METHODOLOGY/PRINCIPAL FINDINGS: Effects of S9 on PI3K-Akt-mTOR pathway were determined by Western blot, immunofluorescence staining and in vitro kinas assay. The interactions between
tubulin and S9 were investigated by polymerization assay, CD, and SPR assay. The potential binding modes between S9 and PI3K, mTOR or
tubulin were analyzed by molecular modeling. Anti-
tumor activity of S9 was evaluated in
tumor cells and in nude mice bearing human
cancer xenografts. S9 abrogated
EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane in human
tumor cells. S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30
kinases. S9 also completely impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by
rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct from compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited
tubulin polymerization via binding to
colchicine-binding site of
tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the
kinase domains of PI3K p110alpha subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with
tubulin. Moreover, S9 induced rapid apoptosis in
tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and
tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of
tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human
tumor xenografts.
CONCLUSIONS/SIGNIFICANCE: Taken together, S9 targets both PI3K-Akt-mTOR signaling and microtubule cytoskeleton, which combinatorially contributes its antitumor activity and provides new clues for anticancer drug design and development.