Nicholas J.E. Starkey

Inhibition of hedgehog/Gli signaling by botanicals: a review of compounds with potential hedgehog pathway inhibitory activities.

The hedgehog (Hh) signaling pathway is an important therapeutic target in cancer; involvement of the Hh pathway has been shown in a variety of cancers including basal cell carcinoma, medulloblastoma, leukemia, and gastrointestinal, breast, prostate, lung, and pancreatic cancers [1-10]. Currently, several Hh pathway inhibitory drugs are in clinical development, and the FDA recently approved Erivedge (vismodegib) from Curis/Genentech [11-15]. These new drugs are effective in many, but not all patients [16]. In fact there are documented reports of tumors developing mutations that confer resistance to the drugs [14, 17-19]. This highlights the importance of finding second generation drugs that can be used on cancers that develop resistance to the first generation Hh inhibitors. Botanicals may serve as the backbone for such research. The gold-standard pathway inhibitor, cyclopamine, is itself a naturally occurring alkaloid found in Veratrum californicum [20]. In this review we will summarize the available literature on botanical compounds in Hh-related studies. In particular we will look at curcumin, genistein, EGCG, resveratrol, quercetin, baicalen, and apigenin along with novel compounds isolated from Southeast Asian plants, such as the potent sub-micromolar gitoxigenin derivatives. Due to the nature of the pathway, most of the research published has focused on functional Gli-transcriptional assays, which we will describe and summarize.

Inhibition of Gli/hedgehog signaling in prostate cancer cells by "cancer bush" Sutherlandia frutescens extract.

Sutherlandia frutescens is a medicinal plant, traditionally used to treat various types of human diseases, including cancer. Previous studies of several botanicals link suppression of prostate cancer growth with inhibition of the Gli/hedgehog (Gli/Hh) signaling pathway. Here we hypothesized the anti‐cancer effect of S. frutescens was linked to its inhibition of the Gli/Hh signaling in prostate cancer. We found a dose‐ and time‐dependent growth inhibition in human prostate cancer cells, PC3 and LNCaP, and mouse prostate cancer cell, TRAMP‐C2, treated with S. frutescens methanol extract (SLE). We also observed a dose‐dependent inhibition of the Gli‐reporter activity in Shh Light II and TRAMP‐C2QGli cells treated with SLE. In addition, SLE can inhibit Gli/Hh signaling by blocking Gli1 and Ptched1 gene expression in the presence of a Gli/Hh signaling agonist (SAG). A diet supplemented with S. frutescens suppressed the formation of poorly differentiated carcinoma in prostates of TRAMP mice. Finally, we found Sutherlandioside D was the most potent compound in the crude extract that could suppress Gli‐reporter in Shh Light II cells. Together, this suggests that the S. frutescens extract may exert anti‐cancer effect by targeting Gli/Hh signaling, and Sutherlandioside D is one of the active compounds.

Inhibition of Hedgehog-Signaling Driven Genes in Prostate Cancer Cells by Sutherlandia frutescens Extract

Sutherlandia frutescens (L) R. Br. (Sutherlandia) is a South African botanical that is traditionally used to treat a variety of health conditions, infections and diseases, including cancer. We hypothesized Sutherlandia might act through Gli/ Hedgehog (Hh)-signaling in prostate cancer cells and used RNA-Seq transcription profiling to profile gene expression in TRAMPC2 murine prostate cancer cells with or without Sutherlandia extracts. We found 50% of Hh-responsive genes can be repressed by Sutherlandia ethanol extract, including the canonical Hh-responsive genes Gli1 and Ptch1 as well as newly distinguished Hh-responsive genes Hsd11b1 and Penk.

