Therapeutic Effect Of KDM6B Inhibitor GSK-J4 Combined With Cisplatin On Ovarian Cancer And Its Mechanism

Background and purpose:Ovarian cancer is one of the three major malignant tumors in the female reproductive system.Due to the difficulty in early diagnosis of ovarian cancer,the mortality rate ranks first in the mortality rate of gynecological malignancies.The 5-year survival rate of patients with early-stage ovarian cancer is about 90%.However,clinically,nearly 75% of ovarian cancer patients are already at an advanced stage(stage III or IV)at the time of diagnosis.In global cancer patient survival data analysis,the five-year survival rate of patients with advanced ovarian cancer is only 30%-40%.At present,the common clinical treatment for ovarian cancer is surgical treatment,supplemented by platinum-based comprehensive treatment after surgery.Therefore,the development of new drugs is necessary to improve the prognosis of ovarian cancer.Histone methylation is one of the key research areas in epigenetics histone modification.It mainly modulates the three-dimensional configuration of DNA in genetic material,thereby regulating gene transcription.The methylation modification of histones is closely related to the occurrence,development,metastasis and recurrence of many malignant tumors.Among them,H3K27 is one of the common sites in the methylation modification of histones.The methylation modification of H3K27(H3K27 trimethylation,namely H3K27me3)can inhibit the transcription of many genes in the genetic material.KDM6B(lysine(K)specific demethylase 6B,also known as JMJD3),is a histone demethylation transferase that can cause the demethylation of H3K27me3,thereby promoting the transcription of multiple genes in the genetic material.Many existing studies have shown that KDM6 B is very active in the process of cell proliferation,differentiation,and apoptosis,and therefore occupies a very important position in the development of embryonic development,inflammatory diseases,tumors and other diseases.In addition,many studies have found that KDM6 B plays a role in promoting cancer in glioma,prostate cancer,kidney cancer,ovarian cancer,melanoma,Hodgkin’s lymphoma and multiple myeloma.However,the specific biological function and molecular mechanism of KDM6 B in ovarian cancer have not been systematically elucidated.GSK-J4 is an inhibitor of the H3K27me3 demethylase KDM6B(also known as JMJD3),which can effectively inhibit the demethylation of H3K27me3.This study aims to explore whether the inhibitor GSK-J4,which targets KDM6 B,can have an anti-tumor effect on ovarian cancer.Materials and Methods:Search the TCGA database to explore the expression of KDM6 B in the nucleus of ovarian cancer cells and its impact on the prognosis of ovarian cancer patients.In vitro experiment: Cell Counting Kit 8(CCK8)cytotoxicity kit was used to detect the proliferation activity of ovarian cancer cells after the combined action of the drugs GSK-J4,cisplatin(DDP)and the two drugs for 24 hours and 48 hours,and calculate the two at the same time Combination Index(CI)of drug action;using Annexin-Ⅴ/PI apoptosis kit and flow cytometry to detect the apoptosis of ovarian cancer cells after the combined action of the drugs GSK-J4,cisplatin(DDP)and the two drugs Death status;Western blot(WB)experiment was used to detect H3K27me3 expression level and apoptosis-related protein expression in ovarian cancer cells after the drug GSK-J4,cisplatin(DDP)and the combination of the two drugs from the protein expression level;flow Cytometry is used to investigate whether GSK-J4 has a cycle-blocking effect on ovarian cancer cells;the colony formation experiment is used to determine the effect of the drug GSK-J4 on the colony-forming ability of ovarian cancer cells;the morphology of ovarian cancer cells after GSK-J4 is observed by cell staining The change of the situation;the cell-climbing immunofluorescence experiment verified that GSK-J4 can increase the sensitivity of ovarian cancer cells to cisplatin;the Transwell test was used to detect the effect of GSK-J4 on the migration and invasion of ovarian cancer cells.In vivo experiment: A2780 human epithelial ovarian cancer cell line was used to construct a human ovarian cancer cell line A2780 celiac tumor model in female BALB/c mice at 4-6 weeks;the ID8 murine epithelial ovarian cancer cell line was used in 6 The murine-derived ovarian cancer cell line ID8 peritoneal tumor model was constructed in female C57BL/6 mice of ～8 weeks.After intraperitoneal injection,observe the inhibitory effect of GSK-J4 on ovarian cancer xenografts and whether it has a synergistic effect in the body after the combined treatment with cisplatin.The expression of cell proliferation(Ki67)and H3K27 methylation level(H3K27me3)in tumor tissues was detected by immunohistochemistry and immunofluorescence techniques.Using the peritoneal flow cytometry technique,explore the changes