ERK1/2 And Sp1 Are Involved In Stress-induced PUMA Expression And Apoptosis In Colorectal Cancer Cells

Background and aimsPUMA(p53 up-regulated modulator of apoptosis) is a direct mediator of p53 and belongs to BH3-only protein family.It plays an important role in stress-induced apoptosis such as chemotherapy,UV radiation,hypoxia or oxidative stress.Recent work suggested that PUMA was involved in apoptosis and tumorigenesis in a p53-dependent and -independent manner.In provious study,we found oxaliplatin and H₂O₂ could induce PUMA expression and apoptosis in colorectal cancer cells,and oxaliplatin induced PUMA expression and apoptosis in a p53 -independent manner.These results suggested that other factors was involved in this regulation.PUMA is a downstream gene of p53 and induces apoptosis in a p53-independent manner.This makes it as a target gene for gene therapy when p53 function is abrogated.Increasing PUMA expression in tumor cells or decreasing PUMA expression in normal cells will benefit the use of higher dose chemotherapeutics during cancer chemotherapy.To make it possible,we must elucidate the signal pathway associated with it.On the other hand,the effects of DNA element,transcription factor and atoxic micromolecular compound on PUMA should be identified.Mitogen-activated protein kinases(MAPKs) are serine/threonine kinases that play an important role in signal transduction from the cell surface to the nucleus. They regulates cell proliferation and apoptosis through regulating gene expression. For example,the interacion between Bcl-_XL and ERK1/2 induced apoptosis in a mitochondria way.So we hypothesis that MAPK signal pathway is involved in PUMA regulation.On the other hand,there are series of p53 and Sp1-binding sites on PUMA promoter and recent work suggested that H₂O₂ regulated apoptosis and angiogenesis through transcription factor including Sp1.This study is to investigate the role of MAPK signaling pathway in oxaliplatin-induced PUMA expression and Sp1 in H₂O₂-induced PUMA expression in colorectal cancer cells.Identification of the molecular components involved in regulating PUMA will benefit the design of molecules(small peptides or chemical inhibitors) targeted on PUMA and may provide new idea in cancer chemotherapy.Methods and results1.Oxaliplatin induced inactivation of ERK1/2 and activation of JNK1/2,but it had no effect on p38 or the expression of p73.Since MAPKs pathway is associated with cell survival and stress-induced apoptosis,we determined the effects of oxaliplatin on the activities of ERK1/2,JNK1/2,p38 MAPK and p73 in colorectal cancer cells.Our data showed that ERK1/2 was inactivated and JNK1/2 was activated by oxaliplatin in a dosedependent manner.However,oxaliplatin had no effect on the expression of p73 expression or p38 activation.2.Suppressing the activation of ERK1/2 enhanced oxaliplatin-induced PUMA expression and apoptosis in a p53-independent manner.Starved cells were treated with JNK inhibitor SP600125 and ERK inhibitor PD98059 separatedly an hour before oxaliplatin was added.PUMA expression was assessed by Western blotting analysis.Our data indicated that PD98059 enhanced oxaliplatin-induced PUMA expression,which was consistent with the result that oxaliplatin inactivated ERK.And SP600125 had no effect on PUMA expression.To confirm this result,we suppressed the activation of ERK1/2 by transient transfection of DN-MEK1 plasmid into the cells and gained the same conclusion.To investigate the role of PD980059 and the relationship between ERK1/2 and p53 status in oxaliplatin-induced apoptosis,LoVo p53 wide-type cells and LoVo p53-/- cells were treated with PD98059 an hour before 2.4μM oxaliplatin was added. Our data suggested that oxaliplatin and PD98059 could induce apoptosis synergistically and there was no significant difference between LoVo p53 wide-type and p53-/- cells.3.PUMA