Cancer Prevention Research Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/ KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the downregulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/ WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G2-M arrest in six p53 mutant-type, highgrade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G2-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G2-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G2-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer. Bladder cancer is a major public health burden. Tumor resection with possible intravesical treatments for superficial disease, and cystectomy or chemotherapy with radiation protocols for invasive bladder cancer have associated limitations. Despite improvement in treatments, patients with advanced bladder cancer still have a significant risk of mortality. Therefore, different and novel approaches need to be considered. Carcinogens such as cigarette smoking and occupational exposure to aromatic amines contribute to >75% of all bladder cancer cases in the United States (1). However, there are many potentially protective phytochemicals contained in plantderived food that are excreted through the urinary tract and concentrated in urine (2). For example, urinary levels of the free form of genistein can be 50 μmol/L or higher, levels at which genistein exhibits effective anticancer activities in vitro (3). Thus, investigation of these phytochemicals for prevention and therapeutic intervention against urinary bladder cancer is plausible. Moreover, prevention and therapeutic intervention by using well-studied, effective phytochemicals could be a newer approach with potentially fewer side effects and, if targeted correctly, more efficacy as compared with current chemotherapeutic agents in cancer management. Consumption of the traditional kava tea preparation, predominately by men, has been reported to correlate with low and uncustomary gender ratios of cancer incidences (more cancer in women than men) in three kava-drinking countries: Fiji, Vanuatu, and Western Samoa, despite the presence of many smokers in these populations (4–7). We have shown that flavokawain A, the major chalcone in kava extracts, exhibits strong antiproliferative and apoptotic effects against human urinary bladder cancer cell lines derived from different stages of bladder cancer (8). In this study, we have shown that flavokawain A differentially induced G1 and G2-M arrests in p53 wild-type RT4 cells and p53 mutant-type T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197, respectively, as the mechanisms for its cell growth–inhibitory effect. In p53 wild-type RT4 cells, flavokawain A inhibits cyclin-dependent kinase-2 (CDK2) kinase activity through the accumulation of p21/ WAF1 (p21) and p27/KIP1 (p27), and down-regulation of Authors' Affiliations: Department of Urology and Chao Family Comprehensive Cancer Center, University of California, Irvine, Orange,