Benzidine Induces Epithelial-Mesenchymal Transition of Human Bladder Cancer Cells through Activation of ERK5 Pathway

  • Sun, Xin (Department of Urology, The Second Affiliated Hospital of Anhui Medical University) ;
  • Zhang, Tao (Department of Urology, The Second Affiliated Hospital of Anhui Medical University) ;
  • Deng, Qifei (Second Department of Urology, Anhui Provincial Children's Hospital) ;
  • Zhou, Qirui (Department of Otolaryngology, The Second Affiliated Hospital of Anhui Medical University) ;
  • Sun, Xianchao (Department of Urology, The Second Affiliated Hospital of Anhui Medical University) ;
  • Li, Enlai (Department of Urology, The Second Affiliated Hospital of Anhui Medical University) ;
  • Yu, Dexin (Department of Urology, The Second Affiliated Hospital of Anhui Medical University) ;
  • Zhong, Caiyun (Department of Nutrition and Food Safety, School of Public Health, Nanjing Medical University)
  • Received : 2017.07.02
  • Accepted : 2017.12.20
  • Published : 2018.03.31


Benzidine, a known carcinogen, is closely associated with the development of bladder cancer (BC). Epithelial-mesenchymal transition (EMT) is a critical pathophysiological process in BC progression. The underlying molecular mechanisms of mitogen-activated protein kinase (MAPK) pathway, especially extracellular regulated protein kinases 5 (ERK5), in regulating benzidine-induced EMT remains unclarified. Hence, two human bladder cell lines, T24 and EJ, were utilized in our study. Briefly, cell migration was assessed by wound healing assay, and cell invasion was determined by Transwell assay. Quantitative PCR and western blot were utilized to determine both gene expressions as well as protein levels of EMT and MAPK, respectively. Small interfering RNA (siRNA) was transfected to further determine ERK5 function. As a result, the migration and invasion abilities were enhanced, epithelial marker expression was decreased while mesenchymal marker expression was increased in human BC cell lines. Meanwhile, benzidine administration led to activation of ERK5 and activator protein 1 (AP-1) proteins, without effective stimulation of the Jun N-terminal kinase (JNK) or p38 pathways. Moreover, Benzidine-induced EMT and ERK5 activation were completely suppressed by XMD8-92 and siRNAs specific to ERK5. Of note, ERK1/2 was activated in benzidine-treated T24 cells, while benzidine-induced EMT could not be reversed by U0126, an ERK1/2 inhibitor, as indicated by further study. Collectively, our findings revealed that ERK5-mediated EMT was critically involved in benzidine-correlated BC progression, indicating the therapeutic significance of ERK5 in benzidine-related BC.


Supported by : National Natural Science Foundation of China


  1. Bakiri, L., Macho-Maschler, S., Custic, I., Niemiec, J., GuioCarrion, A., Hasenfuss, SC., Eger, A., Muller, M., Beug, H., and Wagner, E.F. (2015). Fra-1/AP-1 induces EMT in mammary epithelial cells by modulating Zeb1/2 and $TGF{\beta}$ expression. Cell Death Differ. 22, 336-350.
  2. Baan, R., Straif, K., Grosse, Y., Secretan, B., El. Ghissassi, F., Bouvard, V., Benbrahim-Tallaa, L., Cogliano, V., and WHO International Agency for Research on Cancer Monograph Working Group. (2008). Carcinogenicity of some aromatic amines, organic dyes, and related exposures. Lancet Oncol. 9, 322-323.
  3. Boffetta, P. (2008). Tobacco smoking and risk of bladder cancer. Scand J. Urol. Nephrol. Suppl. 218, 45-54.
  4. Chung, K.T. (2016). Azo dyes and human health: A review. J. Environ. Sci. Health C. Environ. Carcinog. Ecotoxicol. Rev. 34, 233-261.
  5. Geng, H., Zhao, L., Liang, Z., Zhang, Z., Xie, D., Bi, L., Wang, Y., Zhang, T., Cheng, L., Yu, D., et al. (2015). ERK5 positively regulates cigarette smoke-induced urocystic epithelial-mesenchymal transition in SV-40 immortalized human urothelial cells. Oncol Rep. 34, 1581-1588.
