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The effects of low-dose sorafenib on epithelial-mesenchymal transition and multidrug resistance markers in HepG2 cell line

Year 2023, Volume: 9 Issue: 2, 367 - 374, 04.03.2023
https://doi.org/10.18621/eurj.1206680

Abstract

Objectives: Sorafenib is an orally administered tyrosine kinase inhibitor in hepatocellular cancer. Low sorafenib concentrations are attained during pharmacotherapy due to pharmacokinetic profile and patient inadherence. Resistance to treatment is a limitation to improving survival. Underlying mechanisms include epithelial-mesenchymal transition. The aim of the study was to evaluate epithelial-mesenchymal transition and multidrug resistance-related parameters in HepG2 cells following low-dose and short-term sorafenib treatment.

Methods: Epithelial-mesenchymal transition and multidrug resistance-related markers were examined by quantitative PCR, flow cytometry, and confocal laser scanning microscopy.

Results: An increase in epithelial marker E-cadherin and downregulation of mesenchymal markers Vimentin and Snail1 were detected by gene expression analysis. While P-glycoprotein expression increased, multidrug resistance protein 1, and breast cancer resistance protein mRNA levels did not alter after sorafenib treatment. The accumulation of the ABC transporter substrate rhodamine 123 in the cells increased following the treatment, corresponding to a less efficient efflux of rhodamine 123 and a possible effect on other transporters and mechanisms.

Conclusions: The results indicate a protective effect of sorafenib against epithelial-mesenchymal transition and upregulation in P-glycoprotein expression, which is, however, not sufficient to cause less intracellular rhodamine 123 accumulation. The effects of low-dose and short-term sorafenib on epithelial-mesenchymal transition and multidrug resistance-related markers might contribute to enlightening new treatment strategies in hepatocellular cancer.

Supporting Institution

This study was funded by Maltepe University Research Project Council (Grant date: 24/02/2021).

