The incidence of human urinary bladder cancer increases markedly with age, suggesting a mechanistic connection between aging and bladder carcinogenesis and a potential use of anti-aging agents in bladder cancer chemoprevention. Rhodiola rosea, growing in high altitude or cold regions of the world, has been reported to have anti-aging effects in Drosophila. We demonstrated that a R. rosea extract and one of its bioactive components, salidroside, inhibited the growth of bladder cancer cell lines with a minimal effect on nonmalignant bladder epithelial cells TEU-2. Interestingly, the R. rosea extract and salidroside component exhibited a selective ability to inhibit the growth of p53 knockout primary mouse embryo fibroblasts (p53-/- MEFs) compared to their wild-type counterparts. The growth inhibitory effects of the R. rosea extract and salidroside were, however, attenuated in TSC2 and p53 double knock MEFs (TSC2-/-, p53-/- MEFs), suggesting that TSC2 protein is, at least in part, required for the growth inhibitory effects of the R. rosea extract and salidroside. The R. rosea extract and salidroside treatment of UMUC3 cells resulted in an increase of AMP-activated protein kinase (AMPK)-α phosphorylation and a decrease of 4E-BP1 phosphorylation, leading to increased binding of 4E-BP1 to m7 GTP. These results indicate that the R. rosea extract and salidroside inhibit translation initiation. Furthermore, both the R. rosea extract and salidroside treatment of UMUC3 cells caused a significant percentage of cells undergoing autophagy. Therefore, the R. rosea extract and salidroside deserve further study as novel agents for chemoprevention of bladder carcinogenesis.

Aim

Here, we aim to evaluate the chemopreventive efficacy of kava root extracts (KRE) in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and investigate potential molecular targets of kavalactones, the main components of kava.

Methods

TRAMP mice were administrated with KRE formulated food for different periods of time, and then the incidences of high-grade prostatic intraepithelial neoplasia (HG-PIN) and adenocarcinomas and tumor burdens were compared between vehicle control and KRE food fed groups. In addition, the inhibitory effect of the KRE and kavalactones on monoamine oxidase A (MAO-A) and lysine-specific demethylase 1 (LSD1) enzyme activities were examined by commercially available inhibitor screening kits. Histone H3 lysine 9 dimethylation was also evaluated in prostate cancer cells and tumor tissues using Western blotting analysis.

Results

Dietary feeding of 0.3% and 0.6% KRE to TRAMP mice from ages of 6 weeks to 12 weeks inhibited HG-PIN by 43.5% and 59.7%, respectively, and prostate adenocarcinoma by 53.5% and 66.4%, respectively. In addition, 0.6% KRE fed TRAMP mice from ages of 6 weeks to 24 weeks exhibited a significant reduction of genitourinary weight (a surrogate of tumor burden) by 54.5% and reduced body weight gain. Furthermore, the KRE and kavalactones showed a significant inhibition of LSD1 and MAO-A enzyme activities.

Conclusion

Our results suggest that consumption of kava products through diet can delay prostate cancer development and progression and that kavalactones may be a new structure model for developing a potent dual inhibitor of LSD1 and MAO-A.

Flavokawain A (FKA), a major chalcone in the Kava plant, has recently demonstrated promising anti-cancer activities. A systematic evaluation of FKA's safety profile has not been reported before. In this study, male FVB/N mice were fed with an AIN-76A diet or AIN-76A diet supplemented with 0.6% (6 g/kg food) FKA or 0.6% commercial kava root extract (KRE) for three weeks. Dietary feeding of FKA did not affect food consumption and body weight. Histopathological examination of liver, kidney, colon, lung, heart, spleen, and thymus revealed no signs of FKA-induced toxicity. Biochemical serum analysis and histological examination confirmed normal organ function in FKA-treated mice. The cytotoxicity profile showed FKA had minimal side effects on bone marrow and small intestinal epithelial cells compared with Adriamycin. In addition, oral feeding of FKA increased activities of both glutathione S-transferase and quinone reductase in the liver, lung, prostate and bladder tissues of mice. In comparison, dietary feeding of 0.6% KRE increased liver/body weight ratio and decreased spleen, thymus, and testis/body weight ratios, as well as induced nodular proliferation in liver tissues. Therefore, dietary feeding FKA showed no adverse effects on major organ function and homeostasis in mice, suggesting the potential of FKA for chemoprevention study of human cancers.

Background

Recent evidence indicates that insulin resistance in skeletal muscle may be related to reduce mitochondrial number and oxidation capacity. However, it is not known whether increasing mitochondrial number and function improves insulin resistance. In the present study, we investigated the effects of a combination of nutrients on insulin resistance and mitochondrial biogenesis/function in skeletal muscle of type 2 diabetic Goto–Kakizaki rats.