27-Hydroxycholesterol Is an Estrogen Receptor β–Selective Negative Allosteric Modifier of 17β-Estradiol Binding

Estrogens bind to two nuclear estrogen receptor (ER) subtypes, ERα and ERβ, which are expressed in differing amounts in various tissues. The endogenous estrogen, 17β-estradiol (E2), binds to both subtypes with nearly equal affinity and is the prototypical agonist. Selective estrogen receptor modulators (SERMs) may bind to both subtypes with equivalent affinities but have agonist activities in some tissues while having antagonist activities in others. In the present study, we demonstrate that the first reported endogenous SERM, 27-hydroxycholesterol (27-OHC), binds preferentially (>100-fold) to ERβ over ERα. Furthermore, 27-OHC is not able to fully compete with E2 binding, suggesting the two may bind at different sites. We provide an allosteric ternary complex model for the simultaneous binding of 27-OHC and E2 to ERβ, which accurately describes the binding data we have observed. We conclude that 27-OHC is a negative allosteric modifier of E2 binding, with an inhibitor constantof 50 nM and cooperativity factor (α) of 0.036. We also propose an in silico three-dimensional model of the simultaneous binding to guide future experiments. Further study of this unique binding model may allow for the discovery of novel ERβ-selective ligands and potentially explain the lack of effectiveness of ERβ-selective agonists in humans vs preclinical models.

Abstract 2104: Mechanisms for the inhibition of estrogen receptors by estrogen related receptor beta and oxysterols

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have previously shown that Estrogen Related Receptor Beta - Short Form (ERRβ-SF) inhibits the transcriptional activity of the Estrogen Receptors Alpha (ERα) and Beta (ERβ). Our lab and others have also shown the importance of these receptors in prostate cancer (PCa) as well as other ER positive, hormonally responsive, cancers such as breast and uterine. We have recently found, via RNA-Seq anlyses, that the overexpression of ERRβ-SF in the DU-145 PCa cell line changes the expression of many oxysterol metabolizing enzymes. These include, for example, CYP27A1 increased 69% and CYP46A1 increased 300% (although with a very low read number). These enzymes are responsible for the production of 27-hydroxycholesterol (27-OHC) and 24(S)-hydroxycholesterol (24(S)-OHC), respectively. Interestingly, the enzymes that catabolize these cholesterol metabolites were: CYP7A1 - not expressed, CYP7B1 - not expressed, and CYP39A1 which decreased by 63%. We hypothesized, that this represents two potential mechanisms for the inhibition of ER activity by ERRβ-SF. Oxysterol concentrations are increased when ERRβ-SF is overexpressed, this will either: 1) inhibit ERs through the known mechanism of directly binding to and inhibiting ERs, as has been previously shown for 27-OHC, or 2) inhibit by a novel mechanism in which oxysterols bind to ERRβ-SF and increase its ability to bind to and inhibit ER activities. To test these hypotheses, we used a transcriptional assay with an Estrogen Response Element driven Luciferase (ERE-Luc) reporter gene. ERs were expressed independently, or with ERRβ-SF, and treated with various oxysterols in the Ishikawa uterine cancer cell line. Using this assay, we have found that 27-OHC inhibits ER activity with a 4 fold difference between ERα and ERβ (IC50s = 4µM and 1µM, respectively). 24(S)-OHC inhibits ERβ only, with no effect on ERα at concentrations as high as 10µM. Interestingly, 10µM 24(S)-OHC increased the growth of Ishikawa cells >20% within 24hrs treatment under all conditions tested. Unfortunately, due to the overlap in ERE binding by the ERR and ERs, it was difficult to test the proposed novel mechanism using the ERE-Luc assay. To address this we are creating a new FRET based assay to observe the effects of the ligands on the ERR-ER interaction. We are further confirming the endogenous concentrations of the 24(S)-OHC and 27-OHC metabolizing enzymes via qRT-PCR. In conclusion, we have identified a potential molecular mechanism via oxysterols for the inhibitory action of ERRβ-SF on the ERs. This may explain the role of cholesterol metabolites in prostate cancer progression. Citation Format: Nicholas J.E. Starkey, Lu Yuan, Yufei Li, Dennis B. Lubahn. Mechanisms for the inhibition of estrogen receptors by estrogen related receptor beta and oxysterols. [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 2104. doi:10.1158/1538-7445.AM2014-2104

Abstract 1520: Simvastatin alters oxysterol profiles in TRAMP mice.

