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CArG-driven GADD45α activated by resveratrol inhibits lung cancer cells.

Shi Q, Geldenhuys W, Sutariya V, Bishayee A, Patel I, Bhatia D - Genes Cancer (2015)

Bottom Line: Therefore, induction of exogenous GADD45α resulted in growth inhibition.Since natural promoter may have antagonized effects, we tested synthetic promoter that contains either five, six or nine repeats of CArG elements essential in the Egr-1 promoter to drive the expression of GADD45α upon resveratrol treatment.Further analysis confirmed that both synthetic promoter and natural Egr-1 promoter were able to "turn on" the expression of GADD45α when combined with resveratrol, and subsequently led to suppression of cell proliferation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, Ohio, USA ; School of Biomedical Sciences, Kent State University, Kent, Ohio, USA.

ABSTRACT
We report anticarcinogenic effects of suicide gene therapy that relies on the use of resveratrol-responsive CArG elements from the Egr-1 promoter to induce GADD45α. In A549 lung cancer cells, endogenous GADD45α was not induced upon resveratrol treatment. Therefore, induction of exogenous GADD45α resulted in growth inhibition. Resveratrol transiently induced Egr-1 through ERK/JNK-ElK-1. Hence, we cloned natural or synthetic Egr-1 promoter upstream of GADD45α cDNA to create a suicide gene therapy vector. Since natural promoter may have antagonized effects, we tested synthetic promoter that contains either five, six or nine repeats of CArG elements essential in the Egr-1 promoter to drive the expression of GADD45α upon resveratrol treatment. Further analysis confirmed that both synthetic promoter and natural Egr-1 promoter were able to "turn on" the expression of GADD45α when combined with resveratrol, and subsequently led to suppression of cell proliferation and apoptosis.

No MeSH data available.


Related in: MedlinePlus

Both synthetic and natural promoters are able to overexpress GADD45α to induce cell apoptosis when combined with resveratrolA. Cells were co-transfected with luciferase constructs containing either the synthetic CArG promoter (pE5, pE6 and pE9NS) or the natural Egr-1 promoter (pE460) and Renilla luciferase control reporter vector (pRL-TK) followed by resveratrol or non-treatment. Relative luminescence was plotted as the average of triplicate experiment and represented as mean ± SE. B. Level of GADD45α protein was detected by western blotting after cells were transfected with either suicide gene therapy vector (pE460.G45α or pE9NS.G45α) or control vector (pT.Luc) and then treated with or without 100 μM of resveratrol for 24 h. C. MTT assay was performed for cells pre-transfected with pE460.G45α, pE9NS.G45α or pT.Luc and treated with different doses of resveratrol for 24 h. D. Cells were exposed to 100 μM of resveratrol for 24 h after transfected with control or suicide gene therapy constructs. Apoptosis was determined by FACS of Annexin V-FITC and PI stained cells.
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Figure 5: Both synthetic and natural promoters are able to overexpress GADD45α to induce cell apoptosis when combined with resveratrolA. Cells were co-transfected with luciferase constructs containing either the synthetic CArG promoter (pE5, pE6 and pE9NS) or the natural Egr-1 promoter (pE460) and Renilla luciferase control reporter vector (pRL-TK) followed by resveratrol or non-treatment. Relative luminescence was plotted as the average of triplicate experiment and represented as mean ± SE. B. Level of GADD45α protein was detected by western blotting after cells were transfected with either suicide gene therapy vector (pE460.G45α or pE9NS.G45α) or control vector (pT.Luc) and then treated with or without 100 μM of resveratrol for 24 h. C. MTT assay was performed for cells pre-transfected with pE460.G45α, pE9NS.G45α or pT.Luc and treated with different doses of resveratrol for 24 h. D. Cells were exposed to 100 μM of resveratrol for 24 h after transfected with control or suicide gene therapy constructs. Apoptosis was determined by FACS of Annexin V-FITC and PI stained cells.

Mentions: In order to confirm the potential of combining CArG-mediated gene therapy with resveratrol, we tested the resveratrol-responsiveness of synthetic promoters E5 (five repeats of prototype CArG sequence (CCTTATTTGG), E6 (six repeats of prototype CArG sequence) and E9NS (nine repeats of new CArG sequence (CCATATAAGG), and the natural Egr-1 promoter E460 (460 nucleotides upstream of transcription start site). The relative luminescence was monitored by dual-luciferase assay reporter system. All synthetic promoters (E5, E6 and E9NS) were activated 1.75- to 2.25-fold by resveratrol (Figure 5A). Therefore, due to lack of statistical difference in resveratrol-responsiveness amongst these synthetic promoters, further studies only included E9NS promoter [20].