  6. Hoang, V.T., Yan, T.J., Cavanaugh, J.E., Flaherty, P.T., Beckman, B.S., and Burow, M.E. (2017). Oncogenic signaling of MEK5-ERK5. Cancer Lett. 392, 51-59.
  7. Hui, K., Gao, Y., Huang, J., Xu, S., Wang, B., Zeng, J., Fan, J., Wang, X., Yue, Y., Wu, S., et al. (2017). RASAL2, a RAS GTPase-activating protein, inhibits stemness and epithelial-mesenchymal transition via MAPK/SOX2 pathway in bladder cancer. Cell Death Dis. 8, e2600.
  8. IARC (1987). Overall evaluations of carcinogenicity. IARC monographs on the evaluation of carcinogenic risk of chemicals to humans, supplement 7. (Lyon, France: International Agency for Research on Cancer). pp. 440-447.
  9. Kiselyov, A., Bunimovich-Mendrazitsky, S., and Startsev, V. (2016). Key signaling pathways in the muscle-invasive bladder carcinoma: Clinical markers for disease modeling and optimized treatment. Int. J. Cancer 138, 2562-2569.
  10. Liang, Z., Xie, W., Wu, R., Geng, H., Zhao, L., Xie, C., Li, X., Huang, C., Zhu, J., Zhu, M., et al. (2015a). ERK5 negatively regulates tobacco smoke-induced pulmonary epithelial-mesenchymal transition. Oncotarget 6, 19605-19618.
  11. Liang, Z., Xie, W., Wu, R., Geng, H., Zhao, L., Xie, C., Li, X., Zhu, M., Zhu, W., and Zhu, J., et al. (2015b). Inhibition of tobacco smokeinduced bladder MAPK activation and epithelial-mesenchymal transition in mice by curcumin. Int. J. Clin. Exp. Pathol. 8, 4503-4513.
  12. Liang, Z., Wu, R., Xie, W., Xie, C., Wu, J., Geng, S., Li, X., Zhu, M., Zhu, W., and Zhu, J., et al. (2017). Effects of Curcumin on Tobacco Smoke-induced Hepatic MAPK Pathway Activation and Epithelial-Mesenchymal Transition In Vivo. Phytother Res. doi: 10.1002
  13. Liu, F., Zhang, H., and Song, H. (2017). Upregulation of MEK5 by Stat3 promotes breast cancer cell invasion and metastasis. Oncol Rep. 37, 83-90.
  14. Liu, Z., Liu, J., Zhao, L., Geng, H., Ma, J., Zhang, Z., Yu, D., and Zhong, C. (2017). Curcumin reverses benzidine-induced epithelialmesenchymal transition via suppression of ERK5/AP-1 in SV-40 immortalized human urothelial cells. Int. J. Oncol. 50, 1321-1329.
  15. Loveridge, C.J., Mui, E.J., Patel, R., Tan, E.H., Ahmad, I., Welsh, M., Galbraith, J., Hedley, A., Nixon, C., Blyth, K., et al. (2017). Increased T-cell Infiltration Elicited by Erk5 Deletion in a Pten-Deficient Mouse Model of Prostate Carcinogenesis. Cancer Res. 77, 3158-3168.
  16. Lu, J., Zhang, Z.L., Huang, D., Tang, N., Li, Y., Peng, Z., Lu, C., Dong, Z., and Tang, F. (2016). Cdk3-promoted epithelial-mesenchymal transition through activating AP-1 is involved in colorectal cancer metastasis. Oncotarget 7, 7012-7028.
  17. Ma, Q.W., Lin, G.F., Chen, J.G., Guo, W.C., Qin, Y.Q., Golka, K., and Shen, J.H. (2012). N-Acetyltransferase 2 genotype, exfoliated urothelial cells and benzidine exposure. Front Biosci (Elite Ed). 4, 1966-1974.
  18. Min, J., Geng, H., Liu, Z., Liang, Z., Zhang, Z., Xie, D., Wang, Y., Zhang, T., Yu, D., and Zhong, C. (2017). ERK5 regulates tobacco smoke-induced urocystic epithelial-mesenchymal transition in BALB/c mice. Mol. Med. Rep. 15, 3893-3897.
