Selective Induction of Apoptosis in Hepatocellular Carcinoma Cells by AFP Promoter- Driven Apoptin Expression: An In Vitro Gene Therapy Approach

Abstract

Background: Hepatocellular Carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, and developing tumor-selective therapies with minimal off-target toxicity is a major challenge. Alpha-fetoprotein (AFP), which is highly expressed in HCC but absent in most normal adult hepatocytes, is a promising target for selective gene therapy. Apoptin, a viral protein, induces apoptosis preferentially in malignant cells.

Methods: An AFP promoter-driven Apoptin expression construct was generated using the pAdenoVator-IRES-GFP vector and transiently transfected into HepG2 cells. Promoter specificity was evaluated in AFP-negative HeLa and LNCaP cells. Transfection efficiency was assessed by GFP fluorescence microscopy. Cell viability was measured by MTT assay, Apoptin mRNA expression by RT-qPCR, and apoptosis by Annexin V/7-AAD flow cytometry. Experiments were performed in triplicate.

Results: GFP fluorescence was visibly stronger in HepG2 cells than in HeLa or LNCaP cells on qualitative microscopic examination, consistent with AFP promoter selectivity. AFP promoter-driven Apoptin expression significantly reduced HepG2 cell viability to 50.2%±4.3% of control levels (p=0.0317, n=3 independent experiments). RT-qPCR showed a significant increase in Apoptin mRNA expression in transfected HepG2 cells. Flow cytometry showed a significant increase in apoptosis, with approximately 20% more Annexin V-positive cells than controls (p=0.0286, n=3 independent experiments).

Conclusion: AFP promoter-driven Apoptin expression selectively induced apoptosis and reduced viability in HCC cells in vitro. These findings highlight the potential of AFP promoter-based Apoptin gene therapy as a tumor-selective therapeutic approach for HCC and support further evaluation in preclinical models.