Methodology/Principal Findings

We demonstrated that defect of glucose and lipid metabolism is associated with low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle of the diabetic Goto-Kakizaki rats. The treatment of combination of R-α-lipoic acid, acetyl-L-carnitine, nicotinamide, and biotin effectively improved glucose tolerance, decreased the basal insulin secretion and the level of circulating free fatty acid (FFA), and prevented the reduction of mitochondrial biogenesis in skeletal muscle. The nutrients treatment also significantly increased mRNA levels of genes involved in lipid metabolism, including peroxisome proliferator–activated receptor-α (Pparα), peroxisome proliferator–activated receptor-δ (Pparδ), and carnitine palmitoyl transferase-1 (Mcpt-1) and activity of mitochondrial complex I and II in skeletal muscle. All of these effects of mitochondrial nutrients are comparable to that of the antidiabetic drug, pioglitazone. In addition, the treatment with nutrients, unlike pioglitazone, did not cause body weight gain.

Conclusions/Significance

These data suggest that a combination of mitochondrial targeting nutrients may improve skeletal mitochondrial dysfunction and exert hypoglycemic effects, without causing weight gain.

Flavokawain A (FKA) is the predominant chalcone identified from the kava plant. We have previously shown that FKA preferentially inhibits the growth of p53 defective bladder cancer cell lines. Here, we examined whether FKA could inhibit bladder cancer development and progression in vivo in the UPII-SV40T transgenic model that resembles human urothelial cell carcinoma (UCC) with defects in the p53 and the retinoblastoma (Rb) protein pathways. Genotyped UPII-SV40T mice were fed orally with vehicle control (AIN-93M) or FKA (6 g/kg food; 0.6%) for 318 days starting at 28 days of age. More than 64% of the male mice fed with FKA-containing food survived beyond 318 days of age, whereas only about 38% of the male mice fed with vehicle control food survived to that age (P = 0.0383). The mean bladder weights of surviving male transgenic mice with the control diet versus the FKA diet were 234.6 ± 72.5 versus 96.1 ± 69.4 mg (P = 0.0002). FKA was excreted primarily through the urinary tract and concentrated in the urine up to 8.4 μmol/L, averaging about 38 times (males) and 15 times (females) more concentrated than in the plasma (P = 0.0001). FKA treatment inhibited the occurrence of high-grade papillary UCC, a precursor to invasive urothelial cancer, by 42.1%. A decreased expression of Ki67, survivin, and X-linked inhibitor of apoptotic proteins (XIAP) and increased expression of p27 and DR5, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive apoptotic cells were observed in the urothelial tissue of FKA-fed mice. These results suggest a potential of FKA in preventing the recurrence and progression of non-muscle-invasive UCC.

Background

Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown.

Methods

An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods.

Results

FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2.

Conclusion

These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components (kavalactones and flavokawains) on PCa growth and androgen receptor (AR) expression. PCa cell lines (LNCaP, LAPC-4, 22Rv1, C4-2B, DU145 and PC-3) with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones (kawain, 5′6′-dehydrokawain, yangonin, methysticin) and flavokawain B. Expression of AR and its target genes (PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR.

S phase kinase-associated protein 2 (Skp2) has been shown to be required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb) deficient mice. Here we have demonstrated that flavokawain A (FKA), a novel chalcone from the kava plant, selectively inhibited the growth of pRb deficient cell lines and resulted in a proteasome-dependent and ubiquitination-mediated Skp2 degradation. Degradation of Skp2 by FKA was found to be involved in a functional Cullin1, but independent of Cdh1 expression. Further studies have demonstrated that FKA docked into the ATP binding pocket of the precursor cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) complex, inhibited NEDD8 conjugations to both Cullin1 and Ubc12 in PC3 cells and Ubc12 NEDDylation in an in vitro assay. Finally, dietary feeding of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with FKA inhibited the formation of high-grade prostatic intra-epithelial neoplasia lesions (HG-PIN) and prostate adenocarcinomas, reduced the tumor burden and completely abolished distant organ metastasis. Immunohistochemistry studies revealed that dietary FKA feeding resulted in marked anti-proliferative and apoptotic effects via down-regulation of Skp2 and NEDD8 and up-regulation of p27/Kip1 in the prostate of TRAMP mice. Our findings therefore provide evidence that FKA is a promising NEDDylation inhibitor for targeting Skp2 degradation in prostate cancer prevention and treatment.

Background: Aberrations 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. Results: The 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. Conclusion: These 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.

Abstract Background Aberrations 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. Results The 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. Conclusion These 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.