Flavokawain A Reduces Tumor-Initiating Properties and Stemness of Prostate Cancer.

We have previously demonstrated the in vivo chemopreventive efficacy of flavokawain A (FKA), a novel chalcone from the kava plant, in prostate carcinogenesis models. However, the mechanisms of the anticarcinogenic effects of FKA remain largely unknown. We evaluated the effect of FKA on prostate tumor spheroid formation by prostate cancer stem cells, which were sorted out from CD44+/CD133+ prostate cancer cells 22Rv1 and DU145. FKA treatment significantly decreased both the size and numbers of the tumor spheroids over different generations of spheroid passages. In addition, the dietary feeding of FKA-formulated food to Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice bearing CD44+/CD133+ 22Rv1 xenograft tumors resulted in a significant reduction of tumor growth compared to those fed with vehicle control food-fed mice. Furthermore, the expression of stem cell markers, such as Nanog, Oct4, and CD44, were markedly downregulated in both tumor spheroids and tumor tissues. We also observed that FKA inhibits Ubc12 neddylation, c-Myc, and keratin-8 expression in both CD44+/CD133+ prostate tumor spheroids and xenograft tumors. Our results suggest that FKA can reduce the tumor-initiating properties and stemness of prostate cancer, which provides a new mechanism for the chemoprevention efficacy of FKA.

Kava root extracts hinder prostate cancer development and tumorigenesis by involvement of dual inhibition of MAO-A and LSD1.

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.

Gartanin is a novel NEDDylation inhibitor for induction of Skp2 degradation, FBXW2 expression, and autophagy.

Gartanin, a 4-prenylated xanthone, has been identified from the purple mangosteen fruit as a potent growth inhibitor of various cancer cell lines, including prostate cancer. However, much of Gartanin's anticancer mechanism remains unknown. We have discovered that Gartanin docked onto the regulatory subunit of the precursor cell-expressed developmentally downregulated 8 (NEDD8)-activating enzyme (NAE) complex and next to the NEDD8 binding complex, which leads to inhibit NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 NEDDylation in an in vitro assay. The S phase kinase-associated protein (Skp2) and F-box and WD-repeat domain-containing 2 (FBXW2), the NEDD8 family members of E3 ubiqutin ligases, were also downregulated and upregulated by Gartainin, respectively. Knock-down of NEDD8 expression by short harpin (Sh) RNAs blocked or attenuated these effects of Gartainin. Finally, Gartanin demonstrated its ability to inhibit growth of prostate cancer lines via autophagy initiation. Our data support that Gartanin is a naturally occurring NEDDylation inhibitor and deserves further investigation for prostate cancer prevention and treatment.

Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation.

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.

Bioactive heterocycles containing a 3,4,5-trimethoxyphenyl fragment exerting potent antiproliferative activity through microtubule destabilization.

Novel bioactive heterocycles containing a 3,4,5-trimethoxyphenyl fragment as antiproliferative agents by targeting tubulin were synthesized and their preliminary structure activity relationships (SARs) were explored. Among all these chemical agents, 2-(Benzo[d]oxazol-2-ylthio)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (4d) exhibited the potent antiproliferative activity against MGC-803 cells with an IC50 value of 0.45 µM by induction of G2/M pahse arrest and cell apoptosis. In addition, 4d could change the membrane potential (ΔΨ) of the mitochondria against MGC-803 cells. Importantly, 4d acted as a novel tubulin polymerization inhibitor binding to colchicine site with an IC50 value of 3.35 µM.