Silibinin Causes Cell Cycle Arrest and Apoptosis in Human Bladder Transitional Cell Carcinoma Cells by Regulating CDKI-CDK-cyclin Cascade, and Caspase 3 and PARP Cleavages

Bladder cancer is the fourth and eighth most common cancer in men and women in the USA, respectively. Flavonoid phytochemicals are being studied for both prevention and therapy of various human malignancies including bladder cancer. One such naturally occurring flavonoid is silibinin isolated from milk thistle. Here, we assessed the effect of silibinin on human bladder transitional cell carcinoma (TCC) cell growth, cell cycle modulation and apoptosis induction, and associated molecular alterations, employing two different cell lines representing high-grade invasive tumor (TCC-SUP) and high-grade TCC (T-24) human bladder cancer. Silibinin treatment of these cells resulted in a significant dose- and time-dependent growth inhibition together with a G(1) arrest only at lower doses in TCC-SUP cells but at both lower and higher doses in T-24 cells; higher silibinin dose showed a G(2)/M arrest in TCC-SUP cells. In other studies, silibinin treatment strongly induced the expression of Cip1/p21 and Kip1/p27, but resulted in a decrease in cyclin-dependent kinases (CDKs) and cyclins involved in G(1) progression. Silibinin treatment also showed an increased interaction between cyclin-dependent kinase inhibitors (CDKIs)-CDKs and a decreased CDK kinase activity. Further, the G(2)/M arrest by silibinin in TCC-SUP cells was associated with a decrease in pCdc25c (Ser216), Cdc25c, pCdc2 (Tyr15), Cdc2 and cyclin B1 protein levels. In additional studies, silibinin showed a dose- and a time-dependent apoptotic death only in TCC-SUP cells that was associated with cleaved forms of caspase 3 and poly(ADP-ribose) polymerase. Together, these results suggest that silibinin modulates CDKI-CDK-cyclin cascade and activates caspase 3 causing growth inhibition and apoptotic death of human TCC cells, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in bladder cancer pre-clinical models.