Figure 1.
Activation of Akt pathway by pemetrexed.
(A) Akt activation in a time course dependent manner. Human lung adenocarcinoma A549 cells were treated without or with 1 µM pemetrexed for 0, 4, 8, 12, 24, and 48 h, and then cell lysates were isolated. (B) Akt activation occurred in a concentration dependent manner. A549 cells were treated with 0, 0.1, 0.3 and 1 µM pemetrexed for 48 h. After treatment, the levels of total Akt, phosphorylated Akt, total GSK3β, and phosphorylated GSK3β were examined by Western blot analysis. β-Actin was used as an internal loading control. The proportion of S-phase population and apoptotic cells was determined as described in the Materials and Methods section.
Figure 2.
PI3K inhibitors suppress pemetrexed-induced Akt and GSK3β activation, S-phase arrest, cell apoptosis and caspase-3 activation.A549 were pretreated with 10 µM Ly294002 or 3 µM wortmannin (inhibitors of PI3K/Akt) for 2 h, and then 1 µM pemetrexed was added.
(A) the levels of Akt and GSK3β and (B) the cell cycle distribution were determined for 24 h after pemetrexed treatment. (C) Cell apoptosis and (D) the caspase-3 activity were examined after 72 h pemetrexed incubation. **P<0.01, ***P<0.001.
Figure 3.
Knockdown of Akt by siRNA diminishes pemetrexed-induced S-phase arrest and apoptosis.
Cells were transfected with 10 and 25-specific siRNA or scrambled siRNA for 16 h, and then incubated with 1 µM pemetrexed for 24 h; (A) the expression levels of Akt, and (B) cell cycle distribution were examined. (C) Apoptotic cells and (D) caspase-3 activity were assessed after 72 h pemetrexed treatment. ***P<0.001.
Figure 4.
Akt translocates to the nucleus during pemetrexed-induced cell death.
(A) A549 cells were treated with 0, 0.1, 0.3, and 1 µM pemetrexed for 48 h, and (B) A549 cells were treated with 1 µM pemetrexed for 0, 12, 24, and 48 h. After treatment, the subcellular distribution of p-AktS473 was detected by confocal microscopy after immunostaining with anti-phospho-AktS473 and Rhodamine-conjugated secondary antibody. Hoechst 33342 was used to counter stain nuclei, and the images were overlaid to determine the Akt localization within the cell. (C) Inhibiton of Akt activation by Ly294002 and wortmannin blocked Akt nuclear accumulation. A549 cells were pretreated with 10 µM Ly294002 or 3 µM wortmannin for 2 h, and then 1 µM pemetrexed was added for another 48 h. The subcellular distribution of p-AktS473 was detected by immunocytochemistry.
Figure 5.
Inhibition of Akt activation suppresses pemetrexed-induced Cdk2 activation.
(A) Pemetrexed stimulates Cdk2/Cyclin A-associated kinase activity. A549 cells were treated with 0, 0.3, and 1 µM pemetrexed for 24 h, and then protein lysates were isolated. Total protein (500 µg) was incubated with anti-Cdk2 antibody for immunoprecipitation of a Cdk2 kinase complex. Recombinant histone H1 protein was used as a substrate for the Cdk2 kinase activity assay. The levels of immunoprecipitated Cdk2, Cyclin A and Cyclin E were determined by immunoblotting with anti-Cdk2, anti-Cyclin A and anti-Cyclin E antibodies. β-Actin was used as an internal loading control. (B) Pharmacological inhibition of Akt activity decreases Cdk2 activity in pemetrexed-treated cells. A549 cells pretreated with 10 µM Ly294002 or 3 µM wortmannin for 2 h, and then 1 µM pemetrexed was added for another 24 h. The Cdk2 kinase activity and protein level were examined as above mentioned. (C) Knock down Akt expression by Akt specific siRNA reduces Cdk2 kinase activity. A549 cells were transfected with 25 nM of Akt-specific siRNA or scrambled siRNA for 16 h, and then incubated with 1 µM pemetrexed for 24 h, the Cdk2 kinase activity and protein levels were examined as above mentioned.
Figure 6.
Activation of Akt and Cdk2 are occurred in pemetrexed-treated H1299 cells.
(A) Pemetrexed stimulates Akt pathway activation. H1299 cells were treated with 0, 0.1, 0.3 and 1 µM pemetrexed for 48 h. After treatment, the levels of total Akt, phosphorylated Akt, total GSK3β, and phosphorylated GSK3β were examined by Western blot analysis. β-Actin was used as an internal loading control. The proportion of S-phase population and apoptotic cells were determined as described in the Materials and Methods section. (B) Nuclear accumulation of Akt occurred in pemetrexed-treated H1299 cells. Cells were treated with 0, 0.1, 0.3, and 1 µM pemetrexed for 48 h, the subcellular distribution of p-AktS473 was detected by confocal microscopy after immunostaining with anti-phospho-AktS473. Hoechst 33342 was used to counterstain nuclei. (C) Pemetrexed activated Cdk2/Cyclin A-associated kinase in H1299cells. H1299 cells were treated with 0, 0.3, and 1 µM pemetrexed for 24 h, and then protein lysates were isolated. The Cdk2 kinase activity and the levels of Cdk2, Cyclin A and Cyclin E were determined.
Figure 7.
Pharmacological inhibition of Akt reduced pemetrexed-mediated Cdk2 activation, S-phase arrest and apoptosis in H1299 cells.
Cells were pre-treated with wortmannin or Ly294002 for 2 h, and then treated with 1 µM pemetrexed, (A) the Cdk2 kinase activity and protein level were examined and (B) cell cycle distribution analysis was performed after 24 h treatment; (C) apoptotic cells and (D) caspase-3 activity were estimated after 72 h pemetrexed administration.
Figure 8.
Schematic representation of the molecular mechanisms of pemetrexed-induced growth arrest and cell death in human NSCLC cell lines.