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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 induces Egr-1 protein expression; silencing of Egr-1 abrogates the effect of ciglitazone on PDK1 promoter activity, protein expression and cell proliferation. A, Cellular proteins were isolated from H1299 cells treated with ciglitazone (20 μM) for the indicated time period (upper panel) or with metformin (5 mM) for 24 h (lower panel). Afterwards, Western blot analyses were performed for detecting Egr-1, p65 and p53 proteins. B, H1299 cells were transfected with control or AMPKα siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h followed by Western blot. C, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) together with a wild type PDK1 promoter construct for 30 h, then cells were exposed to ciglitazone (20 μM) for an additional 24 h. Insert shows the Western blot result for Egr-1 protein. D, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h, followed by Western blot. E, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposure of 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. The bars represent the mean ± SD of at least four independent experiments for each condition. *Indicates significant difference as compared to the control. **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).
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Figure 5: Ciglitazone induces Egr-1 protein expression; silencing of Egr-1 abrogates the effect of ciglitazone on PDK1 promoter activity, protein expression and cell proliferation. A, Cellular proteins were isolated from H1299 cells treated with ciglitazone (20 μM) for the indicated time period (upper panel) or with metformin (5 mM) for 24 h (lower panel). Afterwards, Western blot analyses were performed for detecting Egr-1, p65 and p53 proteins. B, H1299 cells were transfected with control or AMPKα siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h followed by Western blot. C, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) together with a wild type PDK1 promoter construct for 30 h, then cells were exposed to ciglitazone (20 μM) for an additional 24 h. Insert shows the Western blot result for Egr-1 protein. D, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h, followed by Western blot. E, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposure of 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. The bars represent the mean ± SD of at least four independent experiments for each condition. *Indicates significant difference as compared to the control. **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).

Mentions: We further tested the role of the transcription factors in mediating the effect of ciglitazone on PDK1 expression in human lung carcinoma cells. We showed that ciglitazone significantly induced the expression of Egr-1 protein in a time–dependent manner, while it had little effect on p65 and p53 (Figure 5A, upper panel). Note that a synergy was observed in the combination of ciglitazone and metformin treatment (Figure 5A lower panel). Interestingly, we also found that silencing of AMPKα abolished the effect of ciglitazone on Egr-1 protein expression, further suggesting the critical role of AMPKα activation in this process (Figure 5B). Next, we found that while cells transfected with Egr-1 siRNA slightly increased PDK1 promoter activity at baseline, it greatly antagonized the inhibitory effect of ciglitazone on PDK1 promoter activity. Note that the control siRNA had no effect (Figure 5C, lower panel). Egr-1 siRNA reduced the production of Egr-1 protein (Figure 5C, upper panel). Furthermore, it eliminated the ciglitazone-reduced PDK1 protein expression, whereas the control siRNA had no effect (Figure 5D). Consistent with these findings, we found that cells transfected with Egr-1 siRNA blocked the inhibitory effects of ciglitazone on cell growth (Figure 5E). The control siRNA had no effect.


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 induces Egr-1 protein expression; silencing of Egr-1 abrogates the effect of ciglitazone on PDK1 promoter activity, protein expression and cell proliferation. A, Cellular proteins were isolated from H1299 cells treated with ciglitazone (20 μM) for the indicated time period (upper panel) or with metformin (5 mM) for 24 h (lower panel). Afterwards, Western blot analyses were performed for detecting Egr-1, p65 and p53 proteins. B, H1299 cells were transfected with control or AMPKα siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h followed by Western blot. C, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) together with a wild type PDK1 promoter construct for 30 h, then cells were exposed to ciglitazone (20 μM) for an additional 24 h. Insert shows the Western blot result for Egr-1 protein. D, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h, followed by Western blot. E, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposure of 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. The bars represent the mean ± SD of at least four independent experiments for each condition. *Indicates significant difference as compared to the control. **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Ciglitazone induces Egr-1 protein expression; silencing of Egr-1 abrogates the effect of ciglitazone on PDK1 promoter activity, protein expression and cell proliferation. A, Cellular proteins were isolated from H1299 cells treated with ciglitazone (20 μM) for the indicated time period (upper panel) or with metformin (5 mM) for 24 h (lower panel). Afterwards, Western blot analyses were performed for detecting Egr-1, p65 and p53 proteins. B, H1299 cells were transfected with control or AMPKα siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h followed by Western blot. C, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) together with a wild type PDK1 promoter construct for 30 h, then cells were exposed to ciglitazone (20 μM) for an additional 24 h. Insert shows the Western blot result for Egr-1 protein. D, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h, followed by Western blot. E, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposure of 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. The bars represent the mean ± SD of at least four independent experiments for each condition. *Indicates significant difference as compared to the control. **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).
Mentions: We further tested the role of the transcription factors in mediating the effect of ciglitazone on PDK1 expression in human lung carcinoma cells. We showed that ciglitazone significantly induced the expression of Egr-1 protein in a time–dependent manner, while it had little effect on p65 and p53 (Figure 5A, upper panel). Note that a synergy was observed in the combination of ciglitazone and metformin treatment (Figure 5A lower panel). Interestingly, we also found that silencing of AMPKα abolished the effect of ciglitazone on Egr-1 protein expression, further suggesting the critical role of AMPKα activation in this process (Figure 5B). Next, we found that while cells transfected with Egr-1 siRNA slightly increased PDK1 promoter activity at baseline, it greatly antagonized the inhibitory effect of ciglitazone on PDK1 promoter activity. Note that the control siRNA had no effect (Figure 5C, lower panel). Egr-1 siRNA reduced the production of Egr-1 protein (Figure 5C, upper panel). Furthermore, it eliminated the ciglitazone-reduced PDK1 protein expression, whereas the control siRNA had no effect (Figure 5D). Consistent with these findings, we found that cells transfected with Egr-1 siRNA blocked the inhibitory effects of ciglitazone on cell growth (Figure 5E). The control siRNA had no effect.

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