Abstract #2815: Inhibition of Cap-dependent protein translation concomitant with induction of autophagy through repression of mTOR signaling pathway in PC3 prostate cancer cells by dietary flavonoid fisetin

The mammalian target of rapamycin (mTOR) kinase is an important component of PTEN/PI3K/Akt signaling pathway which is frequently dysregulated in various cancers including prostate cancer (CaP). Recent studies suggest that targeting PTEN/PI3K/Akt and mTOR signaling pathway could be an effective strategy for the treatment of cancer. Moreover, mTOR signaling is involved in CaP progression, especially in transition to hormone refractory disease. Fisetin, 3,7,3',4'-tetrahydroxyflavone, is a naturally occurring flavonoid commonly found in fruits and vegetables such as strawberry, apple, onion, and cucumber. We recently showed that fisetin induces apoptosis and cell cycle arrest in LNCaP human CaP cells and inhibits androgen receptor signaling and tumor growth in vivo. We hypothesized that fisetin may provide chemotherapeutic effects against hormone-independent subtype of CaP. We found that the treatment of androgen-independent and PTEN negative PC3 human CaP cells with fisetin resulted in dose-dependent inhibition of cell growth and mTOR kinase signaling pathway. In fisetin treated cells, we observed dose-dependent suppression of mTOR kinase activity and downregulation of mTOR signaling complex (mTORC) constituents such as Raptor, Rictor, PRAS40, and G\#946;L. Immunoprecipitation experiments confirmed reduced formation of mTORC1 and mTORC2 in fisetin treated cells. Fisetin also activated mTOR repressor TSC2 through inhibition of Akt and activation of AMPK which was induced by low cellular ATP level. Fisetin-mediated inhibition of mTOR resulted in hypophosphorylation and thus activation of translation initiation repressor 4EBP1. The inhibition of mTOR also resulted in the inactivation of S6K70 and its target proteins rpS6 and translation initiation factor eIF4B. The 7-methyl-GTP Sepharose chromatography revealed that fisetin inhibits the formation of protein translation initiation complex that lead to suppression of Cap-dependent translation as assessed by bicistronic luciferase reporter assay. The role of fisetin in autophagy was also determined as inhibition of mTOR is known to induce autophagy. We found that fisetin treatment of cells leads to induction of autophagy as validated by autophagosome staining with monodansylcadaverine and GFP-LC3. Subsequent studies with autophagy inhibitor chloroquine and autophagy essential gene Beclin1 knockout cells indicated that fisetin induces autophagic programmed cell death rather than cytoprotective autophagy. Taken together, we provide evidence that the plant flavonoid fisetin can inhibit mTOR signaling pathway that leads to inhibition of Cap-dependent protein translation and induction of autophagic cell death in PC3 cells. These results suggest that fisetin could be useful as chemotherapeutic agent in treatment of hormone refractory CaP.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2815.