Withaferin A Attenuates Epithelial-Mesenchymal Transition and Cancer Stem Cells Properties in Hepatocellular Carcinoma Cells by Inhibiting the PI3K/AKT Pathway through miR-200c

ABSTRACT

Objectives: The present study was aimed at elucidating the mechanism of action of withaferin A against hepatocellular carcinoma (HCC) cells via the PI3K/AKT signaling pathway. Materials and Methods: MTT (tetrazolium dye) Assay was performed for the relative assessment of the viabilities of cell lines. The effect of withaferin A on the proliferative capability of HCC cells was analyzed using the EdU staining assay, and the colony‑forming potential was assessed with the help of a clonogenic assay. Cell apoptosis was estimated through (Modified Annexin V/Propidium Iodide Apoptosis Assay) staining followed by flow cytometry. Transwell assays were carried out for estimating the migration and invasion of cancer cells. The qRT‑PCR and western blotting, respectively, were used for gene and protein expression studies. Results: Withaferin A selectively inhibited the viability of HCC cells although the viability of normal liver cells was minimally affected. The IC50 of withaferin A against the HepG2 cells was found to be 12 µM as against 150 µM for normal THLE‑2 cells. The treatment with withaferin A significantly minimized the proliferation and colony‑forming potential of cancer cells by inducing cell apoptosis. The percentage of apoptosis increased from 3.8% in control to 20.3% at 24 µM withaferin A. The cancer cell migration and invasion were significantly declined by withaferin A together with the inhibition of Epithelial to mesenchymal transition (EMT) of HCC cells. The withaferin A treatment decreased the expression of carcinoma cell markers CD44, CD90, and EpCAM. The expression of miR‑200c markedly increased under withaferin A treatment and the latter was shown to exert its anti‑cancer effects through miR‑200c‑mediated inhibition of the PI3K/ AKT signaling pathway in HCC cells. Conclusion: In conclusion, withaferin A modulated the expression of miR‑200c in HCC cells to inhibit the PI3K/ AKT pathway and restricted the cancer cell EMT together with the inhibition of in vitro cancer cell growth and viability via induction of apoptosis.

KEYWORDS

• Anticancer
• apoptosis
• hepatocellular carcinoma
• proliferation
• withaferin A

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• DOI10.4103/pm.pm_86_22Copy

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