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

Abstract

BackgroundFlavokawain 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.MethodsAn 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.ResultsFKB 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.ConclusionThese findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.Electronic supplementary materialThe online version of this article (10.1186/s12964-019-0338-2) contains supplementary material, which is available to authorized users.

Chalcone Derivatives: Role in Anticancer Therapy

“…Chalcones have been shown to exhibit anticancer activity through their inhibitory potential against various targets, such as 5α-reductase [ 187 ], aromatase [ 188 ], histone deacetylase inhibitors (HDAC) [ 189 , 190 ], proteasome [ 191 , 192 ], JAK/STAT signaling pathways [ 193 ], cell division cycle 25 (CD25) [ 194 ], cathepsin-K [ 72 , 195 ], topoisomerase-II [ 196 , 197 ], Wnt [ 198 , 199 ], ROS/MAPK [ 200 ], p38 [ 201 , 202 ] and mTOR [ 203 , 204 ]. However, these studies are not covered in this review because not enough evidence has been provided to indicate that these proteins or pathways are direct targets of chalcones.…”

Kava as a Clinical Nutrient: Promises and Challenges

“…Other mechanisms have also been suggested for flavokavain B’s anticancer potential, but with limited data [ 128 , 129 , 130 , 143 ]. For example, Flavokavain B was shown to enhance the anticancer effect of bortezomib in PC3 and C4-2B cell lines, potentially via promoting Skp2 degradation in an ubiquitin and proteasome-dependent manner [ 145 ]. Flavokavain B enhanced the anticancer effects of daunorubicin (DNR) in DNR-resistant acute myeloid leukemia, potentially by modifying the NF-κB activation (10 μM) [ 146 ].…”

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

“…Intriguingly, compared to other areas of the world, incidences of prostate cancer in kava drinking countries, such as Fiji, are very low. However, when Fijian men moved to Australia, their prostate cancer incidence raised by 5.1-fold [13,14] . These results have led us to investigate whether kava consumption can affect prostate cancer development and progression.…”