Abstract
Hepatic fibrosis is known as the accumulation of connective tissue secondary to chronic damage to the liver. Epithelial–mesenchymal transition (EMT) corresponding increase in liver fibrogenesis was shown with immunohistochemistry and PCR-based studies. Suberoylanilide hydroxamic acid (SAHA), a synthetic compound approved as a histone deacetylase inhibitor (HDAC) by the FDA to treat cutaneous T-cell lymphoma is under investigation for the treatment of lung and renal fibrosis. Experimental modeling for hepatic fibrosis can be constructed with an LX2 cell line isolated from human hepatic stellate cells (HSCs). In this study, we aimed to investigate the modulation of SAHA in the pathogenesis of liver fibrosis by detecting the levels of proteins; (E-cadherin (E-cad), N-cadherin (N-cad), Vimentin (Vim), and genes; E-cad, N-cad, Vim, transforming growth factor-beta (TGF-β), alpha-smooth muscle actin (α-SMA), type 1 collagen (COL1A1), type 3 collagen (COL3A1)) that play a significant role in EMT with the LX2 cell line. We also evaluated the action of SAHA with cell proliferation, clonogenic, and migration assay. Cell proliferation was performed by flow cytometry. All the protein levels were determined by Western blot analysis, and gene expression levels were measured by Real-Time PCR. Our study observed that SAHA treatment decreased cell viability, colony formation and migration in LX2 cells. We found that SAHA increased E-cad expression level, while it decreased N-cad, Vim, COL1A1, COL3A1, α-SMA TGF-β genes expression levels. SAHA decreased the level of E-cad, N-cad, and Vim protein levels. We thought that SAHA possesses potent antifibrotic and anti-EMT properties in LX2.
Similar content being viewed by others
References
Elpek, G. Ö. (2014). Cellular and molecular mechanisms in the pathogenesis of liver fibrosis: an update. World Journal of Gastroenterology, 20, 7260–7276.
Higashi, T., Friedman, S. L., & Hoshida, Y. (2017). Hepatic stellate cells as key target in liver fibrosis. Advanced Drug Delivery Reviews, 121, 27–42.
Zhang, C. Y., Yuan, W. G., He, P., Lei, J. H., & Wang, C. X. (2016). Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets. World Journal of Gastroenterology, 22, 10512–10522.
Tsuchida, T., & Friedman, S. L. (2017). Mechanisms of hepatic stellate cell activation. Nature Reviews Gastroenterology & Hepatology, 14, 397–411.
Ning, B., Li, W., Zhao, W., & Wang, R. (2016). Targeting epigenetic regulations in cancer. Acta Biochimica et Biophysica Sinica, 48, 97–109.
Lakshmaiah, K. C., Jacob, L. A., Aparna, S., Lokanatha, D., & Saldanha, S. C. (2014). Epigenetic therapy of cancer with histone deacetylase inhibitors. Journal of Cancer Researche and Therapeutics, 10, 469–478.
Roche, J., & Bertrand, P. (2016). Inside HDACs with more selective HDAC inhibitors. European Journal of Medicinal Chemistry, 121, 451–483.
Richters, A., & Koehler, A. N. (2017). Epigenetic modulation using small molecules—-targeting histone acetyltransferases in disease. Current Medicinal Chemistry, 24, 4121–4150.
Massey, V., Cabezas, J., & Bataller, R. (2017). Epigenetics in liver fibrosis. Seminars in Liver Disease, 37, 219–230.
Hadden, M. J., & Advani, A. (2018). Histone deacetylase inhibitors and diabetic kidney disease. International Journal of Molecular Science, 19, 2630.
Beneden, K. V., Mannaerts, I., Pauwels, M., Branden, C. V., & van Grunsven, L. A. (2013). HDAC inhibitors in experimental liver and kidney fibrosis. Fibrogenesis & Tissue Repair, 6, 1.
Marfurt, J., Chalfein, F., Prayoga, P., Wabiser, F., Kenangalem, E., & Piera, K. A., et al. (2011). Ex vivo activity of histone deacetylase inhibitors against multidrug-resistant clinical isolates of Plasmodium falciparum and P. vivax. Antimicrobial Agents and Chemotherapy, 55, 961–966.
Claveria-Cabello, A., Colyn, L., Arechederra, M., Urman, J. M., Berasain, C., & Avila, M. A., et al. (2020). Epigenetics in liver fibrosis: could HDACs be a therapeutic target? Cells, 9, 2321.
