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Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.

Chang JG, Yang DM, Chang WH, Chow LP, Chan WL, Lin HH, Huang HD, Chang YS, Hung CH, Yang WK - PLoS ONE (2011)

Bottom Line: Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins.We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation.These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research, University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. jgchang@ms.kmuh.org.tw

ABSTRACT
Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could "normalize" the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, 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. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.

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Growth inhibitory and apoptotic/cytotoxic consequences of amiloride-induced AS alterations.(Panel A) Huh-7 cells plated in 6-well dishes at 3×105 cells per well overnight were changed to growth medium containing various concentrations of amiloride; cell numbers were determined one and three days later. Cell counts at 24 hours show growth inhibition by amiloride at 0.4 mM and above. Further cell count decreases were evident at 72 hours due to cell death and detachment in medium containing amiloride at 0.3 mM and above. (Panel B) Cytofluorometric analysis shows amiloride inhibition of cell cycle progression with accumulation of G2/M phase cells. (Panel C) Apoptosis is evident by cytofluorometric detection of annexinV binding in Huh-7 cells treated with 0.5 mM of amiloride. (Panel D) Marked nuclear DNA degradation was observed in Huh-7 cells treated with 0.4 mM amiloride but not with 0.2 mM amiloride, particularly noted at 48 and 72 hours.
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pone-0018643-g007: Growth inhibitory and apoptotic/cytotoxic consequences of amiloride-induced AS alterations.(Panel A) Huh-7 cells plated in 6-well dishes at 3×105 cells per well overnight were changed to growth medium containing various concentrations of amiloride; cell numbers were determined one and three days later. Cell counts at 24 hours show growth inhibition by amiloride at 0.4 mM and above. Further cell count decreases were evident at 72 hours due to cell death and detachment in medium containing amiloride at 0.3 mM and above. (Panel B) Cytofluorometric analysis shows amiloride inhibition of cell cycle progression with accumulation of G2/M phase cells. (Panel C) Apoptosis is evident by cytofluorometric detection of annexinV binding in Huh-7 cells treated with 0.5 mM of amiloride. (Panel D) Marked nuclear DNA degradation was observed in Huh-7 cells treated with 0.4 mM amiloride but not with 0.2 mM amiloride, particularly noted at 48 and 72 hours.

Mentions: Our exon array analysis of amiloride-treated Huh-7 cells (Tables S1 & S2) detected alterations of RNA splicing predominantly of proteins involved in cytokinesis, associated with solute transport, and functions of actin, microtubules, and cytokinesis-related kinases [33]. In addition, the altered RNA splicing of CENPE (centromere protein E 312kDa), CEP250 (centrosomal protein 250kDa) and CEP192 (centrosomal protein 192kDa) would be expected to result in inefficient chromatid separation during the cell cycle. Indeed, proliferation of Huh-7 cells was inhibited by 0.3 mM amiloride or higher in the growth medium (Figure 7A). Furthermore, there was an accumulation of G2/M tetraploid cells beginning at 24 hours, becoming marked at 48 hours (Figure 7B). By light microscopic examination, there were many bi-nucleated and late-stage anaphase cells that were presumably the G2/M tetraploid cell population observed by fluorocytometry. During cell cycle progression and daughter cell separation in the presence of varying concentrations of amiloride, mitotic cells exposed to amiloride at 0.3 mM or higher developed into bi-nucleated forms at 24 hours but subsequently failed to generate separated daughter cells (Figure 6C and 6D). These three observations imply that through altering the RNA AS of genes, amiloride causes disturbance of cytokinesis by inhibiting mitotic cells from passing through late anaphase processes of abscission, division, and separation of daughter cells. Finally, we confirmed a previous report that Huh-7 cells express Prominin/AC133/CD133, an immunological marker of the “cancer stem-like” cells [34], and further found that amiloride at 0.3 mM or more down-regulated CD133 expression Huh-7 cells (Figure S2-A), resulting in suppression of the number and size of cell colony formations (Figure S2-B).


Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.