Due to mounting epidemiological data evidence, we sought to determine if simvastatin, the most widely used cholesterol lowering medication, could alter prostate cancer incidence in the TRAMP mouse model of prostate cancer. We hypothesized that simvastatin would inhibit advanced prostate cancer formation. Two separate studies were performed using high doses of simvastatin (up to 0.050% w/w) or simvastatin plus genistein in a high fat Western diet. While prostate cancer incidence was only moderately reduced, surprising changes in the serum oxysterol profiles of the TRAMP mice were detected. Oxysterols, oxygenated derivatives of cholesterol, have recently been shown to influence human diseases, and here we suggest that five oxysterols may play a role in prostate cancer progression. Ten mice were chosen from each treatment group, and their serum oxysterol profiles were analyzed by LC-MS-MS. The oxysterol that was most responsive to treatment was 24(S)-hydroxycholesterol, reducing significantly in all treatment groups. 24(S)-OHC was reduced from the control at 20 ng/mL to 10 ng/mL with statin treatment (p-value Citation Format: Sara K. Drenkhahn, Glenn A. Jackson, Nicholas J.E. Starkey, Yufei Li, Roxanne E. Gelven, Charles E. Wiedmeyer, Jim D. Browning, Kevin L. Fritsche, Cynthia L. Besch-Williford, Dennis B. Lubahn. Simvastatin alters oxysterol profiles in TRAMP mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1520. doi:10.1158/1538-7445.AM2013-1520

Abstract 1998: Inhibition of hedgehog signaling in prostate cancer by extracts of Sutherlandia

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Prostate cancer possesses long latency periods and is responsive to dietary mediators, making it a target for phytochemoprevention. Many botanical compounds that have been proposed to prevent cancer may potentially work via inhibition of the hedgehog signaling pathway. Here we investigated the potential of Sutherlandia frutescens (also called Lessertia and “cancer bush” in South Africa) to inhibit hedgehog signaling. We hypothesize that the anti-cancer effects of Sutherlandia are due to its inhibition of hedgehog signaling pathway activity. Methods: We evaluated Sutherlandias effects of growth inhibition in vitro both in the human prostate cancer cell line PC3 and mouse prostate cancer cell line TRAMP-C2 by exposing the cells for 72hrs to plant extracts before protein concentrations were measured to evaluate inhibition of growth. To determine hedgehog pathway inhibitory activity, we treated two different cell lines (an NIH3T3 cell line and a TRAMPC2 cell line) stably transfected with a firefly luciferase reporter having Gli response elements in the firefly luciferase promoter. After 48hr exposure to treatment with multiple doses of Sutherlandia, a firefly substrate was added to the cell lysate and the luciferase signal was measured using a Biotek Synergy plate reader. Finally, Sutherlandia was incorporated into a diet and fed to TRAMP (Transgenic Adenocarcinoma Mouse Prostate) mice to examine whether the plant can delay or even inhibit prostate cancer incidence in the TRAMPmouse model. Results: Sutherlandia extracts inhibited growth of both PC3 and TRAMPC2 cells. In addition, hedgehog signaling was inhibited by Sutherlandia in both NIH3T3 and TRAMPC2 reporter cells. Interestingly, in vivo the lowest concentration of Sutherlandia diet had the greatest effect on inhibition of poorly differentiated adenocarcinoma in the TRAMP mice. Conclusions: Sutherlandias inhibition of prostate cancer growth may be via inhibition of hedgehog signaling. Future work to identify in Sutherlandia the active compound having this effect, and the ideal concentration for use in vivo may lead to a means whereby dietary intervention for prostate cancer prevention and/or treatment is possible. 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 1998. doi:1538-7445.AM2012-1998

Abstract 4221: Inhibition of hedgehog signaling by extracts of sutherlandia

Many botanical compounds that have been proposed to prevent cancer may potentially work via inhibition of the hedgehog-signaling pathway. Here we investigated the potential of Sutherlandia frutescens (also called “cancer bush” in South Africa) to prevent and/or treat prostate cancer. We hypothesize that the anti-cancer effects of Sutherlandia are due to its inhibition of hedgehog-signaling pathway activity. To determine hedgehog pathway inhibitory activity, we treated Shh light II cells with multiple doses of a methanol extraction of Sutherlandia and measured Gli1 reporter activities. Results: We found that a methanol extract of Sutherlandia was able to inhibit hedgehog pathway activity in a dose-dependent manner as monitored by Gli reporter assay (IC50=1:4000). Moreover, the Sutherlandia extract can inhibit the growth of human prostate cancer cells PC3 and LNCaP with IC50 of 1:400 and 1:1500 fold dilutions, respectively. At these same extract dilutions normal prostate cancer cell growth was not inhibited. Our data indicate that Sutherlandia contains potent anti-cancer botanicals that have hedgehog inhibitory activity. Conclusion: Our results suggest that this plant offers a potentially cheap and effective alternative for hedgehog-driven cancer therapies. Additionally, Sutherlandia may yield novel targets that potentially could lead to a second generation hedgehog inhibitor, as resistance has been found to the first generation drugs currently in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4221. doi:10.1158/1538-7445.AM2011-4221

