4β-Hydroxywithanolide E Modulates Alternative Splicing of Apoptotic Genes in Human Hepatocellular Carcinoma Huh-7 Cells

Abstract

Alternative splicing is a mechanism for increasing protein diversity from a limited number of genes. Studies have demonstrated that aberrant regulation in the alternative splicing of apoptotic gene transcripts may contribute to the development of cancer. In this study, we isolated 4β-Hydroxywithanolide E (4bHWE) from the traditional herb Physalis peruviana and investigated its biological effect in cancer cells. The results demonstrated that 4bHWE modulates the alternative splicing of various apoptotic genes, including HIPK3, SMAC/DIABLO, and SURVIVIN. We also discovered that the levels of SRSF1 phospho-isoform were decreased and the levels of H3K36me3 were increased in 4bHWE treatment. Knockdown experiments revealed that the splicing site selection of SMAC/DIABLO could be mediated by changes in the level of H3K36me3 in 4bHWE-treated cells. Furthermore, we extended our study to apoptosis-associated molecules, and detected increased levels of poly ADP-ribose polymerase cleavage and the active form of CASPASE-3 in 4bHWE-induced apoptosis. In vivo experiments indicated that the treatment of tumor-bearing mice with 4bHWE resulted in a marked decrease in tumor size. This study is the first to demonstrate that 4bHWE affects alternative splicing by modulating splicing factors and histone modifications, and provides a novel view of the antitumor mechanism of 4bHWE.

Figure 1

4bHWE modulates the alternative splicing of apoptotic gene transcripts. Cells were treated with 4bHWE at the indicated concentration for 24 h. (a) Chemical structure of 4bHWE. (b) Cell viability of Huh-7 cells and human fibroblasts were analyzed through MTT assays. (c) mRNA was extracted and detected using RT-PCR for the alternative splicing of the HIPK3, SMAC/DIABLO, and SURVIVIN transcripts in Huh-7 (second row), A549 (third row), and GBM8401 (fourth row) cells. The splicing isoforms are illustrated in the top row, and their expected PCR products derived through the primers are indicated by arrowheads. The ratios of the densities of the two bands (alternative exon-containing isoforms to alternative exon-lacking isoforms) are presented below each group. Data are the mean of three independent experiments. *p < 0.05 compared with the control. M = marker.

Figure 2

4bHWE affects the splicing factors and histone tail PTMs. Huh-7 cells were treated with the indicated concentration of 4bHWE for 24 h and then harvested. Equal amounts of whole cell lysates (20 μg) were separated using SDS-PAGE and immunoblotted with various antibodies as indicated. (a) SR proteins and hnRNPs. (b) Histone tail PTMs. Tubulin and Histone H3 are shown as internal standards. The fold-change values are presented below each band. Data are the mean of three independent experiments. *p < 0.05 compared with the control. (c) Analysis of the phosphorylation status of SRSF1. Equal amounts of whole cell lysates (20 μg) were incubated with or without three units of CIP at 37 °C for 30 mins, separated by SDS-PAGE, and immunoblotted with various antibodies as indicated. Tubulin is shown as internal standards. Full-length blots are presented in Supplementary Fig. S1.

Figure 3

Effect of SRSF1 knockdown or the reduction of H3K36me3 levels on the alternative splicing of apoptotic gene transcripts. (a) Huh-7 cells were transfected with 40 nM si-SRSF1 in the SRSF1 knockdown, or 40 nM scramble RNA was used as the negative control for 72 h. The efficiency of the SRSF1 knockdown was assessed through Western blotting. (b) mRNAs were extracted and detected using RT-PCR for the alternative splicing of the HIPK3, SMAC/DIABLO, and SURVIVIN transcripts. (c) Huh-7 cells were transfected with 20 nM si-SETD2 for the reduction of H3K36me3 levels or 20 nM scramble RNA was used as the negative control for 72 h, followed by exposure to 10 μM 4bHWE for 24 h. The efficiency of the SETD2 knockdown and the reduction of H3K36me3 levels was assessed through Western blotting. (d) mRNAs were extracted and detected using RT-PCR for the alternative splicing of the HIPK3, SMAC/DIABLO, and SURVIVIN transcripts. Western blotting: equal amounts of whole cell lysates (20 μg) were separated using SDS-PAGE and immunoblotted with various antibodies as indicated. Tubulin and Histone H3 are shown as internal standards. The fold-change values are presented below each band. RT-PCR: the ratios of the densities of the two bands (alternative exon-containing isoforms to alternative exon-lacking isoforms) are presented below each group. Data are the mean of three independent experiments. *p < 0.05 compared with the control. ^a p < 0.05 for the comparison between scramble RNA + 4bHWE and si-SETD2 + 4bHWE. M = marker. Full-length blots are presented in Supplementary Fig. S2.