of M2 macrophages,MDSCs,neutrophils and monocytes in the relevant tumor microenvironment in the abdominal cavity after medication,so as to explore the mechanism of action.HE staining was used to detect the morphological changes of the main organs of the mice,and the blood biochemical indicators of the mice were detected to reflect the toxic and side effects of the drugs.Results:1.By searching the TCGA database,it is found that KDM6 B is moderately expressed in ovarian cancer and is negatively correlated with the prognosis of ovarian cancer patients.2.In vitro :(1)CCK8 cell proliferation inhibition test showed that GSK-J4 single drug can inhibit the proliferation of five kinds of ovarian cancer cells A2780,ID8,A2780/DDP,SKOV3,ES2 in vitro,and with GSK-As the concentration of J4 action increases and the time is prolonged,the relative cell growth rate decreases gradually.Among them,ID8 and A2780 cells were more effective in inhibiting proliferation after GSK-J4 treatment.At the same time,the calculation of the combination index shows that GSK-J4 and cisplatin have a synergistic effect in vitro,and the lowest concentrations of cisplatin at the corresponding time points of 24 h and 48 h are 8.4u M and 4.4u M,respectively.(2)Flow cytometry detection of cell apoptosis experiments show that GSK-J4 single agent and its combination with cisplatin can induce apoptosis of ovarian cancer cells.With the increase of the concentration and time of GSK-J4 action,The proportion of apoptosis in ID8 and A2780 ovarian cancer cells gradually increased;at the same time,the proportion of induced ovarian cancer cell apoptosis when GSK-J4 and cisplatin were used in combination was significantly different from that of GSK-J4 as a single agent.At the same time,Western Blot technology showed that after using GSK-J4 drug,the expression of apoptosis-related protein and H3K27 methylation level in ovarian cancer cells increased.(3)Cell cycle detection by cell flow cytometry experiments show that GSK-J4 can arrest tumor cell cycle in vitro,ID8 cells stagnate in S phase,and A2780 cells stagnate in G1 phase.(4)Cell colony formation experiment found that GSK-J4 can inhibit the formation of ovarian cancer cell colonies in vitro.(5)Through cell-climbing immunofluorescence technology,it is found that GSK-J4 can inhibit the repair of DNA double-strand breaks,thereby increasing the sensitivity of ovarian cancer cells to cisplatin.(6)Transwell experiment shows that GSK-J4 can inhibit the invasion and migration of ID8 and A2780 ovarian cancer cells in vitro.3.In vivo : Two models of mouse-derived ovarian cancer cell line ID8 abdominal tumor model and human-derived ovarian cancer cell line A2780 abdominal tumor model were constructed.(1)In the mouse-derived ovarian cancer cell line ID8 peritoneal tumor model,GSK-J4 single agent and combined with cisplatin have obvious anti-tumor effects.The weight of abdominal tumors in each group at the end of treatment(g): solvent group Compared with the GSK-J4 single-drug group(0.42±0.076 vs 0.329±0.075)and the DDP single-drug group and the combination group(0.108±0.067 vs 0.059±0.021),the tumor tissue was significantly smaller,and there were statistical differences.(2)In the human-derived ovarian cancer cell line A2780 abdominal tumor model,GSK-J4 single agent and combined with cisplatin have obvious anti-tumor effects on it.The weight of abdominal tumors in each group at the end of treatment(g): solvent group Compared with the GSK-J4 single-drug group(4.562±1.008 vs 2.892±1.093)and the DDP single-drug group and the combination group(0.828±0.536 vs 0.175±0.138),the tumor tissue was significantly smaller and the difference was statistically significant.4.Changes in related tumor microenvironment after drug treatment:(1)In the related tumor microenvironment,after the action of GSK-J4,the proportion of M2 type macrophages in the two ovarian cancer cell line models was down-regulated:(1)In the mouse source In the ovarian cancer cell line ID8 abdominal tumor model,the proportion(%)of M2 type macrophages is:(a)On the 17 th day of treatment,the solvent group and the GSK-J4 single-agent group are compared(78.29±2.33 vs59.78±0.535)And the comparison between the DDP single-drug group and the combination group(67.68±0.83 vs 44.73±2.30);(b)on the 47 th day,the solvent group and the GSK-J4 single-drug group(5.80±1.15 vs 2.16±0.395)and The comparison between the DDP single-drug group and the combination-drug group(2.069±0.44 vs0.341±0.05);the differences in each group were statistically significant.(2)In the human-derived ovarian cancer cell line A2780 abdominal tumor model,the proportion of M2 macrophages(%)is: the solvent group and the GSK-J4 single-drug group(45.90±3.01 vs 33.10±2.305)and DDP single The drug group and the combination group were compared(32.89±3.11 vs.20.71±4.05).The difference in the proportion of M2 macrophages in the solvent group and the GSK-J4 single-drug group was statistically significant;although the combination group had M2 macrophages The proportion of phages was lower than that in the DDP group,but the difference was not statistically significant(P=0.0755).