Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with minimal treatment options. In the present work, Withaferin A (WA), a natural steroidal lactone found in <i>Withania somnifera</i> (Solanaceae), was studied to deduce the miRNA expression modulation mediat...
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| Format: | Article |
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MDPI AG
2022-12-01
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| Series: | Metabolites |
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| Online Access: | https://www.mdpi.com/2218-1989/13/1/29 |
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| author | Mohd Shuaib Kumari Sunita Prajapati Sanjay Gupta Shashank Kumar |
| author_facet | Mohd Shuaib Kumari Sunita Prajapati Sanjay Gupta Shashank Kumar |
| author_sort | Mohd Shuaib |
| collection | DOAJ |
| description | Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with minimal treatment options. In the present work, Withaferin A (WA), a natural steroidal lactone found in <i>Withania somnifera</i> (Solanaceae), was studied to deduce the miRNA expression modulation mediated anticancer mode of action in TNBC cells. Small RNA next generation sequencing (NGS) of WA (2 µM) and vehicle (0.1% DMSO)-treated MDA-MB-231 cells revealed a total of 413 differentially expressed miRNAs (DEMs) and demonstrated that WA potentially up-regulates the miR-181c-5p, miR-15a-5p, miR-500b-5p, miR-191-3p, and miR-34a-5p and down-regulates miR-1275, miR-326, miR-1908-5p, and miR-3940-3p among total DEMs. The NGS and qRT-PCR expression analysis revealed a significantly higher expression of miR-181c-5p among the top 10 DEMs. Predicted target genes of the DEMs showed enrichment in cancer-associated gene ontology terms and KEGG signaling pathways. Transient up-expression of mir-181c-5p showed a time-dependent decrease in MDA-MB-231 and MDA-MB-453 cell viability. Co-treatment of miR-181c-5p mimic and WA (at varying concentration) down-regulated cell cycle progression markers (CDK4 and Cyclin D1) at mRNA and protein levels. The treatment induced apoptosis in MDA-MB-231 cells by modulating the expression/activity of Bax, Bcl2, Caspase 3, Caspase 8, Caspase 3/7, and PARP at mRNA and protein levels. Confocal microscopy and Annexin PI assays revealed apoptotic induction in miRNA- and steroidal-lactone-treated MDA-MB-231 cells. Results indicate that the Withaferin A and miRNA mimic co-treatment strategy may be utilized as a newer therapeutic strategy to treat triple-negative breast cancer. |
| first_indexed | 2024-03-09T11:43:40Z |
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| institution | Directory Open Access Journal |
| issn | 2218-1989 |
| language | English |
| last_indexed | 2024-03-09T11:43:40Z |
| publishDate | 2022-12-01 |
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| record_format | Article |
| series | Metabolites |
| spelling | doaj.art-9093d77e2359440dbf7e1f52da0bd8032023-11-30T23:27:51ZengMDPI AGMetabolites2218-19892022-12-011312910.3390/metabo13010029Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer CellsMohd Shuaib0Kumari Sunita Prajapati1Sanjay Gupta2Shashank Kumar3Molecular Signaling & Drug Discovery Laboratory, Department of Biochemistry, Central University of Punjab, Guddha, Bathinda 151401, Punjab, IndiaMolecular Signaling & Drug Discovery Laboratory, Department of Biochemistry, Central University of Punjab, Guddha, Bathinda 151401, Punjab, IndiaDepartment of Urology, Nutrition, Pharmacology and Pathology, Case Western Reserve University, Cleveland, OH 44106, USAMolecular Signaling & Drug Discovery Laboratory, Department of Biochemistry, Central University of Punjab, Guddha, Bathinda 151401, Punjab, IndiaTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with minimal treatment options. In the present work, Withaferin A (WA), a natural steroidal lactone found in <i>Withania somnifera</i> (Solanaceae), was studied to deduce the miRNA expression modulation mediated anticancer mode of action in TNBC cells. Small RNA next generation sequencing (NGS) of WA (2 µM) and vehicle (0.1% DMSO)-treated MDA-MB-231 cells revealed a total of 413 differentially expressed miRNAs (DEMs) and demonstrated that WA potentially up-regulates the miR-181c-5p, miR-15a-5p, miR-500b-5p, miR-191-3p, and miR-34a-5p and down-regulates miR-1275, miR-326, miR-1908-5p, and miR-3940-3p among total DEMs. The NGS and qRT-PCR expression analysis revealed a significantly higher expression of miR-181c-5p among the top 10 DEMs. Predicted target genes of the DEMs showed enrichment in cancer-associated gene ontology terms and KEGG signaling pathways. Transient up-expression of mir-181c-5p showed a time-dependent decrease in MDA-MB-231 and MDA-MB-453 cell viability. Co-treatment of miR-181c-5p mimic and WA (at varying concentration) down-regulated cell cycle progression markers (CDK4 and Cyclin D1) at mRNA and protein levels. The treatment induced apoptosis in MDA-MB-231 cells by modulating the expression/activity of Bax, Bcl2, Caspase 3, Caspase 8, Caspase 3/7, and PARP at mRNA and protein levels. Confocal microscopy and Annexin PI assays revealed apoptotic induction in miRNA- and steroidal-lactone-treated MDA-MB-231 cells. Results indicate that the Withaferin A and miRNA mimic co-treatment strategy may be utilized as a newer therapeutic strategy to treat triple-negative breast cancer.https://www.mdpi.com/2218-1989/13/1/29Withania AanticancerapoptosisphytochemicalMiRNAsequencing |
| spellingShingle | Mohd Shuaib Kumari Sunita Prajapati Sanjay Gupta Shashank Kumar Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells Metabolites Withania A anticancer apoptosis phytochemical MiRNA sequencing |
| title | Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells |
| title_full | Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells |
| title_fullStr | Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells |
| title_full_unstemmed | Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells |
| title_short | Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells |
| title_sort | natural steroidal lactone induces g1 s phase cell cycle arrest and intrinsic apoptotic pathway by up regulating tumor suppressive mirna in triple negative breast cancer cells |
| topic | Withania A anticancer apoptosis phytochemical MiRNA sequencing |
| url | https://www.mdpi.com/2218-1989/13/1/29 |
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