| Background and objectiveOvarian cancer claims the highest mortality rate of gynecologic malignancies. The advanced suffered from an evil five-year survival rate as low as 15% to 20%, in spite of standardized therapy, including content cytoreductive surgery and combined chemotherapy based on platinum and paclitaxel. Chemotherapy resistance remains a major cause of such poor prognosis.Inherent and therapy-induced resistance has been described. Initial drug resistance occurred in about 15%-20% patients with ovarian tumors. Most resistance developed following treated with platinum, with clinical symptoms such as relapse short after the therapy within six months, portion relief during the therapy and the optimal effect-stable disease. The mechanisms of drug-resistance appear to be related to change in gene or in post heredity, resulted from persistent exposed to the medicine, or to the selective growth of the inherent subgroup with property of drug-resistance.Updated mechanisms of platinum-resistance include: 1. decreased intracellular drug accumulation, including less intracellular uptake and more initiative effluence. 2. enchanced detoxification. 3. increased DNA repair. 4. change of the role and behavior of a variety of factors involved in apoptosis, a major mechanism of cisplatin-induced cell death. To make clear the mechanisms of cisplatin-resistance, the first-line therapy, presents significance to overcome clinical resistance and ameliorate the five-year survival rate.In 2003, supported by key foundation of Peking Union Medical College Hospital, we had performed a proteomics analysis on identification of platinum-resistance biomarker for ovarian carcinoma. A resistant ovarian cancer cell line (SKOV3/CDDP) was established in vitro. The other two cell lines, A2780 and 2780/CDDP, was kindly presented. These cells, two human ovarian cancer cisplatin-sensitive cells and the resistant, were analyzed by 2-DE to screen differentially expressed proteins. So annexin A3 was selected as the target protein. The vectors containing sense and antisense cDNA of the target protein were constructed and stable transfection was carried out. The effects on growth and drug resistance of the transfected cells were compared. The changed drug resistance of infected cells indicates there is a direct correlation between Annexin A3 levels and platinum resistance in cell lines in vitro.Chemotherapy is intended to stimulate the altered apoptotic mechanisms in malignant cells. Additional defects in the apoptosis program could lead to the development of chemoresistance in malignant cells. Methods1. The biological characteristics of all the cell lines were compared using light microscopy. Drug sensitivity was monitored by MTT assay. The cell cycle was detected by flow cytometry. The stability of drug resistance of all resistant cell lines was examined at two-week and five-month intervals, respectively, by MTT assay. The expressions of Annexin A3 in the two cell lines, the sensitive and the resistant, were measured by Western blot.2. The retrovirus vectors containing sense and antisense cDNA of the target protein were constructed and stable transfection was carried out. The vectors were verified by PCR, enzymic hydrolysis and sequencing. The virus titer was measured by infecting NIH 3T3 cells. The effects on growth and proliferation and drug sensitivity of the infected cells were detected.3. The cells, infected and uninfected, were exposed to platinum. The difference of the apoptosis rate between the two cells lines were compared by flow cytometry. The expressions of the proteins, including Caspase 8, Caspase 9, Caspase 3 and survivin, were measured.Results1. At the five-month interval, the resistance indexes of SKOV3/CDDP were 1.84±0.37, 2.18±0.19 and 2.07±0.57, while that of A2780/CDDP were2.95±0.26, 3.24±0.56 and 2.96±0.58, respectively. No statistical differences were presented. At the points of 0 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks, the index measurement were repeated, with no statistical differences. Both the two resistant cell lines had some changes in biological characteristics. The expression of Annexin A3 is higher in the resistant.2. The retrovirus vectors verified containing sense and antisense cDNA of the target protein were constructed. The titer of pMSCV-sense anx3 is 1.55×10~5CFU/mL, the other one is 1.78×10~5 CFU/mL. Infected by Annexin A3-expressing vector media, the drug sensitive cells showed 2-6 fold increase in resistance and inhibition of growth and proliferation. Conversely, infected by reverse Annexin A3 vector media, drug resistant cells showed about 1.2-2.2 fold decrease in resistance to these drugs and increased cell growth. All the infected cells had no significant changes in morphology.3. The cells, infected by Annexin A3-expressing vector media, present lower apoptosis rate after the irritation of platinum, while the other not. The expressions of Caspase 9, Caspase 8 and Caspase 3 were higher in the cells infected by pMSCV-antisense anx3, while the expression of survivin is lower in cells A2780. Conclusion1. After cultured in vitro with drug-free media, ovarian cancer platinum-resistant cell lines have not reversed in resistance. They displayed little change in bionomics compared with the primitive cells.2. The increased or decreased drug resistance in infected cells indicates there is a direct correlation between Annexin A3 levels and platinum-resistance.3. Annexin A3 may be a specific protein associated with platinum resistance. Annexin A3 may play a role in the process of apoptosis and finally lead to specific platinum resistance.
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