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

Kübra Sena Baş Topcu

doi:10.23902/trkjnat.1993634

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

Abstract

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.

Keywords

Ethical Statement

Fertilized chicken eggs were used for the CAM assay, and all experiments were terminated before EDD14. Therefore, formal animal ethics approval was not required according to commonly accepted CAM model guidelines.

Thanks

The authors would like to thank Pak Tavuk JSC (Türkiye) for generously providing the fertilized eggs used in the in ovo experiments.

References

Bal-Öztürk, A., Torkay, G., İdil, N., Akar, R. O., Özbaş, Z., & Özkahraman, B. (2024). Propolis-loaded photocurable methacrylated pullulan films: Evaluation of mechanical, antibacterial, biocompatibility, wound healing and pro-angiogenic abilities. International journal of biological macromolecules, 282(Pt 4), 137071. https://doi.org/10.1016/j.ijbiomac.2024.137071
Bray, F., Laversanne, M., Sung, H., Ferlay, J., Siegel, R. L., Soerjomataram, I., & Jemal, A. (2024). Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians, 74(3), 229–263. https://doi.org/10.3322/caac.21834
Chandrashekar, D. S., Bashel, B., Balasubramanya, S. A. H., Creighton, C. J., Ponce-Rodriguez, I., Chakravarthi, B. V. S. K., & Varambally, S. (2017). UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses. Neoplasia (New York, N.Y.), 19(8), 649–658. https://doi.org/10.1016/j.neo.2017.05.002
Chang, W., Jiang, X. P., Jin, S., Li, P. P., Song, S. S., Yuan, P. F., Wei, W., & Lu, J. T. (2022). Synergistic effects of CP-25 and 5-fluorouracil on the hepatocellular carcinoma in vivo and in vitro through regulating activating mitochondrial pathway. Journal of Cancer, 13(3), 1005–1018. https://doi.org/10.7150/jca.54702
Chen, J., Jiang, Y., Hou, M., Liu, C., Liu, E., Zong, Y., Wang, X., Meng, Z., Gu, M., Su, Y., Wang, H., & Fu, J. (2024). Nuclear translocation of plasma membrane protein ADCY7 potentiates T cell-mediated antitumour immunity in HCC. Gut, 74(1), 128–140. https://doi.org/10.1136/gutjnl-2024-332902
Fan, S., Fogg, V., Wang, Q., Chen, X. W., Liu, C. J., & Margolis, B. (2007). A novel Crumbs3 isoform regulates cell division and ciliogenesis via importin beta interactions. The Journal of cell biology, 178(3), 387–398. https://doi.org/10.1083/jcb.200609096
Fang, H., Shi, X., Gao, J., Yan, Z., Wang, Y., Chen, Y., Zhang, J., & Guo, W. (2024). TMEM209 promotes hepatocellular carcinoma progression by activating the Wnt/β-catenin signaling pathway through KPNB1 stabilization. Cell death discovery, 10(1), 438. https://doi.org/10.1038/s41420-024-02207-9
Feng, F., & Zhao, Y. (2024). Hepatocellular Carcinoma: Prevention, Diagnosis, and Treatment. Medical principles and practice : international journal of the Kuwait University, Health Science Centre, 33(5), 414–423. https://doi.org/10.1159/000539349

