Comparative Genomic Hybridization Analysis Of Human Ovarian Carcinoma Cisplatin-resistan Cell COC1/DDP And Its Parent Cell COC1 And Study On The Mechanisms Of Cisplatin Resistance And The Reversal Strategy In COC1/DDP

ObjectiveThe morbidity of ovarian neoplasms is in the rank of the third in female genital malignant tumor, but the first mortality .The cytoreductive surgery and cisplatin-based chemotherapy is the basic treatment principle. Chemotherapy plays a very important role. But the tumor cells can show drug resistance which influence the curative effect and lead to the failure of chemotherapy, the five-year-survival is only 20%-30%. The mechanism of drug-resistance and how to overcome it has become an important problem. It is also concerned by scientists home and abroad. In this study the possible differences in genomic DNA between human ovarian carcinoma cisplatin-resistant cell COC1/DDP and its sensitive cell COCl and the possible significance during the development of ovariancarcinoma resistance to cisplatin were explored. This study was designed to explore the role of apoptosis-associated genes and proteins on the chemoresistance in COC1/DDP. The purpose of this study was to investigate the effect and its mechanism of curcumin on the proliferation, apoptosis and DDP sensitivity of COC1/DDP, and to give the experimental basis for treating refractory ovarian carcinoma with curcumin.Materials and MethodsCOC1 and COC1/DDP were provided by China center for type culture collection. They were cultured in RPMI- 1640 medium supplemental 10% heat-inactive calf serum, 100IU/ml penicillin, 100IU/ml streptomycin in a 5 % CO2 , 37℃ , relative humidity 90% atmosphere until they were in logarithmic growth stage.Part Ⅰ Comparative Genomic Hybridization was applied to investigate the genomic imbalance in COC1/ DDP and COC1 cells.Part Ⅱ The apoptotic ratios of COC1 and COC1/DDP were measured with flow cytometry after treated with different concentration of cisplatin for 48 hours. The expressions of apoptosis-associated genes and proteins Bcl-2, Caspase-3, Smac, Survivin were studied by reverse transcription -polymerase chain reaction (RT-PCR) and Western blot in COC1/DDP and COC1.Part Ⅲ The growth inhibiting rates of COC1/ DDP cells withDDP, curcumin, DDP combined with curcumin were studied by methyl thiazolyl tetrazolium(MTT) method; the apoptic ratios of COC1/ DDP cells with DDP(2.5μg/ml), curcumin(20μmol/l) and DDP(2.5μg/ml) combined with curcumin(20Mmol/l) were measured by flow cytometry ;the expressions of apoptosis-associated genes and proteins Bcl-2, capase-3, Smac, Surviving were studied by RT-PCR and Western blot in COC1/DDP cells.ResultsPart Ⅰ There were extensive chromosome changes in COC1, mainly the gain of 1P21-31 , 2q14-24 , 3q25-29 , 8q22-24, 12p11-12, 19p12q12, 20q12-13 and the loss of 4, 13q22-31, 18q12-21. The COC1/DDP cells, which come from COC1, also showed substantial genomic instability, mainly the gain of 6q21-24, 17q21-25, 18q21-23 and the loss of 10q11-22 , 16q12-22, 17p11-12.Part Ⅱ Using 0, 0. 625, 1. 25, 2. 5, 5, 10μg/ml DDP to stimulate COC1/DDP and COC1 for 48 hours , the apoptic ratios were gradually increased with the concentration of DDP increased . The mRNA and protein expressions of Bcl-2 and Survivin in COC1/DDP cells were significantly higher than those in COC1 cells (P< 0. 05) , contrastly Smac and Caspase-3 were significantly lower than those in COC1 cells (p<0.05).Part Ⅲ The growth inhibiting rates of COC1/DDP had no difference when DDP concentration was below 2. 5μg/ml. But the growthinhibiting rates of COC1/DDP cells were distinctly increased when DDP concentration was above 2. 5μg/ml. 15-30μmol/l curcumin could evidently inhibit the growth of COC1/DDP cells, the effect depending on concentration of curcumin. 1. 25-5μg/ml DDP combined with 20μmol/l curcumin could decrease surviving rates of COC1/DDP cells (P <0. 05), especially 2. 5 μg/ml DDP combined with 20μmol/l curcumin worked. The apoptic ratios of COC1/ DDP cells with DDP combined with curcumin were significantly higher than only DDP and curcumin (p<0.05) . The expressions of Caspase-3 and Same were distinctly higher in DDP combined with curcumin group than only DDP at mRNA and protein level, but Bcl-2 and Smac significantly lower (p<0.05).ConclusionsThere are extensive genomic instabilities in COC1/DDP and COC1 cells. Some genes known or unknown on 6q, 17q, 18q, 10q, 16q and 17p may be involved in the development of ovarian carcinoma resistance to cisplatin.The cisplatin-resistance in human ovarian carcinoma cell lines may be associated with the overexpression of anti-apoptic gene and protein of Bcl-2, Survivin and the downregulation of Caspase-3 and Smac.Curcumin can inhibit the growth of COC1/DDP enhance its DDP sensitivity. This may be associated with syneric effect between curcumin and DDP in inducing apoptosis. Its mechanism may be...