Screening For The Signature Profile Of Cisplatin Resistance In Human Ovarian Cancer By CDNA Microarray

Ovarian cancer is the most lethal of the gynaecological cancers. Despite the introduction of cytoreductive surgery and cisplatin-based chemotherapy for 20 years, reported 5-year survival rates for ovarian cancer patients still vary from 30 to 50%. Ovarian cancer patients have high response rates to initial chemotherapy after cytoreductive surgery, but most will develop acquired resistance to chemotherapy during the course of their treatment, drug resistance in cancer has been a major obstacle to successful chemotherapy. However, now for relapsed and metastatic ovarian cancers, treatment of cancer with chemotherapy is often empirical based more on the histological appearance of the tumor than on an understanding of drug-resistant phenotypes on an individual basis. This treatment usually cause chemotherapy failure and poor prognosis. The availability of techniques of gene expression profiling has allowed to establish correlations between gene expression and drug resistance/sensitivity of tumor cells. If a number of molecular signatures of drug resistance could be identified as predictive markers of drug activity, their use will be progressively implemented for drug selection in patients receiving chemotherapy,allowing thus more rational and personalized chemotherapy.In this study, we have used cDNA microarray to monitor the expression profiles of human ovarian cancer COCi cells that are either transiently treated with cisplatin or stably selected for resistance to cisplatin, aim to identify the signature profile of cisplatin-resistant cells, further to characterize the mechanism of the cytotoxic response and resistance to cisplatin. The main contents were as follows. (1) Fabrication of Expression Profile cDNA Microarrays. At first, we made specific microarray according to standard references, including the following steps: pick up of cDNA clone, probe preparation by PCR amplification, printing of probes to the glass slide and post processes. (2) Monitoring the Expression Profiles of Cisplatin-induced Ovarian Carcinoma COCi Cells by cDNA Microarray. In acquired drug resistance, the emergence of a relapse drug-resistant tumor following chemotherapy suggests that the resistance phenotype may be either induced or selected in a population of tumor cells exposed to chemotherapy. In this study, expression profiles of the human ovarian carcinoma COCi cells transiently treated with cisplatin were examined by microarray. The COCi cells were transiently treated with cisplatin (10 u g/ml) for various times, ranging from 5-10-15 h. Total RNA prepared from these cells was used to synthesize Cy5/Cy3-labeled cDNAs by reverse transcription, followed by hybridization to the human cDNA microarray. The differential expression of genes in cisplatin-induced COCi cells and control COCi cells was confirmed by RT-PCR analysis. Our results show that cisplatin-induced apoptosis of COCi cells persist and augment gradually from 5 to 10 hours after treated with 10 u g/ml cisplatin. apoptotic effects increased in a time-dependent pattern. COCi cells were blocked in G2 phase after treated with cisplatin. Approximately 5 h after treatment with cisplatin, there were a few changes in gene expression. As time progresses following exposure to cisplatin, an increasing number of simultaneous changes in gene expression wereobserved. The patterns of expression of some of these genes exhibited a time-dependent alteration after cisplatin treatment, as indicated by the gradual changes in expression. Approximately 522 genes, representing about 13% of the total DNA elements on the array, had substantially altered levels of expression after induction with cisplatin for 15 h. The biochemical functions of the genes in this expression profile are diverse and include DNA damage repair and transcription factors, apoptotic and antiapoptotic factors, oncogenes and tumor suppressor genes, cell cycle regulators, protein kinases and phosphatases, as well as a large number of metabolic genes. These results suggest that the COCi cellular response and resistance to cisplatin is a complex process involving a diverse group of genes. (3) Gene Expression Profile Analysis of Human Ovarian Carcinoma Cisplatin-Resistant Cell Line COCi/DDP. In this study, we have examined genes that are differentially expressed in cisplatin-induced chemoresistant ovarian cancer cell lines COCi/DDP, as compared with its drugsensitive parent cell lines COCi. COCi/DDP was induced by moderate concentration and intermittent action with cisplatin(4 n g/ml) for 5 month, the differential expression of associated genes between the parental COCi and COCi/DDP cells was determined with cDNA microarray. It was found that COCi/DDP cells are resistant to DDP with its IC50 of DDP is 6.2 times higher than that of COCi, and cross-resistant to many other anticancer agents. Furthermore, COCi/DDP had a prolonged doubling time of 8.9 hours, the number of cells in S-phase was significantly decreased (9.4+1.17)% while those in Gi and G2-phase increased (8.6+1.15)% and (0.9+0.32)%, respectively. Compared to COCi, 143 genes were differentially expressed significantly in COCi/DDP cells, among which 71 genes were up-regulated and 72 genes were down-regulated, these genetic alterations involve a diverse group of genes that have specific functions in apoptosis and antiapoptosis, signal transduction, cell cycle regulation, transcription, DNA repair,Immunology, metabolism. MDR1, MRP and LRP was not found by microarray to be overexpressed in COCi/DDP cells. Further analysis revealed that some of the genes that are overexpressed in the COCi cells were also induced in COQ cells after treatment with cisplatin. These results suggest that cancer cells may recruit and activate the expression of a distinct set of genes in transient induction or when selected for resistance to cisplatin. The expression of this subset of genes may be the molecular signature of cisplatin resistance.For this paper, our results show that transient exposure to cisplatin elicits a gradual change in the expression profile of a large number of genes in human ovarian cancer COQ cells. A subset of these transiently induced genes intersected with a distinct set of genes that are constitutively overexpressed in a cell line stably selected for resistance to cisplatin. This set of genes may represent the signature profile of cisplatin-resistant cells. It was concluded that the cytotoxic response and resistance to cisplatin is multifactorial, in that it likely involves the altered expression of a diverse group of genetic factors influencing various biochemical pathways. By increasing clinical case number and gene number of microarray to be used in the analysis of expression profile of gene cluster affecting anticancer drug resistance and sensitivity of the ovarian cancer, it would be possible to apply microarray analysis to personalization of chemotherapy such as selection of effective chemotherapy protocol and prediction of therapeutic effect in the near future.