Effect of adenovirus-mediated recombinant Buthus martensii Karsch chloride toxin artifact on human glioma U251 cells / 肿瘤研究与临床

ABSTRACT

Objective:To investigate the inhibitory effect of adenovirus-mediated recombinant Buthus martensii Karsch chloride toxin artifact (Ad-rBmK CTa) on human glioma U251 cells and its related mechanisms.Methods:Groups of 3 titer gradients of 3.5×10 9, 7.0×10 9 and 3.5×10 10 pfu/ml Ad-rBmK CTa were set up and applied to U251 cells for 24, 48 and 72 h, and a blank control group (no cells and Ad-rBmK CTa were added) and a negative control group (only U251 cells were added) were set up at the same time. The virus infection status was observed by laser confocal fluorescence microscopy. The cell proliferation in each group was detected by methyl thiazolyl tetrazolium (MTT) assay. The cell cycle and apoptosis in each group were detected by flow cytometry. The expressions of apoptosis-related proteins bax, bcl-2 and caspase-3 were detected by Western blot. Results:The infection rate of Ad-rBmK CTa was over 90% after acting on U251 cells for 24 h. As the titer of Ad-rBmK CTa increased, the proliferation inhibition rate of U251 cells treated for the same hours gradually increased (all P <0.01); with the extension of time, the proliferation inhibition rate of U251 cells treated with the same titer of Ad-rBmK CTa also gradually increased (all P < 0.01). After 7.0×10 9 pfu/ml Ad-rBmK CTa acted on U251 cells for 48 h, the proportion of cells in G 0/G 1 phase was (40.7±0.8)%, and cells in S phase and G 2 phase accounted for (35.7±0.6)% and (23.6±1.4)%, and the difference was statistically significant ( F = 225.119, P < 0.01). When 7.0×10 9 pfu/ml Ad-rBmK CTa acted on U251 cells for 24, 48 and 72 h, the apoptosis rates were (7.4±1.4)%, (19.2±1.7)% and (22.3±1.7)% ( F = 49.470, P < 0.01). After 7.0×10 9 pfu/ml Ad-rBmK CTa acted on U251 cells for 48 h, compared with the negative control group, the expressions of bax and caspase-3 proteins increased, and the expressions of bcl-2 decreased. Conclusions:Ad-rBmK CTa may act on the DNA damage-induced G 1/S detection site to arrest the cell cycle in G 0/G 1 phase, thus inhibiting the proliferation of U251 cells in vitro. However, its induction of apoptosis in U251 cells is not obvious. The mechanism may be related to the direct or indirect inhibition of chloride ion channels.