The Effects Of TMP On The Chemotherapy Sensitivity Of Human Osteosarcoma Cells U-2OS And Its Mechanism

Objective: To observe the chemosensitivities of human osteosarcoma cells U-2OS to the chemotherapeutics through culturing cells with different concentration of tetramethylpyrazine. To study the mechanism of tetramethylpyrazine in regulating chemosensitivities of human osteosarcoma U-2OS cells, in order to facilitate the use of tetramethylpyrazine in the chemotherapy of osteosarcoma. And finally, improve the chemotherapy effectiveness and the prognosis of patients carrying osteosarcoma.Methods: Human osteosarcoma U-2OS cells were dealing with different concentrations of tetramethylpyrazine. The dose response curve was rendered, in order to determine the "non-cytotoxic dose" and "low-cytotoxic dose"of Tetramethylpyrazine to U-2OS. Co-treat the human osteosarcoma U-2OS cells with tetramethylpyrazine ("non-cytotoxic dose" or "low-cytotoxic dose") and different chemotherapeutics. 48 hours later, cell growth rate in each group were measured, the IC50 value and efficiency ratio were calculated to reflect the effect of tetramethylpyrazine on human osteosarcoma U-2OS cell chemosensitivity. The expression level of multidrug resistance-associated genes (MDR1,BCL2 and P53) and its expression product (P-gp, bcl-2 and p53) were measured by using the RT-PCR, flow cytometry and Western blot analysis, in order to demonstrate the mechanism of tetramethylpyrazine in regulating chemosensitivities of human osteosarcoma U-2OS cells.Results: Tetramethylpyrazine was with dose-dependent cell toxicity to human osteosarcoma U-2OS cell. The "non-cytotoxic dose" and "low-cytotoxic dose" of U-2OS cell to Tetramethylpyrazine were 200 ug/ml and 800 ug/ml respectively. The chemosensitivities of human osteosarcoma cells U-2OS to the chemotherapeutics (ADM, MTX, DDP and IFO, etc.) were significantly improved with "Non-cytotoxic dose" and "low-cytotoxic dose" of Tetramethylpyrazine respectively. The efficiency ratio were ADM (3.17-fold and 10.31-fold); MTX (4.28 times and 8.43 times); DDP (4.11 times and 8.33 times); IFO (3.46 times and 8.93 times). Through the RT-PCR, flow cytometry and Western blot analysis, an inhibited expression levels of MDR1 and BCL2 mRNA and their expression products were observed, while the expression levels of p53 mRNA and its expression products were elevated.Conclusions: The "non-cytotoxic dose" and "low-cytotoxic dose"of Tetramethylpyrazine to U-2OS were determined. This facilitated the futher study of tetramethylpyrazine in the human osteosarcoma U-2OS cells. The chemosensitivities of human human osteosarcoma cells U-2OS to the chemotherapeutics (ADM, MTX, DDP and IFO, etc.) were significantly improved with "Non-cytotoxic dose" and "low-cytotoxic dose" of Tetramethylpyrazine respectively, suggesting that co-treatment of the human osteosarcoma U-2OS cells with tetramethylpyrazine and different chemotherapeutics will be expected to the improve the chemotherapy effectiveness and the prognosis of patients carrying osteosarcoma. "Non-cytotoxic dose" and "low-cytotoxic dose"of Tetramethylpyrazine to U-2OS significantly inhibited the expression levels of MDR1 and BCL2 mRNA and their expression products were observed, while the expression levels of p53 mRNA and its expression products were elevated. This suggests that Tetramethylpyrazine may improve the chemosensitivities of human osteosarcoma cells U-2OS through regulating MDR1, BCL2 and P53.