Over the past several decades researchers have focused on finding drugs that specifically affect cancer cells without harming normal cells. Often this research has focused on naturally occurring compounds. Jang et al. (1
) found that resveratrol (Res), a natural product found in grapes, peanuts, and Japanese knotweed, could prevent cancer in a mouse model of melanoma. Following this initial study the anti-cancer effect of Res has been studied in a variety of cancers. In this study we focus on neuroblastoma, a childhood cancer that most often arises in the sympathetic nerves of the adrenal gland and accounts for 15% of the childhood deaths from cancer (2
). Despite aggressive combination therapeutic approaches, more than 60% of the children with high-risk neuroblastoma do not survive due to metastasis, high recurrence and chemo-resistance. Between 20% and 50% of high-risk cases do not respond adequately to induction high-dose chemotherapy and are progressive or refractory. Relapse after completion of frontline therapy is also common. Further, the quality of life of these children is threatened by therapy-induced toxicity (4
). Thus, these children would benefit from a non-toxic compound, such as resveratrol, to alleviate the devastating side effects of standard therapeutics.
mechanistic studies have indicated that Res can activate pathways implicated in apoptosis (5
). However, in mouse xenograft models where Res has been shown to inhibit tumor growth when delivered orally there is little evidence of apoptosis. One possible explanation for the absence of apoptosis in these mouse models is the low bioavailability of Res. The inhibition of tumor growth achieved at lower levels of resveratrol could be explained by other mechanisms of action, including anti-proliferative and anti-angiogenic activities (6
studies in animals and humans indicate that resveratrol is poorly absorbed from the gastrointestinal tract and undergoes extensive first-pass metabolism, mainly glucuronidation and sulfation, in the gut and liver leading to trace amounts of the compound in the serum. The resveratrol metabolites are then dependent on ABC transporters for uptake (7
). Our previous studies in neuroblastoma indicate that despite low bioavailability, resveratrol is effective at inhibiting tumor growth when delivered orally (5
). Indeed, when the serum levels of Res were measured in such rodent studies there was less than 1 μM of Res and twenty fold higher levels of the Res metabolites within half an hour of drug delivery.
The anti-cancer properties of Res could result either from activation of pathways through extra-cellular receptors, or uptake into cells and activation of intra-cellular regulators. Currently, the only evidence for entry of Res into cells comes from cell types that have specialized uptake mechanisms (11
). Determining the subcellular localization of Res would help elucidate potential binding partners and mechanisms whereby Res induces tumor cell death. We therefore want to investigate how Res and its metabolites might differ in cellular uptake, pathway activation, and ultimately in tumor cell death. An early pathway activated by Res, in tumor cells, is calcium signaling by increasing cytoplasmic calcium (12
). Calcium signaling plays a vital role in cell proliferation, angiogenesis and cell death (13
). The versatility of calcium signaling is due to the cell's ability to control the localization and concentration of the signal, and the array of responsive calcium binding proteins in the cell. A sustained increase in [Ca2+
, in tumor cells is thereby associated with the activation of pathways leading to inhibition of tumor cell proliferation as well as cell death (14
). Previous studies (15
) have shown that chelating [Ca2+
with BAPTA-AM inhibits the Res-induced tumor cell death.
In this study we demonstrate that Res enters neuroblastoma cells, induces a calcium signal and ultimately decreases tumor cell viability. Both Res uptake and ER calcium release occur within minutes of drug exposure. Meanwhile, we present evidence using purified derivatives that Res's sulfated and glucuronidated metabolites are not taken up by neuroblastoma cells and, therefore, are incapable of activating calcium signals. Consequently, the Res metabolites have no impact on the viability of these cells. Therefore metabolism diminishes Res's anti-cancer effects. As proof of principle, we demonstrate a method of Res delivery that reduces its rapid metabolism to achieve apoptosis and tumor regression in a neuroblastoma mouse model.