Inositol hexakisphosphate blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting the Akt/NFκB-mediated cell survival pathway

It has been reported that inositol hexakisphosphate (InSP6, phytic acid), a natural product, has an anticancer role. However, there is inadequate information regarding the mechanism by which InSP6 exerts anticancer actions, and the effect requires relatively high concentration of the agent, both of which hinders the usage of InsP(6) as an anticancer drug. In the present study, we investigated the mechanism by which InSP6 acts as an anticancer agent, and tried to reduce the concentration of effective InSP6. Treatment of HeLa cells with InSP6 at 1 mM induced apoptosis, as assessed by counting the cell number, and by Hoechst and TUNEL staining. This is probably mediated by intracellular InSP6 itself and/or the dephosphorylated forms of metabolized InSP6, because incubation of HeLa cells with [H-3]InsP(6) produces dephosphorylated forms such as InsP(4) and InsP(5). Induction of apoptosis by InsP(6) was examined in two ways: inhibition of cell survival signaling and direct induction of apoptosis. Treatment of HeLa cells with tumor necrosis factor (TNF) or insulin stimulated the Akt-nuclear factor kappaB (NFkappaB) pathway, a cell survival signal, which involves the, phosphorylation of Akt and IkappaB, nuclear translocation of NFkappaB and NFkappaB-luciferase transcription activity. InSP6 blocked all these cellular events, but phosphatidylinositol 3-kinase activity was not affected. As well as inhibiting the Akt-NFkappaB pathway, InSP6 itself caused mitochondrial permeabilization, followed by cytochrome c release, which later caused activation of the apoptotic machinery, caspase 9, caspase 3 and poly (ADP-ribose) polymerase. When InSP6 was applied together with histone, the effective concentration to induce apoptosis was similar to10-fold lower. These results revealed that extracellularly applied InsP(6) directly activates the apoptotic machinery as well as inhibits the cell survival signaling, probably by the intracellular delivery followed by a dephosphorylation.