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Repression of phosphoinositide-dependent protein kinase 1 expression by ciglitazone via Egr-1 represents a new approach for inhibition of lung cancer cell growth.

Hann SS, Tang Q, Zheng F, Zhao S, Chen J, Wang Z - Mol. Cancer (2014)

Bottom Line: Moreover, silencing of Egr-1 abrogated the effect of ciglitazone on PDK1 promoter activity and cell growth.On the contrary, overexpression of Egr-1 enhanced the effect of ciglitazone on PDK1 gene promoter activity.Activation of AMPKα by metformin enhances the effect of ciglitazone.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Guangzhou Traditional Chinese Medicine, Guangdong Academy of Traditional Chinese Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, Guangdong Province, China 510120. swhan2010@live.com.

ABSTRACT

Background: Peroxisome proliferator-activated receptors gamma (PPARγ) ligands have been shown to inhibit the growth of non-small cell lung cancer (NSCLC) cells. However, the mechanisms underlying this effect remain incompletely elucidated.

Methods: Cell proliferation and apoptosis were measured by cell viability, MTT and caspase3/7 activity assays. Phosphorylation/protein expression and gene silence/overexpression of AMPKα, phosphoinositide-dependent protein kinase 1 (PDK1), Egr-1 and PPARγ were performed by Western blot and siRNA/transfection assays. Dual-Luciferase Reporter Kit was used to measure the PPAR response elements (PPRE) reporter and PDK1 promoter activities, and ChIP assay was used to detect the Egr-1 protein binding to the DNA site in the PDK1 gene promoter.

Results: We found that ciglitazone, one synthetic PPARγ ligand, inhibited growth and induced apoptosis of NSCLC cells through decreased expression of PDK1, which was not blocked by GW9662 (a specific PPARγ antagonist). Overexpression of PDK1 overcame the effect of ciglitazone on cell growth and caspase 3/7 activity. Ciglitazone increased the phosphorylation of AMPKα and c-Jun N-terminal kinase (JNK), and the inhibitor of AMPK (compound C), but not JNK (SP600125), reversed the effect of ciglitazone on PDK1 protein expression. Ciglitazone reduced PDK1 gene promoter activity, which was not observed in cells exposed to compound C, but not silenced of PPARγ siRNA. Combination of ciglitazone and metformin further reduced PDK1 expression and promoter activity. Furthermore, we showed that ciglitazone induced the protein expression of Egr-1, which was not observed in cells silencing of AMPKα. Moreover, silencing of Egr-1 abrogated the effect of ciglitazone on PDK1 promoter activity and cell growth. On the contrary, overexpression of Egr-1 enhanced the effect of ciglitazone on PDK1 gene promoter activity. ChIP assays demonstrated that ciglitazone induced Egr-1 protein bind to the specific DNA site in the PDK1 gene promoter.

Conclusion: Collectively, our results demonstrate that ciglitazone inhibits PDK1 expression through AMPKα-mediated induction of Egr-1 and Egr-1 binding to the specific DNA site in the PDK1 gene promoter, which is independent of PPARγ. Activation of AMPKα by metformin enhances the effect of ciglitazone. In turn, this leads to inhibition of NSCLC cell proliferation.

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Ciglitazone inhibited PDK1 protein expression independent of PPARγ. A, H1299 and H1650 cells were transfected with control or PPRE X3-TK-luc reporter (from Addgene) for 24 h, followed by treating with ciglitazone for an additional 24 h. Afterwards, the Luciferase reporter activity was measured using Luciferase Assay System (Promega) according to manufacturer's instructions. The bars represent the mean ± SD of at least three independent experiments for each condition. *indicates significant difference as compared to the untreated control group (P < 0.05). B, Cellular protein was isolated from H1299 and H1650 cells cultured for 1 h in the presence or absence of GW9662 (20 μM) before exposing the cells to ciglitazone (20 μM) for an additional 24 h, then subjected to Western blot analysis. C, H1299 cells were transfected with control or PDK1 siRNA (80 nM) for 40 h, followed by exposing the cells to ciglitazone (20 μM) for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit. D-E, H1299 cells were transfected with the control and PDK1 expression vectors using the oligofectamine reagent according to the manufacturer’s instructions. After 24 h of incubation, cells were treated with or without ciglitazone for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit (D). In separate experiment, the relative caspase 3/7 activity (E) is indicated as percentage of untreated control cells. The bars represent the mean ± SD of at least four independent experiments for each condition. Insert on the top panel shows a Western blot for PDK1 protein. *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).
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Figure 2: Ciglitazone inhibited PDK1 protein expression independent of PPARγ. A, H1299 and H1650 cells were transfected with control or PPRE X3-TK-luc reporter (from Addgene) for 24 h, followed by treating with ciglitazone for an additional 24 h. Afterwards, the Luciferase reporter activity was measured using Luciferase Assay System (Promega) according to manufacturer's instructions. The bars represent the mean ± SD of at least three independent experiments for each condition. *indicates significant difference as compared to the untreated control group (P < 0.05). B, Cellular protein was isolated from H1299 and H1650 cells cultured for 1 h in the presence or absence of GW9662 (20 μM) before exposing the cells to ciglitazone (20 μM) for an additional 24 h, then subjected to Western blot analysis. C, H1299 cells were transfected with control or PDK1 siRNA (80 nM) for 40 h, followed by exposing the cells to ciglitazone (20 μM) for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit. D-E, H1299 cells were transfected with the control and PDK1 expression vectors using the oligofectamine reagent according to the manufacturer’s instructions. After 24 h of incubation, cells were treated with or without ciglitazone for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit (D). In separate experiment, the relative caspase 3/7 activity (E) is indicated as percentage of untreated control cells. The bars represent the mean ± SD of at least four independent experiments for each condition. Insert on the top panel shows a Western blot for PDK1 protein. *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).

