Combination chemotherapy with Zyflamend reduced the acquired resistance of bladder cancer cells to cisplatin through inhibiting NFκB signaling pathway

Yanshi Xue,1,* Lin Yang,2 Junzun Li,3 Yilin Yan,4 Qinghui Jiang,5,6 Lan Shen,3 Shuai Yang,3 Bing Shen,4 Ruimin Huang,5,6 Jun Yan,3,7,* Hongqian Guo1 1Department of Urology, Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, China; 2Department of Urology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Institute of Urology, Nanjing University, Nanjing, China; 3MOE Key Laboratory of Model Animals for Disease Study and State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center of Nanjing University, Nanjing, China; 4Department of Urology, Shanghai General Hospital, Shanghai Jiaotong University, Shanghai, China; 5University of Chinese Academy of Sciences, Beijing, China; 6Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China; 7Collaborative Innovation Center of Genetics and Development, Shanghai, China *These authors equally contributed to this work Background: Cisplatin-based chemotherapy is mainstay treatment in urinary bladder cancer (UBC). However, tumor recurrence frequently occurs with the acquisition of cisplatin resistance. We explored the potential effect of a polyherbal preparation, Zyflamend, on UBC cells resistant to cisplatin treatment. Methods: To establish a cisplatin-resistant human bladder cancer cell line, T24 cells were cultured in increasing concentrations of cisplatin for more than 10 months. These cells (T24R) were then treated with different concentrations of Zyflamend, and both proliferation and activity of nuclear factor kappaB (NFκB) signaling pathway were examined. To test the synergistic effect between Zyflamend and cisplatin, we treated T24R cells either with Zyflamend or cisplatin alone, or in combination. Apoptotic effect was evaluated by Annexin V/propidium iodide double staining, and the levels of the proteins involved in cell cycle and anti-apoptosis were examined by Western blotting. Finally, mice with palpable xenograft were treated either with cisplatin and Zyflamend alone or in combination for 28 days before they were sacrificed for measuring the sizes and weights of the tumor tissues. In addition, proliferation and apoptosis markers were examined by immunohistochemistry. Results: Comparing to that in the parental T24 cells, NFκB is constitutively active in cisplatin-resistant T24R cells. Zyflamend is capable of inhibiting the growth of T24, T24R, as well as another UBC cell line J82 in a concentration-dependent manner. Mechanistically, Zyflamend suppresses NFκB-mediated cell proliferation, survival, and invasion/angiogenesis and induces apoptosis. In addition, Zyflamend significantly increased the sensitivity of T24R and J82 cells to cisplatin treatment and these findings were confirmed in T24R xenograft model with reduced proliferation index and decreased expression of RelA and its downstream target MMP9. Conclusion: Zyflamend is capable of counteracting bladder cancer resistance to cisplatin by repressing proliferation and inducing apoptosis through targeting NFκB signaling pathway. Keywords: Zyflamend, cisplatin, bladder cancer, NFκB, acquired resistanc