The Expression And Biological Function Of MTRR On The Effect Of Multiple Drug Resistance Malignant Ovarian Carcinomas

Chapter 1A review on the effect of Folic acid metabolism enzymes expression and biological functions to multiple drug resistance malignant ovarian carcinomasMalignant ovarian carcinomas is a serious threat to women’s lives and health, with the mortality rating in the first place and the morbidity ranking only second to cervical cancer and corpus cancer in gynecologic malignant tumors. Ovarian carcinomas occoured hiddenly, many patients had already have peritoneal metastasis before the onset of symptoms,60%-70% of them were diagnosed at late stage. Currently the best treatment is given priority to surgery, complementary with platinum and paclitaxel combination chemotherapy (first-line chemotherapy), cooperated with radiation therapy, biological therapy, etc. However, at least 60% of the patients end up with acquired drug resistance, and the late stage patients’5-year-survival rate is only 30% to 40%.Multi-resistant ovarian carcinomas greatly influenced the sensitivity of the ovarian carcinomas patients on chemotherapy, which is the main limiting factor for its clinical curative effect. The occurrence of multi-resistant ovarian carcinomas is a complex process of multi-factors, multi-genes, multi-ways, and multi-steps. At present, the study of the mechanism of multi-resistant ovarian carcinomas mainly concentrated in the research of resistant genes and their expression product, multiple drug resistance related enzymes; and other associated materials.In recent years, the study of multi-resistant ovarian carcinomas have also made new progress, such as the change of ovarian carcinomas cell monolayer culture to three-dimensional cultivation; the impact of the tumor microenvironment to multiple drug resistance; the relationship of tumor metastasis and multiple drug resistance. But the mechanism of multi-resistant ovarian carcinomas is very complex, and it has not been fully studied. Further study in clinic to prevent drug resistance, control drug resistance and reverse drug resistance, looking for high-efficiency and low-toxicity new drugs, have become the most urgent problems at present. Folic acid is involved in deoxyribonucleic acid (DNA) synthesis and methylation reaction in the cells, which is the important methyl donor in the body, and can interfere with the DNA synthesis and methylation in tumor formation. The human body can not use the folic acid before a series of enzyme activating it to play a physiological function.Therefore folic acid metabolism enzymes can maintain the stability of the genome, affect the DNA synthesis and repairment, as well as the normal methylation.The changes of folate metabolism enzyme activity can lead to the abnormity of DNA synthesis and repair, abnormal methylation, and genome instability. Folate metabolism enzymes’gene polymorphism, lacking of folate or folic acid metabolism disorder are closely related to the occurrence and development of tumor. At present, the relationship between folate metabolism enzyme gene polymorphism and the tumor is one of the agro-scientific research in the molecular epidemiology.FOLR1, MTRR and DHFR are important enzymes in the folate metabolic pathways, the changes of their activity influence the normal folate metabolism and the biosynthesis of DNA nucleotide triphosphate and DNA methylation reaction, which play an important role in the occurrence and development of tumor.FOLRl is a folate receptor with high affinity, which is located in the endocytic pathway of folic acid mediated by the cell membrane. Some studies have shown that the expression of FOLR1 is significantly higher in the tissue oriented from the epithelial cells, The suppression of FOLR1 increased the cells’ chemosensitivity.MTRR can maintain the methionine synthetases’activity, plays a significant role in the process of the MTR activation, and it can also affect Hcy metabolism and DNA methylation. Researches have shown that decreasing the activity of MTRRA66G can lead to the occurrence of cardiovascular disease, oaf, and tumor.DHFR can degrade the folate,and make it into the tetrahydrofolic acid (active folates), which is a methyl donor. Overexpression of DHFR can increase the cells’resistance.There are many studies at home and abroad investigating the relationship between the folate metabolic enzymes and the tumor, but the results are still of much controversy. Folate metabolism enzyme, as a very promising research, its relationship with ovarian carcinomas development and multi-drug resistance is still not clear, which is worth further exploration.Chapter 2 The clinical significance of FOLR1,MTRR and DHFR on multi-resistant ovarian carcinomasObjective:To investigate the expression level of FOLR1,MTRR and DHFR, and their relationship with the clinical pathologic features.Methods:Detecting and comparing the expression level of FOLR1,MTRR and DHFR by Western Blot, in 30 cases of normal ovarian tissue,50 cases of benign ovarian tumor and 80 cases of malignant ovarian tumor tissues, analyzing the correlation with clinical pathology and multidrug resistance in malignant ovarian carcinomas.Results:(1)The expression level of FOLR1 and MTRR increased successively in normal ovarian tissue, benign ovarian tumor and malignant ovarian tumors (P< 0.05);while the expression of DHFR decreased orderly(P>0.05).