Mentions: We first examined the effect of ciglitazone on growth and apoptosis of lung cancer cells. We found that ciglitazone inhibited growth of lung cancer cell H1650 in the time- and dose-dependent manner, with significant inhibition observed at 20 μM at 48 h (Figure 1A, upper panel). Similar results were also observed in other NSCLC cell lines (Figure 1A, lower panel). We also showed that ciglitazone induced caspase 3/7 activity in H1650 cells indicating increase in apoptosis (Figure 1B). We then examined whether ciglitazone affected the expression of PDK1. We found that ciglitazone inhibited PDK1 protein expression in a time- and dose-dependent manner, with an effective response of 20 μM at 24 h in H1650 cells (Figure 1C). Reduction of PDK1 protein expression by ciglitazone was also found in other NSCLC cell lines (Figure 1D).We then tested whether the effects of ciglitazone on PDK1 were mediated through the activation of PPARγ. We showed that, while ciglitazone increased the PPRE luciferase activity (activation of PPAR) (Figure 2A), the effects of ciglitazone on PDK1 expression were not eliminated in the presence of GW9662, a specific PPARγ antagonist (Figure 2B) and in cells (H1299 and H1650) silencing of PPARγ (not shown). The result suggests that PPARγ-independent signals mediate the effect of ciglitazone on PDK1 protein expression.


Repression of phosphoinositide-dependent protein kinase 1 expression by ciglitazone via Egr-1 represents a new approach for inhibition of lung cancer cell growth.

Hann SS, Tang Q, Zheng F, Zhao S, Chen J, Wang Z - Mol. Cancer (2014)

Ciglitazone inhibited PDK1 protein expression independent of PPARγ. A, H1299 and H1650 cells were transfected with control or PPRE X3-TK-luc reporter (from Addgene) for 24 h, followed by treating with ciglitazone for an additional 24 h. Afterwards, the Luciferase reporter activity was measured using Luciferase Assay System (Promega) according to manufacturer's instructions. The bars represent the mean ± SD of at least three independent experiments for each condition. *indicates significant difference as compared to the untreated control group (P < 0.05). B, Cellular protein was isolated from H1299 and H1650 cells cultured for 1 h in the presence or absence of GW9662 (20 μM) before exposing the cells to ciglitazone (20 μM) for an additional 24 h, then subjected to Western blot analysis. C, H1299 cells were transfected with control or PDK1 siRNA (80 nM) for 40 h, followed by exposing the cells to ciglitazone (20 μM) for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit. D-E, H1299 cells were transfected with the control and PDK1 expression vectors using the oligofectamine reagent according to the manufacturer’s instructions. After 24 h of incubation, cells were treated with or without ciglitazone for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit (D). In separate experiment, the relative caspase 3/7 activity (E) is indicated as percentage of untreated control cells. The bars represent the mean ± SD of at least four independent experiments for each condition. Insert on the top panel shows a Western blot for PDK1 protein. *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).
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Related In: Results  -  Collection

