Bcl-2 siRNA Induces Apoptosis and Enhances the Sensitivity to Arsenic Trioxide in NB4 Cells.

Bcl-2 is the prominent member of a family of proteins responsible for dysregulation of apoptosis, prevention of cancer-cell death, and resistance to chemotherapy and radiotherapy. Overexpression of Bcl-2 protein was shown to result in resistance to a variety of apoptosis-inducing signals in acute leukemia. Arsenic trioxide (As₂0₃)induces clinical remission in acute promyelocytic leukemia with minimal toxicity and apoptosis in APL-derived NB4 cells at low (0.5 to 2 μmol/L) concentration. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. In this study, we studied the effect of transfection with siRNA targeting Bcl-2 in NB4 cells in terms of proliferation and induction of apoptosis. Further,we investigated whether the Bcl-2 siRNA may enhance the sensitivity of NB4 cells to arsenic trioxide. Synthetic siRNA was transferred against Bcl-2 into NB4 cells using Oligofectamine transfection protocol in the presence or absence of arsenic trioxide. RT-PCR and Western blotting analysis of Bcl-2 expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a modified MTT assay, counting cell number. Apoptosis was determined by morphological observation and flow cytometric analysis. When synthetic Bcl-2 siRNA was delivered into NB4 cells, no Bcl-2 transcripts were detected at 24h, and Bcl-2 protein levels were reduced by approximately 72% for 48h after siRNA transfer. Bcl-2 siRNA treatment for 72h led to 36% apoptosis of cells,as compared to control siRNA treatment. Control siRNA had no significant effect on growth and apoptosis of cells. Simultaneous administration of As₂0₃ and Bcl-2 siRNA was able to reduce the mRNA and protein expression of Bcl-2 significantly compared to Bcl-2 siRNA alone demonstrating a synergistic effect of combined As₂0₃ and siRNA treatment (P<0.05). Proliferation in NB4 cells was strongly inhibited (about 86%) following combination treatment for 72h as opposed to only 40% after administration of 0.5μmol/L As₂0₃ alone. When NB4 cells were incubated with 0.5μmol/L As₂0₃ for 72h after siRNA transfer, the percentage of apoptotic cells significantly increased (P<0.05), compared with combined As₂0₃ and control siRNA treatment or As₂0₃ treatment alone, respectively. No significant difference on apoptosis was seen in cells treated with As₂0₃ alone or treated with As₂0₃ and control siRNA. These results show that Bcl-2 siRNA could induce apoptosis and enhance the sensitivity to arsenic trioxide in NB4 cells. Our results suggest that the combination of arsenic trioxide and Bcl-2 siRNA might be an effective anti-leukemia protocol.