Study On Cytochrome P4501B1 And Epithelial Ovarian Cancer Paclitaxel-resistance

Study Background Ovarian carcinoma is the most common cause of death among all gynecologic malignancies. Poor prognosis is due to the late presentation of the disease or having the apparent syndrome during metastasis and the resistant response of ovarian cancer to chemotherapy, 70% cases are advanced ovarian cancer when being diagnosed. Therefore, it is significant value for early prevention, etiologic diagnosis, clinical treatment to searching high susceptibility heredity marker of ovarian cancer, predicting chemotherapy effect by virtue of specially and highly expressed genes, increasing neoplastic cell's sensitivity to anti-cancer drugs. Cytochrome P450(CYP) enzymes are important hemoprotein monooxygenases that catalyze the oxidation of a wide variety of both endogenous and exogenous compounds, such as catalyzing steroid hormone and metabolizing exogenous drugs, sometime inactiving the drug activity. Several cytochrome P450 enzymes are involved in the metabolism of a range of anticancer drugs, including paclitaxel, docetaxel and cyclophosphamide. Cytochrome P450 1B1 , a superfamily members of CYP, is considered a candidated carcinogenic gene of many cancers . The expression of CYPIBI in tumor cells may affect the anti-cancer drugs' sensitivity. Using CYP1B1 particular inhibitor to reverse anti-cancer drug resistance, which may be a new target of reversing drug resistance.Objective To study the correlation between gene mutation of cytochrome P450 1B1 and the susceptibility to ovarian cancer, to investigate the expression of CYPIBI in ovarian cancer , benign tumour of ovary and normal ovarian tissue, aimed to identify the CYPIBI to be a novel target for the development of anticancer therapies. To study the relationship between the expression of CYPIBI and drug-resistant gene SurvivinN MDR-K MRP-1 gene and paclitaxel resistance in ovarian cancer cell lines. Assess CYPIBI inhibitor ANF enhancing paclitaxel cytotoxity in vitro and in vivo in drug-resistant ovarian cell and analyze its mechanisms.Methods a) Allele-specific polymerase chain reaction method was used toanalyze gene polymorphism of CYPIBI in 53 cases of ovarian cancer and 30 normal. Immunohistochemistry method (SP) was used to investigate whether the expression of estrogen receptors-a(ERa), progesterone receptor(PR) are influenced by the CYPIBI genotypes in ovrian cancer. The immumohistochemical way SP was performed to estimate the expression of CYPIBI in 53 cases of epithelial ovarian cancers , 30 cases of benign tumours of ovary, and 19 cases of normal ovarian tissue.b) Using MTT assay to assess inhibitory rate of cancer cells. The RT-PCR and Western blot was used to test the mRNA level of those four genes in ovarian cancer cell lines COCKHO8910-P]VKHO-8910> A2780 and paclitaxel resistant sub-cell line A2780TS. Besides, those four genes' expressive changes were also observed after treated by paclitaxel in different concentration.c) Using MTT assay to observe the CYPIBI inhibitor ANF enhancing paclitaxel cytotoxity of drug-resistant ovarian cell line A2780TS in vitro and in vivoand to analyze its mechanisms. Flow cytometry was used to analyze apoptosis rates and the chances of cell cyclye. RT-PCR and Western blot methods was used to assess CYPIBI expression in different levels and demonstrate its drug-resistancemechanisms.Results a) The frequency of allel C, G on codon 43 2 of CYP1B1 gene showed a significant difference between the ovarian cancer group and the control group , with an odds of 2.71. Statistically significant difference in the frequency of genotype C/C, C/G, G/G was observed between the two groups. Compared with wild-type C/C, the susceptibility of ovarian cancer with the genotypes of homozygotic mutation G/G and heterozygote mutation C/G was increased by 4.53 and 4.43 respectively(PO.01) . Moveover, the positive expression of ER in genotype G/G, C/G was higher than that in genotype C/C, there was a significant difference ? The positive staining rate of CYP1B1 was 92.45% in epithelial ovarian cancers, which was significantly higher than that in benign tumours of ovary (13. 3 3 %) (P<0.01), there is no positive expression in normal ovarian tissue.b) The expression of CYP1B1 and MDR-1 gene in those five ovarian cancer cell lines were associated with paclitaxel IC50 values(r=0.948, r=0.897, p<0.05), while Survivin and MRP-1 gene were not. The mRNA levels of CYP IB 1 and Survivin gene were higher in A2780TS cell line than sensitive cell line A2780. MDR-1 gene was not express in primary cell line A2780, however, it expressed steadily in resistant sub-cell line A2780TS. There is no difference of MRP-1 gene expression in both cell lines (P>0.05) . Paclitaxel has different regulation to CYP1BK Surviving MDR-1 and MRP-1 genes in fiver cell lines.c) Paclitaxel combined with 1 U M, 10uM> 100 u MANF treatment could increase paclitaxel sensitivity in A2780TS cell line by 1.21 >. 2.15> 6.22. Paclitaxelcombined with ANF treatment could increase apoptosis rate and percentage of G2/M phase in A2780TS cell line. After treated by paclitaxel combinded with ANF, the expression of CYP IB 1 gene in mRNA level was no apparent change, while significant decreased in protein level. The rate of successfully inoculation ofsubcutaneous xenograft was 95 %, the tumor inhibitive rate in paclitaxel group and paclitaxel combined with ANF group were 9.05% and 42.79% respectively, there issignificant difference between combined treatment group and control group and single paclitaxel treatment group(p<0.01). There is no significant difference of the weights of nude mouse in each groups. The livers and kidneys of nude mouse had no pathological alterations.Conclusion a) The gene polymorphism of CYPIBI in exon 3 codon 432 mightbe a genetic susceptibility factor for ovarian cancer. The mutation of CYPIBI gene increases the risk of ovarian cancer, and has positive correlation with ER expression.b) The positive expressive rate in epithelial ovarian cancers of CYPIBI was significantly higher than that in benign tumours of ovary, there is no positive expression in normal ovarian tissue. CYPIBI is a novel target for the development of anticancer therapies and for reversing the drug resistance.c) The mRNA expression of CYPIBI and MDR-1 gene in those five ovarian cancer cell lines were associated with paclitaxel IC50 values, while Survivin and MRP-1 gene were not. The mRNA levels of CYPIBI and Survivin gene were higher in A2780TS cell line than sensitive cell line A2780. MDR-1 gene was not express in primary cell line A2780, however, it expressed steadily in resistant sub-cell line A2780TS. There is no difference of MRP-1 gene expression in both cell lines. Paclitaxel has different regulation to CYPIBI > Survivin ^ MDR-1 and MRP-1 genes in fiver cell lines.d) In vitro and in vivo ,CYP1B1 inhibitor ANF could enhance paclitaxel cytotoxity in drug-resistant ovarian cancer cell line A2780TS, ANF could reverse paclitaxel resistance to some extent. ANF has no apprerent side-effect in vivo study.