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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

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.
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pone.0129663.g003: 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.

Mentions: The most recent study demonstrated that Src phosphorylation may facilitate the activation of Akt-mTOR signaling pathway in AML cells [29]. To investigate the potential interaction between Src and mTOR pathways, we analyzed the activation of Src, PI3K, AKT, and mTOR in sequence. Firstly, the immunofluorescence staining of phospho-Src indicated that Src activation was inhibited by Dasatinib and more significantly repressed by the co-treatment with Rapamycin (Fig 3A), suggesting that mTOR inhibition by Rapamycin could synergize with Dasatinib in suppressing Src activation in A549 cells. And this result was further confirmed by western blotting (Fig 3B). In addition, our data demonstrated that the inhibition of Src by Dasatinib could also suppress the activation of PI3K-Akt signaling in A549 cells, which was in agreement with previous reports [29]. More importantly, the inhibitions of Src, PI3K, and AKT were unanimously enhanced by the presence of Rapamycin in comparison with Dasatinib alone, as shown in Fig 3B and 3C. It was worth noting that the result was well correlated with the observations regarding expression of CDK inhibitor and Forkhead box proteins (Fig 2B), which agreed with our speculation on the connection between cell cycle arrest and Src-PI3K-AKT deactivation. On the other hand, the immunostaining and western blotting results indicated that Src activation was inhibited by Rapamycin, to a less extent than Dasatinib. These data implied that mTOR which was conventionally recognized as the responsive target of PI3K/AKT signaling, might also play an important role in the regulation of Src activation.


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)

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.
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Related In: Results  -  Collection

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pone.0129663.g003: 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.
Mentions: The most recent study demonstrated that Src phosphorylation may facilitate the activation of Akt-mTOR signaling pathway in AML cells [29]. To investigate the potential interaction between Src and mTOR pathways, we analyzed the activation of Src, PI3K, AKT, and mTOR in sequence. Firstly, the immunofluorescence staining of phospho-Src indicated that Src activation was inhibited by Dasatinib and more significantly repressed by the co-treatment with Rapamycin (Fig 3A), suggesting that mTOR inhibition by Rapamycin could synergize with Dasatinib in suppressing Src activation in A549 cells. And this result was further confirmed by western blotting (Fig 3B). In addition, our data demonstrated that the inhibition of Src by Dasatinib could also suppress the activation of PI3K-Akt signaling in A549 cells, which was in agreement with previous reports [29]. More importantly, the inhibitions of Src, PI3K, and AKT were unanimously enhanced by the presence of Rapamycin in comparison with Dasatinib alone, as shown in Fig 3B and 3C. It was worth noting that the result was well correlated with the observations regarding expression of CDK inhibitor and Forkhead box proteins (Fig 2B), which agreed with our speculation on the connection between cell cycle arrest and Src-PI3K-AKT deactivation. On the other hand, the immunostaining and western blotting results indicated that Src activation was inhibited by Rapamycin, to a less extent than Dasatinib. These data implied that mTOR which was conventionally recognized as the responsive target of PI3K/AKT signaling, might also play an important role in the regulation of Src activation.

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