(2)In the relevant tumor microenvironment,the proportion of MDSCs cells in the two tumor models was down-regulated after GSKJ4:(1)In the mouse-derived ovarian cancer cell line ID8 peritoneal tumor model,the proportion(%)of MDSCs cells was: On the 47 th day of medication,the proportion(%)of MDSCs cells was: the comparison between the solvent group and the GSK-J4single-drug group(36.69±1.50 vs 19.52±3.59)and the comparison between the DDP single-drug group and the combination group(3.48±1.04)vs 0.89±0.01).Among them,the difference in the proportion of MDSCs in the solvent group and the GSK-J4 singledrug group was statistically significant;although the proportion of MDSCs in the combination group was lower than that in the DDP group,the difference was not statistically significant(P>0.05).(2)In the human-derived ovarian cancer cell line A2780 abdominal cavity tumor model,the proportion(%)of MDSCs cells is: the solvent group and the GSK-J4 single-drug group(7.84±0.92 vs 4.25±1.32)and the DDP single-drug group and Comparison of the combination group(4.72±1.26 vs2.56±1.52).Although the proportion of MDSCs cells decreased in each group comparison,the results were not statistically significant.(3)In the human-derived ovarian cancer cell line A2780 abdominal cavity implantation tumor model,the proportion of neutrophils with negative immune regulation(%)was down-regulated:the solvent group and the GSK-J4 single-drug group were compared(9.23±2.44 vs6.07±1.32)and the comparison between the DDP single-drug group and the combination group(3.42±1.23 vs 3.37±0.92).Although the proportion of neutrophils with negative immune regulation decreased in each group comparison,the results were not statistically significant.(4)In the mouse-derived cell line ID8 ovarian cancer abdominal cavity implantation tumor model,whether in the early or late tumor microenvironment,the proportion(%)of mononuclear cells with immunological negative effects is down-regulated:(a)Medication On day 17,the solvent group was compared with the GSK-J4 single-drug group(27.86±2.27 vs 4.61±0.79)and the DDP single-drug group and the combination group(21.07±4.12 vs 3.23±0.16).The differences in each group were statistically significant.Scientific significance;(b)On the 47 th day of medication,the comparison between the solvent group and the GSKJ4 single-drug group(2.11±0.13 vs 0.96±0.20)and the comparison between the DDP single-drug group and the combination group(0.31±0.02 vs 0.21±0.05).Among them,the difference in the proportion of monocytes with negative immune regulation in the solvent group and the GSK-J4 single-drug group is statistically significant;although the proportion of monocytes with negative immune regulation in the combination group is higher than that of DDP The group declined,but the difference was not statistically significant(P=0.1581).5.Research on drug toxicity and side effects: It was found by HE staining that GSKJ4 had no obvious histopathological changes on the main organs of mice.At the same time,it was found that GSK-J4 had an effect on the heart function,liver and kidney function of mice by detecting blood biochemical indicators.No obvious impact.Conclusion:1.This study found that the KDM6 B inhibitor GSK-J4 alone or in combination with cisplatin has an anti-tumor effect on ovarian cancer,and the combined use of cisplatin is more effective than the single agent: in vitro experiments show that GSK-J4 can inhibit Proliferation of ovarian cancer cells,and has a synergistic effect with cisplatin in ovarian cancer.It can promote cell apoptosis,inhibit cell cycle,induce cell polyploidization,reduce colony forming ability and tumor cell invasion and migration ability,and there is a time-dependent and concentration-dependent.In vivo experiments found that when GSK-J4 single agent or combined with cisplatin acts on human-derived ovarian cancer cell line A2780 abdominal tumor model and mousederived ovarian cell line ID8 abdominal tumor model,both have significant anti-tumor effects without significant toxicity.side effect.2.The possible mechanisms of GSK-J4’s anti-tumor effect on ovarian cancer include:changing the level of H3K27 methylation in tumor cells,causing apoptosis,etc.;at the same time,GSK-J4 has an immune activation effect: reducing the tumor-promoting M2 type giant The proportion of phages and the proportion of immunosuppressive MDSCs,neutrophils and monocytes.3.The KDM6 B inhibitor GSK-J4 has shown therapeutic effects on ovarian cancer in this study,providing a new strategy for the clinical treatment of ovarian cancer,but whether its anti-tumor effect on ovarian cancer has other mechanisms remains to be seen.Further in-depth research.