Project Number

Grant date: 24/02/2021

References

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  • 2. Karbownik A, Szkutnik-Fiedler D, Czyrski A, Kostewicz N, Kaczmarska P, Bekier M, et al. Pharmacokinetic interaction between sorafenib and atorvastatin, and sorafenib and metformin in rats. Pharmaceutics 2020;12:600.
  • 3. Pang Y, Eresen A, Zhang Z, Hou Q, Wang Y, Yaghmai V, et al. Adverse events of sorafenib in hepatocellular carcinoma treatment. Am J Cancer Res 2022;12:2770-82.
  • 4. Tak KY, Nam HC, Choi JY, Yoon SK, Kim CW, Kim HY, et al. Effectiveness of sorafenib dose modifications on treatment outcome of hepatocellular carcinoma: analysis in real-life settings. Int J Cancer 2020;147:1970-8.
  • 5. Labeur TA, Hofsink Q, Takkenberg RB, van Delden OM, Mathôt RAA, Schinner R, et al. The value of sorafenib trough levels in patients with advanced hepatocellular carcinoma - a substudy of the SORAMIC trial. Acta Oncol 2020;59:1028-35.
  • 6. Gurzu S, Kobori L, Fodor D, Jung I. Epithelial mesenchymal and endothelial mesenchymal transitions in hepatocellular carcinoma: a review. BioMed Res Int 2019;2019:2962580.
  • 7. Myong NH. Loss of E-cadherin and acquisition of Vimentin in epithelial-mesenchymal transition are noble indicators of uterine cervix cancer progression. Korean J Pathol 2012;46:341-8.
  • 8. Jou J, Diehl AM. Epithelial-mesenchymal transitions and hepatocarcinogenesis. J Clin Invest 2010;120:1031-4.
  • 9. Duan B, Huang C, Bai J, Zhang YL, Wang X, Yang J, et al. Multidrug resistance in hepatocellular carcinoma. In: JEE T-P, ed. Hepatocellular carcinoma [Internet]. Chapter 8, Brisbane (AU); 2019.
  • 10. Fornari F, Giovannini C, Piscaglia F, Gramantieri L. Elucidating the molecular basis of sorafenib resistance in hcc: current findings and future directions. J Hepatocell Carcinoma 2021;8:741-57.
  • 11. Cabral LKD, Tiribelli C, Sukowati CHC. Sorafenib resistance in hepatocellular carcinoma: the relevance of genetic heterogeneity. Cancers (Basel) 2020;12:1576.
  • 12. Marin JJG, Monte MJ, Macias RIR, Romero MR, Herraez E, Asensio M, et al. Expression of chemoresistance-associated abc proteins in hepatobiliary, pancreatic and gastrointestinal cancers. Cancers (Basel) 2022;14:3524.
  • 13. Chen YA, Ho CL, Ku MT, Hwu L, Lu CH, Chiu SJ, et al. Detection of cancer stem cells by EMT-specific biomarker-based peptide ligands. Sci Rep 2021;11:22430.
  • 14. Dönmez Çakıl Y, Sitar ME, Özünal ZG, Kayalı D, Gülhan Aktas R. Flow cytometric evaluation of cancer stem cell markers in HepG2 cells following sorafenib treatment. Int J Med Biochem 2021;4:200-4.
  • 15. Cho Y, Kim YK. Cancer stem cells as a potential target to overcome multidrug resistance. Front Oncol 2020;10:764.
  • 16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-8.
  • 17. Wang H, Chirshev E, Hojo N, Suzuki T, Bertucci A, Pierce M, et al. The epithelial-mesenchymal transcription factor SNAI1 represses transcription of the tumor suppressor miRNA let-7 in cancer. Cancers (Basel) 2021;13:1469.
  • 18. Tang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, et al. The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther 2020;5:87.
  • 19. Zhou J, Sun H, Wang Z, Cong W, Wang J, Zeng M, et al. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (2019 Edition). Liver Cancer 2020;9:682-720.
  • 20. Yang MH, Chen CL, Chau GY, Chiou SH, Su CW, Chou TY, et al. Comprehensive analysis of the independent effect of twist and snail in promoting metastasis of hepatocellular carcinoma. Hepatology 2009;50:1464-74.
  • 21. Chen YL, Lv J, Ye XL, Sun MY, Xu Q, Liu CH, et al. Sorafenib inhibits transforming growth factor β1-mediated epithelial-mesenchymal transition and apoptosis in mouse hepatocytes. Hepatology 2011;53:1708-18.