Hu, J., Wang, F., Yuan, Y., Zhu, X., Wang, Y., Zhang, Y., Kou, Z., Wang, S., & Gao, S. (2010). Novel importin-alpha family member Kpna7 is required for normal fertility and fecundity in the mouse. The Journal of biological chemistry, 285(43), 33113–33122. https://doi.org/10.1074/jbc.M110.117044
Kang, Y. J., Pan, L., Liu, Y., Rong, Z., Liu, J., & Liu, F. (2025). GEPIA3: Enhanced drug sensitivity and interaction network analysis for cancer research. Nucleic acids research, 53(W1), W283–W290. https://doi.org/10.1093/nar/gkaf423
Kennedy, D. C., Coen, B., Wheatley, A. M., & McCullagh, K. J. A. (2021). Microvascular Experimentation in the Chick Chorioallantoic Membrane as a Model for Screening Angiogenic Agents including from Gene-Modified Cells. International journal of molecular sciences, 23(1), 452. https://doi.org/10.3390/ijms23010452
Krupa, K., Fudalej, M., Cencelewicz-Lesikow, A., Badowska-Kozakiewicz, A., Czerw, A., & Deptała, A. (2024). Current Treatment Methods in Hepatocellular Carcinoma. Cancers, 16(23), 4059. https://doi.org/10.3390/cancers16234059
Lin, Z. (2026). Artificial intelligence and multi-omics revealed the authentic regulation of ADP-ribosylation in modulation Treg/Th17 ecosystem and multi-target therapeutic strategy enrichment for HBV+ Hepatocellular Carcinoma patients. Discover oncology, https://doi.org/10.1007/s12672-026-05487-y
Lin, Y., Zhong, W., Lin, Q., Ye, Y., Li, S., Chen, H., Liu, H., Xu, L., Zhuang, W., Chen, S., Lin, Y., & Wang, Q. (2023). SFPQ promotes the proliferation, migration and invasion of hepatocellular carcinoma cells and is associated with poor prognosis. American journal of cancer research, 13(6), 2269–2284.
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25(4), 402-408. https://doi.org/10.1006/meth.2001.1262
Luo, J., Lu, C., Chen, Y., Wu, X., Zhu, C., Cui, W., Yu, S., Li, N., Pan, Y., Zhao, W., Yang, Q., & Yang, X. (2023). Nuclear translocation of cGAS orchestrates VEGF-A-mediated angiogenesis. Cell reports, 42(4), 112328. https://doi.org/10.1016/j.celrep.2023.112328
Menadi, S., & Cacan, E. (2026). Synergistic Anticancer Effects of Akkermansia muciniphila Combined With 5-Fluorouracil Through BAX/BCL2 Dependent Apoptosis in Colorectal Cancer Cells. Cell biochemistry and function, 44(5), e70234. https://doi.org/10.1002/cbf.70234
Meng, J., Liu, Y., Han, J., Tan, Q., Chen, S., Qiao, K., Zhou, H., Sun, T., & Yang, C. (2017). Hsp90β promoted endothelial cell-dependent tumor angiogenesis in hepatocellular carcinoma. Molecular cancer, 16(1), 72. https://doi.org/10.1186/s12943-017-0640-9
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods, 65(1-2), 55-63. https://doi.org/10.1016/0022-1759(83)90303-4
Sekimoto, N., Suzuki, Y., & Sugano, S. (2017). Decreased KPNB1 Expression is Induced by PLK1 Inhibition and Leads to Apoptosis in Lung Adenocarcinoma. Journal of Cancer, 8(19), 4125–4140. https://doi.org/10.7150/jca.21802
Sheng, K. L., Pridham, K. J., Sheng, Z., Lamouille, S., & Varghese, R. T. (2019). Functional Blockade of Small GTPase RAN Inhibits Glioblastoma Cell Viability. Frontiers in oncology, 8, 662. https://doi.org/10.3389/fonc.2018.00662
Shi, Q., Lin, M., Cheng, X., Zhang, Z., Deng, S., Lang, K., Yang, Z., & Sun, X. (2023). KPNB1-mediated nuclear import in cancer. European journal of pharmacology, 955, 175925. https://doi.org/10.1016/j.ejphar.2023.175925
