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 Amol/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.

These results demonstrate that resveratrol, a nontoxic natural plant compound, inhibits Y79 cell proliferation and stimulates apoptosis through activation of the mitochondrial (intrinsic) apoptotic pathway and may warrant further exploration as an adjuvant to conventional anticancer therapies for retinoblastoma.

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 resveratrol's 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 resveratrol's 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.

These data suggest that resveratrol can inhibit tumor growth and can induce apoptosis via the intrinsic mitochondrial pathway and that by further increasing bioavailability of resveratrol the potency of the drug can be increased, leading to tumor regression. The nontoxic nature of the drug at levels needed for therapy make resveratrol an attractive candidate for the treatment of uveal melanoma.

ABSTRACT

Previous studies have indicated that the presence of an E2F site is not sufficient for G 1 /S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G 0 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 G 1 /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.E2F plays an important role in regulating gene expression at the G 1 /S phase transition in the mammalian cell cycle. Promoters in which E2F sites contribute to transcriptional regulation are found in genes involved in DNA synthesis (such as dihydrofolate reductase, DNA polymerase ␣, and thymidine kinase) as well as in genes that are involved in cell cycle control (such as B-myb, several cyclins, and cdc2). E2F-mediated regulation of many of these genes leads to differential expression in G 0 and S phase, causing low promoter activity in quiescent cells and increased activity at the G 1 /S phase boundary (1). However, certain promoters contain E2F sites that do not confer growth-regulated transcriptional activity (2, 3). Several models have been put forth to explain why certain E2F sites mediate growth regulation and others do not.One such model is based on the fact that E2F is a family of transcription factors of which seven members have been characterized to date. E2F1-5 can heterodimerize with either DP1 or DP2 to create functional E2F activity (4, 5). Some of the E2Fs are present only at certain stages of the cell cycle (such as E2F1), whereas others are constitutively present (such as E2F4). Also, E2F activity is regulated by the Rb family of proteins (4 -9). E2F1-3 preferentially bind Rb, whereas E2F4 and E2F5 mainly bind p107 and p130. Thus, it is possible that growth-regulated promoters have a different composition of E2F protein complexes bound to the promoter DNA than do non-growth-regulated promoters. This model, however, cannot account for results obtained from deletion analyses of growthregulated promoters. For example, we have shown that deletion o...

Object Glioblastoma multiforme (GBM) is an aggressive brain cancer with median survival of less than two years with current treatment. GBM exhibits extensive intra-tumor and inter-patient heterogeneity, suggesting that successful therapies should exert broad anti-cancer activities. Therefore, the natural non-toxic pleiotropic agent, resveratrol, was studied for anti-tumorigenic effects against GBM. Methods Resveratrol’s effects on cell proliferation, sphere-forming ability, and invasion were tested using multiple patient-derived GBM stem-like cell (GSC) lines and established U87 glioma cells, and changes in oncogenic AKT and tumor suppressive p53 were analyzed. Resveratrol was also tested in vivo against U87 glioma flank xenografts using multiple delivery methods, including direct tumor injection. Finally, resveratrol was delivered directly to brain tissue to determine toxicity and achievable drug concentrations in the brain parenchyma. Results Resveratrol significantly inhibited proliferation in U87 glioma and multiple patient-derived GSC lines, demonstrating similar inhibitory concentrations across these phenotypically heterogeneous lines. Resveratrol also inhibited the sphere-forming ability of GSCs, suggesting anti-stem cell effects. Additionally, resveratrol blocked U87 glioma and GSC invasion in an in vitro Matrigel transwell assay at doses similar to those mediating anti-proliferative effects. In U87 glioma cells and GSCs, resveratrol reduced AKT phosphorylation and induced p53 expression and activation that led to transcription of downstream p53 target genes. Resveratrol administration via oral gavage or ad libitum in the water supply significantly suppressed GBM xenograft growth; intra-tumor or peri-tumor resveratrol injection further suppressed growth and approximating tumor regression. Intracranial resveratrol injection resulted in 100-fold higher local drug concentration compared to intravenous delivery, and with no apparent toxicity. Conclusions Resveratrol potently inhibited GBM and GBM stem-like cell growth and infiltration, acting partially via AKT deactivation and p53 induction, and suppressed glioblastoma growth in vivo. The ability of resveratrol to modulate AKT and p53, as well as reportedly many other anti-tumorigenic pathways, is attractive for therapy against a genetically heterogeneous tumor such as GBM. Although resveratrol exhibits low bioavailability when administered orally or intravenously, novel delivery methods such as direct injection (i.e. convection enhanced delivery) could potentially be used to achieve and maintain therapeutic doses in brain. Resveratrol’s non-toxic nature and broad anti-GBM effects make it a compelling candidate to supplement current GBM therapies.

Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca 2+ -binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca 2+ concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca 2+ concentration. However, binding of Ca 2+ to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca 2+ affinity. The addition of N-terminal ALG-2 peptides, residues 1-22 or residues 7-17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca 2+ binding to EF-3, while Ca 2+ binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition. † This work was supported by NIH Grants EY12768 and EY13705 (A.S.P.), EY08061 (K.P.), and EY06603 and EY14239 (J.W.C.) and the Swiss National Science Foundation 31-65071.01 (J.C.), as well as grants from the Retina Research Foundation, Research to Prevent Blindness Inc. (RPB), the University of Wisconsin Comprehensive Cancer Center, and the E. K. Bishop Foundation. L.S. is a recipient of the Cremer Scholarship. A.S.P. is a Jules and Doris Stein RPB Professor. * To whom correspondence should be addressed. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptApoptosis-linked gene-2 (ALG-2) was first identified in a death-trap assay using a mouse T cell hybridoma model (1). These cells when transfected with an antisense ALG-2 expression vector were resistant to T cell receptor-mediated cell death and were partially protected from other agents that normally initiate programmed cell death such as Fas, staurosporine, actinomycin D, and C 2 -ceramide (1). More recent work suggests that ALG-2 might be involved in the apaf1-independent intrinsic apoptotic pathway activated as a result of endoplasmic reticulum (ER) stress (2).ALG-2 contains five putative EF-hand sequences and is a member of the family of PEF 1 (penta-EF-hand) proteins that includes the calpain small subunit, sorcin, and grancalcin (3). In ALG-2, EF-1 and EF-3 are considered the high-affinity Ca 2+ -binding sites, while one low-affinity site...