  • 22. Ha TY, Hwang S, Moon KM, Won YJ, Song GW, Kim N, et al. Sorafenib inhibits migration and invasion of hepatocellular carcinoma cells through suppression of matrix metalloproteinase expression. Anticancer Res 2015;35:1967-76.
  • 23. Dong S, Kong J, Kong F, Kong J, Gao J, Ji L, et al. Sorafenib suppresses the epithelial-mesenchymal transition of hepatocellular carcinoma cells after insufficient radiofrequency ablation. BMC Cancer 2015;15:939.
  • 24. Dong J, Zhai B, Sun W, Hu F, Cheng H, Xu J. Activation of phosphatidylinositol 3-kinase/AKT/snail signaling pathway contributes to epithelial-mesenchymal transition-induced multi-drug resistance to sorafenib in hepatocellular carcinoma cells. PloS One 2017;12:e0185088.
  • 25. Tian X, Yan T, Liu F, Liu Q, Zhao J, Xiong H, et al. Link of sorafenib resistance with the tumor microenvironment in hepatocellular carcinoma: Mechanistic insights. Front Pharmacol 2022;13:991052.
  • 26. van Malenstein H, Dekervel J, Verslype C, Van Cutsem E, Windmolders P, Nevens F, et al. Long-term exposure to sorafenib of liver cancer cells induces resistance with epithelial-to-mesenchymal transition, increased invasion and risk of rebound growth. Cancer Lett 2013;329:74-83.
  • 27. Jiang ZS, Sun YZ, Wang SM, Ruan JS. Epithelial-mesenchymal transition: potential regulator of ABC transporters in tumor progression. J Cancer 2017;8:2319-27.
  • 28. Saxena M, Stephens MA, Pathak H, Rangarajan A. Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters. Cell Death Dis 2011;2:e179.
  • 29. Choi HS, Kim YK, Yun PY. Upregulation of MDR- and EMT-related molecules in cisplatin-resistant human oral squamous cell carcinoma cell lines. Int J Mol Sci 2019;20:3034.
  • 30. Saengkhae C, Loetchutinat C, Garnier-Suillerot A. Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1). Biophys J 2003;85:2006-14.
  • 31. Jouan E, Le Vee M, Denizot C, Da Violante G, Fardel O. The mitochondrial fluorescent dye rhodamine 123 is a high-affinity substrate for organic cation transporters (OCTs) 1 and 2. Fundam Clin Pharmacol 2014;28:65-77.
  • 32. Hu S, Chen Z, Franke R, Orwick S, Zhao M, Rudek MA, et al. Interaction of the multikinase inhibitors sorafenib and sunitinib with solute carriers and ATP-binding cassette transporters. Clin Cancer Res 2009;15:6062-9.
  • 33. Lagas JS, van Waterschoot RA, Sparidans RW, Wagenaar E, Beijnen JH, Schinkel AH. Breast cancer resistance protein and P-glycoprotein limit sorafenib brain accumulation. Mol Cancer Ther 2010;9:319-26.
  • 34. Huang WC, Hsieh YL, Hung CM, Chien PH, Chien YF, Chen LC, et al. BCRP/ABCG2 inhibition sensitizes hepatocellular carcinoma cells to sorafenib. PloS One 2013;8:e83627.
  • 35. Llovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S, et al. Hepatocellular carcinoma. Nat Rev Dis Primers 2021;7:6.
  • 36. Bolondi L, Craxi A, Trevisani F, Daniele B, Di Costanzo GG, Fagiuoli S, et al. Refining sorafenib therapy: lessons from clinical practice. Future Oncol 2015;11:449-65.
  • 37. Nishikawa H, Osaki Y, Endo M, Takeda H, Tsuchiya K, Joko K, et al. Comparison of standard-dose and half‑dose sorafenib therapy on clinical outcome in patients with unresectable hepatocellular carcinoma in field practice: a propensity score matching analysis. Int J Oncol 2014;45:2295-302.
  • 38. Reiss KA, Yu S, Mamtani R, Mehta R, D'Addeo K, Wileyto EP, et al. Starting dose of sorafenib for the treatment of hepatocellular carcinoma: a retrospective, multi-institutional study. J Clin Oncol 2017;35:3575-81.
Year 2023, Volume: 9 Issue: 2, 367 - 374, 04.03.2023
https://doi.org/10.18621/eurj.1206680

