α-Mangostin Promotes In Vitro and In Vivo Degradation of Androgen Receptor and AR-V7 Splice Variant in Prostate Cancer Cells.

A major limitation of current prostate cancer pharmacotherapy approaches is the inability of these compounds to target androgen receptor variants or mutants that develop during prostate cancer progression. The demand for novel therapeutics to prevent, slow, and treat prostate cancer is significant because FDA approved anti-androgens are associated with adverse events and can eventually drive drug-resistant prostate cancer. This study evaluated α-mangostin for its novel ability to degrade the androgen receptor and androgen receptor variants. α-Mangostin is one of more than 70 isoprenylated xanthones isolated from Garcinia mangostana that we have been evaluating for their anticancer potential. Prostate cancer cells treated with α-mangostin exhibited decreased levels of wild-type and mutated androgen receptors. Immunoblot, immunoprecipitation, and transfection experiments demonstrated that the androgen receptor was ubiquitinated and subsequently degraded via the proteasome, which we hypothesize occurs with the assistance of BiP, an ER chaperone protein that we have shown to associate with the androgen receptor. We also evaluated α-mangostin for its antitumor activity and promotion of androgen receptor degradation in vivo. In summary, our study demonstrates that androgen receptor degradation occurs through the novel activation of BiP and suggests a new therapeutic approach for prostate cancer.

Carnosol, a dietary diterpene from rosemary (Rosmarinus officinalis) activates Nrf2 leading to sestrin 2 induction in colon cells.

BACKGROUND: Rosemary is abundant with phytochemicals and has recently been approved as an antioxidant food preservative in the European Union. The safety of rosemary is well established, however, the benefits on gastrointestinal health are less known. Our overall hypothesis is that the phytochemicals in rosemary including carnosol have the potential to promote gastrointestinal health by activation of the antioxidant sestrin-2 when consumed in our diet. METHODS: Colon cells HCT116 and SW480 were treated with carnosol and evaluated by MTT, immunofluorescence, ELISA, and Western blot analysis to understand the modulation of the PERK/Nrf2/Sestrin-2 pathway. RESULTS: Carnosol was found to modulate PERK and increase the concentration of nuclear Nrf2. Furthermore, a downstream marker of Nrf2 expression, Sestrin-2 was shown to be upregulated. CONCLUSION: Based on these observations carnosol modulates the PERK and Nrf2 pathways along with increased expression of sestrin-2, a known stress inducible antioxidant.

Carnosic acid promotes degradation of the androgen receptor and is regulated by the unfolded protein response pathway in vitro and in vivo.

Androgen deprivation therapy in prostate cancer is extremely effective; however, due to the continuous expression and/or mutagenesis of androgen receptor (AR), the resistance to antihormonal therapy is a natural progression. Consequently, targeting the AR for degradation offers an alternate approach to overcome this resistance in prostate cancer. In this study, we demonstrate that carnosic acid, a benzenediol diterpene, binds the ligand-binding domain of the AR and degrades the AR via endoplasmic reticulum (ER) stress-mediated proteasomal degradative pathway. In vitro, carnosic acid treatment induced degradation of AR and decreased expression of prostate-specific antigen in human prostate cancer cell lines LNCaP and 22Rv1. Carnosic acid also promoted the expression of ER proteins including BiP and CHOP in a dose-dependent manner. Downregulation of CHOP by small interfering RNA somewhat restored expression of AR suggesting that AR degradation is dependent on ER stress pathway. Future studies will need to evaluate other aspects of the unfolded protein response pathway to characterize the regulation of AR degradation. Furthermore, cotreating cells individually with carnosic acid and proteasome inhibitor (MG-132) and carnosic acid and an ER stress modulator (salubrinal) restored protein levels of AR, suggesting that AR degradation is mediated by ER stress-dependent proteasomal degradation pathway. Degradation of AR and induction of CHOP protein were also evident in vivo along with a 53% reduction in growth of xenograft prostate cancer tumors. In addition, carnosic acid-induced ER stress in prostate cancer cells but not in normal prostate epithelial cells procured from patient biopsies. In conclusion, these data suggest that molecules such as carnosic acid could be further evaluated and optimized as a potential therapeutic alternative to target AR in prostate cancer.