Lee, S. J., Kim, K. H., & Park, K. K. (2014). Mechanisms of fibrogenesis in liver cirrhosis: the molecular aspects of epithelial-mesenchymal transition. World Journal of Hepatology, 6, 207–216.
Derycke, L. D., & Bracke, M. E. (2004). N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling. The International Journal of Developmental Biology, 48, 463–476.
Eckes, B., Colucci-Guyon, E., Smola, H., Nodder, S., Babinet, C., & Krieg, T., et al. (2000). Impaired wound healing in embryonic and adult mice lacking vimentin. Journal of Cell Science, 113, 2455–2462.
Kalluri, R., & Weinberg, R. A. (2009). The basics of epithelial-mesenchymal transition. Journal of Clinical Investigation, 119, 1420–1428.
Lee, J. M., Dedharü, S., Kalluri, R., & Thompson, E. W. (2006). The epithelial-mesenchymal transition: new insights in signaling, development, and disease. Journal of Cell Biology, 172, 973–981.
Kalluri, R., & Neilson, E. G. (2003). Epithelial-mesenchymal transition and its implications for fibrosis. Journal of Clinical Investigation, 112, 1776–1784.
Kocabayoglu, P., & Friedman, S. L. (2013). Cellular basis of hepatic fibrosis and its role in inflammation and cancer. Frontier Bioscience, 5, 217–230.
Weiskirchen, R., Weimer, J., Meurer, S. K., Kron, A., Seipel, B., & Vater, I., et al. (2013). Genetic characteristics of the human hepatic stellate cell line LX-2. PLoS ONE, 8, 75692.
Díaz, R., Kim, J. W., Hui, J. J., Li, Z., Swain, G. P., & Fong, K. S., et al. (2008). Evidence for the epithelial to mesenchymal transition in biliary atresia fibrosis. Human Pathology, 39, 102–115.
Syn, W. K., Jung, Y., Omenetti, A., Abdelmalek, M., Guy, C. D., & Yang, L., et al. (2009). Hedgehog-mediated epithelial-to-mesenchymal transition and fibrogenic repair in nonalcoholic fatty liver disease. Gastroenterology, 137, 1478–1488.
Wilson, C. L., Mann, D. A., & Borthwick, L. A. (2017). Epigenetic reprogramming in liver fibrosis and cancer. Advanced Drug Delivery Reviews, 121, 124–132.
De Souza, C., & Chatterji, B. P. (2015). HDAC inhibitors as novel anti-cancer therapeutics. Recent Patients on Anticancer Drug Discovery, 10, 145–162.
Wang, Y. C., Yang, X., Xing, L. H., & Kong, W. Z. (2013). Effects of SAHA on proliferation and apoptosis of hepatocellular carcinoma cells and hepatitis B virus replication. World Jounal of Gastroenterology, 19, 5159–5164.
Hibino, S., Saito, Y., Muramatsu, T., Otani, A., Kasai, Y., & Kimura, M., et al. (2014). Inhibitors of enhancer of zeste homolog 2 (EZH2) activate tumor-suppressor microRNAs in human cancer cells. Oncogenesis, 3, e104 doi: 10.1038/oncsis.
Lu, H., Yang, X. F., Tian, X. Q., Tang, S. L., Li, L. Q., & Zhao, S., et al. (2016). The in vitro and vivo anti-tumor effects and molecular mechanisms of suberoylanilide hydroxamic acid (SAHA) and MG132 on the aggressive phenotypes of gastric cancer cells. Oncotarget, 7, 56508–56525.
Srinivas, C., Swathi, V., Priyanka, C., Anjana, D. T., Subba, R. B. V., & Janaki, R. M., et al. (2016). Novel SAHA analogues inhibit HDACs, induce apoptosis and modulate the expression of microRNAs in hepatocellular carcinoma. Apoptosis, 21, 1249–1264.
Bernhart, E., Stuendl, N., Kaltenegger, H., Windpassinger, C., Donohue, N., & Leithner, A., et al. (2017). Histone deacetylase inhibitors vorinostat and panobinostat induce G1 cell cycle arrest and apoptosis in multidrug resistant sarcoma cell lines. Oncotarget, 8, 77254–77267.
Lee, Y. A., Wallace, M. C., & Friedman, S. L. (2015). Pathobiology of liver fibrosis: a translational success story. Gut, 64, 830–841.
