Many botanical compounds have been proposed to prevent cancer. We investigated the cancer treatment and prevention abilities of apigenin, baicalein, curcumin, epigallocatechin 3-gallate (EGCG), genistein, quercetin, and resveratrol both in vivo in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice as well as in vitro in prostate cancer cell lines. In our experiments, these seven compounds act similarly to the Hedgehog antagonist cyclopamine, a teratogenic plant alkaloid, which had been previously shown to "cure" prostate cancer in a mouse xenograft model. With IC 50 values ranging from <1 to 25 μmol/L, these compounds can inhibit Gli1 mRNA concentration by up to 95% and downregulate Gli reporter activity by 80%. We show that four compounds, genistein, curcumin, EGCG, and resveratrol, inhibit Hedgehog signaling as monitored by real-time reverse transcription-PCR analysis of Gli1 mRNA concentration or by Gli reporter activity. Three compounds, apigenin, baicalein, and quercetin, decreased Gli1 mRNA concentration but not Gli reporter activity. Our results show that these compounds are also able to reduce or delay prostate cancer in vivo in TRAMP mice. All seven compounds, when fed in combination as pure compounds or as crude plant extracts, inhibit well-differentiated carcinoma of the prostate by 58% and 81%, respectively. In vitro, we show that all seven compounds also inhibit growth in human and mouse prostate cancer cell lines. Mechanistically, we propose the Hedgehog signaling pathway to be a direct or indirect target of these compounds. These botanicals at pharmacologic concentrations are potentially safer and less expensive alternatives to cyclopamine and its pharmaceutical analogues for cancer therapy. Cancer Res; 70(8); 3382-90. ©2010 AACR.

No abstract

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 cells 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.

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.

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.

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.

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 &lt;0.001). 27-hydroxycholesterol was also reduced in the 0.044% simvastatin w/w dose to 10 ng/mL from 16 ng/mL in the control diet (p-value = 0.025). 5,6-dihydroxycholesterol responded to the 0.044% simvastatin diet lowering to 10 ng/mL as compared to the control of 18 ng/mL (p-value = 0.0002). 4-beta-hydroxycholesterol only responded to a combination of 0.050% simvastatin and 400 mg/kg genistein treatment, lowering from the control of 605 ng/mL down to 340 ng/mL with combination treatment (p-value &lt;0.0001). Lastly 7-keto-cholesterol was reduced by the 0.044% simvastatin treatment, lowering to 44 ng/mL from the control of 67 ng/mL (p-value = 0.004). Future studies will determine how these oxysterols can influence signaling pathways that can affect prostate cancer progression. In summary, we have found an off target effect of simvastatin treatment in TRAMP mice. Although serum cholesterol parameters remain statistically similar, with no observable changes in LDL, HDL, triglycerides, or total cholesterol, serum oxysterol profiles were changed significantly with simvastatin treatment. Potentially one of the pharmacological mechanisms for the beneficial effects of statin treatment on cancer outcomes is not due to their cholesterol lowering abilities, but instead may hinge on their capacity to change the concentration of oxysterols in vivo. 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

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