The most important finding of our study is that treatment with fisetin caused dual inhibition of PI3K/Akt and mTOR signaling in human NSCLC cells. To our knowledge, no other dietary agent at physiologically attainable concentrations has been shown to exert this dual inhibitory effect. Finally, fisetin did not inhibit cell-growth, PI3K/Akt and mTOR signaling in NHBE cells (data not shown). While it remains unclear as to why fisetin behaves differently in cancer cells compared to normal cells, it could be speculated that uptake mechanisms could partially explain this paradox. It is speculated that fisetin is rapidly taken up by cancer cells, while its uptake is slow and regulated in normal cells.
The mTOR pathway has emerged as an important cancer therapeutic target. The discovery of the highly specific and potent mTOR inhibitor rapamycin and its derivatives that specifically inhibit mTOR are now being actively evaluated inclinical trials.33
A potential mechanism of resistance to mTOR inhibitors is caused by a negative feedback loop in which mTOR inhibition leads to AKT activation through upregulation of receptor tyrosine kinases such as platelet derived growth factor receptors34
and insulin receptor substrate-1.35
The relevance of this feedback is underscored by its existence in cancer patients.36
We found that fisetin inhibits the mTOR pathway and keeps the feedback loop in check by also inhibiting the PI3K/Akt pathway and inhibits cell survival and growth ().
In the present study, we have shown for the first time that fisetin inhibited PI3K/Akt and mTOR signaling in human NSCLC cells. Treatment of A549 and H1792 human lung cancer cells with fisetin caused decrease in cell-viability but had minimal effects on NHBE cells. There was also inhibition in the ability of A549 cells to form colonies on treatment with fisetin. Using autodock4, we also found that fisetin bound to two sites on the mTOR target. The binding energies were in the −7 to −8 Kcal/mol range for the binding constant.
Since the discovery of PTEN as a putative tumor suppressor in 1997, its importance as a tumor suppressor has been validated by its mutation and/or loss of expression in a variety of sporadic cancers and its association with Cowden disease, an autosomal dominant cancer syndrome. PTEN plays an important role in multiple cellular functions such as cell metabolism, proliferation and survival. Loss of the tumor suppressor PTEN is common in various kinds of human solid tumors. Therefore, development of genes and materials that regulate PTEN in tumors is one of the important fields in overcoming resistance against anticancer agents.37
The major substrate of the lipid phosphatase activity of PTEN is PIP3 (phosphatidylinositol 3,4,5-triphosphate), an important intracellular second messenger. By dephosphorylating the D3-position of PIP3, PTEN negatively regulates the PI3K pathway and Akt activation and thus suppresses tumorigenesis. We also found that fisetin increased the protein levels of PTEN dose-dependently. AMPK is a member of a metabolite-sensing protein kinase family which plays an essential role as an energy-sensor mainly in ATP-deprived conditions.38
Therefore, AMPK is known to play a major protective role under metabolic stressed conditions. In the activated states, AMPK down-regulates several anabolic enzymes and thus shuts down the ATP-consuming metabolic pathways. Activation of AMPK inhibits mTOR signaling and is associated with inhibition of cancer cell growth.39
Consistent with these studies, we found that fisetin caused inhibition of the phosphorylation of mTOR, upregulation of AMPKα and decrease in the expression of Raptor, Rictor, PRAS40 and GβL causing less formation of both mTORC1 and mTORC2 in lung cancer cells.
Since we observed a decrease in the phosphorylation of mTOR on treatment with fisetin, we investigated the effect of fisetin on PI3K/Akt pathway. Fisetin treatment resulted in the inhibition of the expression of regulatory and catalytic subunits of PI3K and inhibition of the phosphorylation of Akt, suggesting that fisetin-induced decrease in mTOR phosphorylation is dependent on PI3K/Akt pathway as well.
Tuberous sclerosis, an autosomal dominant disorder is caused by mutations of TSC1 and TSC2, which in humans is associated with hamartomatous polyps in multiple tissues and an increased risk of cancers. TSC2 is a tumor suppressor that has been linked to AMPK and it forms an inhibitory complex with TSC1 that binds to and inhibits mTOR, leading to negative regulation of cell size and growth.40
TSC1/TSC2 complex inhibits mTOR activity by activating the GTPase activity of Ras homologue enriched in brain, and both Akt and AMPK converged at TSC1/TSC2 to regulate mTOR activity.41
Fisetin caused inhibition of the phosphorylation of TSC2 and increase in the protein expression of TSC2 consistent with the fact that Akt phosphorylates TSC2 and disrupts the TSC1/TSC2complex, leading to activation of mTOR.42
The ribosomal S6 kinase and the 4E-BP1 are the two major downstream signaling pathways of mTOR and have a role in control of protein translation.43
The phosphorylation of 4EBP-1 by mTOR results in the release of a cap-binding protein eIF4E, which is held inactive when bound to the hypophosphorylated 4EBP-1 complex.4, 44
Studies have shown that higher levels of eIF4E are found in many transformed cell-lines and various cancers overexpress eIF4E.45
Seki et al.
have shown that eIF4E seems increased in peripheral lung adenocarcinomas and suggests a correlation between the magnitude of the eIF4E increase and the invasiveness of the tumors.46
Fisetin-treated cells showed decreased phosphorylation of mTOR protein expression and its downstream targets (4EBP1, eIF-4E, p70S6K), suggesting the effect of fisetin on mTOR signaling. To confirm that these effects are mediated in part through mTOR signaling, we have shown that when fisetin was added to rapamycin-treated cells, there was further downregulation in the phosphorylation of these proteins. To further validate this, we silenced mTOR and found that it caused decrease in the phosphorylation of the downstream targets of mTOR which was further augmented by the addition of fisetin, suggesting that these effects are in part, due to mTOR signaling and fisetin is likely to have other modes of action, as is the case for other dietary agents. Taken together, these findings show that fisetin, a natural dietary flavonoid inhibits PI3K/Akt and mTOR signaling in human non-small cell lung cancer cells and could be developed as a potential lung cancer chemopreventive/chemotherapeutic agent.