Mitogen-Activated Protein Kinase Inhibitors Increase Sensitivity Of Olaparib-Resistant Ovarian Cancer

Background:Ovarian cancer,as one of the most common and malignant reproductive system tumor,it positions the third place immediately behind cervical cancer and uterine cancer.However,epithelial ovarian cancer is the first cause of cancer-related death in all gynecological tumors.According to histopathological analysis,ovarian cancer roughly categorized into epithelial ovarian carcinoma,germ cell tumor,sex cord stromal tumor and metastatic tumor.However,the ovarian cancer is often under-diagnosed at early stage in clinical practice due to atypical symptoms and lack of reliable and sensitive markers.The available clinical treatments towards ovarian cancer incorporate surgical treatment,radiotherapy and chemotherapy.For most of the aggressive and advanced tumor patients,surgical operation with surgical removal and chemotherapy is an appropriate and feasible management routine,however,surgical operation is it not suitable to the advanced cases owning to the multiple metastasis.At least 80%of chemotherapy eventually develops into drug resistance,which directly affects the effectiveness of chemotherapy and survival rate.Application of radiotherapy in ovarian cancer therapy is greatly limited in view of the resultant infertility and can only be used as a palliative local therapy for patients with recurrent cancer.The immune-checkpoint based therapeutics are also under investigations and exploitations.In general,the prognosis of ovarian cancer patients is relatively moderate and the overall 5-year survival rate is around 45%The next generation sequencing towards cancer patients has indicated that about 10%of ovarian tumors are linked to the hereditary genetic mutations of BRCA1 and BRCA2 genes.Olaparib,also known as AZD-2281(trade name Lynparza),is an FDA-approved targeted therapy for germline BRCA mutated advanced ovarian cancer patients,who have received three or more prior lines of chemotherapy.Olaparib is a poly ADP ribose polymerase(PARP)inhibitor,which specifically inhibits PARP and blockades the DNA repair process.The BRCA mutated ovarian cancer have manifested increased reliance on PARP and therefore vulnerable to PARP inhibition.The early clinical trials indicated a significantly favorable response to olaparib in BRCA mutated ovarian cancer patients.However,a few proportion of targeted patients are intrinsically resistant to the treatment of olaparib due to some unrecognized mechanisms.Moreover,like most of the targeted anti-tumor drugs,the developing resistance is now also observed in olaparib therapy.Therefore,to have a better understanding the molecular mechanism underlying drug resistance and seek for the potential compounds to surmount olaparib resistance are in urgent need.Here we try to understand the underlying molecular events and resistance mechanism of Olaparib resistance,and seek to search for the potential combinational therapeutics to conquer the intrinsic olaparib resistance in human ovarian cancer.Part Ⅰ:Inhibition of Olapani resistance in ovarian cancer cells by MAPKs inhibitors in vitroResearch purposes:1.To construct ovarian cancer cell lines that can develop resistance to Olaparib,which will be convenient for the following experiments;2.To investigate the expression of MAPK pathway-related molecules P38 and JNK in Olaparib-resistant ovarian cancer cells;3.To investigate the effect of MAPKs inhibitors on the proliferation,invasion,autophagy and epithelial-mesenchymal transition in Olaparib-resistant ovarian cancer cells.Research methods:1.The SKOV3 and A2780 cell lines were treated with different concentrations of Olaparib,and the activity of ovarian cancer cells treated with Olaparib at different time points was detected by MTT assay;and Cell Counting Kit-8(CCK-8)K was also used to detect the proliferation of different ovarian cancer cell lines.2.Western blotting assay was used to detect the expression of MAPK pathway-related molecules P38,JNK,ERK and ERK5 in olpapan sensitive and drug-resistant ovarian cancer cells,and to explore whether MAPK signaling pathway is involved in ovarian cancer drug resistance.3.The CCK-8 kit was used to detect the effect of P38-specific inhibitor SB202190 and JNK specific inhibitor SP600125 on the proliferation of ovarian cancer drug-resistant cells after single treatment or combination therapy of ovarian cancer-resistant cells.4.The metastatic capacity were performed using Transwell chamber to observe the effect of P38 specific inhibitor SB202190 and JNK specific inhibitor SP600125 on the invasion of ovarian cancer-resistant ovarian cancer cells after combined treatment with ovarian cancer drug-resistant cells.5.The autophagy process of ovarian cancer resistant cells treated with P38 specific inhibitor SB202190 and JNK specific inhibitor SP600125 was detected by western blotting and confocal microscopy assays.6.The expression of