; and preparation of formulations that upregulate ROS.

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