Abstract


Background: Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR)
pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid
of a-methylene-c-lactone and 2-phenyl indole compound, possessed potent activity against this pathway.


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 p110a 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.