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Background  Osteosarcoma is the most prevalent primary malignant bone tumor, but treatment is difficult and prognosis remains poor. Recently, large-dose 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.    Methods  In our study, trypan blue dye exclusion assay was performed to investigate the optimal conditions for the sensitization of osteosarcoma U2OS 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 U2OS cells in the present of shock wave and methotrexate. To explore the mechanism, P2X7 receptor in U2OS 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 U2OS cells with up to 450 shock wave pulses at 7 kV or up to 200 shock wave pulses at 14 kV had little effect on cell viability. However, this shock wave treatment significantly promoted the uptake of Calcein and Lucifer Yellow CH by osteosarcoma U2OS 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 shock wave treatment increased the extracellular concentration of ATP and that KN62, an inhibitor of P2X7 receptor reduced the capacity methotrexate-induced apoptosis.    Conclusions  Our results suggest that shock wave treatment promotes methotrexate-induced apoptosis by altering cell membrane permeability in a P2X7 receptor-dependent manner. Shock wave treatment may thus represent a possible adjuvant therapy for osteosarcoma.

Shock wave-induced ATP release from osteosarcoma U2OS cells promotes cellular uptake and cytotoxicity of methotrexate