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Rapamycin Enhances the Anti-Cancer Effect of Dasatinib by Suppressing Src/PI3K/mTOR Pathway in NSCLC Cells.

Chen B, Xu X, Luo J, Wang H, Zhou S - PLoS ONE (2015)

Bottom Line: In this study, we found that Rapamycin dramatically enhanced Dasatinib-induced cell growth inhibition and cell cycle G1 arrest in human lung adenocarcinoma A549 cells without affecting apoptosis.The synergistic effects were consistently correlated with the up-regulation of cyclin-dependent kinases inhibitor proteins, including p16, p19, p21, and p27, as well as the repression of Cdk4 expression and nuclear translocation.Restraining Src and mTOR with small interfering RNA in A549 cells further confirmed that the Src/PI3K/mTOR Pathway played a crucial role in enhancing the anticancer effect of Dasatinib.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Shanghai Pulmonary Hospital, Shanghai, China; School of Medicine Cancer Institute, Tongji University, Shanghai, China.

ABSTRACT
Src and the mammalian target of rapamycin (mTOR) signaling are commonly activated in non-small cell lung cancer (NSCLC) and hence potential targets for chemotherapy. Although the combined use of Src inhibitor Dasatinib with other chemotherapeutic agents has shown superior efficacy for cancer treatment, the mechanisms that lead to enhanced sensitivity of Dasatinib are not completely understood. In this study, we found that Rapamycin dramatically enhanced Dasatinib-induced cell growth inhibition and cell cycle G1 arrest in human lung adenocarcinoma A549 cells without affecting apoptosis. The synergistic effects were consistently correlated with the up-regulation of cyclin-dependent kinases inhibitor proteins, including p16, p19, p21, and p27, as well as the repression of Cdk4 expression and nuclear translocation. Mechanistic investigations demonstrated that FoxO1/FoxO3a and p70S6K/4E-BP1, the molecules at downstream of Src-PI3K-Akt and mTOR signaling, were significantly suppressed by the combined use of Dasatinib and Rapamycin. Restraining Src and mTOR with small interfering RNA in A549 cells further confirmed that the Src/PI3K/mTOR Pathway played a crucial role in enhancing the anticancer effect of Dasatinib. In addition, this finding was also validated by a series of assays using another two NSCLC cell lines, NCI-H1706 and NCI-H460. Conclusively, our results suggested that the combinatory application of Src and mTOR inhibitors might be a promising therapeutic strategy for NSCLC treatment.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0129663.g001: 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.

Mentions: As shown in Fig 1A, Dasatinib at pharmacologically relevant levels markedly decreased the cell number of A549 cells in a concentration-dependent manner. Remarkably, the co-treatment with Rapamycin (50 and 100 nM) significantly enhanced Dasatinib-mediated growth inhibition in A549 cells, in that 10 nM of Dasatinib plus 100 nM of Rapamycin could achieve equal magnitudes of anticancer activity than Dasatinib alone at the concentration of 50 nM. Results about the temporal effects of Dasatinib with Rapamycin indicated that the A549 cell proliferation was significantly inhibited by Dasatinib (10 nM) through 96 h of treatment (Fig 1B). Importantly, the co-treatment with Rapamycin (100 nM) further decreased the cell numbers at as early as 48 h, suggesting the marked synergistic efficacy to the treatment of Dasatinib itself. However, Rapamycin alone showed minor suppression on the growth curve of cancer cells.


Rapamycin Enhances the Anti-Cancer Effect of Dasatinib by Suppressing Src/PI3K/mTOR Pathway in NSCLC Cells.

Chen B, Xu X, Luo J, Wang H, Zhou S - PLoS ONE (2015)

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.
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pone.0129663.g001: 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.
Mentions: As shown in Fig 1A, Dasatinib at pharmacologically relevant levels markedly decreased the cell number of A549 cells in a concentration-dependent manner. Remarkably, the co-treatment with Rapamycin (50 and 100 nM) significantly enhanced Dasatinib-mediated growth inhibition in A549 cells, in that 10 nM of Dasatinib plus 100 nM of Rapamycin could achieve equal magnitudes of anticancer activity than Dasatinib alone at the concentration of 50 nM. Results about the temporal effects of Dasatinib with Rapamycin indicated that the A549 cell proliferation was significantly inhibited by Dasatinib (10 nM) through 96 h of treatment (Fig 1B). Importantly, the co-treatment with Rapamycin (100 nM) further decreased the cell numbers at as early as 48 h, suggesting the marked synergistic efficacy to the treatment of Dasatinib itself. However, Rapamycin alone showed minor suppression on the growth curve of cancer cells.

Bottom Line: In this study, we found that Rapamycin dramatically enhanced Dasatinib-induced cell growth inhibition and cell cycle G1 arrest in human lung adenocarcinoma A549 cells without affecting apoptosis.The synergistic effects were consistently correlated with the up-regulation of cyclin-dependent kinases inhibitor proteins, including p16, p19, p21, and p27, as well as the repression of Cdk4 expression and nuclear translocation.Restraining Src and mTOR with small interfering RNA in A549 cells further confirmed that the Src/PI3K/mTOR Pathway played a crucial role in enhancing the anticancer effect of Dasatinib.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Shanghai Pulmonary Hospital, Shanghai, China; School of Medicine Cancer Institute, Tongji University, Shanghai, China.

ABSTRACT
Src and the mammalian target of rapamycin (mTOR) signaling are commonly activated in non-small cell lung cancer (NSCLC) and hence potential targets for chemotherapy. Although the combined use of Src inhibitor Dasatinib with other chemotherapeutic agents has shown superior efficacy for cancer treatment, the mechanisms that lead to enhanced sensitivity of Dasatinib are not completely understood. In this study, we found that Rapamycin dramatically enhanced Dasatinib-induced cell growth inhibition and cell cycle G1 arrest in human lung adenocarcinoma A549 cells without affecting apoptosis. The synergistic effects were consistently correlated with the up-regulation of cyclin-dependent kinases inhibitor proteins, including p16, p19, p21, and p27, as well as the repression of Cdk4 expression and nuclear translocation. Mechanistic investigations demonstrated that FoxO1/FoxO3a and p70S6K/4E-BP1, the molecules at downstream of Src-PI3K-Akt and mTOR signaling, were significantly suppressed by the combined use of Dasatinib and Rapamycin. Restraining Src and mTOR with small interfering RNA in A549 cells further confirmed that the Src/PI3K/mTOR Pathway played a crucial role in enhancing the anticancer effect of Dasatinib. In addition, this finding was also validated by a series of assays using another two NSCLC cell lines, NCI-H1706 and NCI-H460. Conclusively, our results suggested that the combinatory application of Src and mTOR inhibitors might be a promising therapeutic strategy for NSCLC treatment.

No MeSH data available.


Related in: MedlinePlus