Abstract 5685: The effects of spinach and two of its components, spinacetin and patuletin, on the treatment of prostate cancer

Prostate cancer possesses long latency periods and is responsive to dietary mediators, making it a target for phytochemoprevention. It has been reported that spinach consumption can reduce the incidence of prostate cancer leading our lab to look at structures of compounds present in spinach. We chose to study spinacetin and patuletin, two novel isoflavones found in spinach. We hypothesized that these spinach compounds would inhibit prostate cancer in vitro in the mouse prostate cancer cell line, TRAMP-C2, and that a spinach-containing diet would reduce cancer incidence in our TRAMP mouse model. Methods: We first isolated a crude spinacetin-containing fraction and patuletin-containing fraction from whole spinach leaves using HPLC column purification. A further purified extract was also isolated for each compound. Using these extracts we determined the effect of the spinach compounds on growth of TRAMP-C2 cells, and the effect that they have on the concentration of Gli1, an indicator of hedgehog signaling, as measured by RT-PCR and a Gli1-luciferase reporter assay. We also tested ground dried spinach at 0.2% and 2% incorporated into a casein-based diet fed to B6/FVB TRAMP mice, and then evaluated the development of prostate cancer histologically. Results: The extracts, each containing 0.1% spinacetin or patuletin, both inhibit prostate cancer cell growth in vitro, with the patuletin extract inhibiting approximately 30% and the spinacetin extract inhibiting nearly 50%. In addition, the purified compounds each inhibited Gli1 expression in both TRAMP-C2 (as measured by RTPCR) and Shh Light II cells (as measured by luciferase assay), with patuletin having an effect at 300nM and spinacetin at 1μM. The 2% spinach diet led to a 50% decrease in the incidence of well-differentiated carcinoma, but had no apparent effect on the more aggressive poorly-differentiated carcinoma. In conclusion, spinach and its compounds are capable of inhibiting prostate cancer growth and incidence in in vitro and in vivo mouse models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5685.

Abstract 5705: Inhibition of hedgehog-signaling by extracts of the Sutherlandia and elderberry

Many botanical compounds, which have been proposed to prevent cancer, may potentially work via inhibition of the hedgehog-signaling pathway. Here we investigated the potential of Sutherlandia and elderberry to prevent and/or treat prostate cancer by inhibition of the hedgehog-signaling pathway. To determine hedgehog pathway inhibitory activity, we treated Shh light II cells with multiple doses of Sutherlandia extract or elderberry extract, in a Gli1 reporter assay to determine the extracts9effects on Gli1 concentration. The Sutherlandia extract was prepared by methanol extraction of Sutherlandia frutescens, while the elderberry extract was prepared by ethanol extraction of Sambucus nigra berries. We hypothesized that the compounds canavanine, from Sutherlandia, and cyanidin-3-glucoside, from elderberry, were the active compounds able to inhibit hedgehog signaling. Results: We found that a 1:2000 dilution of Sutherlandia extract and a 1:100 dilution of elderberry extract are able to inhibit hedgehog pathway activity by 99% and 94%, respectively, as monitored by Gli reporter assay. Moreover, cyanidin-3- glucoside (5uM, 10uM and 50uM) can inhibit hedgehog activity by 44%,62% and 81% respectively, and canavanine (1uM and 10uM) can inhibit hedgehog activity by 5% and 15% respectively. These data indicate that, while these two compounds are weak hedgehog inhibitor compounds, they are unlikely to be the major active hedgehog inhibitory compounds in Sutherlandia and elderberry. Conclusion: Our results suggest that these two plants offer a potentially cheap and effective alternative to cyclopamine for cancer therapies. Additionally, these plants may yield novel targets that potentially could lead to a second generation hedgehog inhibitor, if resistance is found to the first generation drugs currently in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5705.