Involvement Of MMP-2in Low Concentration Of Adriamycin Resistance Dependent On MEK/ERK Signal Pathway In Human Osteosarcoma MG-63Cells

Objective1. To induce and establish stable adriamycin osteosarcoma cell line MG-63which resistance to low concentration of adriamycin, and research resistant mechanism of the chemotherapeutic drug for the further study of the gene therapy.2. To investigate the effect of adriamycin and PD98059on the MEK/ERK1/2signal way in stable adriamycin osteosarcoma cell line MG-63.3. To design and induct the siRNA of MMP2, transfect the siRNA in the adriamycin osteosarcoma cell line MG-63by using siRNA technology, investigate the expression of ERK1/2activated protein after the transfection.Methods1. The MG-63cells were cultured by ADM from the dose of50ng/mL and then cells were all stable living in the medium of ADM (200ng/mL) after4months. Experimental groups were ADM-MG-63(100ng/mL) and ADM-MG-63(200ng/mL) and the control group was the normal MG-63cell line. We used the MTT, transwell, flow cytometry, Annexin V/PI double staining to investigate the cell proliferation, invasion, cell cycle and apoptosis in vitro.2. All the cells were blocked by PD98059. RT-PCR and western blot assay tested the mRNA and the protein of MMP2before and after the blocking.3. Design and induct three siRNAs of MMP2. We choosed the best siRNA of the three siRNAs, tested the best concertrations of20nM,50nM,100nM and time points of24h,48h,72h after the transfection. Then we tested the expression of MMP2of the three siRNAs by RT-PCR and western blot. The chosen siRNA was the experimental group, meanwhile the positive group, negative group and the blank group were given by the company. After the siRNA silenced the MMP2expression, we tested the expression of p-ERK.Results1. ADM-induced cancer cells had characteristic that they can stably live and passage in the medium of ADM (200ng/mL) after four months. Compared with the non drug resistant strain of osteosarcoma, the early apoptosis, later apoptosis and the total apoptosis were significantly down-regulated. Cells also had the characteristic that they slowed the proliferation but they were more aggressive than they were before.2. We found that ADM up-regulated p-ERKl/2in a dose-dependent manner. P-ERK1/2began to increase at the ADM dose of100ng/mL. To further confirm the relationship, we also tested the expression of MMP2. Western blot showed that at the ADM dose of200ng/mL, the MMP2was significantly up-regulated. The Western blot found that PD98059can effectively block the MEK/ERK signal way, meanwhile down-regulate p-ERKl/2and MMP2in a dose-dependent manner. PD98059took effect especially at the doses more than20μmol/L.3. Cancer cells were transfected with fluorescent-siRNA at the time points of24h,48h and72h and the concentrations of20nM,50nM,100nM. Finally the best condition of transfection was50nM and at the time point of48h. There were decreases in three siRNAs, among which siRNA2showed great efficiency by silencing. Western blot showed a substantially higher activation of ERK1/2phosphorylation when MMP2protein expression was silenced after transfected with siRNA.Conclusion1. Our findings showed culture of osteosarcoma cells in the presence of ADM leaded to the stable adriamycin osteosarcoma cell line, which cells resistance to low concentration of adriamycin. The new cell line was the basis of the further study of drug resistant mechanism in vitro and the gene therapy.2. The results also suggested that MEK/ERK was indeed involved in the expression of MMP2. We speculated that MEK/ERK signal pathway modulated the levels of MMP2expression.3. Transfection was successfully done in our experiment. The siRNA was transfected in the inner of cancer cells and conbined with the mRNA of MMP2. Cancer cells with siRNA silenced MMP2expression. After the transfection, p-ERKl/2was up-regulated. It had proven that certain connection between ERK1/2and MMP2indeed existed or might exist a negative feedback loop.