The Influence Of Copper On Cytotoxicity Of DDP On Oral Carcinoma Cells

Background: Malignant tumors seriously hurt human health. Surgery, radiotherapy, chemotherapy or combination with these several methods is ordinary therapy in clinic. Surgery and radiotherapy have little effect on metastatic cells. Because of drug resistance, chemotherapy often cause tumor recur. As an essential metal element of eukaryotic organisms, copper has close realation with tumors. It has been identified that many tumor patients have high serum copper and abnormality of copper metabolism and distribution in the prosess of tumor chemotherapy. It is also found that copper has close realation with DDP: the influx transporter of copper CTR1 and the enflux transporter of copper ATP7B also transport DDP. While The realationship between fluctucation of copper concentration in blood and chemotherapeutic responses remains unclear in patients with primary or acquired drug resistant tumor. Object: The aim of our study was to determine the influence of copper on cytotoxicity of tumor cells at the cell lever by a series of tests and search a new method to solve drug resistance of DDP. Our study also tried to build a new method to analyze the influence of drugs on cells continuously fast and accurately useing a modern reporter gene (GFP). Method: DDP was adopted to study the influence of copper on cytotoxicity of oral tumor cells (Tca8113 cell comes from human tongue, Acc-2 and Acc-3 cell come from human salivary) in the experiment. With MTT assay, copper acetate was used to investigate the cytotoxicity of copper on different origin of oral tumor cell lines including. The flow cytometric analysis was used to detect the influence of copper on apoptosis of tumor cells and cell cycle. Through liposome transfection to transfect the combinant plasmid (HSP/GFP) into Tca8113 and Acc-3 cells and used G418 to select clone stably expressing GFP genes. Resulte: MTT showed that in presence of 20～400μM copper acetate, the proliferation inhibition of three tumor cells was significantly enhanced. In presence of 25μM copper acetate combined with various doses of DDP, the result showed that copper acetate enhanced the proliferation inhibition of Tca8113 cell and Acc-2 cell than that exposed to DDP alone; as to Acc-3 cell, copper acetate enhanced the proliferation inhibition of cell exposed to less than 25μM DDP and decreases exposed to more than 25μM DDPcompared with rqual dose of DDP.In presence of 25μM copper acetate combined with various doses of DDP, the IC50 of Tca8113 cell was 2.72±0.38 μM/L, it is much lower than that treated with DDP alone which was 10.84±1.02 μM/L. While for Acc-2 and Acc-3 cells, the IC50 of them exposed to 25μM copper acetate combined with various doses of DDP were only a little lower than that treated with DDP alone, which changed from 5.2±1.3 μM/L and 5.12±0.38 μM/L to 6.88±1.02 μM/L and 7.37±0.71 μM/L. The flow cytometric analysis was used to detect the influence of copper on apoptosis of tumor cells and cell cycle. The result showed that 25μM copper acetate increased the apoptosis of cells exposed to 7.5μM DDP, while decreased the apoptosis under 25μM DDP slightly; it was also showed that 25μM copper acetate slowed down tumor cell to move to S phase combined with 7.5μM DDP while increased tumor cell to move to S phase combined with 25μM DDP. HSP/GFP can be transfected into tumor cells. The transfected cells were observed green fluorescent after 42oC heat shocked for 3h and the transfected efficiency and fluorescent were more enhanced overnight.The green fluorescent could maintain 4 days. The clone stably expressing GFP could be obtained by G418 selection for 40 days. Conclusion: Copper enhanced the proliferation inhibition of low dose DDP influence tumor cells; low dose of copper increased apoptosis of tumor cells exposed to DDP and change cell cycle of tumor cells exposed to DDP. Using G418 to select the transfected cells could obtain a new cell line that can contribute to analyze the influence of drugs on cells continuously fast and accurately.