Paul R. van Ginkel

Resveratrol Inhibits Tumor Growth of Human Neuroblastoma and Mediates Apoptosis by Directly Targeting Mitochondria

Purpose: Neuroblastoma is an aggressive childhood disease of the sympathetic nervous system. Treatments are often ineffective and have serious side effects. Because resveratrol, a natural plant product, has been reported to have limited toxicity at chemotherapeutic levels, we investigated its efficacy in the treatment of neuroblastoma as well as its underlying mechanism of action. Experimental Design: Resveratrol was tested in mouse xenograft models of human neuroblastoma and in vitro using human cell lines. Results: Resveratrol inhibited the outgrowth of tumors by as much as 80%. The bioavailability of the drug in serum was in the low micromolar range (2-10 μmol/L) and no accumulation was observed in tumor tissue. When resveratrol levels were increased by peritumor injection, rapid tumor regression occurred. Resveratrol decreased tumor cell viability in vitro by 75% to 90%, resulting from an inhibition of cell proliferation and an induction of apoptosis. Loss of mitochondrial membrane potential was an early response to resveratrol. In addition, resveratrol treatment of isolated mitochondria also led to depolarization, suggesting that the drug may target mitochondria directly. Following depolarization, resveratrol caused the release of cytochrome c and Smac/Diablo from the mitochondria and subsequently the activation of caspase-9 (4- to 8-fold) and caspase-3 (4- to 6-fold). Conclusions: These studies indicate that, despite low bioavailability, resveratrol is effective at inhibiting tumor growth. Elevated levels of resveratrol enhance its antitumor potency leading to tumor regression, associated with widespread tumor cell death, the underlying mechanism of which involves the direct activation of the mitochondrial intrinsic apoptotic pathway.

Resveratrol: Challenges in Translation to the Clinic — A Critical Discussion

Low cancer survival rates and the serious side effects often associated with current chemotherapeutics highlight the need for new and effective nontoxic anticancer agents. Since 1997 when Jang and colleagues first described resveratrols ability to inhibit carcinogenesis, it has consistently proven effective at tumor inhibition in diverse human cancer models. This finding has raised the hope that resveratrol would pioneer a novel class of nontoxic chemotherapeutics. As a consequence of initial basic and preclinical studies, resveratrol is now being extensively promoted in the unregulated nutraceutical sector. However, some fundamental aspects of resveratrols action need to be understood before it can be developed into a clinically viable anticancer drug. These areas pertain to the key mechanism(s) by which resveratrol potentiates its antitumor effects. Current research suggests that these mechanisms might be through novel pathways, requiring an understanding of cellular uptake, sentinel targets, and in vivo biological networks. The metabolism of resveratrol and its bioavailablity also warrant further consideration in light of recent in vitro and in vivo studies. Finally, we need to appreciate the sorts of information about resveratrol that may translate between different disease entities. We present a critical discussion of these issues and suggest important experiments that could pave the way to the successful translation of resveratrol to the clinic.Clin Cancer Res; 16(24); 5942–8. ©2010 AACR.

The identification and differential expression of calcium-binding proteins associated with ocular melanoma

Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.

E2F-MEDIATED GROWTH REGULATION REQUIRES TRANSCRIPTION FACTOR COOPERATION

Previous studies have indicated that the presence of an E2F site is not sufficient for G1/S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G0 and S phase. We have now utilized the E2F1 minimal promoter to test several hypotheses that could account for these observations. To test the hypothesis that G1/S phase regulation is achieved via E2F-mediated repression of a strong promoter, a variety of transactivation domains were brought to the E2F1 minimal promoter. Although many of these factors caused increased promoter activity, growth regulation was not observed, suggesting that a general repression model is incorrect. However, constructs having CCAAT or YY1 sites or certain GC boxes cloned upstream of the E2F1 minimal promoter displayed E2F site-dependent regulation. Further analysis of the promoter activity suggested that E2F requires cooperation with another factor to activate transcription in S phase. However, we found that the requirement for E2F to cooperate with additional factors to achieve growth regulation could be relieved by bringing the E2F1 activation domain to the promoter via a Gal4 DNA binding domain. Our results suggest a model that explains why some, but not all, promoters that contain E2F sites display growth regulation.

