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Solanum Incanum Extract Downregulates Aldehyde Dehydrogenase 1-Mediated Stemness and Inhibits Tumor Formation in Ovarian Cancer Cells.

Wu YH, Chiu WT, Young MJ, Chang TH, Huang YF, Chou CY - J Cancer (2015)

Bottom Line: Furthermore, the SR-T100 IC50 in chemoresistant cells was similar to the IC50 in chemosensitive cells.Additionally, SR-T100 increased cisplatin and paclitaxel sensitivity in chemoresistant cells.Using microarray analyses, immunoblotting, luciferase activity, and chromatin immunoprecipitation (ChIP) assays, we showed that SR-T100 suppressed the expression of c/EBPβ and COL11A1, and its promoter activity, in resistant cells, but not sensitive cells.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University and Hospital, Tainan, Taiwan.

ABSTRACT
Solanum incanum extract (SR-T100), containing the active ingredient solamargine, can induce apoptosis via upregulation of tumor necrosis factor receptor expression and activation of the mitochondrial apoptosis pathway, and has therapeutic effects in patients with actinic keratosis. Here, we evaluate the novel molecular mechanisms underlying SR-T100-regulated stemness and chemoresistance. The concentration of SR-T100 that inhibited 50% cell viability (IC50) was lower in ovarian cancer cells than in nonmalignant cells. Furthermore, the SR-T100 IC50 in chemoresistant cells was similar to the IC50 in chemosensitive cells. Additionally, SR-T100 increased cisplatin and paclitaxel sensitivity in chemoresistant cells. SR-T100 downregulated the expression of stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), Notch1, and FoxM1, and reduced sphere formation in ovarian cancer cells. Using microarray analyses, immunoblotting, luciferase activity, and chromatin immunoprecipitation (ChIP) assays, we showed that SR-T100 suppressed the expression of c/EBPβ and COL11A1, and its promoter activity, in resistant cells, but not sensitive cells. SR-T100, paclitaxel, and cisplatin inhibited the growth of A2780CP70 cells in mouse xenografts, as compared to the vehicle control, and the combination of cisplatin and SR-T100 was more effective than either treatment alone. SR-T100 may represent a potential therapeutic adjunct to chemotherapy for ovarian cancer treatment.

No MeSH data available.


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SR-T100 downregulated c/EBPβ and COL11A1 in chemoresistant ovarian cancer cells. (A) Left panel: Cells were treated with SR-T100 (2.5 or 5 µg/ml) for 48 h, and cell lysates were collected for western blotting. Right panel: COL11A1 and c/EBPβ protein expression in cells treated with SR-T100 (5 µg/ml) and cisplatin (10 µM) for 48 days. (B) A2780CP70 cells transfected with the indicated COL11A1 promoter constructs were treated with SR-T100 and cisplatin for 48 h. The luciferase activity was measured and normalized to Renilla luciferase activity. All experiments were performed in triplicate. (C) A ChIP assay was performed to evaluate the binding of c/EBPβ to the COL11A1 promoter after treatment of A2780CP70 and A2780 cells for 48 h with varying cisplatin concentrations.
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Figure 3: SR-T100 downregulated c/EBPβ and COL11A1 in chemoresistant ovarian cancer cells. (A) Left panel: Cells were treated with SR-T100 (2.5 or 5 µg/ml) for 48 h, and cell lysates were collected for western blotting. Right panel: COL11A1 and c/EBPβ protein expression in cells treated with SR-T100 (5 µg/ml) and cisplatin (10 µM) for 48 days. (B) A2780CP70 cells transfected with the indicated COL11A1 promoter constructs were treated with SR-T100 and cisplatin for 48 h. The luciferase activity was measured and normalized to Renilla luciferase activity. All experiments were performed in triplicate. (C) A ChIP assay was performed to evaluate the binding of c/EBPβ to the COL11A1 promoter after treatment of A2780CP70 and A2780 cells for 48 h with varying cisplatin concentrations.

Mentions: To identify the genes regulated by SR-T100, A2780CP70 cells were treated with 2.5 or 5 µg/ml SR-T100, and a gene expression array was performed. Microarray analysis showed that 197 genes (66 upregulated and 131 downregulated) were differentially expressed between SR-T100-treated and control cells (Tables 1 and 2). Among these genes, c/EBPβ and COL11A1 were among the most downregulated genes. Our recent findings show that COL11A1 plays a dual role in EOC tumor progression and chemoresistance, and that the c/EBPβ binding site on the COL11A1 promoter is a major determinant of cisplatin- and paclitaxel-dependent COL11A1 activation 29. Our results showed that the expression of c/EBPβ and COL11A1 was enhanced by cisplatin, and that this elevated expression was inhibited by SR-T100 in chemoresistant A2780CP70 cells. In contrast, the expression level of c/EBPβ and COL11A1 was largely unaffected in chemosensitive A2780 cells by either cisplatin or SR-T100 treatment (Fig. 3A). In addition, the increased promoter activity by cisplatin in the region between -541 and -203 on COL11A1, which is important for the transcriptional regulation by cisplatin and paclitaxel 29, was decreased in SR-T100-treated A2780CP70 cells, but not in A2780 cells (Fig. 3B). ChIP assays further indicated that the binding of c/EBPβ to the COL11A1 promoter was inhibited by SR-T100 alone and SR-T100 plus cisplatin (Fig. 3C).


