Fig 1.
Rapamycin enhanced the inhibiting effect of Dasatinib on cell proliferation and cell cycle progression in A549 cells.
(A) The concentration-dependent effect of Rapamycin on the anticancer activity of Dasatinib in A549 cells. As detailed in the “Methods”, A549 cells were treated with vehicle control (0.1% DMSO) or Dasatinib (5, 10, 25, and 50 nM) in the presence and absence of Rapamycin (20, 50, and 100 nM). Viable cell numbers were analyzed at 72 h after the co-treatment. (B) Effects of Rapamycin on the temporal changes of Dasatinib-induced growth inhibition in A549 cells. Cells were treated with vehicle control (0.1% DMSO), Dasatinib (10 nM) with or without Rapamycin (100 nM). Cell numbers were measured at 0, 24, 48, 72, and 96 h after the treatment. (C) Effects of Dasatinib and Rapamycin on the cell cycle distribution. A549 cells were treated with Dasatinib (10 nM) or Rapamycin (100 nM) for 96 h and analyzed by flow cytometry after PI staining. Results are expressed as the average percentage of cells at G0/G1, S, and G2/M phase from three independent experiments. (D) Effects of Dasatinib and Rapamycin on the apoptosis in A549 cells. Cells were treated with Dasatinib (10 nM) or Rapamycin (100 nM) for 96 h and the apoptotic rates were determined by flow cytometry with Annexin-V and PI staining. Columns, mean of three determinations; bars, SD. * p < 0.05, ** p < 0.01.
Fig 2.
Rapamycin potentiated Dasatinib to up-regulate CDK inhibitor proteins and thereby down-regulate Cdk4.
A549 cells were treated with vehicle control (0.1% DMSO) or Dasatinib (10 nM) in the presence and absence of Rapamycin (100 nM) for 24 h. (A) Relative expression of CDK inhibitor proteins (p16, p19, p21 and p27) at mRNA level. Columns, mean of three determinations; bars, SD. * p < 0.05, ** p < 0.01. (B) Expression of CDK inhibitor proteins, CDKs and FoxOs determined by western blotting. (D) Localization of Cdk4 determined by immunofluorescence staining. Representative pictures indicated staining of Cdk4 (red), nucleus (blue), and the merged images.
Fig 3.
Dasatinib synergized with Rapamycin in the inhibition of Src/PI3K/Akt/mTOR signaling in A549 cells.
A549 cells were treated with vehicle control (0.1% DMSO), Dasatinib (10 nM) with or without Rapamycin (100 nM) for 24 h. Homogenate proteins (20 μg) from the whole cell lysate was collected and used for western blotting. (A) Activation of Src determined by immunofluorescence staining. Representative pictures indicated staining of Src (red), nucleus (blue), and the merged images. (B) The activation of Src, PI3K, and Akt determined by western blotting. (C) Densitometry quantification of Src, PI3K and Akt phosphorylation in A549 cells. (D) The activation of mTOR signaling determined by western blotting. (E) Densitometry quantification of mTOR, p70S6K and 4E-BP1 phosphorylation in A549 cells. The data presented average relative phosphorylation ratios to the untreated control from three independent experiments. Columns, mean of three determinations; bars, SD. * p < 0.05, ** p < 0.01.
Fig 4.
Repression of mTOR and Src by siRNAs facilitated cell cycle arrest and growth inhibition in A549 cells.
(A) The activation levels of PI3K, AKT, and mTOR determined by western blotting. A549 cells were transfected with si-Src, si-mTOR, or control siRNA for 8 h, followed by a prolonged incubation of 24 h. Homogenate proteins (20 μg) from the whole cell lysate was collected and used for western blotting. (B) The expression of CDK inhibitor proteins (p16, p19, p21, and p27), FoxO1, and Cdk4 determined by western blotting. (C) Effects of si-mTOR and si-Src on the cell cycle progression. A549 cells were transfected with si-Src, si-mTOR, or control siRNA for 8 h, followed by an prolonged incubation of 24 h. Then the cells were collected and analyzed by flow cytometry with PI staining. Results are expressed as the average percentage of cells at G0/G1, S, and G2/M phase from three independent experiments. Results were expressed as the average percentage of cells at G0/G1, S, and G2/M phase from three independent experiments. (D) Effects of si-mTOR and si-Src on the temporal changes of cell proliferation. A549 ells were transfected with si-Src, si-mTOR, or control siRNA for 8 h, and then incubated with normal medium for 72 h. Cell numbers were measured at 0, 24, 48, and 72 h after the transfection. The data was presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01.