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Figure 2: Ciglitazone inhibited PDK1 protein expression independent of PPARγ. A, H1299 and H1650 cells were transfected with control or PPRE X3-TK-luc reporter (from Addgene) for 24 h, followed by treating with ciglitazone for an additional 24 h. Afterwards, the Luciferase reporter activity was measured using Luciferase Assay System (Promega) according to manufacturer's instructions. The bars represent the mean ± SD of at least three independent experiments for each condition. *indicates significant difference as compared to the untreated control group (P < 0.05). B, Cellular protein was isolated from H1299 and H1650 cells cultured for 1 h in the presence or absence of GW9662 (20 μM) before exposing the cells to ciglitazone (20 μM) for an additional 24 h, then subjected to Western blot analysis. C, H1299 cells were transfected with control or PDK1 siRNA (80 nM) for 40 h, followed by exposing the cells to ciglitazone (20 μM) for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit. D-E, H1299 cells were transfected with the control and PDK1 expression vectors using the oligofectamine reagent according to the manufacturer’s instructions. After 24 h of incubation, cells were treated with or without ciglitazone for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit (D). In separate experiment, the relative caspase 3/7 activity (E) is indicated as percentage of untreated control cells. The bars represent the mean ± SD of at least four independent experiments for each condition. Insert on the top panel shows a Western blot for PDK1 protein. *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).
Mentions: We first examined the effect of ciglitazone on growth and apoptosis of lung cancer cells. We found that ciglitazone inhibited growth of lung cancer cell H1650 in the time- and dose-dependent manner, with significant inhibition observed at 20 μM at 48 h (Figure 1A, upper panel). Similar results were also observed in other NSCLC cell lines (Figure 1A, lower panel). We also showed that ciglitazone induced caspase 3/7 activity in H1650 cells indicating increase in apoptosis (Figure 1B). We then examined whether ciglitazone affected the expression of PDK1. We found that ciglitazone inhibited PDK1 protein expression in a time- and dose-dependent manner, with an effective response of 20 μM at 24 h in H1650 cells (Figure 1C). Reduction of PDK1 protein expression by ciglitazone was also found in other NSCLC cell lines (Figure 1D).We then tested whether the effects of ciglitazone on PDK1 were mediated through the activation of PPARγ. We showed that, while ciglitazone increased the PPRE luciferase activity (activation of PPAR) (Figure 2A), the effects of ciglitazone on PDK1 expression were not eliminated in the presence of GW9662, a specific PPARγ antagonist (Figure 2B) and in cells (H1299 and H1650) silencing of PPARγ (not shown). The result suggests that PPARγ-independent signals mediate the effect of ciglitazone on PDK1 protein expression.

Bottom Line: Moreover, silencing of Egr-1 abrogated the effect of ciglitazone on PDK1 promoter activity and cell growth.On the contrary, overexpression of Egr-1 enhanced the effect of ciglitazone on PDK1 gene promoter activity.Activation of AMPKα by metformin enhances the effect of ciglitazone.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Guangzhou Traditional Chinese Medicine, Guangdong Academy of Traditional Chinese Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, Guangdong Province, China 510120. swhan2010@live.com.

ABSTRACT

Background: Peroxisome proliferator-activated receptors gamma (PPARγ) ligands have been shown to inhibit the growth of non-small cell lung cancer (NSCLC) cells. However, the mechanisms underlying this effect remain incompletely elucidated.

Methods: Cell proliferation and apoptosis were measured by cell viability, MTT and caspase3/7 activity assays. Phosphorylation/protein expression and gene silence/overexpression of AMPKα, phosphoinositide-dependent protein kinase 1 (PDK1), Egr-1 and PPARγ were performed by Western blot and siRNA/transfection assays. Dual-Luciferase Reporter Kit was used to measure the PPAR response elements (PPRE) reporter and PDK1 promoter activities, and ChIP assay was used to detect the Egr-1 protein binding to the DNA site in the PDK1 gene promoter.

Results: We found that ciglitazone, one synthetic PPARγ ligand, inhibited growth and induced apoptosis of NSCLC cells through decreased expression of PDK1, which was not blocked by GW9662 (a specific PPARγ antagonist). Overexpression of PDK1 overcame the effect of ciglitazone on cell growth and caspase 3/7 activity. Ciglitazone increased the phosphorylation of AMPKα and c-Jun N-terminal kinase (JNK), and the inhibitor of AMPK (compound C), but not JNK (SP600125), reversed the effect of ciglitazone on PDK1 protein expression. Ciglitazone reduced PDK1 gene promoter activity, which was not observed in cells exposed to compound C, but not silenced of PPARγ siRNA. Combination of ciglitazone and metformin further reduced PDK1 expression and promoter activity. Furthermore, we showed that ciglitazone induced the protein expression of Egr-1, which was not observed in cells silencing of AMPKα. Moreover, silencing of Egr-1 abrogated the effect of ciglitazone on PDK1 promoter activity and cell growth. On the contrary, overexpression of Egr-1 enhanced the effect of ciglitazone on PDK1 gene promoter activity. ChIP assays demonstrated that ciglitazone induced Egr-1 protein bind to the specific DNA site in the PDK1 gene promoter.

Conclusion: Collectively, our results demonstrate that ciglitazone inhibits PDK1 expression through AMPKα-mediated induction of Egr-1 and Egr-1 binding to the specific DNA site in the PDK1 gene promoter, which is independent of PPARγ. Activation of AMPKα by metformin enhances the effect of ciglitazone. In turn, this leads to inhibition of NSCLC cell proliferation.

Show MeSH
Related in: MedlinePlus