Shireen, Z., Saha, S., Das, U., Ghosh, S., Adhikary, A., Banerjee, K., & Dey, S. (2025). Autophagy-Apoptosis synchronization: A mechanism of black grape anthocyanins mediated chemosensitization of 5-FU in HepG2 hepatocellular carcinoma cells. Medical oncolog, 43(2), 106. https://doi.org/10.1007/s12032-025-03177-3
Singh, R., Letai, A., & Sarosiek, K. (2019). Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nature reviews. Molecular cell biology, 20(3), 175–193. https://doi.org/10.1038/s41580-018-0089-8
Soderholm, J. F., Bird, S. L., Kalab, P., Sampathkumar, Y., Hasegawa, K., Uehara-Bingen, M., Weis, K., & Heald, R. (2011). Importazole, a small molecule inhibitor of the transport receptor importin-β. ACS chemical biology, 6(7), 700–708. https://doi.org/10.1021/cb2000296
Stelma, T., & Leaner, V. D. (2017). KPNB1-mediated nuclear import is required for motility and inflammatory transcription factor activity in cervical cancer cells. Oncotarget, 8(20), 32833–32847. https://doi.org/10.18632/oncotarget.15834
Tang, Z., Li, C., Kang, B., Gao, G., Li, C., & Zhang, Z. (2017). GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic acids research, 45(W1), W98–W102. https://doi.org/10.1093/nar/gkx247
Topcu, K. S. B., & Cacan, E. (2025). Twist1 Regulates the Immune Checkpoint VISTA and Promotes the Proliferation, Migration and Progression of Pancreatic Cancer Cells. Journal of cellular and molecular medicine, 29(9), e70586. https://doi.org/10.1111/jcmm.70586
Wang, T., Huang, Z., Huang, N., Peng, Y., Gao, M., Wang, X., & Feng, W. (2019). Inhibition of KPNB1 Inhibits Proliferation and Promotes Apoptosis of Chronic Myeloid Leukemia Cells Through Regulation of E2F1. OncoTargets and therapy, 12, 10455–10467. https://doi.org/10.2147/OTT.S210048
Wang, Y., & Deng, B. (2023). Hepatocellular carcinoma: molecular mechanism, targeted therapy, and biomarkers. Cancer metastasis reviews, 42(3), 629–652. DOI: https://doi.org/10.1007/s10555-023-10084-4
Yang, J., Guo, Y., Lu, C., Zhang, R., Wang, Y., Luo, L., Zhang, Y., Chu, C. H., Wang, K. J., Obbad, S., Yan, W., & Li, X. (2019). Inhibition of Karyopherin beta 1 suppresses prostate cancer growth. Oncogene, 38(24), 4700–4714. https://doi.org/10.1038/s41388-019-0745-2
Yang, L., Hu, B., Zhang, Y., Qiang, S., Cai, J., Huang, W., Gong, C., Zhang, T., Zhang, S., Xu, P., Wu, X., & Liu, J. (2015). Suppression of the nuclear transporter-KPNβ1 expression inhibits tumor proliferation in hepatocellular carcinoma. Medical oncology, 32(4), 128. https://doi.org/10.1007/s12032-015-0559-1
Yao, C., Wu, S., Kong, J., Sun, Y., Bai, Y., Zhu, R., Li, Z., Sun, W., & Zheng, L. (2023). Angiogenesis in hepatocellular carcinoma: mechanisms and anti-angiogenic therapies. Cancer biology & medicine, 20(1), 25–43. https://doi.org/10.20892/j.issn.2095-3941.2022.0449
Zhang, W., Tang, Y., Yang, P., Chen, Y., Xu, Z., Qi, C., Huang, H., Liu, R., Qin, H., Ke, H., Huang, C., Xu, F., Pang, P., Zhao, Z., Shan, H., & Xiao, F. (2024). TMX2 potentiates cell viability of hepatocellular carcinoma by promoting autophagy and mitophagy. Autophagy, 20(10), 2146–2163. https://doi.org/10.1080/15548627.2024.2358732
Zhang, X. H., Liu, Y. X., Jia, M., Han, J. S., Zhao, M., Ji, S. P., & Li, A. M. (2014). Oridonin inhibits tumor growth in glioma by inducing cell cycle arrest and apoptosis. Cellular and Molecular Biology, 60(6), 29-36.
Zhu, Z. C., Liu, J. W., Li, K., Zheng, J., & Xiong, Z. Q. (2018). KPNB1 inhibition disrupts proteostasis and triggers unfolded protein response-mediated apoptosis in glioblastoma cells. Oncogene, 37(22), 2936–2952. https://doi.org/10.1038/s41388-018-0180-9

