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 G 1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation 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 G 2 -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 G 2 -M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G 2 -M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G 2 -M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.

BackgroundAberrations in the Wnt pathway have been reported to be involved in the metastasis of prostate cancer (PCa) to bone. We investigated the effect and underlying mechanism of a naturally-occurring Wnt inhibitor, WIF1, on the growth and cellular invasiveness of a bone metastatic PCa cell line, PC3.ResultsThe WIF1 gene promoter was hypermethylated and its expression down-regulated in the majority (7 of 8) of PCa cell lines. Restoration of WIF1 expression in PC-3 cells resulted in a decreased cell motility and invasiveness via up-regulation of epithelial markers (E-cadherin, Keratin-8 and-18), down-regulation of mesenchymal markers (N-cadherin, Fibronectin and Vimentin) and decreased activity of MMP-2 and -9. PC3 cells transfected with WIF1 consistently demonstrated reduced expression of Epithelial-to-Mesenchymal Transition (EMT) transcription factors, Slug and Twist, and a change in morphology from mesenchymal to epithelial. Moreover, WIF1 expression significantly reduced tumor growth by approximately 63% in a xenograft mouse model. This was accompanied by an increased expression of E-cadherin and Keratin-18 and a decreased expression of vimentin in tumor tissues.ConclusionThese data suggest that WIF1 regulates tumor invasion through EMT process and thus, may play an important role in controlling metastatic disease in PCa patients. Blocking Wnt signaling in PCa by WIF1 may represent a novel strategy in the future to reduce metastatic disease burden in PCa patients.

Limited success has been achieved in extending the survival of patients with metastatic and hormone-refractory prostate cancer (HRPC). There is a strong need for novel agents in the treatment and prevention of HRPC. We have shown that flavokawain B (FKB), a kava chalcone, is about 4-to 12-fold more effective in reducing the cell viabilities of androgen receptor (AR)-negative, HRPC cell lines DU145 and PC-3 than AR-positive, hormone-sensitive prostate cancer cell lines LAPC4 and LNCaP, with minimal effect on normal prostatic epithelial and stromal cells. FKB induces apoptosis with an associated increased expression of proapoptotic proteins: death receptor-5, Bim and Puma and a decreased expression of inhibitors of apoptosis protein: XIAP and survivin. Among them, Bim expression was significantly induced by FKB as early as 4 hr of the treatment. Knockdown of Bim expression by short-hairpin RNAs attenuates the inhibitory effect on anchorage-dependent andindependent growth and caspase cleavages induced by FKB. These findings suggest that the effect of FKB, at least in part, requires Bim expression. In addition, FKB synergizes with TRAIL for markedly enhanced induction of apoptosis. Furthermore, FKB treatment of mice bearing DU145 xenograft tumors results in tumor growth inhibition and increases Bim expression in tumor tissues. Together, these results suggest robust mechanisms for FKB induction of apoptosis preferentially for HRPC and the potential usefulness of FKB for prevention and treatment of HRPC in an adjuvant setting.

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G 1 arrest. The G 1 arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G 1 arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and B-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G 1 arrest include (a) SKP2 downregulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.

Docetaxel is currently the most effective drug for the treatment of castration-resistant prostate cancer (CRPC), but it only extends life by an average of 2 months. Lycopene, an antioxidant phytochemical, has antitumor activity against prostate cancer (PCa) in several models and is generally safe. We present data on the interaction between docetaxel and lycopene in CRPC models. The growth-inhibitory effect of lycopene on PCa cell lines was positively associated with insulin-like growth factor I receptor (IGF-IR) levels. In addition, lycopene treatment enhanced the growth-inhibitory effect of docetaxel more effectively on DU145 cells with IGF-IR high expression than on those PCa cell lines with IGF-IR low expression. In a DU145 xenograft tumor model, docetaxel plus lycopene caused tumor regression, with a 38% increase in antitumor efficacy (P = .047) when compared with docetaxel alone. Lycopene inhibited IGF-IR activation through inhibiting IGF-I stimulation and by increasing the expression and secretion of IGF-BP3. Downstream effects include inhibition of AKT kinase activity and survivin expression, followed by apoptosis. Together, the enhancement of docetaxel's antitumor efficacy by lycopene supplementation justifies further clinical investigation of lycopene and docetaxel combination for CRPC patients. CRPC patients with IGF-IR-overexpressing tumors may be most likely to benefit from this combination.

Lizards have highly directional ears, owing to strong acoustical coupling of the eardrums and almost perfect sound transmission from the contralateral ear. To investigate the neural processing of this remarkable tympanic directionality, we combined biophysical measurements of eardrum motion in the Tokay gecko with neurophysiological recordings from the auditory nerve. Laser vibrometry shows that their ear is a two-input system with approximately unity interaural transmission gain at the peak frequency (∼ 1.6 kHz). Median interaural delays are 260 μs, almost three times larger than predicted from gecko head size, suggesting interaural transmission may be boosted by resonances in the large, open mouth cavity (Vossen et al. 2010). Auditory nerve recordings are sensitive to both interaural time differences (ITD) and interaural level differences (ILD), reflecting the acoustical interactions of direct and indirect sound components at the eardrum. Best ITD and click delays match interaural transmission delays, with a range of 200-500 μs. Inserting a mold in the mouth cavity blocks ITD and ILD sensitivity. Thus the neural response accurately reflects tympanic directionality, and most neurons in the auditory pathway should be directional.

Although lizards have highly sensitive ears, it is difficult to condition them to sound, making standard psychophysical assays of hearing sensitivity impractical. This paper describes non-invasive measurements of the auditory brainstem response (ABR) in both Tokay geckos (Gekko gecko; nocturnal animals, known for their loud vocalizations) and the green anole (Anolis carolinensis, diurnal, non-vocal animals). Hearing sensitivity was measured in 5 geckos and 7 anoles. The lizards were sedated with isoflurane, and ABRs were measured at levels of 1 and 3% isoflurane. The typical ABR waveform in response to click stimulation showed one prominent and several smaller peaks occurring within 10 ms of the stimulus onset. ABRs to brief tone bursts revealed that geckos and anoles were most sensitive between 1.6-2 kHz and had similar hearing sensitivity up to about 5 kHz (thresholds typically 20-50 dB SPL). Above 5 kHz, however, anoles were more than 20 dB more sensitive than geckos and showed a wider range of sensitivity (1-7 kHz). Generally, thresholds from ABR audiograms were comparable to those of small birds. Best hearing sensitivity, however, extended over a larger frequency range in lizards than in most bird species.

During female mate choice, both the male's phenotype and resources (e.g. his nest) contribute to the chooser's fitness. Animals other than humans are not known to advertise resource characteristics to potential mates through vocal communication; although in some species of anurans and birds, females do evaluate male qualities through vocal communication. Here, we demonstrate that calls of the male Emei music frog (Babina dauchina), vocalizing from male-built nests, reflect nest structure information that can be recognized by females. Inside-nest calls consisted of notes with energy concentrated at lower frequency ranges and longer note durations when compared with outside-nest calls. Centre frequencies and note durations of the inside calls positively correlate with the area of the burrow entrance and the depth of the burrow, respectively. When given a choice between outside and inside calls played back alternately, more than 70 per cent of the females (33/47) chose inside calls. These results demonstrate that males of this species faithfully advertise whether or not they possess a nest to potential mates by vocal communication, which probably facilitates optimal mate selection by females. These results revealed a novel function of advertisement calls, which is consistent with the wide variation in both call complexity and social behaviour within amphibians.