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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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H2O2 regulated PUMA expression at transcriptional level. (A, B) Dose and time response of LoVo cells treated with H2O2. PUMA mRNA expression was detected by RT-PCR using 0.5 μg total RNA. Beta-actin mRNA was amplified as an internal control. (C) LoVo cells were transiently transfected with the -336/+157 PUMA-luc reporter plasmid (0.5 μg). H2O2 (0.64 mM) was added to the indicated samples 24 hours post-transfection and exposure was continued 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, *p < 0.05) from at least two independent expression performed in duplicate are shown in the form of a bargraph.
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Figure 2: H2O2 regulated PUMA expression at transcriptional level. (A, B) Dose and time response of LoVo cells treated with H2O2. PUMA mRNA expression was detected by RT-PCR using 0.5 μg total RNA. Beta-actin mRNA was amplified as an internal control. (C) LoVo cells were transiently transfected with the -336/+157 PUMA-luc reporter plasmid (0.5 μg). H2O2 (0.64 mM) was added to the indicated samples 24 hours post-transfection and exposure was continued 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, *p < 0.05) from at least two independent expression performed in duplicate are shown in the form of a bargraph.

Mentions: To investigate whether H2O2 increases PUMA expression at the mRNA level, we treated LoVo cells with a dose and time range of H2O2 and performed a standard RT-PCR experiment. As seen in Figure 2A and 2B, H2O2 increased PUMA mRNA in a dose- and time- dependent manner. Therefore, the data suggested that H2O2 up-regulated PUMA expression at transcriptional level.


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)

H2O2 regulated PUMA expression at transcriptional level. (A, B) Dose and time response of LoVo cells treated with H2O2. PUMA mRNA expression was detected by RT-PCR using 0.5 μg total RNA. Beta-actin mRNA was amplified as an internal control. (C) LoVo cells were transiently transfected with the -336/+157 PUMA-luc reporter plasmid (0.5 μg). H2O2 (0.64 mM) was added to the indicated samples 24 hours post-transfection and exposure was continued 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, *p < 0.05) from at least two independent expression performed in duplicate are shown in the form of a bargraph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570657&req=5

Figure 2: H2O2 regulated PUMA expression at transcriptional level. (A, B) Dose and time response of LoVo cells treated with H2O2. PUMA mRNA expression was detected by RT-PCR using 0.5 μg total RNA. Beta-actin mRNA was amplified as an internal control. (C) LoVo cells were transiently transfected with the -336/+157 PUMA-luc reporter plasmid (0.5 μg). H2O2 (0.64 mM) was added to the indicated samples 24 hours post-transfection and exposure was continued 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, *p < 0.05) from at least two independent expression performed in duplicate are shown in the form of a bargraph.
Mentions: To investigate whether H2O2 increases PUMA expression at the mRNA level, we treated LoVo cells with a dose and time range of H2O2 and performed a standard RT-PCR experiment. As seen in Figure 2A and 2B, H2O2 increased PUMA mRNA in a dose- and time- dependent manner. Therefore, the data suggested that H2O2 up-regulated PUMA expression at transcriptional level.

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