Fig 5.
Rapamycin enhanced the inhibiting effect of Dasatinib on invasion and migration in A549 cells.
(A) Representative images (upper panel) and quantification (lower panel) of invading cells. As detailed by “cell invasion assay” in the “Methods”, A549 cells were treated with DMSO (0.1%), Dasatinib (10 nM) alone or in combination with Rapamycin (100 nM) for 24 h. The numbers of invading cells were counted under microscope and the data was presented as mean ± SD from three independent experiments, * p < 0.05, ** p < 0.01. (B) Representative images of wound healing assays in A549 cells that were treated with DMSO (0.1%), Dasatinib (10 nM) or Rapamycin (100 nM) for 18 h. (C) The expression of MMP-2/9 and E-cadherin determined by western blotting in A549 cells that were treated with DMSO (0.1%), Dasatinib (10 nM) or Rapamycin (100 nM) for 24 h.
Fig 6.
Rapamycin enhanced the inhibiting effect of Dasatinib on cell proliferation and cell cycle progression in other NSCLC cells.
(A) Rapamycin enhanced Dasatinib-induced growth inhibition in NCI-H1706 and NCI-H460 cells. NCI-H1706 and NCI-H460 cells were treated with vehicle control (0.1% DMSO), Dasatinib (10 nM) with or without Rapamycin (100 nM). Cell numbers were measured at 0, 24, 48, 72, and 96 h after the treatment. (B) Effects of Dasatinib and Rapamycin on the cell cycle progression. NCI-H1706 and NCI-H460 cells were treated with Dasatinib (10 nM) or Rapamycin (100 nM) for 96 h and analyzed by flow cytometry after PI staining. Results are expressed as the average percentage of cells at G0/G1, S, and G2/M phase from three independent experiments. Columns, mean of three determinations; bars, SD. * p < 0.05, ** p < 0.01.
Fig 7.
Dasatinib synergized with Rapamycin in up-regulating CDK inhibitors through Src/PI3K/Akt/mTOR pathway in other NSCLC cells.
NCI-H1706 and NCI-H460 cells were treated with vehicle control (0.1% DMSO) or Dasatinib (10 nM) in the presence and absence of Rapamycin (100 nM) for 24 h. (A) Relative expression of CDK inhibitor proteins (p16, p19, p21 and p27) at mRNA level. Columns, mean of three determinations; bars, SD. * p < 0.05, ** p < 0.01. (B) The activation of Src, PI3K, AKT, and mTOR determined by western blotting. NCI-H1706 and NCI-H460 cells were treated with vehicle control (0.1% DMSO) or Dasatinib (10 nM) in the presence and absence of Rapamycin (100 nM) for 24 h. Homogenate proteins (20 μg) from the whole cell lysate was collected and used for western blotting.
Fig 8.
A schematic model for the anti-cancer function of Dasatinib and Rapamycin mediated by the Src-PI3K-Akt-mTOR pathway in NSCLC cells.
Dasatinib-induced Src inhibition further suppressed the activation of mTOR via PI3K-Akt signaling pathway. The inhibition of mTOR by Rapamycin potentially facilitates Dasatinib-induced Src deactivation (dash line), thereby results in the enhanced up-regulation of CDK inhibitor proteins and cell cycle arrest via PI3K/AKT pathway. Meanwhile, the combination of Dasatinib with Rapamycin induces inhibition of cell migration and invasion through suppressing Src/PI3K/AKT signaling.