Emetine regulates the alternative splicing of Bcl-x through a protein phosphatase 1-dependent mechanism

Abstract

Exon 2 of the Bcl-x gene undergoes alternative splicing in which the Bcl-xS splice variant promotes apoptosis in contrast to the anti-apoptotic splice variant Bcl-xL. In this study, the regulation of the alternative splicing of pre-mRNA of Bcl-x was examined in response to emetine. Treatment of different types of cancer cells with emetine dihydrochloride downregulated the level of Bcl-xL mRNA with a concomitant increase in the mRNA level of Bcl-xS in a dose- and time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), blocked emetine-induced alternative splicing in contrast to okadaic acid, a specific inhibitor of PP2A in cells, demonstrating a PP1-mediated mechanism. Our finding on the regulation of RNA splicing of members of the Bcl-2 family in response to emetine presents a potential target for cancer treatment.

Fig. 1. Chemical Structures

A. Emetine; B. Cycloheximide (CHX).

Fig. 2. Emetine regulates Bcl-x splicing in C33A cells

C33A cells were treated with emetine. Total RNA was extracted and analyzed by RT-PCR for the alternative splicing of Bcl-x. A. Alternative splicing of Exon 2 in the Bcl-x gene produces larger Bcl-xL and smaller Bcl-xS. B. Decrease of Bcl-xL and increase of Bcl-xS is correlated with the emetine concentration. C. Cycloheximide (CHX) also regulates exon 2 splicing in the Bcl-x gene. D. Ratio of Bcl-xL/Bcl-xS. Each experiment was repeated at least three times. Results from all experiments consistently show that emetine and cycloheximide (CHX) modulate exon 2 splicing in the Bcl-x gene. CHX: cycloheximide.

Fig. 3. Emetine regulates Bcl-x splicing in MCF-7 breast cancer cells and PC3 prostate cancer cells

Total RNA was extracted from MCF-7 and PC3 cells that were treated with emetine. Splicing of exon 2 in the Bcl-x gene was analyzed by RT-PCR. A. RT-PCR results from MCF-7 cells treated with different concentrations of emetine. B. RT-PCR results from PC3 cells treated with different concentrations of emetine. C. Reduction of Bcl-xL and increase of Bcl-xS were observed and quantified. All experiments were repeated at least three times and consistently showed that emetine regulated exon 2 splicing of the Bcl-x gene in MCF-7 and PC3 cells.

Fig. 4. Emetine regulates Bcl-x splicing in A549 lung cancer cells

A549 cells were treated with emetine. Total RNA was extracted and analyzed by RT-PCR for the alternative splicing of Bcl-x. A & C. Reduction of Bcl-xL and increase of Bcl-xS is correlated with the emetine concentration. B & D. Cells were treated with 1 μM of emetine for different durations. RT-PCR was carried out to quantify alternative splicing of Bcl-x. All experiments were repeated at least three times.

Fig. 5. Calyculin A but not okadaic acid blocks effects of emetine on Bcl-x splicing

Cells were pretreated with either 5 μM of calyculin A, an inhibitor of both protein phosphatase1 (PP1) and protein phosphatase 2A (PP2A) or 5 μM of okadaic acid, a selective PP2A inhibitor and then exposed to 1.0 μM of emetine for 24 hours. RT-PCR was carried out. The results suggest that PP1 not PP2A mediates the effects of emetine on the alternative splicing of Bcl-x. A. Effects of calyculin A in C33A cells. B. Effects of calyculin A in PC3 cells. All experiments were repeated at least three times. C. Effects of calyculin A or okadaic acid on Bcl-x splicing mediated by emetine were quantified and plotted. Ratios of Bcl-xL/Bcl-xS in C33A cells: no treatment, 12.0; with emetine, 4.5; with okadaic acid, 12.0; with emetine and okadaic acid, 5.8; with calyculin A, 12.0; with emetine and calyculin A, 11.8. Ratios of Bcl-xL/Bcl-xS in PC3 cells: no treatment, 7.9; with emetine, 3.0; with okadaic acid, 7.0; with emetine and okadaic acid, 3.1; with calyculin A, 7.0; with emetine and calyculin A, 7.0. +: With emetine, okadaic acid or calyculin A. 0: Without emetine, okadaic acid or calyculin A.