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Sp1 is involved in H2O2-induced PUMA gene expression and apoptosis in colorectal cancer cells.

Wang X, Wang J, Lin S, Geng Y, Wang J, Jiang B - J. Exp. Clin. Cancer Res. (2008)

Bottom Line: In previous study, we found PUMA (p53-upregulated modulator of apoptosis) played an important role in oxaliplatin-induced apoptosis.Furthermore, induction of PUMA promoter activity by H2O2 was abrogated by PFT-alpha (a p53 inhibitor) and Mithramycin A (a Sp1 inhibitor), as compared with PFT-alpha alone.Mithramycin A and PFT-alpha also reduced H2O2-induced apoptosis synergistically and abrogated the expression of procaspase 3 and procaspase 9.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, PR China. sunwingwxy@163.com

ABSTRACT

Background: Reactive oxygen species (ROS) are intricately involved in tumor progression through effects on proliferation, apoptosis and metastasis. But how ROS works is not well understood. In previous study, we found PUMA (p53-upregulated modulator of apoptosis) played an important role in oxaliplatin-induced apoptosis. In the present study, we detect the role of PUMA in H2O2-induced apoptosis in colorectal cancer cells and investigate the potential mechanism.

Methods and results: We showed that H2O2 stimulated the activity of a 493 PUMA promoter reporter gene construct. Suppressing the expression of PUMA abrogated H2O2-induced apoptosis. Deletion of the Sp1-binding sites also decreased the transactivation of PUMA promoter by H2O2. Furthermore, induction of PUMA promoter activity by H2O2 was abrogated by PFT-alpha (a p53 inhibitor) and Mithramycin A (a Sp1 inhibitor), as compared with PFT-alpha alone. To determine the effects of Sp1 on PUMA in H2O2-induced apoptosis, procaspase 3, procaspase 9 and procaspase 8 expression was assessed. Mithramycin A and PFT-alpha also reduced H2O2-induced apoptosis synergistically and abrogated the expression of procaspase 3 and procaspase 9.

Conclusion: Our findings suggest that PUMA plays a role in H2O2-induced apoptosis, and that Sp1 works together with p53 in the regulation of H2O2-induced PUMA expression and apoptosis in colorectal cancer cells. This study provides important regulatory insights in the mechanisms of ROS in colorectal cancer.

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Sp1 is required for the transactivation of the human PUMA promotor by H2O2. (A) LoVo cells were transiently transfected with reporter plasmids (0.5 μg). 0.64 mM H2O2 was added 24 hours post-transfection for 15 minutes. The pSVβ-Galactosidase plasmid (0.1 μg) was included in each sample for normalization of transfection variability. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD), from at least two independent experiments performed in duplicate (*p > 0.05,-336/+157 -126/-25PUMA-Luc vs-136/+157 PUMA-Luc, #p < 0.05,-36/+157PUMA-Luc vs-336/+157 -126/-25PUMA-Luc). (B) Mithr.A (200 ng/ml) was added to LoVo cells. Extracts from the treated or untreated LoVo cells were subjected to immunoblotting using antibodies against p53, Sp1 and actin. (C) LoVo cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of endogenous p53, Sp1 and actin as indicated by immunoblotting. (D, E) LoVo and HCT 116 cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of PUMA as indicated by immunoblotting. (F) H2O2 -induced PUMA expression was abrogated by transfecting Sp1 siRNA. LoVo cells were transfected with control siRNA or Sp1 siRNA and H2O2 was added 24 hours post-transfection. PUMA expression was detected by Western blotting analysis 48 hours post-transfection. (G) LoVo cells were transiently transfected with the -336/+157 PUMA-Luc reporter plasmid, 0.64 mM H2O2 was added 24 hours post-transfection and remained for 15 minutes. Mithr.A (200 ng/ml) and/or PFT-α (20 μM) was added an hour before H2O2. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD.) from at least two in dependent expression performed in duplicate are shown in the form of a bargraph, *p < 0.05, significant difference between cells treated as indicated in Figure 4G, # p > 0.05, no significant difference between cells treated as indicated in Figure 4G (factor analysis, n = 6).
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Figure 4: Sp1 is required for the transactivation of the human PUMA promotor by H2O2. (A) LoVo cells were transiently transfected with reporter plasmids (0.5 μg). 0.64 mM H2O2 was added 24 hours post-transfection for 15 minutes. The pSVβ-Galactosidase plasmid (0.1 μg) was included in each sample for normalization of transfection variability. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD), from at least two independent experiments performed in duplicate (*p > 0.05,-336/+157 -126/-25PUMA-Luc vs-136/+157 PUMA-Luc, #p < 0.05,-36/+157PUMA-Luc vs-336/+157 -126/-25PUMA-Luc). (B) Mithr.A (200 ng/ml) was added to LoVo cells. Extracts from the treated or untreated LoVo cells were subjected to immunoblotting using antibodies against p53, Sp1 and actin. (C) LoVo cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of endogenous p53, Sp1 and actin as indicated by immunoblotting. (D, E) LoVo and HCT 116 cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of PUMA as indicated by immunoblotting. (F) H2O2 -induced PUMA expression was abrogated by transfecting Sp1 siRNA. LoVo cells were transfected with control siRNA or Sp1 siRNA and H2O2 was added 24 hours post-transfection. PUMA expression was detected by Western blotting analysis 48 hours post-transfection. (G) LoVo cells were transiently transfected with the -336/+157 PUMA-Luc reporter plasmid, 0.64 mM H2O2 was added 24 hours post-transfection and remained for 15 minutes. Mithr.A (200 ng/ml) and/or PFT-α (20 μM) was added an hour before H2O2. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD.) from at least two in dependent expression performed in duplicate are shown in the form of a bargraph, *p < 0.05, significant difference between cells treated as indicated in Figure 4G, # p > 0.05, no significant difference between cells treated as indicated in Figure 4G (factor analysis, n = 6).

