Knockdown of ERK/Slug signals sensitizes HL-60 Leukemia cells to Cytarabine via upregulation of PUMA.

OBJECTIVE To study the mechanism of early chemosensitivity and later chemoresistance of Cytarabine (Cyt) to HL-60 cells to regulate ERK/Slug signal pathway. MATERIALS AND METHODS HL-60 cells were stably transfected with NF-κBp65 siRNA，P53 up-regulated modulator of apoptosis (PUMA) siRNA (PUMA siRNA) and Slug siRNA respectively or exposed to 25 nM U0126 for 24h, then the cells were exposed to Cyt (250 nM) respectively. In different time point after Cyt treatment, the activity of NF-κB and the expression of Erk1/2, NF-κBp65, Slug and PUMA protein were detected. The cells survival rate and apoptotic index were detected by MTT and TUNEL methods. RESULTS After the HL-60 cells were exposed to Cyt (250 nM) for 24 h, the activity of NF-κB reached maximum value, and reached minimum value for 48 h. The expressions of NF-κBp65 and PUMA reached maximum value after exporser to Cyt (250 nM) for 24 h, and reached minimum value after 48 h. Erk1/2 phosphorylation and Slug expression began to increase after exporser to Cyt (250 nM) for 24 h, and reached maximum value for 48 h. The cells survival rate was decreased, and arrived to minimum value (29.5 ± 4.6, F = 12.74, p < 0.01). The cells apotosis rate was increased, and arrived to maximum value (14.2 ± 2.8, F = 15.02, p < 0.01). By inhibiting NF-κBp65 or PUMA by siRNA transfection, the cells survival rate was enhanced and the apotosis rate was significantly declined after exporsed to Cyt (250 nM). When NF-κBp65 was inhibited, the expression of PUMA was decreased. By inhibiting Erk1/2 phosphorylation and Slug expression, the cells survival rate began to decrease for 24 h, and reached the fewest at 48 h after exporsed to Cyt (250 nM) (19.8 ± 2.7, F = 11.4, p < 0.01 and 17.4 ± 0.6, F = 15.3, p < 0.01). The cells apotosis rate began to increase from 24 h, and reach a head at 48 h after exporser to Cyt (250 nM) (28.6 ± 4.7, F = 9.84, p < 0.01 and 27.6 ± 6.4, F = 10.31, p < 0.01. After Erk1/2 phosphorylation inhibited, Slug expression was lowered and PUMA expression was significantly increased. The changes are most distinct after the cells were exporser to Cyt (250 nM) for 48 h. By Slug inhibition had no effect on Erk1/2 phosphorylation, but increase the expression of PUMA. CONCLUSIONS The chemosensitiveness of HL-60 cells to Cyt were relation to the up-regulation of PUMA induced by NF-κB. The chemoresistance to Cyt was related to the up-regulation of Slug induced by Erk1/2, which inhibited the expression of PUMA.