Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: Findings from 3-D melanoma skin equivalents and computational modeling

Abstract

The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways.

Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma

“…We previously showed that the dietary flavonoid fisetin decreased Mitf levels in 451Lu melanoma cells through interference with Wnt/β-catenin signaling and inhibited tumor growth in a xenograft model (20). Employing cell free systems and 3-D melanoma skin equivalent constructs, we next demonstrated, that fisetin binds with much higher affinity to p70S6K and mTOR in comparison to AKT, to execute its growth inhibitory effect on cancer cells (27). The aim of the current study was to further dissect the molecular and biochemical pathways activated in fisetin-induced cytotoxicity and subsequent growth inhibition in human melanoma cells.…”

Exploring the molecular targets of dietary flavonoid fisetin in cancer

“…We had reported previously that fisetin negatively regulated the growth of human melanoma cells through disruption of Wnt/β-catenin signaling and decreased Mitf levels [31]. We extended these studies in melanoma cells and evaluated the relative binding affinities of fisetin to kinases involved in growth and proliferation [32]. Our studies in melanoma monolayers and 3D melanoma skin equivalent model demonstrated that fisetin targets p70S6K and mTOR (Figure 1) and exerts an inhibitory effect on the growth of human melanoma cells through direct binding to these kinases [32].…”

“…We extended these studies in melanoma cells and evaluated the relative binding affinities of fisetin to kinases involved in growth and proliferation [32]. Our studies in melanoma monolayers and 3D melanoma skin equivalent model demonstrated that fisetin targets p70S6K and mTOR (Figure 1) and exerts an inhibitory effect on the growth of human melanoma cells through direct binding to these kinases [32]. Interestingly, fisetin was found to have a very low binding affinity to Akt suggesting that the decreased phosphorylation of Akt observed upon fisetin treatment was mediated through its effect on interrelated pathways [32].…”

“…Our studies in melanoma monolayers and 3D melanoma skin equivalent model demonstrated that fisetin targets p70S6K and mTOR (Figure 1) and exerts an inhibitory effect on the growth of human melanoma cells through direct binding to these kinases [32]. Interestingly, fisetin was found to have a very low binding affinity to Akt suggesting that the decreased phosphorylation of Akt observed upon fisetin treatment was mediated through its effect on interrelated pathways [32]. Recently, fisetin was shown to inhibit growth and proliferation of human melanoma cells both in vitro and in vivo in combination with the BRAF inhibitor sorafenib [33].…”

Phytochemicals for the Management of Melanoma

“…Fisetin treatment also promoted mesenchymal to epithelial transition (MET) by decreasing mesenchymal marker proteins and increasing epithelial marker proteins [108]. Syed et al [111, 112] observed downregulation of Wnt/β-catenin, PI3K/AKT, mTOR, and microphthalmia-associated transcription factor (MITF) signaling proteins in melanoma cell lines and in a three-dimensional human skin equivalent melanoma model. These findings show that fisetin is a phytochemical with promising anti-melanoma activities.…”