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Pterostilbene exerts antitumor activity via the Notch1 signaling pathway in human lung adenocarcinoma cells.

Yang Y, Yan X, Duan W, Yan J, Yi W, Liang Z, Wang N, Li Y, Chen W, Yu S, Jin Z, Yi D - PLoS ONE (2013)

Bottom Line: PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells.DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment.The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an City, China.

ABSTRACT
Although pterostilbene (PTE) has been shown to have potent antitumor activities against various cancer types, the molecular mechanisms of these activities remain unclear. In this study, we investigated the antitumor activity of PTE against human lung adenocarcinoma in vitro and in vivo and explored the role of the Notch1 signaling pathway in this process. PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells. Additionally, PTE exhibited strong antitumor activity, as evidenced not only by a reduced mitochondrial membrane potential (MMP) and a decreased intracellular glutathione content but also by increases in the apoptotic index and the level of reactive oxygen species (ROS). Furthermore, PTE treatment induced the activation of the Notch1 Intracellular Domain (NICD) protein and activated Hes1. DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment. The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment. In summary, lung adenocarcinoma cells may enhance Notch1 activation as a protective mechanism in response to PTE treatment. Combining a gamma secretase inhibitor with PTE treatment may represent a novel approach for treating lung adenocarcinoma by inhibiting the survival pathways of cancer cells.

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The effects of PTE combined with Notch1 siRNA on the viability, apoptotic index and Notch1 signaling of lung adenocarcinoma cells.(A) Cell viability was assessed using the MTT assay and was expressed as an OD value. (B) Apoptosis was detected by the TUNEL assay (×200) and was expressed as the apoptotic index. TUNEL staining was performed to stain the nuclei of the apoptotic cells (green), and DAPI was used to stain the nuclei of all cells (blue). The apoptotic index was expressed as the number of green cells/the total number of cells counted×100%. (C) Representative Western blot results are shown. The results are expressed as the mean ± SEM, n = 6. **P<0.01 compared with the control siRNA group, ##P<0.01 compared with the PTE 6 µM group, $$P<0.01 compared with the Notch siRNA PTE 6 µM group. PTE, pterostilbene; OD, optical density.
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pone-0062652-g007: The effects of PTE combined with Notch1 siRNA on the viability, apoptotic index and Notch1 signaling of lung adenocarcinoma cells.(A) Cell viability was assessed using the MTT assay and was expressed as an OD value. (B) Apoptosis was detected by the TUNEL assay (×200) and was expressed as the apoptotic index. TUNEL staining was performed to stain the nuclei of the apoptotic cells (green), and DAPI was used to stain the nuclei of all cells (blue). The apoptotic index was expressed as the number of green cells/the total number of cells counted×100%. (C) Representative Western blot results are shown. The results are expressed as the mean ± SEM, n = 6. **P<0.01 compared with the control siRNA group, ##P<0.01 compared with the PTE 6 µM group, $$P<0.01 compared with the Notch siRNA PTE 6 µM group. PTE, pterostilbene; OD, optical density.

Mentions: Because the gamma secretase complex cleaves multiple transmembrane receptors other than Notch1 (including the other Notch receptors, Notch2, 3 and 4), it was necessary to confirm that the sensitization effect of DAPT was mediated by Notch1. We used Notch1 siRNA (to specifically inhibit Notch1) to determine if the sensitization effect observed for DAPT could be replicated. We transfected the cells with Notch1 siRNA for 48 h to decrease expression of NICD (Figure 7C), and this treatment also decreased the expression of Hes1 (Figure 7C). Compared with the cells in the control group, the A549 cells in the Notch1 siRNA-treated group had a decreased viability (P<0.01, compared with the control group, Figure 7A). Similar to the results obtained with DAPT, the combination of Notch1 siRNA and PTE decreased the cell viability significantly (P<0.01, compared with the PTE 6 µM group or the Notch1 siRNA group, Figure 7A). In addition, the co-treatment of A549 cells with Notch1 siRNA and PTE significantly increased the percentage of apoptotic cells (P<0.01, compared with the PTE 6 µM group or the Notch1 siRNA group, Figure 7B).


