| Ovarian cancer is a common malignant tumor of the female reproductive system,which can occur at any age.Among gynecological tumors,ovarian cancer has the highest fatality rate[1].Due to the insidious incidence of ovarian cancer and the lack of effective early diagnosis technology,70%of ovarian cancer is diagnosed in the late stage of the disease[2]Currently,the main clinical treatment measures are surgery+chemotherapy+maintenance therapy(PARP inhibitors),among which chemotherapy is still the key method of ovarian cancer treatment.About 70%of epithelial ovarian cancer is sensitive to platinum combined with paclitaxel chemotherapy.However,with the increase in the number of chemotherapy,platinum resistance and uncontrollable progression of the disease eventually occur[3],resulting in a 5-year survival rate of only about 30%for patients with advanced ovarian cancer[4].Cisplatin is a commonly used anti-tumor chemotherapy drug,which is clinically used for the treatment of various solid tumors[5].Currently,it is generally believed that cisplatin kills tumor cells mainly by causing DNA damage,damaging DNA replication and transcription,resulting in cytotoxicity,and indirectly initiating the endogenous apoptosis pathway mediated by the mitochondrial pathway,in which the activation of Bcl-2/BAX family and Caspase family proteins play a crucial role[6].Cisplatin is a dose-concentration dependent anti-tumor drug,but due to its serious neurotoxicity,otonephrotoxicity and other serious side effects,the long-term application of large doses of platinum drugs is limited.In addition,platinum resistance gradually appears in the long-term treatment process,which limits the use of platinum drugs.It is necessary to find drugs to be used in combination with platinum drugs to improve the efficacy,delay the drug resistance and reduce the dose to alleviate the side effects.In recent years,with the application of molecular targeted drugs in the treatment of malignant tumors,the key enzyme in the folic acid metabolic pathway as the target of tumor treatment has attracted more and more attention.Pemetrexed is a new kind of multiple targets for drug folate metabolism[7],In vitro studies have shown that permetrexide mainly inhibits thymidylate synthase(TS),and it also inhibits the activities of dihydrofolic acid reductase(DHFR)and glyceramide nucleotide formyltransferase,leading to the synthesis of purines and pyrimidines,thus inhibiting DNA replication and blocking cells in the S phase.A number of clinical studies have shown that pemetrexed combined with chemotherapy drugs has antitumor activity against malignant pleural mesothelioma,lung cancer,breast cancer and other tumors[8].Pemetrexed enters the cell through the folate binding protein transport system on the cell cembrane[9],A and its anti-tumor effect may be related to the level of folate receptor expression.Currently,there are three subtypes of folic acid receptor[10],namely folic acid receptor-a(FR-a),folic acid receptor-?(FR-?),and folic acid receptor-y(FR-y).Among them,high expression of FR-a is associated with tumor progression and chemotherapy resistance in humans[11].FR-a,located in the extracellular membrane[12],Folic acid is transported into cells by pinocytosis,and is involved in the synthesis of purines and pyrimidines by thymidine synthase(TS).YU et al[11]showed that FR-a was not expressed or low in the serum and tissues of normal ovaries and benign ovarian lesions,but was highly expressed in epithelial ovarian cancer,most of which was expressed in serous ovarian cancer.By enhancing the synthesis of purines and pyrimidines,high expression of FR-a promotes DNA replication and damage repair in tumor cells,leading to drug resistance in tumor cells.Data suggests that FR-? expression may be an effective biomarker of pemetrexed antitumor therapy[13].When combined with cisplatin,on the one hand,DNA damage caused by cisplatin could not be repaired and the sensitivity of cisplatin chemotherapy was enhanced.On the other hand,tumor cell proliferation is inhibited by blocking DNA replication[14].The purpose of this study was to study the effect of the anti-folate metabolism drug pemetrexed combined with cisplatin on the proliferation inhibition?apoptosis and invasion of SKOV3 cells in ovarian cancer and the relevant mechanism,providing a new idea for the treatment of clinical ovarian cancer.purposeMRNA and protein levels of FR-? and thymidine synthase(TS)in Chinese hamster ovarian epithelial cells CHO and ovarian cancer SKOV3 cells were detected by qRT-PCR and Western Blot,The effects of pemetrexed combined with cisplatin on the proliferation,apoptosis and invasion of ovarian cancer SKOV3 cells and their possible mechanisms were studied.Research object and experimental method1.Subjects:Chinese hamster ovarian epithelial cells CHO,ovarian cancer SKOV3 cells.2.Research methods:mRNA,protein