Figure 4

Effect of 4bHWE on AKT kinase, SRPK1, SRPK2, and PP1. (a) Huh-7 cells were treated with the indicated concentration of 4bHWE for 24 h. The proteins were analyzed through Western blotting. (b) and (c) Huh-7 cells were treated with 5 μM AKT inhibitor for 1 h and then exposed to 10 μM 4bHWE for 24 h. The proteins and mRNA were analyzed through Western blotting and RT-PCR, respectively. (d) and (e) Huh-7 cells were treated with 20 nM okadaic acid for 1 h and then exposed to 10 μM 4bHWE for 24 h. The proteins and mRNA were analyzed through Western blotting and RT-PCR, respectively. Western blotting: equal amounts of whole cell lysates (20 μg) were separated using SDS-PAGE and immunoblotted with various antibodies as indicated. Tubulin and Histone H3 are shown as internal standards. The fold-change values are presented below each band. RT-PCR: mRNA was extracted and detected for the alternative splicing of the HIPK3, SMAC/DIABLO, and SURVIVIN transcripts. The ratios of the densities of the two bands (alternative exon-containing isoforms to alternative exon-lacking isoforms) are presented below each group. Data are the mean of three independent experiments. *p < 0.05 compared with the control. ^a p < 0.05 for the comparison between 4bHWE and AKT inhibitor + 4bHWE. ^b p < 0.05 for the comparison between 4bHWE and okadaic acid + 4bHWE. AI = AKT inhibitor; OA = okadaic acid; M = marker. Full-length blots are presented in Supplementary Fig. S3.

Figure 5

4bHWE affects genome-wide alternative splicing detected using RNA sequencing. Huh-7 cells were treated with (a) 0.1% DMSO and (b) 10 μM 4bHWE for 24 h prior to RNA sequencing. Grey bars indicate the number of alternative splicing events and white bars indicate the number of genes in which alternative splicing events occurred. The number shown above the bar is the event. This experiment was performed once. (c) Huh-7 cells were treated with the indicated concentration of 4bHWE for 24 h and then harvested. mRNAs were extracted and detected using RT-PCR for the alternative splicing of APAF1, CCAR1, and RIPK1 transcripts. The splicing isoforms are illustrated in the left column, and their expected PCR products using the primers are indicated by arrowheads. The ratios of the densities of the two bands (alternative exon-containing isoforms to alternative exon-lacking isoforms) are presented below each group. Data are the mean of three independent experiments. *p < 0.05 compared with the control. M = marker.

Figure 6

4bHWE arrested the cell cycle at the G₂/M phase and induced apoptosis. Huh-7 cells were treated with 4bHWE at the indicated concentration for 24 h and then stained with PI. (a) DNA content was analyzed using flow cytometry. (b) Histogram of the cell cycle distribution. (c) Histogram of the apoptotic sub-G₁ population. (d) Huh-7 cells were treated with the indicated concentration of 4bHWE for 24 h and then harvested. Equal amounts of whole cell lysates (20 μg) were separated using SDS-PAGE and immunoblotted with various antibodies as indicated. Tubulin is shown as an internal standard. The fold-change values are presented below each band. (e) Effect of a pan-caspase inhibitor on 4bHWE-induced apoptosis in Huh-7 cells. Cells were pretreated with 20 μM pan-caspase inhibitor for 1 h, and then treated with 10 μM 4bHWE for 24 h. Cell viability was determined using MTT assay. Data are the mean of three independent experiments. *p < 0.05 compared with the control. Full-length blots are presented in Supplementary Fig. S4.

Figure 7

Inhibitory effect of 4bHWE on tumor growth in a xenograft animal model. Male nude mice bearing Huh-7 cell tumors were treated with a solvent (control) or 4bHWE (11.6 μg/10 g) for 21 days. (a) Representative picture of tumor growth in xenograft nude mice administered solvent (left) and 4bHWE (right). Scale bar = 1 cm. (b) Tumor volumes were measured after therapy was initiated. (c) Histogram of tumor weight. *p < 0.05 compared with the control.

Figure 8

Hypothetical schematic diagram of 4bHWE-induced alternative splicing in Huh-7 cells.