Multimodal actions of the phytochemical sulforaphane suppress both AR and AR-V7 in 22Rv1 cells: Advocating a potent pharmaceutical combination against castration-resistant prostate cancer

Abstract

Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (P<0.05). Immunoblot analyses showed that SFN (5–20 µM) rapidly decreases both AR-FL and AR-V7 levels, and immunofluorescence microscopy (IFM) depicted decreased AR in both cytoplasm and nucleus with SFN treatment. SFN increased both ubiquitination and proteasomal activity in 22Rv1 cells. Studies using a protein synthesis inhibitor (cycloheximide) or a proteasomal inhibitor (MG132) indicated that SFN increases both ubiquitin-mediated aggregation and subsequent proteasomal-degradation of AR proteins. Previous studies reported that SFN inhibits the chaperone activity of heat-shock protein 90 (Hsp90) and induces the nuclear factor erythroid-2-like 2 (Nrf2) transcription factor. Therefore, we investigated whether the Hsp90 inhibitor, ganetespib (G) or the Nrf2 activator, bardoxolone methyl (BM) can similarly suppress AR levels in 22Rv1 cells. Low doses of G and BM, alone or in combination, decreased both AR-FL and AR-V7 levels, and combined exposure to G+BM sensitized 22Rv1 cells to ENZ. Therefore, adjunct treatment with the phytochemical SFN or a safe pharmaceutical combination of G+BM may be effective against CRPC cells, especially those expressing AR-V7.

Targeting Crosstalk between Nrf-2, NF-κB and Androgen Receptor Signaling in Prostate Cancer

“…We recently showed that sulforaphane (SFN) can increase the efficacy of antiandrogens like bicalutamide and enzalutamide by degrading AR in androgen dependent as well as androgen independent PCa cells [ 108 ]. We also showed that SFN can increase the efficacy of enzalutamide in enzalutamide resistant PCa cell line by degrading both AR-FL as well as AR-V7 [ 109 ].…”

“…We recently showed that bardoxolone methyl (BM) which is a potent Nrf-2 inducer can increase the efficacy of enzalutamide in an enzalutamide-resistant PCa cell line (CWR22Rv1) by degrading both AR-FL as well as AR-V7 further underscoring the AR suppressive function of Nrf-2 [ 109 ]. This corroborates the fact that AR downregulation and Nrf-2 activation are the major pathways responsible for the anticancer efficacy of phytochemicals like SFN [ 38 , 39 , 40 , 41 , 42 ] and CUR [ 43 , 44 , 45 , 46 , 47 ] in PCa.…”

Mesenchymal stem cells are attracted to latent HIV-1-infected cells and enable virus reactivation via a non-canonical PI3K-NFκB signaling pathway

“…Changes in migratory ability of ASCs were measured using Scratch wound assays 62 . Briefly, ASCs were allowed to generate a confluent monolayer in 12-well petri dishes.…”

“…Immunoblot analysis was performed according to our previous published protocols 62 . Briefly, cells were lysed in RIPA buffer from Santa Cruz biosciences (Santa Cruz, CA).…”

Natural Compounds in Prostate Cancer Prevention and Treatment: Mechanisms of Action and Molecular Targets

“…Other natural products that have been demonstrated to trigger similar inhibitory effects on the AR axis are sulforaphane [62][63][64][65], epigallocatechin-3-gallate (EGCG) [66,67], ginsenosides [68][69][70][71], silymarin [72], berberine [73], honokiol [74], and celastrol [75].…”