Chang JG, Yang DM, Chang WH, Chow LP, Chan WL, Lin HH, Huang HD, Chang YS, Hung CH, Yang WK - PLoS ONE (2011)

Growth inhibitory and apoptotic/cytotoxic consequences of amiloride-induced AS alterations.(Panel A) Huh-7 cells plated in 6-well dishes at 3×105 cells per well overnight were changed to growth medium containing various concentrations of amiloride; cell numbers were determined one and three days later. Cell counts at 24 hours show growth inhibition by amiloride at 0.4 mM and above. Further cell count decreases were evident at 72 hours due to cell death and detachment in medium containing amiloride at 0.3 mM and above. (Panel B) Cytofluorometric analysis shows amiloride inhibition of cell cycle progression with accumulation of G2/M phase cells. (Panel C) Apoptosis is evident by cytofluorometric detection of annexinV binding in Huh-7 cells treated with 0.5 mM of amiloride. (Panel D) Marked nuclear DNA degradation was observed in Huh-7 cells treated with 0.4 mM amiloride but not with 0.2 mM amiloride, particularly noted at 48 and 72 hours.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111415&req=5

pone-0018643-g007: Growth inhibitory and apoptotic/cytotoxic consequences of amiloride-induced AS alterations.(Panel A) Huh-7 cells plated in 6-well dishes at 3×105 cells per well overnight were changed to growth medium containing various concentrations of amiloride; cell numbers were determined one and three days later. Cell counts at 24 hours show growth inhibition by amiloride at 0.4 mM and above. Further cell count decreases were evident at 72 hours due to cell death and detachment in medium containing amiloride at 0.3 mM and above. (Panel B) Cytofluorometric analysis shows amiloride inhibition of cell cycle progression with accumulation of G2/M phase cells. (Panel C) Apoptosis is evident by cytofluorometric detection of annexinV binding in Huh-7 cells treated with 0.5 mM of amiloride. (Panel D) Marked nuclear DNA degradation was observed in Huh-7 cells treated with 0.4 mM amiloride but not with 0.2 mM amiloride, particularly noted at 48 and 72 hours.
Mentions: Our exon array analysis of amiloride-treated Huh-7 cells (Tables S1 & S2) detected alterations of RNA splicing predominantly of proteins involved in cytokinesis, associated with solute transport, and functions of actin, microtubules, and cytokinesis-related kinases [33]. In addition, the altered RNA splicing of CENPE (centromere protein E 312kDa), CEP250 (centrosomal protein 250kDa) and CEP192 (centrosomal protein 192kDa) would be expected to result in inefficient chromatid separation during the cell cycle. Indeed, proliferation of Huh-7 cells was inhibited by 0.3 mM amiloride or higher in the growth medium (Figure 7A). Furthermore, there was an accumulation of G2/M tetraploid cells beginning at 24 hours, becoming marked at 48 hours (Figure 7B). By light microscopic examination, there were many bi-nucleated and late-stage anaphase cells that were presumably the G2/M tetraploid cell population observed by fluorocytometry. During cell cycle progression and daughter cell separation in the presence of varying concentrations of amiloride, mitotic cells exposed to amiloride at 0.3 mM or higher developed into bi-nucleated forms at 24 hours but subsequently failed to generate separated daughter cells (Figure 6C and 6D). These three observations imply that through altering the RNA AS of genes, amiloride causes disturbance of cytokinesis by inhibiting mitotic cells from passing through late anaphase processes of abscission, division, and separation of daughter cells. Finally, we confirmed a previous report that Huh-7 cells express Prominin/AC133/CD133, an immunological marker of the “cancer stem-like” cells [34], and further found that amiloride at 0.3 mM or more down-regulated CD133 expression Huh-7 cells (Figure S2-A), resulting in suppression of the number and size of cell colony formations (Figure S2-B).

Bottom Line: Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins.We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation.These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research, University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. jgchang@ms.kmuh.org.tw

ABSTRACT
Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could "normalize" the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, 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. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.

Show MeSH
Related in: MedlinePlus