Research Of Shock Wave Assisted With Methotrexate In Apoptosis-inducing Of Human Osteosarcoma U2OS Cells

Background: Osteosarcoma is the most prevalent primary malignant bone tumor,but treatment is difficult and prognosis remains poor.Recently,largedose chemotherapy has been shown to improve outcome but this approach can cause many side effects.Minimizing the dose of chemotherapeutic drugs and optimizing their curative effects is a current goal in the management of osteosarcoma patients.Balancing the dose of chemotherapeutic drugs to reduce side effects and maintain the curative efficacy of chemotherapeutics remains a challenge in the care for osteosarcoma patients.Recent advances suggest that shock wave treatment may sensitize osteosarcoma cells to chemotherapeutic drugs.In this study,we treated human osteosarcoma U2 OS cells with methotrexate in the presence or absence of shock wave and assessed cell viability.The underlying mechanisms was also explored.Methods: In our study,trypan blue dye exclusion assay was performed to investigate the optimal conditions for the sensitization of osteosarcoma U2 OS cells.Cellular uptake of the fluorophores Lucifer Yellow CH dilithium salt and Calcein was measured by qualitative and quantitative methods.Human MTX ELISA Kit and MTT assay were used to assess the outcome for osteosarcoma U2 OS cells in the present of shock wave and methotrexate.To explore the mechanism,P2X7 receptor in U2 OS cells was detected by immunofluorescence and the extracellular ATP levels was detected by ATP assay kit.All data were analyzed using SPSS17.0 statistical software.Comparisons were made with t test between two groups.Results: Treatment of human osteosarcoma U2 OS cells with up to 450 shock wave pulses at 7 k V or up to 200 shock wave pulses at 14 k V had little effect on cell viability.However,this shock wave treatment significantly promoted the uptake of Calcein and Lucifer Yellow CH by osteosarcoma U2 OS cells.Importantly,shock wave treatment also significantly enhanced the uptake of the chemotherapy drug methotrexate and increased the rate of methotrexate-induced apoptosis.We found that P2X7 was highly expressed on the surface of U2 OS cells,shock wave treatment increased the extracellular concentration of ATP and that KN62,an inhibitor of P2X7 receptor reduced the capacity methotrexate-induced apoptosis.In addition,we found that ATP had a significant effect on cell viability with concentrations ≥100μM only,while the highest ATP efflux concentration by shock wave was 1.6μM,indicating that ATP and P2X7 receptors do not directly induce apoptosis following shock wave.Conclusions: Our results suggest that shock wave could transiently permeabilize plasma membrane of human osteosarcoma U2 OS cells to allow entry of macromolecular substance.Shock wave promote U2 OS cell uptake of the chemotherapeutic drug MTX,and enhances MTX-induced cytotoxicity.P2X7 was highly expressed on the surface of U2 OS cells.Shock wave induce ATP release by several pathways.We thus speculate that shock wave promote the release of intracellular ATP,which in turn binds P2X7 receptors that contribute to the opening of cell membrane channels that allow entrance of MTX into cells and enhance MTX-induced apoptosis.Shock wave treatment may thus represent a possible adjuvant therapy for osteosarcoma.Therefore,after chemotherapy,osteosarcoma patients may be treated by local tumor shock wave therapy to promote the uptake of chemotherapy drugs by osteosarcoma cells.This strategy may reduce the dosage of chemotherapy drugs,alleviate side effects of chemotherapy drugs,but also improve the treatment outcome of osteosarcoma.