Pterostilbene exerts antitumor activity via the Notch1 signaling pathway in human lung adenocarcinoma cells.

Yang Y, Yan X, Duan W, Yan J, Yi W, Liang Z, Wang N, Li Y, Chen W, Yu S, Jin Z, Yi D - PLoS ONE (2013)

The effects of PTE combined with Notch1 siRNA on the viability, apoptotic index and Notch1 signaling of lung adenocarcinoma cells.(A) Cell viability was assessed using the MTT assay and was expressed as an OD value. (B) Apoptosis was detected by the TUNEL assay (×200) and was expressed as the apoptotic index. TUNEL staining was performed to stain the nuclei of the apoptotic cells (green), and DAPI was used to stain the nuclei of all cells (blue). The apoptotic index was expressed as the number of green cells/the total number of cells counted×100%. (C) Representative Western blot results are shown. The results are expressed as the mean ± SEM, n = 6. **P<0.01 compared with the control siRNA group, ##P<0.01 compared with the PTE 6 µM group, $$P<0.01 compared with the Notch siRNA PTE 6 µM group. PTE, pterostilbene; OD, optical density.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643961&req=5

pone-0062652-g007: The effects of PTE combined with Notch1 siRNA on the viability, apoptotic index and Notch1 signaling of lung adenocarcinoma cells.(A) Cell viability was assessed using the MTT assay and was expressed as an OD value. (B) Apoptosis was detected by the TUNEL assay (×200) and was expressed as the apoptotic index. TUNEL staining was performed to stain the nuclei of the apoptotic cells (green), and DAPI was used to stain the nuclei of all cells (blue). The apoptotic index was expressed as the number of green cells/the total number of cells counted×100%. (C) Representative Western blot results are shown. The results are expressed as the mean ± SEM, n = 6. **P<0.01 compared with the control siRNA group, ##P<0.01 compared with the PTE 6 µM group, $$P<0.01 compared with the Notch siRNA PTE 6 µM group. PTE, pterostilbene; OD, optical density.
Mentions: Because the gamma secretase complex cleaves multiple transmembrane receptors other than Notch1 (including the other Notch receptors, Notch2, 3 and 4), it was necessary to confirm that the sensitization effect of DAPT was mediated by Notch1. We used Notch1 siRNA (to specifically inhibit Notch1) to determine if the sensitization effect observed for DAPT could be replicated. We transfected the cells with Notch1 siRNA for 48 h to decrease expression of NICD (Figure 7C), and this treatment also decreased the expression of Hes1 (Figure 7C). Compared with the cells in the control group, the A549 cells in the Notch1 siRNA-treated group had a decreased viability (P<0.01, compared with the control group, Figure 7A). Similar to the results obtained with DAPT, the combination of Notch1 siRNA and PTE decreased the cell viability significantly (P<0.01, compared with the PTE 6 µM group or the Notch1 siRNA group, Figure 7A). In addition, the co-treatment of A549 cells with Notch1 siRNA and PTE significantly increased the percentage of apoptotic cells (P<0.01, compared with the PTE 6 µM group or the Notch1 siRNA group, Figure 7B).

Bottom Line: PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells.DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment.The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an City, China.

ABSTRACT
Although pterostilbene (PTE) has been shown to have potent antitumor activities against various cancer types, the molecular mechanisms of these activities remain unclear. In this study, we investigated the antitumor activity of PTE against human lung adenocarcinoma in vitro and in vivo and explored the role of the Notch1 signaling pathway in this process. PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells. Additionally, PTE exhibited strong antitumor activity, as evidenced not only by a reduced mitochondrial membrane potential (MMP) and a decreased intracellular glutathione content but also by increases in the apoptotic index and the level of reactive oxygen species (ROS). Furthermore, PTE treatment induced the activation of the Notch1 Intracellular Domain (NICD) protein and activated Hes1. DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment. The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment. In summary, lung adenocarcinoma cells may enhance Notch1 activation as a protective mechanism in response to PTE treatment. Combining a gamma secretase inhibitor with PTE treatment may represent a novel approach for treating lung adenocarcinoma by inhibiting the survival pathways of cancer cells.

Show MeSH
Related in: MedlinePlus