Kynureninase Involving In Cisplatin Resistance Of Cervical Adenocarcinoma And Its Potential Mechanism

Cervical cancer is one of the most common malignancies in women and is still a serious public health problem in China.With the popularization of "two cancers"screening,the early detection rate of cervical cancer has greatly increased,and its overall morbidity and mortality have decreased,however,the rising incidence of cervical adenocarcinoma and its younger onset age cannot be ignored.Cisplatinbased concurrent chemoradiotherapy is the standard treatment for locally advanced cervical adenocarcinoma.However,cervical adenocarcinoma is less sensitive to radiotherapy,and there is no standard treatment for patients with advanced cervical adenocarcinoma if they are cisplatin resistance and their prognosis is extremely poor.Therefore,to explore the molecular mechanism of cisplatin resistance in cervical adenocarcinoma can not only provide theoretical basis for overcoming its resistance,but also provide a new way to improve the prognosis of patients with cervical adenocarcinoma.Based on the difficulties of clinical treatment,we have found out the differentially expressed kynureninase(KYNU)mRNA in cervical adenocarcinoma cells(HeLa/DDP)and cervical adenocarcinoma cisplatin resistance cells(HeLa/DDP)by gene chip.In addition,in cervical adenocarcinoma tissues,the expressions of KYNU and P-glycoprotein(P-gp)in the chemo-resistance group were significantly higher than those in the chemo-sensitive group,which showed a significant positive correlation.Meanwhile,the high expression of KYNU predicted a worse prognosis,suggesting that KYNU may be involved in cisplatin resistance of cervical adenocarcinoma.KYNU is a key enzyme in the metabolic pathway of tryptophan.A large number of studies have shown that KYNU plays an important role in carcinogenesis,progression and chemo-resistance.However,the role of KYNU in cisplatin resistance and malignant progression of cervical adenocarcinoma and its potential mechanism remain unclear.In this study,real-time quantitative PCR(qPCR)and Western blot(WB)were applied to verify the expression of KYNU mRNA and protein in HeLa and HeLa/DDP cells.Then we constructed a stable HeLa/DDP cell line with KYNU mRNA knock-down,and detected the expression changes of KYNU and P-gp protein by WB.The effects of KYNU knockdown on biological behavior of HeLa/DDP cells in cisplatin were detected by relevant functional tests.Finally,we established a nude mouse model with transplanted tumor and performed cisplatin intrabitoneal chemotherapy to those nude mice.The efficacy of cisplatin chemotherapy in nude mice after KYNU knockdown was observed.Immunohistochemistry was used to detect the expression KYNU,P-gp,glutathione s-transferase π(GST-π),multidrug resistance associated protein 1(MRP1),pro-apoptosis regulatory protein Bax,anti-apoptosis proteins Bcl-2 and e-cadherin CD34 in transplanted tumor tissue of nude mouse,in hope of clarifying the influence of KYNU knockdown on cisplatin resistance of cervical adenocarcinoma and its malignant progression,and to preliminarily explore its potential mechanism,so as to provide a possible new target and approach for the reversal of cisplatin resistance in cervical adenocarcinoma.Part I Influence of KYNU knockdown on the expression of KYNU and P-gp protein and biological behavior of cisplatin-resistance cell line of cervical adenocarcinomaObjective:To investigate the influence of KYNU knockdown on KYNU and P-gp protein expression and biological behavior in cisplatin-resistance cell line of cervical adenocarcinoma.Methods:Real-time quantitative PCR(qPCR)and Western blot(WB)were applied to verify the expression of KYNU gene and protein in HeLa and HeLa/DDP cells.Then we constructed stable HeLa/DDP cells with KYNU mRNA knock-down,and detected the expression changes of KYNU and P-gp protein by WB.Invasion and migration ability of HeLa/DDP cells in cisplatin after KYNU knockdown were detected by Transwell,cell proliferation was detected by CCK-8 cell viability assay and cell apoptosis was detected by flow cytometry assay.Results:1.The expression of KYNU mRNA and protein in HeLa/DDP was significantly higher than that in HeLa cells,and the difference was statistically significant(P<0.05).2.KYNU knockdown cell lines(GV248-KYNU)and negative control cell lines(GV248-NC)showed green fluorescence 24h after lentivirus infection,and the fluorescence expression was the strongest 72h later.The transfection efficiency was 99%after 2μg/mL purinomycin screening.The expressions