Gartanin, an isoprenylated xanthone from the mangosteen fruit (Garcinia mangostana), is an androgen receptor degradation enhancer.

SCOPE: Androgen receptor (AR) has been a target of prostate cancer for nearly seven decades. In the last several years there has been an interest in identifying compounds that promote degradation of the androgen receptor. In the present study, gartanin, an isoprentylated xanthone in the mangosteen fruit, was evaluated for enhancing AR degradation, and inducing the unfolded protein response pathway. METHODS AND RESULTS: The interaction of gartanin with the ligand-binding domain was characterized using a fluorescence polarization cell-free assay and cell-based FRET assay. Western blot analysis identified modulation of ER stress markers (BiP, PERK, IRE1, and CHOP) along with androgen receptor degradation. A computation simulation was performed to identify possible orientations of gartanin with the ligand-binding domain. Utilizing a cell-free and cell-based FRET assays gartanin was found to interact with the ligand-binding domain through a solely antagonist interaction. Interestingly, inhibition of CHOP, a critical component of the ER stress pathway, was observed to stabilize AR. CONCLUSIONS: Gartanin is an isoprenylated xanthone that promotes AR degradation with evidence suggesting this process is critically regulated by the unfolded protein response pathway.

Diterpenes from rosemary (Rosmarinus officinalis): Defining their potential for anti-cancer activity.

Recently, rosemary extracts standardized to diterpenes (e.g. carnosic acid and carnosol) have been approved by the European Union (EU) and given a GRAS (Generally Recognized as Safe) status in the United States by the Food and Drug Administration (FDA). Incorporation of rosemary into our food system and through dietary selection (e.g. Mediterranean Diet) has increased the likelihood of exposure to diterpenes in rosemary. In consideration of this, a more thorough understanding of rosemary diterpenes is needed to understand its potential for a positive impact on human health. Three agents in particular have received the most attention that includes carnosic acid, carnosol, and rosmanol with promising results of anti-cancer activity. These studies have provided evidence of diterpenes to modulate deregulated signaling pathways in different solid and blood cancers. Rosemary extracts and the phytochemicals therein appear to be well tolerated in different animal models as evidenced by the extensive studies performed for approval by the EU and the FDA as an antioxidant food preservative. This mini-review reports on the pre-clinical studies performed with carnosic acid, carnosol, and rosmanol describing their mechanism of action in different cancers.

Inhibition of CHOP accentuates the apoptotic effect of α-mangostin from the mangosteen fruit (Garcinia mangostana) in 22Rv1 prostate cancer cells.

The mangosteen (Garcinia mangostana) fruit has been a popular food in Southeast Asia for centuries and is increasing in popularity in Western countries. We identified α-Mangostin as a primary phytochemical modulating ER stress proteins in prostate cancer cells and propose that α-Mangostin is responsible for exerting a biological effect in prostate cancer cells. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells procured from two patients undergoing radical prostatectomy were treated with α-Mangostin and evaluated by RT-PCR, Western blot, fluorescent microscopy and siRNA transfection to evaluate ER stress. Next, we evaluated α-Mangostin for microsomal stability, pharmacokinetic parameters, and anti-cancer activity in nude mice. α-Mangostin significantly upregulated ER stress markers in prostate cancer cells. Interestingly, α-Mangostin did not promote ER stress in prostate epithelial cells (PrECs) from prostate cancer patients. CHOP knockdown enhanced α-Mangostin-induced apoptosis in prostate cancer cells. α-Mangostin significantly suppressed tumor growth in a xenograft tumor model without obvious toxicity. Our study suggests that α-Mangostin is not the only active constituent from the mangosteen fruit requiring further work to understand the complex chemical composition of the mangosteen.

