2,6-BIS-(4-hydroxy-3-methoxybenzylidine) cyclohexanone (BHMC) induces apoptosis and activates the P38 MAPK in hepatoma HEPG2 cells

Introduction Hepatocellular carcinoma (HCC) is the most common type of liver cancer, marked by limited treatment options and poor survival rates. Curcumin, the main component of turmeric, is a powerful chemo preventive agent with good tolerability and low toxicity, though its bioavailability is poor. To address this, 2,6-Bis-(4‑hydroxy-3-methoxybenzylidene) cyclohexanone (BHMC) was synthesized as a curcumin analog to improve bioavailability. However, the molecular mechanisms of BHMC's action remain unclear. This study aims to investigate the effects of BHMC on cell cycle analysis and apoptosis in Human hepatocellular carcinoma cell (Hep G2 Cell Lines), as well as to examine the molecular mechanisms underlying BHMC's actions in these hepatoma cells. Methods Cell viability following incubation in complete DMEM was determined utilizing the MTT assay. BHMC's apoptotic effect was confirmed at 24 and 48 h through Annexin V staining. Cell cycle analysis was conducted using flow cytometry. RT‑qPCR analysis was used to investigate the RNA concentrations of HepG2 cells treated with BHMC by detecting expression levels of Ras homolog family member A (Rho A), Matrix Metalloproteinase 2 and 9 (MMP‑2, and MMP‑9). Western Blot analysis was used to assess if BHMC regulates mitogen-activated protein kinase (MAPK) and p38 (phosphorylated and dephosphorylated) in HepG2 cells. Results BHMC exhibited significant potency against HepG2 compared to Normal human foreskin fibroblast cell lines (Hs-27), with a relative IC50 of 21 µM after 24 h. The percentage of apoptosis escalated in a concentration and time-dependent manner. Flow cytometry analysis revealed cell cycle arrest in the S-phase. The current investigation elucidating that the expression of the Ras homolog gene family, member A, as well as matrix metalloproteinases-2 and-9 in HepG2 cells, has diminished. Furthermore, the phosphorylation of p38 mitogen-activated protein kinase increased in a dose-dependent manner in response to BHMC treatment. Conclusion BHMC demonstrated cytotoxic effects on HepG2 cells, as evidenced by increased apoptosis rates. Additionally, BHMC induced cell cycle arrest in the S-phase. Our findings indicate that BHMC activates p38 MAPK, which plays a key role in triggering apoptosis, inhibiting invasion, and limiting metastasis. Consequently, BHMC holds potential as a promising anticancer therapy for hepatoma.

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