Cancer Cell. phase and both apoptosis and autophagy. Targeting Akt as a key protein of PI3K/Akt/mTOR pathway with multiple drugs might represent a new and promising pharmacological strategy for treatment of T-ALL patients. treatment with MK-2206, GSK690693 and Perifosine could lead to a modulation of PI3K/Akt/mTOR pathway, we checked the phosphorylation status of key components of this signaling cascade in our panel of more responsive cell lines. In particular we analyzed p-Akt, its downstream target, GSK3 /, and TTP-22 the ribosomal protein S6 kinase, readout of mTORC1 activity, after 30 min of drugs exposure. GSK690693 and Perifosine were used at 1/2 of the IC50 concentration, whereas MK-2206 was used at 1/5 of IC50, since half of MK-2206 IC50 concentration was enough to completely abolish the Ser 473 Akt phosphorylation already at 30 minutes. Akt phosphorylation was affected in different ways by single drug administration: in all cell lines MK-2206 very significantly reduced p-Akt, Perifosine only slightly reduced it and GSK690693 on the contrary increased the protein phosphorylation. The latter one is an already described phenomenon . This increase of Akt phosphorylation diminished the observable effect of double or triple compound combination, since p-Akt was not significantly reduced, unless when using MK-2206 in double exposure (Fig. ?(Fig.3A3A). Open in a separate window Figure 3 Multiple Akt inhibition affects PI3K/Akt/mTOR pathway and the Akt inhibition is time-dependentA Western blot analysis of Akt drug sensitive T-ALL cell lines for total and phosphorylated form of Akt and of its downstream substrate GSK3 / and of mTOR downstream target S6. Samples were treated for 30 minutes with GSK690693, MK-2206 and Perifosine, alone or in double or triple combinations. B Akt protein inhibition as detected by its phosphorylation status. MOLT-4 and JURKAT cells were treated for 30 minutes with a combination of a fixed concentration of GSK690693 and three different concentrations of MK-2206. C Akt phosphorylation levels in cells treated with 7 M Perifosine at different time points. D p-Akt status in MOLT-4 and JURKAT cells pre-treated for 6 h with 7 M Perifosine followed by GSK690693 and MK-2206 administered for 30 minutes. Twenty-five g of protein was blotted to each lane. -actin served as a loading control. TTP-22 For all panel one representative experiment of three is shown as well as cell lines are representative also of the others if not shown. On the contrary, even after such a short time of treatment, in all of the four cell lines it was very evident the efficacy of the multiple hit on Akt. The triple administration of the drugs completely abolished the phosphorylation on the downstream targets, Ser 21/9 p-GSK3 / and Ser 235/236 p-S6, with a much superior efficacy of the triple exposure when compared with the single or with the different double combinations (Fig. ?(Fig.3A).3A). The total amount of the proteins was unchanged in all the treatments (Fig. ?(Fig.3A3A). Pre-treatment with Perifosine enhance synergistic effect Given that the GSK690693 drug alone led to Ser 473 p-Akt increase, whereas MK-2206 alone almost turn off the signal, we sought to explore if we can find a compound combination capable of synergistically dephosphorylate Akt. We first tested if there is any concentration capable to modulate Akt phosphorylation in JURKAT and MOLT-4 cells. Therefore GSK690693 was administered at 1/2 of the IC50 value (0.1 M for JURKAT and 0.15 M for MOLT-4 cells) and MK-2206 was contemporary given at increasing concentrations (0.3C0.5C1 M). After 30 minutes of exposure, Western blot was performed. The best drug combination to observe p-Akt modulation resulted to be 1 M MK-2206 for JURKAT and 0.5 M for MOLT-4 cells (Fig. ?(Fig.3B3B). We then analyzed by Western blot the phosphorylation levels of Akt after treatment with 7 M Perifosine at different time points. In both cell lines the drug affected in a time-dependent manner the Ser 473 Akt phosphorylation (Fig. ?(Fig.3C3C). Finally, we merged the two Cd69 previous assays pre-treating cells for 6 h with Perifosine before a 30 min administration of GSK690693 and MK-2206. As shown in (Fig. ?(Fig.3D),3D), in 6 h Perifosine pre-treated cells, the administration of GSK690693 reduced Ser 473 p-Akt hyperphosphorylation. The combination of all three drugs allowed to obtain a full Akt dephosphorylation in both MOLT-4 and JURKAT cells, thus showing that full Akt inhibition TTP-22 with low drug doses is not only concentration but also time and drug TTP-22 sequence dependent. The triple Akt inhibition induces.
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