Abstract

Project Number

Grant date: 24/02/2021

References

  • 1. Keating GM. Sorafenib: a review in hepatocellular carcinoma. Target Oncol 2017;12:243-53.
  • 2. Karbownik A, Szkutnik-Fiedler D, Czyrski A, Kostewicz N, Kaczmarska P, Bekier M, et al. Pharmacokinetic interaction between sorafenib and atorvastatin, and sorafenib and metformin in rats. Pharmaceutics 2020;12:600.
  • 3. Pang Y, Eresen A, Zhang Z, Hou Q, Wang Y, Yaghmai V, et al. Adverse events of sorafenib in hepatocellular carcinoma treatment. Am J Cancer Res 2022;12:2770-82.
  • 4. Tak KY, Nam HC, Choi JY, Yoon SK, Kim CW, Kim HY, et al. Effectiveness of sorafenib dose modifications on treatment outcome of hepatocellular carcinoma: analysis in real-life settings. Int J Cancer 2020;147:1970-8.
  • 5. Labeur TA, Hofsink Q, Takkenberg RB, van Delden OM, Mathôt RAA, Schinner R, et al. The value of sorafenib trough levels in patients with advanced hepatocellular carcinoma - a substudy of the SORAMIC trial. Acta Oncol 2020;59:1028-35.
  • 6. Gurzu S, Kobori L, Fodor D, Jung I. Epithelial mesenchymal and endothelial mesenchymal transitions in hepatocellular carcinoma: a review. BioMed Res Int 2019;2019:2962580.
  • 7. Myong NH. Loss of E-cadherin and acquisition of Vimentin in epithelial-mesenchymal transition are noble indicators of uterine cervix cancer progression. Korean J Pathol 2012;46:341-8.
  • 8. Jou J, Diehl AM. Epithelial-mesenchymal transitions and hepatocarcinogenesis. J Clin Invest 2010;120:1031-4.
  • 9. Duan B, Huang C, Bai J, Zhang YL, Wang X, Yang J, et al. Multidrug resistance in hepatocellular carcinoma. In: JEE T-P, ed. Hepatocellular carcinoma [Internet]. Chapter 8, Brisbane (AU); 2019.
  • 10. Fornari F, Giovannini C, Piscaglia F, Gramantieri L. Elucidating the molecular basis of sorafenib resistance in hcc: current findings and future directions. J Hepatocell Carcinoma 2021;8:741-57.
  • 11. Cabral LKD, Tiribelli C, Sukowati CHC. Sorafenib resistance in hepatocellular carcinoma: the relevance of genetic heterogeneity. Cancers (Basel) 2020;12:1576.
  • 12. Marin JJG, Monte MJ, Macias RIR, Romero MR, Herraez E, Asensio M, et al. Expression of chemoresistance-associated abc proteins in hepatobiliary, pancreatic and gastrointestinal cancers. Cancers (Basel) 2022;14:3524.
  • 13. Chen YA, Ho CL, Ku MT, Hwu L, Lu CH, Chiu SJ, et al. Detection of cancer stem cells by EMT-specific biomarker-based peptide ligands. Sci Rep 2021;11:22430.
  • 14. Dönmez Çakıl Y, Sitar ME, Özünal ZG, Kayalı D, Gülhan Aktas R. Flow cytometric evaluation of cancer stem cell markers in HepG2 cells following sorafenib treatment. Int J Med Biochem 2021;4:200-4.
  • 15. Cho Y, Kim YK. Cancer stem cells as a potential target to overcome multidrug resistance. Front Oncol 2020;10:764.
  • 16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-8.
  • 17. Wang H, Chirshev E, Hojo N, Suzuki T, Bertucci A, Pierce M, et al. The epithelial-mesenchymal transcription factor SNAI1 represses transcription of the tumor suppressor miRNA let-7 in cancer. Cancers (Basel) 2021;13:1469.
  • 18. Tang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, et al. The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther 2020;5:87.
  • 19. Zhou J, Sun H, Wang Z, Cong W, Wang J, Zeng M, et al. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (2019 Edition). Liver Cancer 2020;9:682-720.
  • 20. Yang MH, Chen CL, Chau GY, Chiou SH, Su CW, Chou TY, et al. Comprehensive analysis of the independent effect of twist and snail in promoting metastasis of hepatocellular carcinoma. Hepatology 2009;50:1464-74.
  • 21. Chen YL, Lv J, Ye XL, Sun MY, Xu Q, Liu CH, et al. Sorafenib inhibits transforming growth factor β1-mediated epithelial-mesenchymal transition and apoptosis in mouse hepatocytes. Hepatology 2011;53:1708-18.
  • 22. Ha TY, Hwang S, Moon KM, Won YJ, Song GW, Kim N, et al. Sorafenib inhibits migration and invasion of hepatocellular carcinoma cells through suppression of matrix metalloproteinase expression. Anticancer Res 2015;35:1967-76.