N-CADHERIN,a-SMA in epithelial-mesenchymal transition process in ovarian cancer resistant cells treated with P38 specific inhibitor SB202190 and JNK specific inhibitor SP600125 was detected by western blotting..Research results:1.Successfully constructed Olaparib-resistant ovarian cancer cell lines:SKOV3-R and A2780-R.The half-inhibitory concentration(IC50)of Olaparib on drug-resistant A2780-R cells was increased from 16±5μM in parental A2780-P cells to 415±12μM by MTT assay.The IC50 value of drug-resistant SKOV3-R cells was increased from 25±17μm in parental SKOV3-R cells to 688±22 μM.This indicates that less effect of Olaparib on cell activation of resistant cell lines was observed than that of the parental cells.The proliferation of different cell lines after Olaparib treatment was detected by CCK-8 method.It was found that the treatment with Olaparib significantly inhibited the proliferation of the parental SKOV3-P and A2780-P cell lines after increasing days of cell culture(p<0.05).No effect on the proliferation of the resistant cell lines was observed.Combined with the MTT assay,it was confirmed that Olaparib-resistant ovarian cancer cell lines were successfully constructed.2.Determination of MAPKs pathway-related molecules in Olaparib-resistant ovarian cancer cells by western blotting assayAfter comparing the expression changes of the MAPK signaling pathway-related molecules in A2780-R cell line resistant to Olaparib and the parental sensitive cell line A2780-P,we found that the expression of P38 and JNK molecules was significantly increased in A2780-R cells(p<0.05),but no significant changes were observed in the expression of ERK and ERK5.It is suggested that the MAPK signaling pathway may be involved in the resistance of Olaparib in A2780-R cell lines.At the same time,we also analyzed the expression changes of MAPK signaling pathway-related molecules in SKOV3-R cell line and the parental sensitive cell line SKOV3-R,and observed that the expression of P38 and JNK molecules in A2780-R cells increased significantly(p<0.05).However,there was no significant change in the expression of ERK and ERK5.It is suggested that the MAPK signaling pathway may be involved in the resistance of Olaparib in SKOV3-R cell lines.3.Effect of MAPKs pathway P38 Specific inhibitor SB202190 and JNK Specific inhibitor SP600125 on the proliferation of ovarian cancer Olaparib-resistant cellsWe used different concentration gradients of P38-specific inhibitor SB202190 or JNK-Specific inhibitor SP600125 at 0μM,5 μM,10μM and 20μM,and add them when culture A2780-R and SKOV3-R cell lines,which are resistant to Olaparib treatment.After detect the proliferation of different cell lines at different time points,we determined that the optimal concentration of the two inhibitors that can inhibit cell proliferation was 1 0 μM.The usage of SB202190 or SP600125 alone could inhibit the growth of A2780-R and SKOV3-R cells moderately,but the combination of the two inhibitors inhibited the cell proliferation significantly(p<0.05).4.Effect of combined treatment of P38 specific inhibitor SB202190 and JNK specific inhibitor SP600125 on the invasion of Olaparib-resistant cell linesInvasion experiments were carried out by Transwell chamber method,and the number of invading cells of ovarian cancer resistant lines A2780-R and SKOV3-R after combined treatment with P38 Specific inhibitor SB202190 and JNK Specific inhibitor SP600125 was observed under microscope.The number of ovarian cancer resistant cells in the SB202190 and SP600125 combination treatment groups was significantly less than that in the untreated control cells.It shows that the combination of SB202190 and SP600125 reduces the invasion ability of Olaparib-resistant ovarian cancer cells significantly.5.Effect of combined treatment of P38 specific inhibitor SB202190 and JNK specific inhibitor SP600125 on the autophagy flux of Olaparib-resistant cellsGFP-labeled LC3 was transfected into Olaparib-resistant ovarian cancer cell lines A2780-R and SKOV3-R,and western blotting assay was used to detect the expression of LC3-phosphatidylethanolamine conjugate(LC3-Ⅱ)in the Olaparib-resistant cell lines after combination therapy of P38-Specific inhibitor SB202190 and JNK-Specific inhibitor SP600125.The expression of LC3-II was enhanced in combination therapy group compared with the untreated group.The autophagy structure of GFP-labeled LC3 was observed with the fluorescence microscope.We found that the ovarian cancer resistant tumor cells had more autophagy structures than the control cells after combined treatment(P<0.05).These results indicate that combination therapy with P38 and JNK inhibitors increases autophagy flux in Olaparib-resistant A2780-R and SKOV3-R cells.6.Effect of combined treatment of P38 Specific inhibitor SB202190 and JNK Specific inhibitor SP600125 on epithelial-mesenchymal transition in Olaparib-resistant cellsThe expression of epithelial-mesenchymal transition