and TS mRNA and protein levels of Chinese hamster ovarian epithelial cells and ovarian cancer SKOV3 cells were detected by qRT-PCR and Western Blot.The effects of MTT,Annexin V-FITC and PI double staining combined with flow cytometry and Transwell on proliferation,apoptosis and invasion of SKOV3 cells were detected by pemetrexed,cisplatin and pemetrexed combined with cisplatin.Western blot assay was used to detect the effects of pemetrexed,cisplatin and combination on the expression of BAX and Bcl-2 proteins.Meanwhile,Western blot was used to detect the changes of TS and FR-?before and after the addition of pemetrexed in SKOV3 cells.3.Statistical methods:SPSS24.0 was used for statistical analysis.The normal distribution measurement data were expressed as x±s,while the non-normal distribution quantitative data were expressed as M(P25-P75).The two groups of quantitative data were analyzed by two independent samples t test.The effects of pemetrexed,cisplatin and combined use on SKOV3 invasion and apoptosis were analyzed by one-way anova,with the test level=0.05.Result1.In ovarian cancer SKOV3 cells,FR-? protein(0.46±0.04)and TS protein(0.34±0.03)were significantly different from FR-? protein(0.12±0.02)and TS protein(0.19±0.02)in Chinese hamster ovarian epithelial cells CHO(P<0.001).The differences of FR-? mRNA(3.73±1.50)and TS mRNA(1.77±0.48)in ovarian cancer SKOV3 cells were statistically significant(P<0.01)compared with those in Chinese hamster ovarian epithelial cells(0.90±0.38)and TS mRNA(0.75±0.29).2.In vitro,SKOV3 was treated with pemetrexed at different concentrations(10mg/L,20mg/L,40mg/L,80mg/L,160mg/L,320mg/L,640mg/L,1280mg/L),and SKOV3 was treated with cisplatin at different concentrations(0.75mg/L,1.5mg/L,3mg/L,6mg/L,12mg/L).The results showed that the inhibitory effect of pemetrexed and cisplatin on SKOV3 cell proliferation was dose concentration dependent.The IC50 value of pemetrexed was(597.53±113.27)mg/L,and the IC50 value of cisplatin was(8.23±0.79)mg/L.The proliferation inhibition rate of 6mg/L cisplatin was(25.57±0.94)%,the proliferation inhibition rate of 320mg/L pemetrexed was(34.82±1.64)%,and the combined inhibition rate of the two was(50.00±1.40)%,the difference was statistically significant(P<0.001).6mg/L cisplatin and 320mg/L pemetrixed were selected as the following concentrations.3.The invasion results showed that the average number of invasive cells in the pemetrexed group was(53.11±4.17),the average number of invasive cells in the cisplatin group was(43.89±3.69),the average number of invasive cells in the pemetrexed + cisplatin group was(26.22±2.99),and the average number of invasive cells in the control group was(73.44±6.16),the single factor variance analysis results showed that combination,compared to a single drug groups statistically significant difference(P<0.001).The difference between the single drug group and the control group was statistically significant(P<0.001).4.Apoptosis results showed that the apoptosis rate of pemetrexed group was(17.46±0.84)%,that of cisplatin group was(20.50±0.93)%,The apoptosis rate of the combined treatment increased to(36.41±2.07)%,The results of one-way analysis of variance showed that the apoptosis rate of the combined drug group was significantly higher than that of the single drug group,and the difference was statistically significant(P<0.001).The difference between the single drug group and the control group was statistically significant(P<0.001).5.Western blot results showed that Bcl-2 protein was detected in cisplatin group(0.25±0.03),pemetrixed group(0.31±0.02),cisplatin + pemetrixed group(0.12±0.01),and control group(0.49±0.03).BAX protein was found in cisplatin group(0.42±0.04),pemetrexed group(0.37±0.03),cisplatin + pemetrexed group(0.61±0.04),and control group(0.16±0.02).The expression levels of BAX and Bcl-2 in the combination group were statistically significant compared with that in the cisplatin group(P<0.001).6.Western blot results showed that the difference of TS protein in SKOV3 cells(0.34±0.03)was statistically significant(P<0.001)when compared with TS protein in SKOV3 cells(0.25±0.02),while there was no significant change in FR-?.Conclusion1.Pemetrexed inhibits ovarian cancer SKOV3 cell proliferation,induces apoptosis and weakens cell invasion,and has a synergistic effect in combination with cisplatin.2.In SKOV3 cells of ovarian cancer,FR-? and TS protein were highly expressed,while pemetrexed decreased the expression of TS protein.3.Pemetrexed combined with cisplatin may inhibit DNA replication and damage repair by reducing the expression of TS protein in folate metabolism pathway,and activating endogenous apoptosis pathway mediated by mitochondrial pathway,thus contributing to sensitization of cisplatin chemotherapy and providing new ideas for the treatment of ovarian cancer. |
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