CArG-driven GADD45α activated by resveratrol inhibits lung cancer cells.

Shi Q, Geldenhuys W, Sutariya V, Bishayee A, Patel I, Bhatia D - Genes Cancer (2015)

Both synthetic and natural promoters are able to overexpress GADD45α to induce cell apoptosis when combined with resveratrolA. Cells were co-transfected with luciferase constructs containing either the synthetic CArG promoter (pE5, pE6 and pE9NS) or the natural Egr-1 promoter (pE460) and Renilla luciferase control reporter vector (pRL-TK) followed by resveratrol or non-treatment. Relative luminescence was plotted as the average of triplicate experiment and represented as mean ± SE. B. Level of GADD45α protein was detected by western blotting after cells were transfected with either suicide gene therapy vector (pE460.G45α or pE9NS.G45α) or control vector (pT.Luc) and then treated with or without 100 μM of resveratrol for 24 h. C. MTT assay was performed for cells pre-transfected with pE460.G45α, pE9NS.G45α or pT.Luc and treated with different doses of resveratrol for 24 h. D. Cells were exposed to 100 μM of resveratrol for 24 h after transfected with control or suicide gene therapy constructs. Apoptosis was determined by FACS of Annexin V-FITC and PI stained cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482243&req=5

Figure 5: Both synthetic and natural promoters are able to overexpress GADD45α to induce cell apoptosis when combined with resveratrolA. Cells were co-transfected with luciferase constructs containing either the synthetic CArG promoter (pE5, pE6 and pE9NS) or the natural Egr-1 promoter (pE460) and Renilla luciferase control reporter vector (pRL-TK) followed by resveratrol or non-treatment. Relative luminescence was plotted as the average of triplicate experiment and represented as mean ± SE. B. Level of GADD45α protein was detected by western blotting after cells were transfected with either suicide gene therapy vector (pE460.G45α or pE9NS.G45α) or control vector (pT.Luc) and then treated with or without 100 μM of resveratrol for 24 h. C. MTT assay was performed for cells pre-transfected with pE460.G45α, pE9NS.G45α or pT.Luc and treated with different doses of resveratrol for 24 h. D. Cells were exposed to 100 μM of resveratrol for 24 h after transfected with control or suicide gene therapy constructs. Apoptosis was determined by FACS of Annexin V-FITC and PI stained cells.
Mentions: In order to confirm the potential of combining CArG-mediated gene therapy with resveratrol, we tested the resveratrol-responsiveness of synthetic promoters E5 (five repeats of prototype CArG sequence (CCTTATTTGG), E6 (six repeats of prototype CArG sequence) and E9NS (nine repeats of new CArG sequence (CCATATAAGG), and the natural Egr-1 promoter E460 (460 nucleotides upstream of transcription start site). The relative luminescence was monitored by dual-luciferase assay reporter system. All synthetic promoters (E5, E6 and E9NS) were activated 1.75- to 2.25-fold by resveratrol (Figure 5A). Therefore, due to lack of statistical difference in resveratrol-responsiveness amongst these synthetic promoters, further studies only included E9NS promoter [20].

Bottom Line: Therefore, induction of exogenous GADD45α resulted in growth inhibition.Since natural promoter may have antagonized effects, we tested synthetic promoter that contains either five, six or nine repeats of CArG elements essential in the Egr-1 promoter to drive the expression of GADD45α upon resveratrol treatment.Further analysis confirmed that both synthetic promoter and natural Egr-1 promoter were able to "turn on" the expression of GADD45α when combined with resveratrol, and subsequently led to suppression of cell proliferation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, Ohio, USA ; School of Biomedical Sciences, Kent State University, Kent, Ohio, USA.

ABSTRACT
We report anticarcinogenic effects of suicide gene therapy that relies on the use of resveratrol-responsive CArG elements from the Egr-1 promoter to induce GADD45α. In A549 lung cancer cells, endogenous GADD45α was not induced upon resveratrol treatment. Therefore, induction of exogenous GADD45α resulted in growth inhibition. Resveratrol transiently induced Egr-1 through ERK/JNK-ElK-1. Hence, we cloned natural or synthetic Egr-1 promoter upstream of GADD45α cDNA to create a suicide gene therapy vector. Since natural promoter may have antagonized effects, we tested synthetic promoter that contains either five, six or nine repeats of CArG elements essential in the Egr-1 promoter to drive the expression of GADD45α upon resveratrol treatment. Further analysis confirmed that both synthetic promoter and natural Egr-1 promoter were able to "turn on" the expression of GADD45α when combined with resveratrol, and subsequently led to suppression of cell proliferation and apoptosis.

No MeSH data available.


Related in: MedlinePlus