Pharmacokinetic characterization of mangosteen (Garcinia mangostana) fruit extract standardized to α-mangostin in C57BL/6 mice.

Previously, we have reported the pharmacokinetic (PK) properties of α-mangostin in mice. For this study, we evaluated the PK profile of α-mangostin using a standardized mangosteen extract in C57BL/6 mice. The primary objective was to determine the PK properties of α-mangostin when administered as an extract. This experiment was designed to test our primary hypothesis that α-mangostin in an extract should achieve a desirable PK profile. This is especially relevant as dietary supplements of mangosteen fruit are regularly standardized to α-mangostin. Mice received 100 mg/kg of mangosteen fruit extract orally, equivalent to 36 mg/kg of α-mangostin, and plasma samples were analyzed over a 24-hour period. Concentrations of α-mangostin were determined by liquid chromatography-tandem mass spectrometry. In addition, we evaluated the stability in the presence of phase I and phase II enzymes in liver and gastrointestinal microsomes. Furthermore, we identified evidence of phase II metabolism of α-mangostin. Further research will be required to determine if less abundant xanthones present in the mangosteen may modulate the PK parameters of α-mangostin.

Rosemary (Rosmarinus officinalis) extract modulates CHOP/GADD153 to promote androgen receptor degradation and decreases xenograft tumor growth.

The Mediterranean diet has long been attributed to preventing or delaying the onset of cardiovascular disease, diabetes and various solid organ cancers. In this particular study, a rosemary extract standardized to carnosic acid was evaluated for its potential in disrupting the endoplasmic reticulum machinery to decrease the viability of prostate cancer cells and promote degradation of the androgen receptor. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells procured from two different patients undergoing radical prostatectomy were treated with standardized rosemary extract and evaluated by flow cytometry, MTT, BrdU, Western blot and fluorescent microscopy. A significant modulation of endoplasmic reticulum stress proteins was observed in cancer cells while normal prostate epithelial cells did not undergo endoplasmic reticulum stress. This biphasic response suggests that standardized rosemary extract may preferentially target cancer cells as opposed to "normal" cells. Furthermore, we observed standardized rosemary extract to decrease androgen receptor expression that appears to be regulated by the expression of CHOP/GADD153. Using a xenograft tumor model we observed standardized rosemary extract when given orally to significantly suppress tumor growth by 46% compared to mice not receiving standardized rosemary extract. In the last several years regulatory governing bodies (e.g. European Union) have approved standardized rosemary extracts as food preservatives. These results are especially significant as it is becoming more likely that individuals will be receiving standardized rosemary extracts that are a part of a natural preservative system in various food preparations. Taken a step further, it is possible that the potential benefits that are often associated with a "Mediterranean Diet" in the future may begin to extend beyond the Mediterranean diet as more of the population is consuming standardized rosemary extracts.

Selective modulation of endoplasmic reticulum stress markers in prostate cancer cells by a standardized mangosteen fruit extract.

The increased proliferation of cancer cells is directly dependent on the increased activity of the endoplasmic reticulum (ER) machinery which is responsible for protein folding, assembly, and transport. In fact, it is so critical that perturbations in the endoplasmic reticulum can lead to apoptosis. This carefully regulated organelle represents a unique target of cancer cells while sparing healthy cells. In this study, a standardized mangosteen fruit extract (MFE) was evaluated for modulating ER stress proteins in prostate cancer. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells (PrECs) procured from two patients undergoing radical prostatectomy were treated with MFE. Flow cytometry, MTT, BrdU and Western blot were used to evaluate cell apoptosis, viability, proliferation and ER stress. Next, we evaluated MFE for microsomal stability and anti-cancer activity in nude mice. MFE induced apoptosis, decreased viability and proliferation in prostate cancer cells. MFE increased the expression of ER stress proteins. Interestingly, MFE selectively promotes ER stress in prostate cancer cells while sparing PrECs. MFE suppressed tumor growth in a xenograft tumor model without obvious toxicity. Mangosteen fruit extract selectively promotes endoplasmic reticulum stress in cancer cells while sparing non-tumorigenic prostate epithelial cells. Furthermore, in an in vivo setting mangosteen fruit extract significantly reduces xenograft tumor formation.