Lee, U. E., & Friedman, S. L. (2011). Mechanisms of hepatic fibrogenesis. Best Practice & Research Clinical Gastroenterology, 25, 195–206.
Friedman, S. L., Sheppard, D., Duffield, J. S., & Violette, S. (2013). Therapy for fibrotic diseases: nearing the starting line. Science Translational Medicine, 5, 167.
Wang, W., Yan, M., Ji, Q., Lu, J., Ji, Y., & Ji, J. (2015). Suberoylanilide hydroxamic acid suppresses hepatic stellate cells activation by HMGB1 dependent reduction of NF-κB1. Peer Journal, 3, e1362 https://doi.org/10.7717/peerj.1362.
Rao, S. S., Zhang, X. Y., Shi, M. J., Xiao, Y., Zhang, Y. Y., & Wang, Y. Y., et al. (2016). Suberoylanilide hydroxamic acid attenuates paraquat-induced pulmonary fibrosis by preventing Smad7 from deacetylation in rats. Journal of Thoracic Disease, 8, 2485–2494.
Zhang, X., Liu, H., Hock, T., Thannickal, V. J., & Sanders, Y. Y. (2013). Histone deacetylase inhibition downregulates collagen 3A1 in fibrotic lung fibroblasts. International Journal of Molecular Science, 14, 19605–19617.
Wang, Y., Zhao, L., Jiao, F. Z., Zhang, W. B., Chen, Q., & Gong, Z. J. (2018). Histone deacetylase inhibitor suberoylanilide hydroxamic acid alleviates liver fibrosis by suppressing the transforming growth factor-β1 signal pathway. Hepatobiliary & Pancreatic Disease International, 17, 423–429.
Munker, S., Wu, Y. L., Ding, H. G., Liebe, R., & Weng, H. L. (2017). Can a fibrotic liver afford epithelial-mesenchymal transition? World Journal of Gastroenterology, 23, 4661–4668.
Choi, S. S., & Diehl, A. M. (2009). Epithelial-to-mesenchymal transitions in the liver. Hepatology, 50, 2007–2013.
Larue, L., & Bellacosa, A. (2005). Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3’ kinase/AKT pathways. Oncogene, 24, 7443–7454.
Wells, R. G. (2010). The epithelial-to-mesenchymal transition in liver fibrosis: here today, gone tomorrow? Hepatology, 51, 737–740.
Pinzani, M. (2011). Epithelial-mesenchymal transition in chronic liver disease: fibrogenesis or escape from death? Journal of Hepatology, 55, 459–465.
Park, J. H., Park, B. & Park, K. K. (2017). Suppression of hepatic epithelial-to-mesenchymal transition by melittin via blocking of TGFβ/Smad and MAPK-JNK signaling pathways. Toxins (Basel), https://doi.org/10.3390/toxins9040138.
Yoshikawa, M., Hishikawa, K., Marumo, T., & Fujita, T. (2007). Inhibition of histone deacetylase activity suppresses epithelial-to-mesenchymal transition induced by TGF-beta1 in human renal epithelial cells. Journal of The American Society of Nephrology, 18, 58–65.
Choi, S. Y., Kee, H. J., Kurz, T., Hansen, F. K., Ryu, Y., & Kim, G. R., et al. (2016). Class I HDACs specifically regulate E-cadherin expression in human renal epithelial cells. Journal of Cellular and Molecular Medicine, 20, 2289–2298.
Author contributions
Concept—G.B.; Design—G.B., M.Ö.; Supervision—G.B.; Resource—G.B.; Materials—G.B., M.Ö., H.A.; Data Collection and/or Processing—G.B., M.Ö., H.A.; Analysis and/or Interpretation—G.B., M.B.; Literature Search—G.B., M.Ö.; Writing—G.B., M.Ö.; Critical Reviews—G.B., M.B.
Funding
The authors declared that this study had received no financial support.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Ethics committee approval
Ethics committee approval is not needed since it is a cell culture study.
Informed consent
No informed consent is needed since it is a cell culture study.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Özel, M., Baskol, M., Akalın, H. et al. Suberoylanilide Hydroxamic Acid (SAHA) Reduces Fibrosis Markers and Deactivates Human Stellate Cells via the Epithelial–Mesenchymal Transition (EMT). Cell Biochem Biophys 79, 349–357 (2021). https://doi.org/10.1007/s12013-021-00974-1
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12013-021-00974-1