(2) According to FIGO clinical stage for ovarian carcinomas,the expression levels of FOLRland MTRR in stage Ⅰ-Ⅱ were lower than that of stage Ⅲ-Ⅳ, and the levels of well-differentiated were lower than that of poorly differentiated.The differences were both statistically significant (P< 0.05). For DHFR,the expression differences had no statistical significance in clinical stage and the tissue differentiation grade (P>0.05).(3) For ovarian carcinomas, the FOLR-1 expression level of the platinum-resistant were less than the platinum-sensitive, the differences were statistically significant (P<0.05).After chemotherapy the FOLR-1 levels of progressive ovarian carcinomas were lower than those in remission,and the difference was statistically significant (P< 0.05). The expression levels of MTRR,DHFR in the two aspects displayed as above were quite the contrary (P<0.05). (4)1)The FOLR-1 levels of the mucinous ovarian carcinomas were lower than that of the serous, and the difference was statistically significant (P<0.05); The FOLR-1 levels of ovarian carcinomas with lymph nodes and distant organs metastasis were higher than those without metastasis, and the difference was statistically significant (P<0.05);However,the FOLR-1 levels had no significant association with pathological classification, amounts of ascites and metastasis to the omentum (P> 0.05).2) The MTRR levels of the serous ovarian carcinomas were higher than that of the mucinous, and the difference was statistically significant (P<0.05); The MTRR levels of ovarian carcinomas with distant organs metastasis were higher than those without metastasis, and the difference was statistically significant (P<0.05);However,the MTRR levels had no significant association with lymph nodes metastasis, amounts of ascites and metastasis to the omentum (P>0.05).3) The DHFR levels of the serous ovarian carcinomas were higher than that of the mucinous and endometrioid, and the difference was statistically significant (P<0.05); The DHFR levels of omentum metastatic ovarian carcinomas were lower than those without metastasis, and the difference was statistically significant (P<0.05);However,the DHFR levels had no significant association with lymph nodes and distant organs metastasis and amounts of ascites (P> 0.05). (5) 1)ROC curve determining the FOLR1 levels and the MTRR levels associated drug resistance of malignant ovarian tumors demonstrated that the maximum Youden index respectively were 4.725 and 1.345, and MTRR levels showed obvious correlation with median survival time (P<0.05). COX multivariate analysis denied the FOLR1 and MTRR levels as the independent prognostic factor.2)ROC curve determining the DHFR levels associated nature of ovarian tumors demonstrated that the maximum Youden index was 0.250 and the DHFR levels showed no correlation with median survival time (P>0.05). COX multivariate analysis denied the DHFR level as an independent prognostic factor.Conclusions:The FOLR1 and MTRR levels of normal ovarian tissues and benign ovarian tumors were lower than that of ovarian carcinomas, while the levels of DHFR was on the contrary.which suggested FOLR-1 and MTRR could be the new biomarkers for early diagnosis of ovarian carcinomas. Meanwhile, the FOLR-1 levels in ovarian carcinomas were low for the platinum-resistant but high for the platinum-sensitive, which suggested that FOLR1 associated with multidrug resistance of ovarian carcinomas and could be a potential target for judging multidrug resistance. The levels of MTRR in ovarian carcinomas were high for the platinum-resistant but low for the platinum-sensitive, which suggested that the MTRR expression level can be used as one of the potential biomarker to predict the tumor properties and platinum multi-resistant. The levels of DHFR in ovarian carcinomas were high for the platinum-resistant but low for the platinum-sensitive, which suggested that the DHFR participated in the normal cellular physiological metabolism,whose expression level was associated with the multi-resistant ovarian carcinomas and can be used as a potential biomarker.Chapter 3 Biological effect of the MTRR gene on SKOV3 cells with experimental study in vitroObjective:To construct and identify the lentiviral vector expressing the gene of MTRR, and explore the biological