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4?-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 4?-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.

SUBMITTER: Lee CC

PROVIDER: S-EPMC5544667 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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

Lee Chien-Chin CC Chang Wen-Hsin WH Chang Ya-Sian YS Liu Ting-Yuan TY Chen Yu-Chia YC Wu Yang-Chang YC Chang Jan-Gowth JG

Scientific reports 20170804 1

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 apo ...[more]

PMID: 28779122

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Project description:Screening small molecules and drugs for activity to modulate alternative splicing, we found that amiloride, distinct from four other intracellular pH-affecting analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts in human hepatocellular carcinoma Huh-7 cells. To elucidate the underlying mechanisms, our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF and also decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, while increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulated kinases and up-regulated phosphatases in the signal pathways known to affect the splicing factor protein phosphorylation. The amiloride effects of splicing factor protein hypo-phosphorylation and“normalized”oncogenic RNA splicing were both abrogated by pre-treatment with a PP1 inhibitor. We then performed global exon array analysis of Huh-7 cells treated with amiloride for 24 hours. Using gene array chips (Affymetrix GeneChip® Human Exon 1.0 ST Array of >518000 exons of 42974 genes) for exon array analysis (set parameters of correlation coefficient ≥ 0.7, splicing index ≤ -1.585 , and log2 ratio ≤ -1.585), we found that amiloride influenced the splicing patterns of 551 genes involving at least 584 exons, which included 495 known protein-coding genes involving 526 exons, many of which play key roles in functional networks of ion transport, extracellular matrix, cytoskeletons and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of abnormal RNA splicing for cancer therapeutics. Target was prepared from 3 biological replicates of 0.5mM amiloride-treatment for 24hr and 3 control untreatment from Huh7 cell line and hybridized to the Affymetrix Human Exon 1.0 ST Array. The values of the hybridized probesets were normalized and analyzed for gene expression using dChip2010 software.

Dataset Information

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

Publications

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

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