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

Abstract

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 Withania somnifera (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 (2µM) 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.

Loss of miR‐6844 alters stemness/self‐renewal and cancer hallmark(s) markers through CD44‐JAK2‐STAT3 signaling axis in breast cancer stem‐like cells

“…GAPDH was used for normalization (Supporting Information: Table 1). QuantStudio (R) 3 Real‐Time PCR machine (Applied Biosystems, Thermo Fisher Scientific USA) was used for RT‐PCR analysis following protocol: start at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 45 s, and elongation at 72°C for 45 s. The reaction was repeated for 40 amplification cycles 19,20 …”

Piper chaba, an Indian spice plant extract, inhibits cell cycle G1/S phase transition and induces intrinsic apoptotic pathway in luminal breast cancer cells

“…Deregulation of the cell cycle permits unrestricted cell division in cancer cells. Therapeutically targeting cell cycle has long been considered a promising anticancer approach 4 . Breast tumor cells undergo alterations in cell cycle component that results in abnormal distribution of cell cycle phase(s).…”

“…The most recent generation of anticancer drugs for breast cancer that target the cell cycle includes CDK4/6 inhibitors. Cyclin D1 functions as an allosteric regulator of cyclin dependent kinase 4 and 6 (CDK4/CDK6) to regulate cell cycle transition from G1 to S phase 4,5 . Overexpression of cyclin D1 leads to dysregulated CDK activity and ultimately rapid cell growth under restricted mitogenic signaling conditions.…”

“…Cyclin D1 functions as an allosteric regulator of cyclin dependent kinase 4 and 6 (CDK4/CDK6) to regulate cell cycle transition from G1 to S phase. 4,5 Overexpression of cyclin D1 leads to dysregulated CDK activity and ultimately rapid cell growth under restricted mitogenic signaling conditions. The critical role of cyclin D1-CDK4 complex in cell cycle progression regulation and the overexpression of cyclin D1-CDK4 makes this complex an attractive target for cancer treatment in several human tumors.…”

Editorial: Targeting triple negative breast cancer by natural compounds

“…Treatment decisions for advance-stage breast cancer are guided by the expression of three major therapeutic targets, viz. estrogen receptor-α (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) (Shuaib et al, 2022). While ER-positive breast cancers are treated with ER antagonists such as tamoxifen and aromatase inhibitors, HER2-positive tumors are treated with HER2 inhibitors such as herceptin.…”