effect and the functional pathways after downregulating the MTRR gene on SKOV3 cells.Methods:Screening the shRNA with the best interference effect, then cloning it into the vector plasmid pSicoR, and further confirming it by sequencing. After the combinant plasmid pSicoR and its helper plasmid were co-transfected to the virus packaging cells 293T, SKOV3 cells were infected with the virus particles and then sorted by flow cytometry. Finally the expression of MTRR gene was detected by RT-PCR and Western Blotting. Detecting the proliferation ability, the IC50 value to cisplatin, the growth curve under different concentrations of cisplatin and different time, and the colony forming ability in 3 groups cells: SKOV3/DDP、shNC-SKOV3/DDP、shMTRR-SKOV3/DDP. Meanwhile, the three groups were acted by different cisplatin concentrations after 48h, flow cytometry was used to detect cell cycle distribution. Results:The MTRR lentiviruses interfere vector was successfully constructed, SKOV3/DDP cells were effectively infected with the lentiviruses carrying pSicoR -MTRR gene. There’s stable downexpression of MTRR in the infected SKOV3 cells. Compared the shMTRR-SKOV3/DDP group to the other two control groups:1) the shMTRR-SKOV3/DDP group showed significantly dcreased growth rate as well as the IC50 values to cisplatin.2)The growth inhibition rate of the experimental group increased, meanwhile the sensitivity to cisplatin was increased after downregulatin MTRR.3) Cisplatin-induced cell cycle was arrested in GO/G1 phase,the rate was significantly increased.Conclusion:MTRR silencing could reduce the growth ability of SKOV3/DDP cells. Cell cycle arrested in GO/G1 phase.Chapter4 Effect of MTRR on apoptosis and autophagy pathways in multi-resistant epithelial ovarian cancer SKOV3/DDPObjective:To explore the effect of down-regulated MTRR on the apoptosis pathway Caspase, Bcl-2 family and the autophagy pathways PI3K-AKT1 in cisplatin-resistant SKOV3/DDP cells, so that offering a possible approach for the MTRR to reverse the multi-resistant ovarian cancer, providing a beneficial evidence for clinic.Methods:The three groups cell were acted by different cisplatin concentrations after 48h, flow cytometry was used to detect cell apoptosis. Laser scanning confocal microscopy was used to detect LC3B distribution; Western blot was used to detect the expression of protein LC3B-Ⅱ,LC3B-Ⅰ,p62 and calculateing the ration of LC3B-Ⅱ/LC3B-Ⅰ. The shMTRR-SKOV3/DDP group cells were induced to autophagy by Rapamycin, then detecting the survival rate and apoptosis rate acted by cisplatin; Transmission electron microscope was used to observe the autophagy formed in the three groups of cells. Induced the 3 groups cells (SKOV3/DDP、 shNC-SKOV3/DDP、shMTRR-SKOV3/DDP) by a certain concentration of cisplatin(4(4μg/ml) under different time,then detecting the protein expression of Caspase, Bcl-2 family in apoptosis pathway and the key proteins in PI3K-AKT1 autophagy pathways by western blot, getting the time when the proteins’expression changed. Next, the 3 groups cells were acted by a certain concentration of cisplatin(4μg/ml) under a certain time,western blot was used to detect the expression variation of the key proteins.Results:In shMTRR-SKOV3/DDP cells cisplatin-induced apoptosis rate was increased. The aggregation of LC3B-Ⅱ was decreased in cisplatin-induced cells under laser confocal microscope In shMTRR-SKOV3/DDP cells. The western blot showd that the ratio of LC3B-Ⅱ/LC3B-I was dereased and the p62 was overexpression in cisplatin-induced cells. The shMTRR-SKOV3/DDP group cells were induced to autophagy by Rapamycin, the cells’ survival rate went up and the apoptosis rate went down acted by cisplatin. The autophagy couldn’t be observed by the transmission electron microscope in shMTRR-SKOV3/DDP cells. The 3 groups cells were induced by a certain concentration of cisplatin(4μg/ml) after 48h,compared with the other two control groups, the experimental group of shMTRR-SKOV3/DDP cells showed that:l) in the apoptosis pathway, the expression level of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP were significantly increased (P<0.05),and Bcl-2 was decreased (P<0.05).2)in the autophagy pathway, p-AKT1and p-mTOR were significantly increased (P<0.05),but AKT1 and mTOR had no significant variation.Conclusion:MTRR silencing significantly increased cisplatin-induced apoptosis and reduced the autophagy induced by cisplatin in SKOV3/DDP cells. Down-regulation of MTRR enhanced the chemosensitivity of cisplatin-resistant ovarian cancer cells by activating caspase and Bcl-2 apoptosis family and inhibiting the PI3K-AKT1 autophagy pathway,which had provided an important guideline for us to further research in the mechanism of signal transduction pathways in the development of multi-resistant ovarian cancer and for the application in clinic.