Polyphenols from the Mediterranean herb rosemary (Rosmarinus officinalis) for prostate cancer.

The Mediterranean diet is rich in fruits and vegetables and has been associated with a variety of health benefits including cancer prevention. One aspect of the diet that has not received enough attention is Mediterranean herbs. Specifically, rosemary and its polyphenolic diterpenes (carnosic acid and carnosol) are known to possess anti-oxidant activity that may be beneficial for cancer control. Herein, we describe the in vitro and in vivo studies carried out towards understanding the molecular mechanisms of carnosic acid and carnosol leading to inhibition of prostate cancer. The reported findings suggest that these polyphenols target multiple signaling pathways involved in cell cycle modulation and apoptosis. Further work is required to understand its potential for health promotion and potential drug discovery for prostate cancer chemoprevention.

Single dose oral pharmacokinetic profile of α-mangostin in mice.

The mangosteen fruit (Garcinia mangostana) is a rich source of dietary xanthones with the most prominent being α-mangostin. Dietary xanthones have been reported to have a variety of health promoting properties. Until now, in vivo studies on the pharmacokinetic profile of α-mangostin are limited. For this study we employed an LC/MS/MS assay to determine the pharmacokinetic properties of α-mangostin suspension in cottonseed oil in C57BL/6 Mice. Mice were administered 100 mg/kg of α-mangostin by oral gavage and the plasma levels were analyzed over a 24 hour period. We observed the degree of exposure (i.e. area under the curve) of α-mangostin to be 5,736 nmol/L/hr and the maximum plasma concentration was 1,382 nmol/L. Furthermore, we provide evidence that α-mangostin undergoes glucuronidation into monoglucuronide and diglucuronide metabolites. Our study demonstrated that α-mangostin when administered in cotton seed oil to mice at a dose equivalent to 615 mg in a 90kg human adult achieves an approximate maximum plasma concentration of 1,300 nmol/L and is detectable for up to 24 hours. Further research is needed to understand the relationship between the pharmacokinetic properties of α-mangostin following oral administration and reported health benefits.

α-Mangostin, a xanthone from mangosteen fruit, promotes cell cycle arrest in prostate cancer and decreases xenograft tumor growth.

There is a need to characterize promising dietary agents for chemoprevention and therapy of prostate cancer (PCa). We examined the anticancer effect of α-mangostin, derived from the mangosteen fruit, in human PCa cells and its role in targeting cell cycle-related proteins involved in prostate carcinogenesis. Using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, we found that α-mangostin significantly decreases PCa cell viability in a dose-dependent manner. Further analysis using flow cytometry identified cell cycle arrest along with apoptosis. To establish a more precise mechanism of action, we performed a cell free biochemical kinase assay against multiple cyclins/cyclin-dependent kinases (CDKs) involved in cell cycle progression; the most significant inhibition in the cell free-based assays was CDK4, a critical component of the G1 phase. Through molecular modeling, we evaluated α-mangostin against the adenosine triphosphate-binding pocket of CDK4 and propose three possible orientations that may result in CDK4 inhibition. We then performed an in vivo animal study to evaluate the ability of α-mangostin to suppress tumor growth. Athymic nude mice were implanted with 22Rv1 cells and treated with vehicle or α-mangostin (100 mg/kg) by oral gavage. At the conclusion of the study, mice in the control cohort had a tumor volume of 1190 mm(3), while the treatment group had a tumor volume of 410 mm(3) (P < 0.01). The ability of α-mangostin to inhibit PCa in vitro and in vivo suggests α-mangostin may be a novel agent for the management of PCa.