Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and in vivo through induction of cell cycle arrest at the G2-M phase.

Abstract

e16024 Background: Protein Kinase D (PKD) is implicated in tumor growth, death, invasion, and progression. CRT0066101 is an inhibitor of PKD and has antitumor activity in several types of carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer remain unknown. Methods: The MTS assay was used to evaluate the ability of CRT0066101 to inhibit cellular proliferation in bladder cancer cells. Cell cycle was analyzed by flow cytometry. Protein expression and phosphorylation were assessed by western blotting. Results: We showed that CRT0066101 suppressed the proliferation and migration of 4 bladder cancer cell lines in vitro. We also demonstrated that CRT0066101 inhibited tumor growth in an in vivo mouse model of bladder cancer. To verify the role of PKD in bladder tumor, we found that PKD2 was highly expressed in 8 bladder cancer lines and that RNA interference-mediated silencing of the PKD2 gene dramatically reduced bladder cancer growth in vitro and in vivo, suggesting that the effect of the compound in bladder cancer is mediated through inhibition of PKD2. This notion was confirmed by demonstrating that the levels of PKD2 and phospho-PKD2 (Ser-876) were markedly decreased in CRT0066101-treated bladder cancer. In addition, our cell cycle analysis by flow cytometry revealed that CRT0066101 arrested bladder cancer cells at the G2-M phase. We further validated these data by immunoblotting showing that treatment of bladder carcinoma cells with CRT0066101 downregulated the expression of cyclin B1, cdc2 and cdc25C, but elevated the levels of p27kip1, gadd45a, chk1/2, and wee1. Finally, CRT0066101 was found to increase the phosphorylation of cdc2 and cdc25C, which lead to reduction in cdc2-cyclin B1 activity. Conclusions: These novel findings suggest that CRT0066101 inhibits bladder cancer growth through modulating the cell cycle G2 checkpoint and inducing cell cycle G2-M arrest, which lead to blockade of cell cycle progression. QQL and IH contributed equally to this work.