Gail Singer Harrison

A study of high‐dose oral silybin‐phytosome followed by prostatectomy in patients with localized prostate cancer

Silibinin is a polyphenolic flavonolignan derived from milk thistle (Silybum marianium) with anti‐oxidant properties. The purpose of the current trial was to determine the tissue and blood effects of high‐dose silybin‐phytosome in prostate cancer patients.

Chemopreventive effects of silymarin and silibinin on N-butyl-N-(4-hydroxybutyl) nitrosamine induced urinary bladder carcinogenesis in male ICR mice.

Effective strategies are lacking for the management of urinary bladder cancer for which smoking is a potential risk factor. Herein, we evaluated chemoprevention of urinary bladder cancer by natural chemopreventive agents, silymarin and silibinin, in a preclinical animal (ICR mouse) model of bladder cancer induced by tobacco smoke carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN). Mice were fed p.o. with saline or OH-BBN (0.05%, w/v) in drinking water for 6 weeks or with silymarin or silibinin (200 mg/kg body weight for both) starting 1 week before OH-BBN exposure for 51 weeks. Silymarin and silibinin strongly arrested OH-BBN–induced tumor progression at the stage of mucosal dysplasia with a striking reduction in papillary nodular dysplasia as well as invasive carcinoma. Some silymarin- or silibinin-treated mice developed no urothelial lesions in spite of OH-BBN exposure. Immunohistochemical analyses at study conclusion revealed that silymarin and silibinin decreased cell proliferation by 42% (P < 0.001) and 44% (P < 0.001) and increased apoptosis by 4-fold (P < 0.05) and 6-fold (P < 0.05) in OH-BBN–induced urothelium, respectively. Antiproliferative and apoptotic effects of silymarin and silibinin were associated with decreases in (a) cyclin D1 protein level and extracellular signal–regulated kinase-1/2 phosphorylation and in (b) protein levels of survivin and nuclear phospho-p65 (Ser276 and Ser536), respectively. Together, these results suggest that silymarin and silibinin inhibit chemically induced urinary bladder tumor growth and progression possibly by inhibiting cell proliferation and enhancing apoptosis. [Mol Cancer Ther 2007;6(12):3248–55]

Silibinin synergizes with mitoxantrone to inhibit cell growth and induce apoptosis in human prostate cancer cells

The purpose of these experiments was to assess the synergistic activity of silibinin with chemotherapy agents in clinical use against prostate cancer. Silybin‐phytosome, a commercially available formulation containing silibinin, has recently been studied in a phase I clinical trial. The silibinin doses used in the present study are clinically achievable based on the preliminary phase I data. DU145, PC‐3 and LNCaP prostate cancer cells were seeded in 96‐well plates in triplicate. Twenty‐four hours later, silibinin (10, 20 and 40 μM) and either mitoxantrone or docetaxel were added to the designated wells. Seventy‐two hours post‐treatment, cell viability was determined with a tetrazolium‐based assay. The combination index (CI) for determination of a synergistic effect was calculated, with values of <0.9 indicating synergy and values >1.1 antagonism. Apoptosis was also assessed using a luminescent assay after 72 hr of treatment with media alone, silibinin, mitoxantrone, or silibinin plus mitoxantrone. Silibinin showed a synergistic effect with mitoxantrone, as measured by reduction in cell viability. The CI values ranged from 0.413 to 2.650 for the combination of silibinin and mitoxantrone; in contrast, treatment with docetaxel and silibinin showed little or no synergy, with CI values of 0.898–4.469. In concordance with these findings, the addition of silibinin increased the level of apoptosis compared to mitoxantrone alone, particularly in the PC‐3 cells. The combination of silibinin and mitoxantrone exhibits a pattern of synergy in reducing cell viability with increased apoptosis. These data are important in the planning of future clinical applications of silibinin.

Binding and Cytotoxicity of Conjugated and Recombinant Fusion Proteins Targeted to the Gonadotropin-Releasing Hormone Receptor

Pokeweed antiviral protein (PAP) is a plant-derived, highly potent ribosome inactivating protein that causes inhibition of protein translation and rapid cell death. We and others have delivered this protein to various cell types, including cancer cells, using hormones to specifically target cells bearing the hormone receptor. Here, we compare binding and cytotoxicity of GnRH-PAP hormonotoxins prepared either by protein conjugation (GnRH-PAP conjugate) or through recombinant DNA technology (GnRH-PAP fusion). Although GnRH-PAP conjugate protein bound specifically to and caused cell death in cells bearing the gonadotropin-releasing hormone (GnRH) receptor, we could not detect binding or cytotoxicity using two different versions of the fusion protein in receptor-positive cells. We conclude that generation of an active GnRH-PAP fusion protein may not be feasible either because both ends of the GnRH molecule are required for receptor binding, but only the NH2 terminus is free in the fusion protein and/or that more potent analogues of GnRH (inclusion of which is not feasible in the fusion protein) are needed for efficient targeting. In contrast, the GnRH-PAP conjugate shows promise as a novel anticancer agent, capable of targeting cancer cells expressing the GnRH receptor such as prostate, breast, ovarian, endometrial, and pancreatic cells. It may also be useful as a therapeutic agent to eliminate pituitary gonadotrophs, eliminating the need for chronic GnRH analogue administration to treat hormone-sensitive diseases.

