Effect Of Low-dose Shock Wave In Combination With Methotrexate On Apoptosis In Jurkat Celts

Chemotherapy plays an important role in the treatment of cancer. But some of thosedrugs are difficult to pass through the cell membranes, such as methotrexate （MTX）.Thedoses of the drug are too large to bear the negative effects for many patients. Previousresearch has shown that low-dose shock waves （LDSWs） could increase the permeability ofcell membranes and induce the cells release macromolecules, such as the ATP, into theextracellular. Based on the above, our experiments are intended to observe the effect of MTXcombined with LDSWs on apoptosis of Jurkat cells in vitro. It is expected to search a newcomprehensive method of tumor for clinical work.We cultured Jurkat cells in vitro. The cells The cells were exposed to different LDSWsimpulses at an energy flux density of0.18mJ/mm2and cell viability was assayed using trypanblue dye exclusion. Jurkat cells were incubated with various concentrations of MTX or MTXcombined with LDSWs. MTT assay was performed to evaluate the proliferation inhibitoryeffect. Apoptotic rates were measured with flow cytometry. Jurkat cells were incubated withP2X₇antibody and was mesured with immunofluorescence technique. The next, Jurkat cellswere pretreated with with KN-62（a purinergic receptor antagonist with specificity for theP2X₇receptor subtype） and MTX. The cells were then subjected to LDSWs treatment and thecontrol group without KN-62. Then the apoptotic rates were measured with flow cytometry.And next the cells were incubated with MTX, the experimental group were incubated withMTX combined with LDSWs and the intracellular MTX concentration was measured withELISA kit. At last, the expression of caspase-3/8/9mRNA was measured with RT-PCR.Results: death rates of Jurkat cells subjected to＜400LDSWs impulses remained at＜ 5%. Compared with MTX group, both the inhibit rates and apoptotic rates of MTX combinedwith LDSWs group were increased significantly （P<0.05）. Red fluorescence was expressedby Jurkat cells. Compared with control group, apoptotic rates of KN-62group were increasedsignificantly （P<0.05）. Compared with control group, the intracellular MTX concentration ofthe combination group compared to the MTX content in Jurkat cells, these two groups wereincreased significantly （P <0.05）. The expression of caspase-3/8/9mRNA of the experimentalgroup was higher than the control group.Conclusion: We observed that the death rate of Jurkat cells subjected to <400LDSWsimpulses remained at <5%. As compared with control group, both the inhibitory rates and theapoptotic rates of the experimental group were increased significantly. LDSWs could induceJurkat cells apoptosis by increasing the intracellular MTX concentration. Jurkat cellsexpressed P2X₇receptor, which was combined with ATP to induce Jurkat cells apoptosis.MTX with LDSWs mediated Jurkat cells apoptosis through the internal and external pathwaysof apoptosis involving caspase-3/8/9.