  • 23. Dong S, Kong J, Kong F, Kong J, Gao J, Ji L, et al. Sorafenib suppresses the epithelial-mesenchymal transition of hepatocellular carcinoma cells after insufficient radiofrequency ablation. BMC Cancer 2015;15:939.
  • 24. Dong J, Zhai B, Sun W, Hu F, Cheng H, Xu J. Activation of phosphatidylinositol 3-kinase/AKT/snail signaling pathway contributes to epithelial-mesenchymal transition-induced multi-drug resistance to sorafenib in hepatocellular carcinoma cells. PloS One 2017;12:e0185088.
  • 25. Tian X, Yan T, Liu F, Liu Q, Zhao J, Xiong H, et al. Link of sorafenib resistance with the tumor microenvironment in hepatocellular carcinoma: Mechanistic insights. Front Pharmacol 2022;13:991052.
  • 26. van Malenstein H, Dekervel J, Verslype C, Van Cutsem E, Windmolders P, Nevens F, et al. Long-term exposure to sorafenib of liver cancer cells induces resistance with epithelial-to-mesenchymal transition, increased invasion and risk of rebound growth. Cancer Lett 2013;329:74-83.
  • 27. Jiang ZS, Sun YZ, Wang SM, Ruan JS. Epithelial-mesenchymal transition: potential regulator of ABC transporters in tumor progression. J Cancer 2017;8:2319-27.
  • 28. Saxena M, Stephens MA, Pathak H, Rangarajan A. Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters. Cell Death Dis 2011;2:e179.
  • 29. Choi HS, Kim YK, Yun PY. Upregulation of MDR- and EMT-related molecules in cisplatin-resistant human oral squamous cell carcinoma cell lines. Int J Mol Sci 2019;20:3034.
  • 30. Saengkhae C, Loetchutinat C, Garnier-Suillerot A. Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1). Biophys J 2003;85:2006-14.
  • 31. Jouan E, Le Vee M, Denizot C, Da Violante G, Fardel O. The mitochondrial fluorescent dye rhodamine 123 is a high-affinity substrate for organic cation transporters (OCTs) 1 and 2. Fundam Clin Pharmacol 2014;28:65-77.
  • 32. Hu S, Chen Z, Franke R, Orwick S, Zhao M, Rudek MA, et al. Interaction of the multikinase inhibitors sorafenib and sunitinib with solute carriers and ATP-binding cassette transporters. Clin Cancer Res 2009;15:6062-9.
  • 33. Lagas JS, van Waterschoot RA, Sparidans RW, Wagenaar E, Beijnen JH, Schinkel AH. Breast cancer resistance protein and P-glycoprotein limit sorafenib brain accumulation. Mol Cancer Ther 2010;9:319-26.
  • 34. Huang WC, Hsieh YL, Hung CM, Chien PH, Chien YF, Chen LC, et al. BCRP/ABCG2 inhibition sensitizes hepatocellular carcinoma cells to sorafenib. PloS One 2013;8:e83627.
  • 35. Llovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S, et al. Hepatocellular carcinoma. Nat Rev Dis Primers 2021;7:6.
  • 36. Bolondi L, Craxi A, Trevisani F, Daniele B, Di Costanzo GG, Fagiuoli S, et al. Refining sorafenib therapy: lessons from clinical practice. Future Oncol 2015;11:449-65.
  • 37. Nishikawa H, Osaki Y, Endo M, Takeda H, Tsuchiya K, Joko K, et al. Comparison of standard-dose and half‑dose sorafenib therapy on clinical outcome in patients with unresectable hepatocellular carcinoma in field practice: a propensity score matching analysis. Int J Oncol 2014;45:2295-302.
  • 38. Reiss KA, Yu S, Mamtani R, Mehta R, D'Addeo K, Wileyto EP, et al. Starting dose of sorafenib for the treatment of hepatocellular carcinoma: a retrospective, multi-institutional study. J Clin Oncol 2017;35:3575-81.
There are 38 citations in total.

Details

Primary Language English
Subjects Biochemistry and Cell Biology (Other)
Journal Section Original Articles
Authors

Yaprak Dönmez Çakıl 0000-0002-4605-1167

Zeynep Akbulut 0000-0002-7526-8496

Gamze Demirel 0000-0002-5501-3736

Ranan Gülhan 0000-0002-4474-7371

Zeynep Ozunal 0000-0002-3060-1507

Project Number Grant date: 24/02/2021
Publication Date March 4, 2023
Submission Date November 18, 2022
Acceptance Date February 3, 2023
Published in Issue Year 2023 Volume: 9 Issue: 2

Cite

AMA Dönmez Çakıl Y, Akbulut Z, Demirel G, Gülhan R, Ozunal Z. The effects of low-dose sorafenib on epithelial-mesenchymal transition and multidrug resistance markers in HepG2 cell line. Eur Res J. March 2023;9(2):367-374. doi:10.18621/eurj.1206680

e-ISSN: 2149-3189 


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