Epidermal growth factor receptor mediates silibinin-induced cytotoxicity in a rat glioma cell line.

Silibinin, derived from milk thistle extract, has been shown to inhibit growth factor receptor-mediated mitogenic and cell survival signaling, and to alter cell cycle regulators. Alteration in pathways regulating cell growth likely account for silibinin’s inhibition of tumor growth. Since the epidermal growth factor receptor (EGFR) is a key regulator in cell signaling pathways, in the present study we directly tested the hypothesis that the EGFR plays a key role in mediating silibinin cytotoxicity to cancer cells. We generated a cell line, 9L-EGFR, which stably expressed human EGFR; the parental rat glioma cell line, 9L, does not contain endogenous EGFR message or protein. Our results show that expression of EGFR was both necessary and sufficient for conferring toxicity in response to silibinin in 9L-EGFR cells. Addition of silibinin was shown to inhibit EGFR activation by EGF in 9L-EGFR cells. These studies support the hypothesis that silibinin toxicity to cancer cells involves the EGFR signaling pathway. The findings presented here provide a rationale for understanding the growth inhibition effect of silibinin in cancer cells, and warrant further investigation into the effect of silibinin on specific pathways of cell signaling mediated by the EGF receptor.

Abstract 3695: Oligonol, a novel lychee fruit-derived low-molecular weight procyanidin extract, inhibits angiogenesis: Implications for its use as angiopreventive agent against cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Neo-angiogenesis, formation of new blood vessels from pre-existing vessels, is an essential hallmark for cancer growth and progression. Accordingly, several anti-angiogenic drugs including VEGF and VEGFR2 inhibitors have been either tested clinically or already approved by FDA for the treatment of various malignancies. Despite their strong anti-angiogenic efficacy, these drugs are costly, have significant side-effects and tumors quickly develop resistance, providing only marginal survival benefits. This scenario clearly warrants the need to identify non-toxic, affordable and effective alternative angiogenesis inhibitors to target cancer growth and progression. In this regard, an increasing number of natural compounds have shown anti-angiogenic activity with little or no side effects, and angioprevention of cancer through these chemopreventive agents is emerging as an exciting alternative approach. Accordingly, here, for the first time we investigated the anti-angiogenic efficacy of oligonol using human umbilical vein endothelial cells (HUVEC) as a model. Oligonol is a novel oligomerized low molecular weight procyanidin extract from lychee fruit and has shown good bioavailability in humans. Our results showed that oligonol (10-20 microgram/ml) strongly inhibits proliferation of HUVEC by 34-96%, 59-96% and 60% to complete inhibition after 24, 48 and 72 hrs of its exposure, respectively. In the studies examining its effect on cell cycle progression and apoptosis induction in HUVEC, we found that oligonol (10-20 microgram/ml) causes cell cycle arrest at G1 phase and induces apoptosis in HUVEC in a dose-dependent manner. Western blot analyses revealed that oligonol increases Cip1/p21 levels and Bad/Bcl2 ratio, decreases survivin and p-Akt levels, and activates caspase 3 and 9 followed by PARP cleavage. In other studies, oligonol (5-15 microgram/ml) strongly inhibited the capillary-like tube formation as well as disrupted pre-formed tubes by HUVEC on matrigel. Importantly, oligonol also inhibited VEGF-induced growth and tube formation by HUVEC. In addition, oligonol strongly inhibited the invasiveness of HUVEC in transwell assay and the migratory potential of HUVEC in wound healing assay by 40-95% and 58% to complete inhibition at 5, 10 & 15 microgram/ml doses, respectively. Importantly, we also observed that oligonol treatment inhibits phosphorylation of STAT3(Tyr705), Src(Tyr416) and FAK(Y576/577) which are known to regulate proliferation, migration and invasion of endothelial cells. Taken together, our findings clearly demonstrated the anti-angiogenic potential of oligonol, suggesting the use of this non-toxic and highly bioavailable agent for clinical cancer conditions. 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 3695. doi:10.1158/1538-7445.AM2011-3695