Details

Primary Language

English

Subjects

Cell Development, Proliferation and Death, Cell Metabolism

Journal Section

Research Article

Authors

Kübra Sena Baş Topcu ^*
0000-0003-3161-1042
Türkiye

Early Pub Date

October 9, 2026

Publication Date

October 15, 2026

Submission Date

July 14, 2026

Acceptance Date

October 5, 2026

Published in Issue

Year 2026 Volume: 27 Number: 2

DOI

https://doi.org/10.23902/trkjnat.1993634

IZ

https://izlik.org/JA28KY89KX

Cite

RIS / Bibtex

APA

Baş Topcu, K. S. (2026). Importazole suppresses proliferation and modulates KPNB1 and Bax/Bcl-2 expression in HepG2 hepatocellular carcinoma cells: bioinformatic, in vitro, and in ovo analyses. Trakya University Journal of Natural Sciences, 27(2), 202-212. https://doi.org/10.23902/trkjnat.1993634

AMA

1.Baş Topcu KS. Importazole suppresses proliferation and modulates KPNB1 and Bax/Bcl-2 expression in HepG2 hepatocellular carcinoma cells: bioinformatic, in vitro, and in ovo analyses. Trakya Univ J Nat Sci. 2026;27(2):202-212. doi:10.23902/trkjnat.1993634

Chicago

Baş Topcu, Kübra Sena. 2026. “Importazole Suppresses Proliferation and Modulates KPNB1 and Bax Bcl-2 Expression in HepG2 Hepatocellular Carcinoma Cells: Bioinformatic, in Vitro, and in Ovo Analyses”. Trakya University Journal of Natural Sciences 27 (2): 202-12. https://doi.org/10.23902/trkjnat.1993634.

EndNote

Baş Topcu KS (October 1, 2026) Importazole suppresses proliferation and modulates KPNB1 and Bax/Bcl-2 expression in HepG2 hepatocellular carcinoma cells: bioinformatic, in vitro, and in ovo analyses. Trakya University Journal of Natural Sciences 27 2 202–212.

IEEE

[1]K. S. Baş Topcu, “Importazole suppresses proliferation and modulates KPNB1 and Bax/Bcl-2 expression in HepG2 hepatocellular carcinoma cells: bioinformatic, in vitro, and in ovo analyses”, Trakya Univ J Nat Sci, vol. 27, no. 2, pp. 202–212, Oct. 2026, doi: 10.23902/trkjnat.1993634.

ISNAD

Baş Topcu, Kübra Sena. “Importazole Suppresses Proliferation and Modulates KPNB1 and Bax Bcl-2 Expression in HepG2 Hepatocellular Carcinoma Cells: Bioinformatic, in Vitro, and in Ovo Analyses”. Trakya University Journal of Natural Sciences 27/2 (October 1, 2026): 202-212. https://doi.org/10.23902/trkjnat.1993634.

JAMA

1.Baş Topcu KS. Importazole suppresses proliferation and modulates KPNB1 and Bax/Bcl-2 expression in HepG2 hepatocellular carcinoma cells: bioinformatic, in vitro, and in ovo analyses. Trakya Univ J Nat Sci. 2026;27:202–212.

MLA

Baş Topcu, Kübra Sena. “Importazole Suppresses Proliferation and Modulates KPNB1 and Bax Bcl-2 Expression in HepG2 Hepatocellular Carcinoma Cells: Bioinformatic, in Vitro, and in Ovo Analyses”. Trakya University Journal of Natural Sciences, vol. 27, no. 2, Oct. 2026, pp. 202-1, doi:10.23902/trkjnat.1993634.

Vancouver

1.Kübra Sena Baş Topcu. Importazole suppresses proliferation and modulates KPNB1 and Bax/Bcl-2 expression in HepG2 hepatocellular carcinoma cells: bioinformatic, in vitro, and in ovo analyses. Trakya Univ J Nat Sci. 2026 Oct. 1;27(2):202-1. doi:10.23902/trkjnat.1993634