Sara K. Drenkhahn

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.

Antiproliferative and Antiestrogenic Activities of Bonediol an Alkyl Catechol from Bonellia macrocarpa

The purpose of this study was to investigate antiproliferative activity of bonediol, an alkyl catechol isolated from the Mayan medicinal plant Bonellia macrocarpa. Bonediol was assessed for growth inhibition of androgen-sensitive (LNCaP), androgen-insensitive (PC-3), and metastatic androgen-insensitive (PC-3M) human prostate tumor cells; toxicity on normal cell line (HEK 293) was also evaluated. Hedgehog pathway was evaluated and competitive 3H-estradiol ligand binding assay was performed. Additionally, antioxidant activity on Nrf2-ARE pathway was evaluated. Bonediol induced a growth inhibition on prostate cancer cell lines (IC50 from 8.5 to 20.6 µM). Interestingly, bonediol binds to both estrogen receptors (ERα (2.5 µM) and ERβ (2.1 µM)) and displaces the native ligand E2 (17β-estradiol). No significant activity was found in the Hedgehog pathway. Additionally, activity of bonediol on Nrf2-ARE pathway suggested that bonediol could induce oxidative stress and activation of detoxification enzymes at 1 µM (3.8-fold). We propose that the compound bonediol may serve as a potential chemopreventive treatment with therapeutic potential against prostate cancer.

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 2399: Simvastatin inhibits advanced prostate cancer in TRAMP mice

Controversial literature suggests the potential off-label benefit of statins in the prevention of breast and prostate cancers. As millions of Americans are currently taking statins to lower cholesterol and prevent heart disease, we wanted to test if simvastatin could inhibit prostate carcinogenesis in the TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) model. We hypothesized that simvastatin would inhibit the most aggressive form of cancer in the model, poorly differentiated carcinoma (PDC), similar to what has been seen in epidemiological studies. Mice were fed a Western Diet to mimic the high-fat diet common among men in the United States (n=25 per group). Two additional groups were fed the Western diet supplemented with either 0.025% or 0.050% w/w simvastatin. The control mice on the Western diet had an increase of PDC when compared to a low-fat AIN93 casein based diet (48% vs. 32%). While the 0.025% simvastatin Western diet reduced PDC incidence from 48% to 38%, the 0.050% simvastatin Western diet drastically reduced PDC incidence from 48% to 16% when compared to Western controls (p=0.0153 by Chi square analysis). Changes in serum profiles analyzing total cholesterol, LDL, HDL, and triglycerides did not correlate to the reduction in PDC incidence. In conclusion, our results show that simvastatin can reduce the most aggressive stage of prostate cancer in the TRAMP model and supports the observation that simvastatin reduces the risk of developing advanced prostate cancer. 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 2399. doi:10.1158/1538-7445.AM2011-2399

Abstract 1711: Regulation of hedgehog signaling by the estrogen receptors

The likelihood of developing prostate cancer increases with age; hence with the increasing lifespan of Americans the prevalence of prostate cancer is also increasing. Prostate cancer is responsive to endocrine mediators and while estrogen therapy is a well-known classic treatment regiment for prostate cancer, the inhibition of hedgehog signaling has more recently been reported to be important for prostate cancer therapy. Our lab is interested in the role of hedgehog signaling in prostate cancer development and progression. Despite the fact that several nuclear receptor ligands including estrogen, androgen, oxysterols, progesterone, and vitamin d can affect hedgehog signaling, very few nuclear receptors have been shown to directly affect the pathway. While it has been published the Liver X Receptor (LXR) has been shown to negatively regulate hedgehog signaling, we have found that another nuclear receptor class, the Estrogen Receptors, are also able to modulate the hedgehog signaling pathway and may play a regulatory role on hedgehog pathway activity in prostate cancer. We hypothesize that the Estrogen Receptors are involved in mediating hedgehog signaling in prostate cancer, and compounds that bind to the estrogen receptors can modulate hedgehog signaling. Recent work in our lab examining phytoestrogens’ inhibitory effect on the pathway led us to study the roles of the Estrogen Receptors and estrogenic compounds on hedgehog signaling. We have found that 17β-estradiol (E2) can inhibit the hedgehog signaling pathway, monitored by real-time RT-PCR analysis of Gli1 mRNA in the TRAMP-C2 mouse prostate cancer cell line. Surprisingly the ER antagonist ICI 182,780 was also able to inhibit Gli1 mRNA. While the compounds alone do not significantly reduce cell growth, when E2 and ICI 182,780 are added together they significantly inhibit cell growth in the human prostate cancer cell line PC3M. We also have found by western blot analysis that E2 treatments can reduce Gli1 protein concentrations in TRAMP-C2 cells. Similar results were seen in the Shh Light II cell line, a mouse embryonic fibroblast cell line with a stably transfected 8xGliBS-luciferase construct. In summary, our research has shown that estrogenic treatment can reduce hedgehog signaling in prostate cancer models and that the estrogen receptors potentially play a key role in regulating the hedgehog pathway. By understanding how the hedgehog signaling pathway functions in prostate cancer we may be able to generate therapies that include standard hormone and chemotherapies coupled with new hedgehog therapies currently in the pipeline to create new treatments for men afflicted with prostate cancer. Potentially, this work may lead to second generation hedgehog pathway inhibitors that are needed as resistance is developing to the first generation hedgehog pathway inhibitors now 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 1711.

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.