Mentions: The promoter of PUMA gene contains a cluster of GC-rich motifs flanked by two p53-recognition motifs at the 5' end. The p53 sites are located at distal regions relative to the proximal Sp1 sites. The family member Sp1 has been shown to mediate oxidative stress-induced gene transcription. To determine the role of Sp1 in H2O2-induced PUMA expression, we examined the activity of a series of PUMA promoter truncation mutants[16] after H2O2 treatment Compared with cells transfected with -336/+157 PUMA-Luc, the transactivation level of cells transfected with -336/+157 -126/-25PUMA-Luc was lower, but the difference was not significant (p > 0.05). Transfection with the -36/+157PUMA-Luc vector induced a significant decrease of transactivation (#p < 0.05, Figure 4A).


Sp1 is involved in H2O2-induced PUMA gene expression and apoptosis in colorectal cancer cells.

Wang X, Wang J, Lin S, Geng Y, Wang J, Jiang B - J. Exp. Clin. Cancer Res. (2008)

Sp1 is required for the transactivation of the human PUMA promotor by H2O2. (A) LoVo cells were transiently transfected with reporter plasmids (0.5 μg). 0.64 mM H2O2 was added 24 hours post-transfection for 15 minutes. The pSVβ-Galactosidase plasmid (0.1 μg) was included in each sample for normalization of transfection variability. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD), from at least two independent experiments performed in duplicate (*p > 0.05,-336/+157 -126/-25PUMA-Luc vs-136/+157 PUMA-Luc, #p < 0.05,-36/+157PUMA-Luc vs-336/+157 -126/-25PUMA-Luc). (B) Mithr.A (200 ng/ml) was added to LoVo cells. Extracts from the treated or untreated LoVo cells were subjected to immunoblotting using antibodies against p53, Sp1 and actin. (C) LoVo cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of endogenous p53, Sp1 and actin as indicated by immunoblotting. (D, E) LoVo and HCT 116 cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of PUMA as indicated by immunoblotting. (F) H2O2 -induced PUMA expression was abrogated by transfecting Sp1 siRNA. LoVo cells were transfected with control siRNA or Sp1 siRNA and H2O2 was added 24 hours post-transfection. PUMA expression was detected by Western blotting analysis 48 hours post-transfection. (G) LoVo cells were transiently transfected with the -336/+157 PUMA-Luc reporter plasmid, 0.64 mM H2O2 was added 24 hours post-transfection and remained for 15 minutes. Mithr.A (200 ng/ml) and/or PFT-α (20 μM) was added an hour before H2O2. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD.) from at least two in dependent expression performed in duplicate are shown in the form of a bargraph, *p < 0.05, significant difference between cells treated as indicated in Figure 4G, # p > 0.05, no significant difference between cells treated as indicated in Figure 4G (factor analysis, n = 6).
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Figure 4: Sp1 is required for the transactivation of the human PUMA promotor by H2O2. (A) LoVo cells were transiently transfected with reporter plasmids (0.5 μg). 0.64 mM H2O2 was added 24 hours post-transfection for 15 minutes. The pSVβ-Galactosidase plasmid (0.1 μg) was included in each sample for normalization of transfection variability. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD), from at least two independent experiments performed in duplicate (*p > 0.05,-336/+157 -126/-25PUMA-Luc vs-136/+157 PUMA-Luc, #p < 0.05,-36/+157PUMA-Luc vs-336/+157 -126/-25PUMA-Luc). (B) Mithr.A (200 ng/ml) was added to LoVo cells. Extracts from the treated or untreated LoVo cells were subjected to immunoblotting using antibodies against p53, Sp1 and actin. (C) LoVo cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of endogenous p53, Sp1 and actin as indicated by immunoblotting. (D, E) LoVo and HCT 116 cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of PUMA as indicated by immunoblotting. (F) H2O2 -induced PUMA expression was abrogated by transfecting Sp1 siRNA. LoVo cells were transfected with control siRNA or Sp1 siRNA and H2O2 was added 24 hours post-transfection. PUMA expression was detected by Western blotting analysis 48 hours post-transfection. (G) LoVo cells were transiently transfected with the -336/+157 PUMA-Luc reporter plasmid, 0.64 mM H2O2 was added 24 hours post-transfection and remained for 15 minutes. Mithr.A (200 ng/ml) and/or PFT-α (20 μM) was added an hour before H2O2. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD.) from at least two in dependent expression performed in duplicate are shown in the form of a bargraph, *p < 0.05, significant difference between cells treated as indicated in Figure 4G, # p > 0.05, no significant difference between cells treated as indicated in Figure 4G (factor analysis, n = 6).
Mentions: The promoter of PUMA gene contains a cluster of GC-rich motifs flanked by two p53-recognition motifs at the 5' end. The p53 sites are located at distal regions relative to the proximal Sp1 sites. The family member Sp1 has been shown to mediate oxidative stress-induced gene transcription. To determine the role of Sp1 in H2O2-induced PUMA expression, we examined the activity of a series of PUMA promoter truncation mutants[16] after H2O2 treatment Compared with cells transfected with -336/+157 PUMA-Luc, the transactivation level of cells transfected with -336/+157 -126/-25PUMA-Luc was lower, but the difference was not significant (p > 0.05). Transfection with the -36/+157PUMA-Luc vector induced a significant decrease of transactivation (#p < 0.05, Figure 4A).

Bottom Line: In previous study, we found PUMA (p53-upregulated modulator of apoptosis) played an important role in oxaliplatin-induced apoptosis.Furthermore, induction of PUMA promoter activity by H2O2 was abrogated by PFT-alpha (a p53 inhibitor) and Mithramycin A (a Sp1 inhibitor), as compared with PFT-alpha alone.Mithramycin A and PFT-alpha also reduced H2O2-induced apoptosis synergistically and abrogated the expression of procaspase 3 and procaspase 9.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, PR China. sunwingwxy@163.com

ABSTRACT

Background: Reactive oxygen species (ROS) are intricately involved in tumor progression through effects on proliferation, apoptosis and metastasis. But how ROS works is not well understood. In previous study, we found PUMA (p53-upregulated modulator of apoptosis) played an important role in oxaliplatin-induced apoptosis. In the present study, we detect the role of PUMA in H2O2-induced apoptosis in colorectal cancer cells and investigate the potential mechanism.

Methods and results: We showed that H2O2 stimulated the activity of a 493 PUMA promoter reporter gene construct. Suppressing the expression of PUMA abrogated H2O2-induced apoptosis. Deletion of the Sp1-binding sites also decreased the transactivation of PUMA promoter by H2O2. Furthermore, induction of PUMA promoter activity by H2O2 was abrogated by PFT-alpha (a p53 inhibitor) and Mithramycin A (a Sp1 inhibitor), as compared with PFT-alpha alone. To determine the effects of Sp1 on PUMA in H2O2-induced apoptosis, procaspase 3, procaspase 9 and procaspase 8 expression was assessed. Mithramycin A and PFT-alpha also reduced H2O2-induced apoptosis synergistically and abrogated the expression of procaspase 3 and procaspase 9.

Conclusion: Our findings suggest that PUMA plays a role in H2O2-induced apoptosis, and that Sp1 works together with p53 in the regulation of H2O2-induced PUMA expression and apoptosis in colorectal cancer cells. This study provides important regulatory insights in the mechanisms of ROS in colorectal cancer.

Show MeSH
Related in: MedlinePlus