Importazole suppresses proliferation and modulates KPNB1 and Bax/Bcl-2 expression in HepG2 hepatocellular carcinoma cells: bioinformatic, in vitro, and in ovo analyses

Background: Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality and is associated with limited therapeutic options and poor prognosis at advanced stages. Although aberrant activation of importin-β-dependent nuclear transport has been reported in several malignancies, the clinical and therapeutic relevance of Karyopherin subunit beta 1 (KPNB1) in HCC remains poorly characterized.Aims: This study aimed to evaluate the expression and clinical relevance of KPNB1 in HCC and to investigate the cytotoxic, apoptotic, and anti-angiogenic effects of importazole (IPZ), a selective KPNB1 inhibitor.Methods: KPNB1 expression and its association with clinicopathological parameters and patient survival were assessed using Gene Expression Profiling Interactive Analysis 3 (GEPIA3) and University of Alabama at Birmingham Cancer (UALCAN). No human tissue samples were collected or experimentally analyzed in the study. The effects of IPZ on HepG2 hepatocellular carcinoma cells were evaluated in comparison with human umbilical vein endothelial cells (HUVEC), using 5-fluorouracil (5-FU) as a reference agent. Cytotoxicity was assessed over a concentration range of 1–100 µM, and 10 µM IPZ was used for 48 h for subsequent cellular experiments. Colony-forming capacity was evaluated, and KPNB1 and apoptosis-related gene expression, including the Bax/Bcl-2 ratio, were analyzed by RT-qPCR. Anti-angiogenic activity was examined using the in ovo chorioallantoic membrane (CAM) model.Results: Bioinformatic analyses demonstrated increased KPNB1 mRNA expression in tissues with HCC compared with normal liver tissues and revealed associations with clinicopathological characteristics and patient survival. IPZ exhibited greater cytotoxicity in HepG2 (IC50: 9.73 µM) than in HUVEC cells (IC50: 13.88 µM), and significantly reduced HepG2 colony formation. RT-qPCR analysis showed that IPZ significantly reduced KPNB1 mRNA expression and increased the Bax/Bcl-2 ratio approximately twofold in HepG2 cells (p ≤ 0.0001). In the in ovo CAM model, IPZ significantly reduced vessel area and total vessel length (p ≤ 0.01), whereas the reduction in total junction number was not statistically significant.Conclusion: KPNB1 mRNA expression is increased in HCC and may have clinical relevance in disease progression and prognosis. In the HepG2 HCC cell model, IPZ suppressed cell proliferation and colony formation, modulated KPNB1 and apoptosis-related gene expression, and reduced vascular parameters in the in ovo CAM model. These findings support further investigation of IPZ and KPNB1-associated nuclear transport as potential therapeutic approaches in HCC.