played a role in ERK inhibitor-enhanced apoptosis in oxaliplatin-treated colorectal cancer cells.Having shown that PD98059 can enhance oxaliplatin-induced PUMA expression and apoptosis in colorectal cancer cells,we subsequently determined the role of PUMA in PD98059 enhanced apoptosis.Apoptosis was evaluated by Hoechst 33258 dye.When PUMA expression was suppressed by stable transfecting PUMA anti-sense vector,apoptosis induced by oxaliplatin and PD98059 was significantly reduced in LoVo PUMA_AS cells comparing with that in the control puma wide-type cells(stable transfecting pcDNA3.1-).4.Sp1 was required for the transactivation of the human PUMA promoter by p53.To determine the role of Sp1 in H₂O₂-induced PUMA expression,we examined the activity of a series of PUMA promoter trunction mutants after H₂O₂ treated. Compared with cells transfected with -336/+157 PUMA-Luc,the transactivation level of cells transfected with -336/+157â–³-126/-25PUMA-Luc was lower,but the difference was not significant.Compared with cells transfected with -336/+157 PUMA-Luc,the transactivation level of cells transfected with -36/+157PUMA-Luc was significant lower.To identify the effects of Mithr.A on p53 and Sp1 expression,Mithr.A (200ng/ml) was added to LoVo cells 24h before p53 and Sp1 expression were assessed by Western blotting analysis.Our data showed Mithr.A had no effects on p53 expression and suppress the expression of Sp1.Mithr.A also abrogate the Sp1 expression-induced by H₂O₂.Similarly,PFT-α(20μM) abrogated p53 expression induced by H₂O₂.To determine the effects of Mithr.A and PFT-αon PUMA expression,Mithr.A and PFT-alpha were added an hour before H₂O₂ treated.Our data suggested that PUMA expression was suppressed by Mithr.A and PFT-αseparated or in combination.To understand the mechanism of Mithr.A and PFT-αaction,we tested the ability of Mithr.A and PFT-αto inhibit PUMA promoter activation induced by H₂O₂.Mithr.A caused an decrease in PUMA promoter activity induced by H₂O₂ but the difference is not significant with untreated cells,while PFT-α.caused an significant decrease.Compared with PFT-αalone,Mithr.A also caused an significant decrease in PUMA promoter acitvity in combination with PFT-α.5.H₂O₂-induced apoptosis and apoptosis-associated gene expression in colorectal cancer cells were abrogated by Mithr.A and PFT-α.To test whether an decrease in the expression of PUMA contributed to an decrease in the apoptosis of the cells,apoptosis assay was proceeded.We next assessed the impact of Mithr.A and PFT-αtreatment on H₂O₂-induced apoptosis in colorectal cancer cells.As shown in Fig.4A,Mithr.A caused an decrease in H₂O₂-induced apoptosis but the difference was not significant with untreated cells, while PFT-α.caused an significant decrease.Compared with PFT-αalone,Mithr.A also caused an significant decrease in H₂O₂-induced apoptosis in combination with PFT-α.To assess the impact of Mithr.A and PFT-αtreatment on these events,total cell extracts were prepared from H₂O₂,Mithr.A and PFT-αtreated cells,procaspase 3, procaspase 9 and procaspase 8 level was monitored by Western blotting analysis. Increased level of procaspase 3 and 9 were observed in Mithr.A and PFT-αtreated cells and Mithr.and PFT-αhave no effects on procaspase 8.Conclusions1.Our results showed that PUMA played an important role in oxaliplatin-induced apoptosis in colorectal cancer cells and ERK1/2 was also involved in oxaliplatin-induced PUMA expression and apoptosis in a p53-independent manner,but not JNK1/2 or p38MAPK.2.A novel mechanism of PUMA stimulation by H₂O₂ in colorectal cancer cells had been domonstrated.The results suggested that H₂O₂-induced up-regulation of PUMA was partly due to Sp1 transcription factor except for p53.These results may provide an insight into the molecular control of H₂O₂-induced PUMA expressoin in colorectal cancer cells through Sp1 binding sites.