Resveratrol Metabolites Do Not Elicit Early Pro-apoptotic Mechanisms in Neuroblastoma Cells

Resveratrol, a nontoxic polyphenol, has been shown to inhibit tumor growth in a xenograft mouse model of neuroblasoma. However, resveratrol is rapidly metabolized, mainly to its glucuronidated and sulfated derivatives. This study demonstrates that resveratrol alone, and not the glucuronidated or sulfated metabolites, is taken up into tumor cells, induces a rise in [Ca(2+)](i), and ultimately leads to a decrease in tumor cell viability. A new water-soluble resveratrol formulation was delivered directly at the site of the tumor in a neuroblastoma mouse model. The amount of unmodified resveratrol associated with the tumor increased more than 1000-fold. The increase of unmodified resveratrol associated with the tumor resulted in tumor regression. The number of residual tumor cells that remained viable also decreased as the ratio of the metabolites relative to unmodified resveratrol declined.

Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells.

Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death.

The Sustained Delivery of Resveratrol or a Defined Grape Powder Inhibits New Blood Vessel Formation in a Mouse Model of Choroidal Neovascularization

The objective of this study was to determine whether resveratrol or a defined, reconstituted grape powder can attenuate the formation of new blood vessels in a mouse model of choroidal neovascularization (CNV). To accomplish this objective, C57BL/6J mice were randomized into control or treatment groups which received either resveratrol or grape powder by daily oral gavage, resveratrol or grape powder delivered ad libitum through the drinking water, or resveratrol by slow release via implanted osmotic pumps. A laser was used to rupture Bruch’s membrane to induce CNV which was then detected in sclerochoroidal eyecups stained with antibodies against intercellular adhesion molecule-2. CNV area was measured using fluorescence microscopy and Image J software. Ad libitum delivery of both resveratrol and grape powder was shown to significantly reduce the extent of CNV by 68% and 57%, respectively. Parallel experiments conducted in vitro demonstrated that resveratrol activates p53 and inactivates Akt/protein kinase B in choroidal endothelial cells, contributing to its anti-proliferative and anti-migratory properties. In addition resveratrol was shown to inhibit the formation of endothelial cell networks, augmenting its overall anti-angiogenic effects. The non-toxic nature of resveratrol makes it an especially attractive candidate for the prevention and/or treatment of CNV.

Use of Combination Therapy with Cisplatin and Calcitriol in the Treatment of Y-79 Human Retinoblastoma Xenograft Model

Background: Retinoblastoma is the most common primary malignant intraocular neoplasm of childhood. The poor outcomes of patients with metastatic retinoblastoma have encouraged the search for new therapies. In the current study, the efficacy of combination therapy with calcitriol and cisplatin in athymic mice with subcutaneous Y-79 human retinoblastoma tumours was assessed. Methods: 60 athymic mice were subcutaneously injected with human Y79 retinoblastoma cells. Animals were randomised into four groups: group 1, 50 μg of cisplatin; group 2, 0.05 μg of calcitriol; group 3, 0.05 μg of calcitriol and 50 μg of cisplatin; group 4, control. The cisplatin was administered once a week, and the calcitriol was given five times a week. Results: There was a significant inhibition of tumour growth in animals treated with the combination therapy of calcitriol and cisplatin as compared with controls and cisplatin alone (p = 0.0001 and p = 0.0041 respectively). In terms of toxicity, serum calcium levels were increased, but there was no mortality and minimal nephrotoxicity in any of the groups. Conclusion: The present study shows that cisplatin given in combination with calcitriol may be a viable multidrug therapy option in the treatment of high-risk retinoblastoma.

[7] Calcium-binding proteins and their assessment in ocular diseases

Publisher Summary Calcium-binding proteins play critical roles in the normal function and assorted pathologies of many cells including those of ocular origin. Interpretation of the vast array of calcium signals impinging on the cell depends on spatiotemporal information about the sources of the signals as well as the localization of specific target molecules. Often these cellular targets are calcium-binding proteins that alter their configuration on the chelation of calcium and in turn bind to and alter the activity of other biologically relevant molecules such as enzymes or ion channels. As the concentration of intracellular calcium diminishes on illumination, guanylate cyclase-activating protein 1 (GCAP1) stimulates guanylate cyclase to synthesize cyclic GMP, thus opening cation channels in the plasma membrane of the photoreceptor cell outer segment and reestablishing the dark resting potential of the cell. Aside from studies of phototransduction, these and other calcium-binding proteins are involved in ocular pathologies arising in humans. This chapter presents an assessment of these calcium-binding proteins and their role in ocular diseases.

Calcium-Binding Proteins in Degenerative and Cancer-Related Diseases of the Eye

Calcium-binding proteins purportedly are involved in a variety of human pathologies, but the supporting evidence often is circumstantial. In contrast, studies of visual function have provided clear examples of how defects in either the expression or function of a calcium-binding protein can manifest as a human disease (Polans et al., 1996).