markers N-CADHERIN,a-SMA in Olaparib-resistant cells treated with P38 Specific inhibitor SB202190 and JNK Specific inhibitor SP600125 was determined by western blotting.It was found that the combined treatment of SB202190 and SP600125 significantly reduced the expression of N-CADHERIN and a-SMA in drug-resistant cells,indicating that the combination of SB202190 and SP600125 inhibits the epithelial-to-mesenchymal transition progression in Olaparib-resistant ovarian cancer cell lines A2780-R and SKOV3-R.Conclusions:The Olaparib-resistant ovarian cancer cell lines SKOV3-R and A2780-R were successfully constructed in the experiment,and the expression of P38 and JNK in MAPKs signaling pathway were up-regulated in drug-resistant cells.The combination of the P38-specific inhibitor SB202190 and the JNK-specific inhibitor SP600125 in the MAPKs signaling pathway inhibited the proliferation,invasion and epithelial-mesenchymal transition progression of the Olaparib-resistant ovarian cancer cell lines SKOV3-R and A2780-R,while increased autophagy flux in the Olaparib-resistant ovarian cancer cell lines SKOV3-R and A2780-R.Part II:In vivo study of MAPKs inhibitors in reversing Olaparib resistance in ovarian cancer cellsResearch purposes:1.Establishing an ovarian cancer xenograft model in nude mice;2.To investigate the effect of MAPKs inhibitor treatment on the proliferation of Olaparib-resistant ovarian cancer cells in vivo.Research methods:A single cell suspension of tumor cells(107 cells/mL)was resuspended in HEPES after trypsinizing the SKOV3-R cell line.100 μL of this cell suspension was thoroughly mixed with an equal amount of basement membrane extract on ice,and then the mixture was inoculated into bilateral flanks of the nude mouse.Then the recipient mice in the experimental group were intraperitoneally injected with MAPKs pathway P38 specific inhibitor SB202190 and JNK specific inhibitor SP600125,and the control group was injected with the same amount of saline.The tumor growth status of the mice was observed every 5 days and the length and width of the tumor were measured at the sanme time.According to the calculation formula to determine the tumor volume:volume(mm3)=(width)2(mm2)×length(mm)/2,we could observed the tumor volume at different time points to establish a xenograft model of ovarian cancer in nude mice,and to figure out whether the MAPKs pathway inhibitors SB202190 and SP600125 could inhibit the proliferation of olrapani-resistant SKOV3-R cell line in vivo.At the same time,we also used A2780-R ovarian cancer to construct the nude mouse ovarian cancer tumor model in the same way,and tried to evaluate whether the MAPKs pathway inhibitors SB202190 and SP600125 could inhibit the proliferation of olrapani-resistant A2780-R cell line in vivo.Research results:Nude mice were inoculated with Olaparib-resistant ovarian cancer cell lines A2780-R and SKOV3-R,and they lived well with normal activity.The tumor usually began to appear around 2 weeks.The morphology of the transplanted tumor constructed with A2780-R and SKOV3-R is relatively regular and the texture is relatively hard.After analysing theXenograft model constructed with A2780-R,we found that the tumor volume of the combined treatment with the P38-specific inhibitor SB202190 and the JNK-specific inhibitor SP600125 began to decrease compared with the control mice at day 15.Then we calculate the tumor volum at day 30,and observed that the tumor volum in combined treatment group with SB202190 and SP600125(about 350 mm3)was smaller than the untreated control group(about 800 mm3),with P<0.05.This indicates that the MAPKs pathway inhibitors SB202190 and SP600125 have an inhibitory effect on the proliferation of the Olaparib-resistant A2780-R cell line in vivo,which is quite in agreement with the in vitro results.At the same time,we also analyzed the xenografts constructed with the SKOV3-R cell line,and found that the tumor volume of the combined treatment with the P38-specific inhibitor SB202190 and the JNK-specific inhibitor SP600125 also began to decrease compared with the control mice at day 15.Then we calculate the tumor volum at day 30,and observed that the tumor volum in combined treatment group with SB202190 and SP600125(about 400 mm3)was smaller than the untreated control group(about 1000 mm3),with P<0.05.This indicates that the MAPKs pathway inhibitors SB202190 and SP600125 also have an inhibitory effect on the proliferation of the Olaparib-resistant SKOV3-R cell line in vivo,which will significantly inhibit tumor growth.These results suggest that MAPKs inhibitors have the potential to treat Olaparib-resistant ovarian cancer.Conclusions:The combined treatment of P38-specific inhibitor SB202190 and JNK-specific inhibitor SP600125 in the MAPKs signaling pathway could inhibit the proliferation of Olaparib-resistant ovarian cancer cell lines SKOV3-R and A2780-R in mice,and show the potential to treat Olaparib-resistant ovarian cancer.