title: 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.
authors.affiliation: Department of Dermatology, University of Wisconsin, Madison, USA.
authors.forename: Deeba N
authors.initials: DN
authors.lastname: Syed
authors.affiliation: Department of Dermatology, University of Wisconsin, Madison, USA.
authors.forename: Jean-Christopher
authors.initials: JC
authors.lastname: Chamcheu
authors.affiliation: Department of Dermatology, University of Wisconsin, Madison, USA.
authors.forename: Mohammad Imran
authors.initials: MI
authors.lastname: Khan
authors.affiliation: Department of Chemistry and Pharmacy, University of Sassari, 07100 Sassari, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Sechi
authors.affiliation: Department of Dermatology, University of Wisconsin, Madison, USA.
authors.forename: Rahul K
authors.initials: RK
authors.lastname: Lall
authors.affiliation: Department of Dermatology, University of Wisconsin, Madison, USA.
authors.forename: Vaqar M
authors.initials: VM
authors.lastname: Adhami
authors.affiliation: Department of Dermatology, University of Wisconsin, Madison, USA. Electronic address: hmukhtar@wisc.edu.
authors.forename: Hasan
authors.initials: H
authors.lastname: Mukhtar
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D045744:Cell Line, Tumor; D003198:Computer Simulation; D005419:Flavonoids / Q000494:pharmacology*; D044948:Flavonols; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008545:Melanoma / Q000188:drug therapy*; D008954:Models, Biological; D011485:Protein Binding; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D005419:Flavonoids; D044948:Flavonols; C546842:MTOR protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; C017875:fisetin
keywords: AKT; Fisetin; Melanoma; mTOR; p70S6K