of KYNU and P-gp protein in GV248-KYNU group were significantly lower than those in HeLa/DDP group and GV248-NC group,the differences were statistically significant(all P<0.05),while there were no significant differences in KYNU and P-GP protein expressions between GV248-NC group and HeLa/DDP group(all P>0.05).3.After KYNU knockdown,the invasion,migration and proliferation of HeLa/DDP cells in cisplatin environment were significantly reduced(all P<0.05),while the apoptosis rate of HeLa/DDP cells was significantly increased(P<0.05).Conclusion:After KYNU knockdown,the expression of KYNU and P-gp protein in HeLa/DDP cell was down-regulated;meanwhile,the invasion,migration and proliferation ability of HeLa/DDP was decreased,while apoptosis rate was increased.These results suggest that KYNU may be involved in P-gp mediated cisplatin resistance of cervical adenocarcinoma and play a role in its malignant progression.Part Ⅱ Effect of KYNU knockdown on the efficacy of cisplatin chemotherapy in nude mice and its potential mechanismObjective:To explore the effect of KYNU knockdown on the efficacy of cisplatin chemotherapy in nude mice and its potential mechanism.Methods:1.GV248-KYNU,GV248-NC and HeLa/DDP nude mice with transplanted tumor were constructed,and were divided into GV248-KYNU-cisplatin group,GV248-NC-cisplatin group,HeLa/DDP-cisplatin group and HeLa/DDP-normal saline(NS)group according to whether cisplatin chemotherapy was performed later,with 8 female nude mice in each group.The size of subcutaneous tumor was measured with vernimetric caliper,and when the transplanted tumors grow to about 100 mm3,cisplatin chemotherapy was performed intraperitoneally,with a dose of 3 mg/Kg every other day for seven times in total.The mice in HeLa/DDP-NS group were injected intraperitoneally with normal saline.The volume of transplanted tumor and the weight of nude mice were measured every other 3 days until their natural death.The growth curve was plotted according to the volume changes of the transplanted tumor and survival analysis was performed based on their survival days.2.The nude mice were dissected to observe whether there was tumor metastasis.The tumor specimens were removed for paraffin section,and the pathological changes of transplanted tumor were observed by HE staining.Expression of KYNU,P-gp,GST-π,MRP1,Bax,Bcl-2 and CD34 were detected by immunohistochemistry.Results:1.The model of subcutaneous transplanted tumor was successfully constructed in nude mice.The mice in HeLa/DDP-NS group showed the fastest growth rate and the largest tumor volume.The mice in the GV248-KYNU-cisplatin group had the slowest growth rate and the smallest tumor size,and the differences were statistically significant compared with HeLa/DDP and GV248-NC groups(all P<0.05).However,there were no significant differences in tumor growth rate and tumor volume between GV248-NC-cisplatin group and HeLa/DDP-cisplatin group(all P>0.05).2.There were no significant differences in body weight and overall survival time among HeLa/DDP-cisplatin,GV248-KYNU-ciplatin and GV248-NC-cisplatin groups.3.No tumor metastasis was observed in all groups of nude mice.Compared with the other three groups,the mice in GV248-KYNU-cisplatin group showed lighter nuclear staining,smaller tumor nests,less atypia,and more nuclear mitosis,flake coagulation and focal necrotic substances.However,the HeLa/DDP-NS group,HeLa/DDP-Cisplatin group and GV248-NC group had more tumor cell,vigorous growth,more nuclear mitosis,and larger and abundant cancer nests.4.After KYNU knockdown,the expressions of KYNU,CD34 and drug-resistance related proteins P-pg,MRP1 and GST-π in GV248-KYNU-cisplatin group were significantly down-regulated compared with HeLa/DDP-cisplatin group and GV248-NC-cisplatin group(all P<0.05).The expression of pro-apoptosis regulatory protein Bax in GV248-KYNU-cisplatin group was significantly higher than that in HeLa/DDP-cisplatin group and GV248-NC-cisplatin group(all P<0.05),The expression of anti-apoptosis protein Bcl-2 in GV248-KYNU-cisplatin group was up-regulated a little than other two groups but the difference was not statistically significant.Conclusion:After KYNU knockdown,the sensitivity of nude mouse with transplanted tumor to cisplatin was increased,and the tumor growth rate was slowed down.KYNU knockdown may increase the sensitivity of tumor bearing mice to cisplatin and promote tumor cell apoptosis by up-regulating the expression of pro-apoptosis regulatory protein Bax and down-regulating the expression of CD34 and P-gp,MRP1 and GST-π.