Abstract 1586: Tumor regression in a 3-D melanoma model by the dietary flavonoid fisetin is associated with inhibition of Akt/mTOR signaling

Abstract

Abstract Melanoma, one of the most aggressive cancers with a relatively high propensity for metastasis, is a leading cause of death among young adults. Surgery remains the cornerstone of curative treatment in earlier stages. Once the disease has metastasized the prognosis is grim as metastatic melanoma does not respond to most systemic treatments. The Akt/mTOR signaling, constitutively activated in melanoma, serves as a convergence point for growth stimuli that contribute to the initiation and maintenance of melanoma growth. Accumulating data suggest that natural phenolic compounds including flavonoids, tannins and others play an important role in cancer prevention and treatment. We recently showed that a flavonoid fisetin, found in fruits and vegetables such as strawberries, cucumbers etc. inhibits melanoma cell proliferation in monolayer cell cultures and xenograft mouse model (Syed et al., JID 2011). To establish the relevance of this work to human melanoma, we determined the effect of fisetin on melanoma growth employing a three dimensional (3-D) human skin equivalent melanoma model comprising of A375 melanoma cells, cultured with epidermal keratinocytes and dermal fibroblasts. The melanoma constructs were treated with fisetin (80 µM) every alternate day for 16 days and cross-section of the constructs were taken at four day interval. H&E-stained images of cross-sections of untreated constructs at days 12 and 16 showed nests of tumor cells and many invading disseminated cells. In sharp contrast, fisetin-treated constructs exhibited significantly less melanocytic lesions. Notably, fisetin caused no detectable damage to the keratinocytes, fibroblasts, or skin morphology present in this model. Immunohistochemical analysis of these constructs showed significant decrease in the phosphorylated form of Akt and mTOR proteins in fisetin treated compared to control sections. Western blot analysis of fisetin treated melanoma cell (A375 and 451Lu) monolayer cell cultures further showed a decrease in the protein expression of PI3K (p85 and p110), inhibition of phosphorylation of Akt, mTOR, and the downstream targets p70S6K, eIF-4E and 4E-BP1. Fisetin increased the binding of TSC (tumor suppressor complex)-1 to TSC-2 thereby stabilizing TSC-2 and downregulated the protein expression of Rheb, a positive regulator of mTOR signaling. Xenograft studies in mice implanted with 451Lu melanoma cells showed concomitant suppression of Akt and mTOR phosphorylation and upregulation of TSC-1/2, associated with reduced tumor growth in the fisetin treated group. Collectively, our studies indicate that fisetin targets both Akt and mTOR signaling which in turn decreases the viability of melanoma cells. Our data suggest a key role of Akt and mTOR in melanoma progression and identify fisetin as a modulator of these targets. Fisetin, therefore can be a useful agent for slowing the progression of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1586. doi:1538-7445.AM2012-1586