  19. Ramsay, A.K., McCracken, S.R., Soofi, M., Fleming, J., Yu, A.X., Ahmad, I., Morland, R., Machesky, L., Nixon, C., and Edwards, D.R. (2011). ERK5 signalling in prostate cancer promotes an invasive phenotype. Br. J. Cancer 104, 664-672.
  20. Rovida, E., Di. Maira, G., Tusa, I., Cannito, S., Paternostro, C., Navari, N., Vivoli, E., Deng, X., Gray, N.S., Esparis-Ogando, A., et al. (2015). The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma. Gut 64, 1454-1465.
  21. Salinas-Sanchez, A.S., Serrano-Oviedo, L., Nam-Cha, S.Y., Roche-Losada, O., Sanchez-Prieto, R., and Gimenez-Bachs, J.M. (2017). Prognostic value of the VHL, $HIF-1{\alpha}$, and VEGF signaling pathway and associated MAPK (ERK1/2 andERK5) pathways in clear-cell renal cell carcinoma. a long-term study. Clin Genitourin Cancer pii: S1558- 7673(17)30153-2.
  22. Sawhney, R.S., Liu, W., and Brattain, M.G. (2009). A novel role of ERK5 in integrin-mediated cell adhesion and motility in cancer cells via Fak signaling, J. Cell Physiol. 219, 152-161.
  23. Siegel, R.L., Miller, K.D., and Jemal, A. (2016). Cancer statistics, 2016. CA. Cancer J. Clin. 66, 7-30.
  24. Sun, X., Deng, Q., Liang, Z., Zhang, Z., Zhao, L., Geng, H., Xie, D., Wang, Y., Yu, D., and Zhong, C. (2016). Curcumin reverses benzidine-induced cell proliferation by suppressing ERK1/2 pathway in human bladder cancer T24 cells. Exp. Toxicol. Pathol. 68, 215-222.
  25. Sun, X., Deng, Q., Liang, Z., Liu, Z., Geng, H., Zhao, Li., Zhou, Q., Liu, J., Ma, J., Wang, D., et al. (2017). Cigarette smoke extract induces epithelial-mesenchymal transition of human bladder cancer T24 cells through activation of ERK1/2 pathway. Biomed. Pharmacother. 86, 457-465.
  26. Trop-Steinberg, S., and Azar, Y. (2017). AP-1 Expression and its Clinical Relevance in Immune Disorders and Cancer. Am. J. Med. Sci. 353, 474-483.
  27. Weistenhofer, W., Blaszkewicz, M., Bolt, H.M., and Golka, K. (2008). N-acetyltransferase-2 and medical history in bladder cancer cases with a suspected occupational disease (BK 1301) in Germany. J. Toxicol. Environ. Health A. 71, 906-910.
  28. Won, K.J., Im, J.Y., Kim, B.K., Ban, H.S., Jung, Y.J., Jung, K.E., Won, M. (2017). Stability of the cancer target DDIAS is regulated by the CHIP/HSP70 pathway in lung cancer cells. Cell Death Dis. 8, e2554.
  29. Wu, J., Cui, H., Zhu, Z., and Wang, L. (2016). MicroRNA-200b-3p suppresses epithelial-mesenchymal transition and inhibits tumor growth of glioma through down-regulation of ERK5. Biochem. Biophys. Res. Commun. 478, 1158-1164.
  30. Yeung, K.T., and Yang. J. (2017). Epithelial-mesenchymal transition in tumor metastasis. Mol. Oncol. 11, 28-39.
  31. Yoshinaga, T., Uwabe, K., Naito, S., Higashino, K., Nakano, T., Numata, Y., and Kihara, A. (2016). AM251 suppresses epithelialmesenchymal transition of renal tubular epithelial Cells. PLoS One 11, e0167848.
  32. Zhao, L., Geng, H., Liang, Z., Zhang, Z., Zhang, T., Yu, D., and Zhong, C. (2015). Benzidine induces epithelial-mesenchymal transition in human uroepithelial cells through ERK1/2 pathway. Biochem. Biophys. Res. Commun. 459, 643-649.
  33. Zhuang, K., Zhang, J., Xiong, M., Wang, X., Luo, X., Han, L., Meng, Y., Zhang, Y., Liao, W., and Liu, S. (2016). CDK5 functions as a tumor promoter in human colorectal cancer via modulating the ERK5-AP-1 axis. Cell Death Dis. 7, e2415.