Anna Slusarz

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.

Effects of nanosecond pulsed electric fields on the human prostate cancer cell line LNCaP

The effects of nanosecond pulsed electric fields (nsPEF) on the human prostate cancer cell line LNCaP was investigated for changes in protein and DNA concentrations as well as for the 11beta hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme activity. Electric fields with intensities of 32.5 kV/cm and pulse widths of 60 ns were applied to cancer cells in trains of 1, 5, and 10 pulses. Protein concentrations were determined through a Bio-Rad DC protein assay, while Hoechst 33258 was used to quantify DNA concentrations. The analyses showed that protein concentrations decreased by 18.77% and 19.04% for 1 and 10 pulses, respectively, while DNA concentrations decreased by 5.29%, 8.95%, and 18.36% for 1, 5, and 10 pulses, respectively, as compared to control groups. The 11betaHSD2 enzyme activity in LNCaP cells also decreased following exposure, thus supporting our initial hypothesis that ultrashort pulses affect intracellular structures; thus, a decrease in the enzyme activity could result in enhancing the anti-proliferative actions of glucocorticoids.

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 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.