Homoharringtonine regulates the alternative splicing of Bcl-x and caspase 9 through a protein phosphatase 1-dependent mechanism

Abstract

Background: Homoharringtonine (HHT) is a natural alkaloid with potent antitumor activity, but its precise mechanism of action is still poorly understood. Methods: We examined the effect of HHT on alternative splicing of Bcl-x and Caspase 9 in various cells using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The mechanism of HHT-affected alternative splicing in these cells was investigated by treatment with protein phosphatase inhibitors and overexpression of a protein phosphatase. Results: Treatment with HHT downregulated the levels of anti-apoptotic Bcl-xL and Caspase 9b mRNA with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-xS and Caspase 9a in a dose- and time-dependent manner. Calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), significantly inhibited the effects of HHT on the alternative splicing of Bcl-x and Caspase 9, in contrast to okadaic acid, a specific inhibitor of PP2A. Overexpression of PP1 resulted in a decrease in the ratio of Bcl-xL/xS and an increase in the ratio of Caspase 9a/9b. Moreover, the effects of HHT on Bcl-x and Caspase 9 splicing were enhanced in response to PP1 overexpression. These results suggest that HHT-induced alternative splicing of Bcl-x and Caspase 9 is dependent on PP1 activation. In addition, overexpression of PP1 could induce apoptosis and sensitize MCF7 cells to apoptosis induced by HHT. Conclusion: Homoharringtonine regulates the alternative splicing of Bcl-x and Caspase 9 through a PP1-dependent mechanism. Our study reveals a novel mechanism underlying the antitumor activities of HHT.