Gongbo Li

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.

Polyphenols from the mangosteen (Garcinia mangostana) fruit for breast and prostate cancer

The mangosteen (Garcinia mangostana) is a tropical fruit native to Southeast Asia and has long been reported to contain multiple health promoting properties. This fruit is an abundant source of xanthones, a class of polyphenolic compounds with a distinctive tricyclic aromatic ring system and is largely responsible for its biological activities including anti-cancer activity. Herein we describe the anti-cancer activity and mechanisms of mangosteen polyphenolic xanthones including α-Mangostin against breast cancer and prostate cancer. So far, extracts and individual xanthones have been found to induce apoptosis and inhibit proliferation on cancer cells in vitro and in vivo. Based on the reported findings there is clear evidence 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 and breast 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.

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.

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.

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.

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.

Abstract 4122: Anti-cancer activity of rosemary extract in colon cancer cells: Involvement of Nrf2 and ERK pathways

Background: The Mediterranean herb rosemary has been used for culinary purposes and for their medicinal properties for millennia. Rosemary and its polyphenolic diterpenes (carnosol and carnosic acid) are known to possess anti-oxidant activity that may be beneficial for cancer control. Colorectal cancer is the second leading cause of death from cancer in the United States. Nutrition, particularly intake of vegetables and certain plant components, has been reported to have a major role in colon cancer risk reduction. In our current study, we investigated the anticancer effects of rosemary extract and its two main active ingredients carnosol and carnosic acid on colon cancer cell lines to understand the mechanism. Next, we evaluated the anti-cancer activity of rosemary extract in a nude mice model. Methods: Two human colon cancer cell lines, HCT116 and SW480, were treated with rosemary extract, carnosol or carnosic acid. MTT, TUNEL and ELISA were used to evaluate cell viability and apoptosis. Western blot and immunofluorescence were used to detect proteins involved in apoptosis. We also evaluated the anti-cancer activity of rosemary extract in nude mouse inoculated with HCT116 cells. Results: Rosemary extract, carnosol and carnosic acid, increased apoptosis, decreased viability in colon cancer cell lines. Rosemary extract, carnosol and carnosic acid significantly upregulated the expression of Nrf2 and ERK. Rosemary extract also effectively suppressed tumor growth in a xenograft tumor model without affecting body weight. Conclusions: Rosemary extract inhibits colon cancer cell growth in a xenograft mouse model without observable toxicity. Furthermore, rosemary extract, carnosol and carnosic acid induce the expression of Nrf2 and ERK pathways in colon cancer cells, suggesting further research is needed to evaluate its potential as an anti-cancer and/or chemoprevention agent. Citation Format: Miao Yan, Gongbo Li, Sakina M. Patiwala, Emily Householter, Jeremy J. Johnson. Anti-cancer activity of rosemary extract in colon cancer cells: Involvement of Nrf2 and ERK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4122. doi:10.1158/1538-7445.AM2014-4122

Abstract 2574: Mangosteen fruit extract promotes endoplasmic reticulum stress through upregulation of CHOP/GADD153 in prostate cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The mangosteen fruit (Garcinia mangostana) is native to Southeast Asia and has been reported to contain a variety of health promoting properties that include antioxidant, anti-inflammatory, anti-microbial and anti-diabetic activities. The principle constituents isolated from the mangosteen fruit are the xanthones with over 50 unique entities isolated to date. Prostate cancer (PCa) is the second most common cancer-related death in adult males and is ideal for evaluating promising cancer preventive agents because it develops at old age and is usually asymptomatic until advanced. This suggests that even a slight delay in the diagnosis of prostate cancer could have a profound public health impact. In our current study, we investigated the anticancer effects of a highly characterized mangosteen fruit extract on PCa cell lines to understand the mechanism of endoplasmic reticulum stress. Next, we evaluated the anticancer activity of mangosteen fruit extract in an athymic nude mouse model. Methods: Two human prostate cancer cell lines, 22Rv1 and LNCaP, were treated with a standardized mangosteen fruit extract. Flow cytometry, MTT, BrdU, and siRNA were used to evaluate cell apoptosis, cell viability and cell proliferation. Whole cell lysates were used to detect protein expression changes of androgen receptor (AR), pro-apoptotic protein Bax and other proteins involved in cell cycle or ER stress following treatment with mangosteen fruit extract. Next, we evaluated mangosteen fruit extract for anti-cancer activity in Athymic (nu/nu) nude mice inoculated with 22Rv1 cells. These mice received either mangosteen fruit extract or vehicle. In addition, a pharmacokinetics assay of mangosteen fruit extract, complete blood count (CBC), and serum biochemistry were performed on wild type C57BL6 mice for possible toxicity. Results: Our results demonstrate that mangosteen fruit extract effectively induced apoptosis, decreased cell viability and inhibited cell proliferation in multiple prostate cancer cell lines. Mangosteen fruit extract treatment induced a dose-dependent AR down-regulation and Bax up-regulation, induced G1 cell cycle arrest, and significantly increased the expression of ER stress proteins, such as PERK, CHOP and IRE1 in PCa cells. In addition, mangosteen fruit extract treatment significantly suppressed tumor growth in a xenograft tumor model and did not alter body weight, complete blood count or serum biochemistry in normal mice. Conclusion: Our results demonstrate that mangosteen fruit extract upregulated the ER stress proteins CHOP, PERK and IRE1. Mangosteen fruit extract treatment effectively suppressed PCa cell growth in a xenograft mouse model without obvious toxicity, suggesting further research is needed to determine its potential as a cancer preventive and/or therapeutic agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2574. doi:1538-7445.AM2012-2574