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Archive > Volume 23 Issue 3 > 2016,23(3):343-349. DOI:10.3872/j.issn.1007-385X.2016.03.008 Prev Next
Basic Research
Silencing ERK enhances susceptibility of A375 melanoma cells to cell apoptosis induced by TRAIL
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Abstract:
Objective:To investigate the effect of extra cellular signal-regulated kinase （ERK） knockdown on melanoma cell apoptosis mediated by TNF related apoptosis inducing ligand (TRAIL) and the underlying mechanisms. Methods: A375 cells were infected with lentivirus carrying either scramble shRNA (SC shRNA) or ERK (shRNA) for 48 hours and then treated with recombinant TRAIL protein (100 μg/ml) for another 6 hours. Cells were harvested and stained with PI; Flow cytometry was used to detect cell cycle, apoptosis, and the expression of TRAIL receptor; Western blotting was used to measure the expression level of relevant proteins. Results: Knockdown of either ERK1 or ERK2 by isotype specific shRNA resulted in G1-phase growth arrest of A375 cells（cell percentageat G1-phase in control group was 71%, compared to 85%, 90%, 81% respectively in ERK1, ERK2 and ERK1+2 shRNA groups, P<0.01). Accordingly, the A375 cell percentage at S-phase was 11% in control group, compared to around 2% in various ERK shRNA groups (P<0.001); the G2/M cell percentage was 17% in control group, compared to the around 3% in various ERK shRNA groups (P<0.01). The change of cell cycle was accompanied by up-regulation of P21 and P27 proteins, and down-regulation of cyclin D1 level, however no obvious cell apoptosis was observed. Treatment of scramble shRNA together with TRAIL caused about 15% of cell apoptosis,in contrast, the combined treatment of ERK shRNA and TRAIL increased cell apoptosis rate up to 40%~60% (P<0.01). Silencing of ERK enhanced DR4 expression on TRAIL receptor (from 32% to 75%~80%, P<0.01), but not DR5 expression. Furthermore, directly silencing ERK resulted in inhibited expression of Glut 1 and hexokinase Ⅱ, which are involved in the unique glucose metabolism of tumor cells. Conclusion: Directly silencing ERK with specific shRNA inhibited tumor cell growth and enhanced tumor cell apoptosis mediated by TRAIL. A combination of increased DR4 expression and impaired glucose metabolism is likely to contribute to the synergistic effect seen with TRAIL and ERK shRNA treatment in melanoma cells.
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Project supported by National Natural Science Foundation of China (No.81241148)
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