Solanum Incanum Extract Downregulates Aldehyde Dehydrogenase 1-Mediated Stemness and Inhibits Tumor Formation in Ovarian Cancer Cells.

Wu YH, Chiu WT, Young MJ, Chang TH, Huang YF, Chou CY - J Cancer (2015)

SR-T100 downregulated c/EBPβ and COL11A1 in chemoresistant ovarian cancer cells. (A) Left panel: Cells were treated with SR-T100 (2.5 or 5 µg/ml) for 48 h, and cell lysates were collected for western blotting. Right panel: COL11A1 and c/EBPβ protein expression in cells treated with SR-T100 (5 µg/ml) and cisplatin (10 µM) for 48 days. (B) A2780CP70 cells transfected with the indicated COL11A1 promoter constructs were treated with SR-T100 and cisplatin for 48 h. The luciferase activity was measured and normalized to Renilla luciferase activity. All experiments were performed in triplicate. (C) A ChIP assay was performed to evaluate the binding of c/EBPβ to the COL11A1 promoter after treatment of A2780CP70 and A2780 cells for 48 h with varying cisplatin concentrations.
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Related In: Results  -  Collection

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Figure 3: SR-T100 downregulated c/EBPβ and COL11A1 in chemoresistant ovarian cancer cells. (A) Left panel: Cells were treated with SR-T100 (2.5 or 5 µg/ml) for 48 h, and cell lysates were collected for western blotting. Right panel: COL11A1 and c/EBPβ protein expression in cells treated with SR-T100 (5 µg/ml) and cisplatin (10 µM) for 48 days. (B) A2780CP70 cells transfected with the indicated COL11A1 promoter constructs were treated with SR-T100 and cisplatin for 48 h. The luciferase activity was measured and normalized to Renilla luciferase activity. All experiments were performed in triplicate. (C) A ChIP assay was performed to evaluate the binding of c/EBPβ to the COL11A1 promoter after treatment of A2780CP70 and A2780 cells for 48 h with varying cisplatin concentrations.
Mentions: To identify the genes regulated by SR-T100, A2780CP70 cells were treated with 2.5 or 5 µg/ml SR-T100, and a gene expression array was performed. Microarray analysis showed that 197 genes (66 upregulated and 131 downregulated) were differentially expressed between SR-T100-treated and control cells (Tables 1 and 2). Among these genes, c/EBPβ and COL11A1 were among the most downregulated genes. Our recent findings show that COL11A1 plays a dual role in EOC tumor progression and chemoresistance, and that the c/EBPβ binding site on the COL11A1 promoter is a major determinant of cisplatin- and paclitaxel-dependent COL11A1 activation 29. Our results showed that the expression of c/EBPβ and COL11A1 was enhanced by cisplatin, and that this elevated expression was inhibited by SR-T100 in chemoresistant A2780CP70 cells. In contrast, the expression level of c/EBPβ and COL11A1 was largely unaffected in chemosensitive A2780 cells by either cisplatin or SR-T100 treatment (Fig. 3A). In addition, the increased promoter activity by cisplatin in the region between -541 and -203 on COL11A1, which is important for the transcriptional regulation by cisplatin and paclitaxel 29, was decreased in SR-T100-treated A2780CP70 cells, but not in A2780 cells (Fig. 3B). ChIP assays further indicated that the binding of c/EBPβ to the COL11A1 promoter was inhibited by SR-T100 alone and SR-T100 plus cisplatin (Fig. 3C).

Bottom Line: Furthermore, the SR-T100 IC50 in chemoresistant cells was similar to the IC50 in chemosensitive cells.Additionally, SR-T100 increased cisplatin and paclitaxel sensitivity in chemoresistant cells.Using microarray analyses, immunoblotting, luciferase activity, and chromatin immunoprecipitation (ChIP) assays, we showed that SR-T100 suppressed the expression of c/EBPβ and COL11A1, and its promoter activity, in resistant cells, but not sensitive cells.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University and Hospital, Tainan, Taiwan.

ABSTRACT
Solanum incanum extract (SR-T100), containing the active ingredient solamargine, can induce apoptosis via upregulation of tumor necrosis factor receptor expression and activation of the mitochondrial apoptosis pathway, and has therapeutic effects in patients with actinic keratosis. Here, we evaluate the novel molecular mechanisms underlying SR-T100-regulated stemness and chemoresistance. The concentration of SR-T100 that inhibited 50% cell viability (IC50) was lower in ovarian cancer cells than in nonmalignant cells. Furthermore, the SR-T100 IC50 in chemoresistant cells was similar to the IC50 in chemosensitive cells. Additionally, SR-T100 increased cisplatin and paclitaxel sensitivity in chemoresistant cells. SR-T100 downregulated the expression of stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), Notch1, and FoxM1, and reduced sphere formation in ovarian cancer cells. Using microarray analyses, immunoblotting, luciferase activity, and chromatin immunoprecipitation (ChIP) assays, we showed that SR-T100 suppressed the expression of c/EBPβ and COL11A1, and its promoter activity, in resistant cells, but not sensitive cells. SR-T100, paclitaxel, and cisplatin inhibited the growth of A2780CP70 cells in mouse xenografts, as compared to the vehicle control, and the combination of cisplatin and SR-T100 was more effective than either treatment alone. SR-T100 may represent a potential therapeutic adjunct to chemotherapy for ovarian cancer treatment.

No MeSH data available.


Related in: MedlinePlus