膀胱灌注chidamide透過Axl信號路徑協同mitomycin C治療基因毒性致癌物誘導之膀胱癌

張乃文¹、張雅娟²、余家瑩²、王紹全^1,2、陳順郎^1,2、宋文瑋^1,2,*

¹中山醫學大學附設醫院泌尿科；²中山醫學大學醫學系

Synergistic effect of intravesical instillation of chidamide and mitomycin C treatment through Axl signaling in genotoxic carcinogen–induced bladder cancer

Nai-Wen Chang¹, Ya-Chuan Chang², Chia-Ying Yu², Shao-Chuan Wang^1,2, Sung-Lang Chen^1,2, Wen-Wei Sung^1,2,*

¹Department of Urology, Chung Shan Medical University Hospital, Taichung, Taiwan, 40201; ²School of Medicine, Chung Shan Medical University, Taichung, Taiwan, 40201; ^*Corresponding author

Purpose:

Intravesical instillation (IVI) of chemotherapy drugs or Bacillus Calmette-Guerin (BCG) can prevent tumor recurrence. However, BCG has been out of stock in recent years; therefore, alternative treatments are needed. Variations occur in genes associated with histone modifications during tumorigenesis, and histone deacetylases (HDACs) play a key role. This suggests that HDAC inhibitors like chidamide might have a potential therapeutic role or even a synergistic effect with chemotherapy drugs in the IVI treatment of bladder cancer.

Materials and Methods:

The cytotoxicity of a combination of chidamide and mitomycin C was investigated in bladder cancer cell lines via colorimetric assays. Cell cycle and cell death analyses were performed by Hoechst staining and flow cytometry. Related molecular pathways were screened via protein arrays and further confirmed by western blotting. The synergistic effect of IVI of chidamide and mitomycin C was examined in vivo in an N-methyl-N-nitrosourea (MNU)-induced rat bladder cancer model, using the Ki67 index for quantitative analysis. Related signaling pathways were investigated by immunohistochemistry. The statistical significance of differences was determined by Student’s t test; p < 0.05 was considered statistically significant.

Results:

A synergistic cytotoxic effect of chidamide and mitomycin C was confirmed in the T24 and UMUC3 cell lines (treatment duration 1 hour, combination index <0.6). Significant induction of cell apoptosis was confirmed by increases in Annexin V+ cells and condensed nuclei, indicating apoptotic cells, observed with Hoechst staining (p<0.05). The protein arrays identified the Axl signaling pathway as a key target of the synergistic effect. Significant suppression of Axl and its downstream molecules were confirmed in both cell lines. In vivo, rats with MNU-induced bladder cancer receiving IVI of chidamide plus mitomycin C had a significantly reduced Ki67 index and lower expression of Axl and its downstream molecules compared with mitomycin C-treated or untreated MNU-induced rats (p<0.05).

Conclusion:

Our study provided evidence for a synergistic effect of IVI of chidamide and mitomycin C in reducing MNU-induced bladder cancer through the Axl signaling pathway. This combination therapy might provide new insights for further clinical trials.