Project description:Withaferin A isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to concentrations of Withaferin A (WA) which can be detected systemically in in vivo experiments. Whereas WA revealed attenuation of multiple cancer hallmarks the withanolide analogue Withanone (WN), did not exert any of the described effects in these cells at comparable concentrations. Pathway enrichment analysis identified that WA targeted relevant cancer programs related to cell death, cell cycle and proliferation, which could functionally be validated flow cytometrically and by real-time proliferation xCELLigence assay. With respect to cell motility, invasion and metastasis processes, WA was found to strongly decrease MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This is further supported by gene expression changes which demonstrate increased expression of a validated breast cancer metastasis suppressor gene (BRMS1) and concomitant decreased expression of extracellular matrix-degrading proteases (PLAU, PLAT, ADAM8), cell adhesion molecules (integrins, laminins) as well as pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R). Subsequent in silico network analysis listed key node transcription factors regulating these processes (p53, E2F1, CDKN2A, NRF2) and revealed the role of chromatin modifying enzymes (p300, JARID1B) as central master switches, linking multiple anticancer activities of WA. Furthermore, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking in account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy resistant triple negative breast cancer, WA based therapeutic strategies hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer. Total RNA from 2 different cell lines treated with WA, are compared to their untreated states

Project description:Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer.

Project description:Withaferin A isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to concentrations of Withaferin A (WA) which can be detected systemically in in vivo experiments. Whereas WA revealed attenuation of multiple cancer hallmarks the withanolide analogue Withanone (WN), did not exert any of the described effects in these cells at comparable concentrations. Pathway enrichment analysis identified that WA targeted relevant cancer programs related to cell death, cell cycle and proliferation, which could functionally be validated flow cytometrically and by real-time proliferation xCELLigence assay. With respect to cell motility, invasion and metastasis processes, WA was found to strongly decrease MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This is further supported by gene expression changes which demonstrate increased expression of a validated breast cancer metastasis suppressor gene (BRMS1) and concomitant decreased expression of extracellular matrix-degrading proteases (PLAU, PLAT, ADAM8), cell adhesion molecules (integrins, laminins) as well as pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R). Subsequent in silico network analysis listed key node transcription factors regulating these processes (p53, E2F1, CDKN2A, NRF2) and revealed the role of chromatin modifying enzymes (p300, JARID1B) as central master switches, linking multiple anticancer activities of WA. Furthermore, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking in account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy resistant triple negative breast cancer, WA based therapeutic strategies hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer. Total RNA from 2 different cell lines treated with WA, are compared to their untreated states

Project description:Withaferin A isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to concentrations of Withaferin A (WA) which can be detected systemically in in vivo experiments. Whereas WA revealed attenuation of multiple cancer hallmarks the withanolide analogue Withanone (WN), did not exert any of the described effects in these cells at comparable concentrations. Pathway enrichment analysis identified that WA targeted relevant cancer programs related to cell death, cell cycle and proliferation, which could functionally be validated flow cytometrically and by real-time proliferation xCELLigence assay. With respect to cell motility, invasion and metastasis processes, WA was found to strongly decrease MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This is further supported by gene expression changes which demonstrate increased expression of a validated breast cancer metastasis suppressor gene (BRMS1) and concomitant decreased expression of extracellular matrix-degrading proteases (PLAU, PLAT, ADAM8), cell adhesion molecules (integrins, laminins) as well as pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R). Subsequent in silico network analysis listed key node transcription factors regulating these processes (p53, E2F1, CDKN2A, NRF2) and revealed the role of chromatin modifying enzymes (p300, JARID1B) as central master switches, linking multiple anticancer activities of WA. Furthermore, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking in account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy resistant triple negative breast cancer, WA based therapeutic strategies hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer.

Dataset Information

Metabolic alterations in mammary cancer prevention by withaferin A in a clinically relevant mouse model.

Background

Methods

Results

Conclusions

Publications

Metabolic alterations in mammary cancer prevention by withaferin A in a clinically relevant mouse model.

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