【作者】 王新颖；

【导师】 姜泊； 王继德；

【作者基本信息】 南方医科大学 ， 消化内科， 2008， 博士

【摘要】 研究背景与目的PUMA（p53 up-regulated modulator of apoptosis）是Bcl-2家族BH-3亚家族中的促凋亡成员,他可被p53快速诱导且具有强大的促凋亡作用,在化疗药物、放射线、紫外线、氧化等多种应激诱导肿瘤细胞凋亡的过程中发挥着重要的作用。近年来研究证实其在p53依赖和非依赖途径的细胞凋亡启动与肿瘤发生过程中发挥着重要作用,可能是调控肿瘤细胞凋亡的最终靶点之一,其具体调控机制及利用其诱导凋亡的性质在抗肿瘤治疗中发挥作用也成为国内外学者关注的问题。在前期工作中我们发现在肠癌LoVo细胞中奥沙利铂和过氧化氢均可以诱导PUMA蛋白的表达,从而导致肠癌LoVo细胞发生凋亡,并且奥沙利铂诱导PUMA表达致肠癌细胞凋亡的过程可以不依赖于p53的表达。在p53凋亡诱导通路中,PUMA位于p53的下游,而且还可以不依赖于p53诱导凋亡,这就有可能在p53功能发生缺陷或外源导入p53诱导凋亡效果不好的肿瘤中以PUMA为治疗靶点进行基因治疗,从而为肿瘤的基因治疗提供新的思路。但要使其成为可能,一方面需要明确PUMA的作用通路,阐明它与其他相关基因相互作用的机制;另一方面需要寻找到能导致PUMA激活的DNA元件、转录因子及其他无毒性的小分子物质。这样就可以利用这些物质激活PUMA在肿瘤细胞内强表达,或特异性地阻断PUMA在化疗可能导致损伤的正常细胞中的表达,充许医生用更高剂量的抗癌药治疗病人。在众多与肿瘤细胞增殖、分化、凋亡密切相关的因子中,丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）信号通路具有至关重要的作用。该信号通路包括一系列高度同源的蛋白激酶,这些激酶在将环境刺激传导到核的过程中起着关键作用,他们能够通过调节基因表达而参与一系列细胞的活动。例如:ERK1/2和JNK1/2可以分别通过影响Bcl-_XL和Bcl-2而通过线粒体途径引起凋亡,而PUMA正是通过与Bcl-_XL、Bcl-2、Bad的相互作用而介导肿瘤细胞的凋亡。另一方面,在研究转录调控对这一过程的影响时我们发现PUMA启动子的5’端靠近p53结合位点附近尚存在着一系列的Sp1家族成员的结合位点。其它有研究表明,过氧化氢可以通过影响包括Sp1在内的转录因子而调控肿瘤细胞的凋亡和血管生成。因此,本研究主要探讨丝裂原活化蛋白激酶（mitogen-activated proteinkinase,MAPK）信号通路及转录因子Sp1（specificity protein1）在应激（奥沙利铂和过氧化氢）引起的PUMA表达致肠癌细胞凋亡过程中的作用及可能的调控机制。本研究将初步阐明PUMA在介导奥沙利铂和过氧化氢诱导肠癌细胞凋亡过程中的调控机制,研究结果将为未来大肠癌的分子靶向治疗提供理论基础和新的思路。主要方法和结果1.奥沙利铂可以磷酸化激活JNK1/2,抑制ERK1/2的磷酸化,而对p38MAPK的磷酸化无影响,而对p53的同源类似物p73的表达无影响。分别给予1.2、2.4、4.8μmol/L的奥沙利铂处理LoVo细胞,24h后蛋白印迹法检测磷酸化JNK、ERK1/2、p38MAPK、p73蛋白的表达。结果表明,在奥沙利铂处理的细胞中,磷酸化JNK蛋白的表达增加,磷酸化ERK蛋白的表达降低,并呈剂量依赖关系,而磷酸化p38MAPK及p73蛋白的表达无明显变化。2.抑制ERK1/2的活性可以协同奥沙利铂诱导PUMA的表达,而JNK抑制剂对这一诱导作用无影响。采用ERK1/2的特异性抑制剂PD98059,JNK1/2的特异性抑制剂SP600125分别抑制两者的功能,利用蛋白印迹法检测其对奥沙利铂诱导PUMA表达的影响。结果表明,在PD98059与奥沙利铂联合处理组中,PUMA的表达较奥沙利铂单纯处理组显著增加,提示PD98059可以加强奥沙利铂对PUMA表达的诱导,而SP600125则对奥沙利铂的诱导作用无明显影响;利用转染MEK1的显性负性失活突变体（DN-MEK-1）抑制ERK1/2的激活后,检测奥沙利铂对PUMA表达的影响,结果进一步证实了抑制ERK的磷酸化激活可以协同奥沙利铂诱导PUMA蛋白的表达。3.奥沙利铂与ERK抑制剂的协同诱导作用可以不依赖于p53的表达。分别采用p53不同表型的细胞株LoVo,SW1116及p53抑制剂来探讨在p53功能发生障碍时这种协同作用是否存在。结果提示奥沙利铂与ERK抑制剂的协同诱导作用可以依赖或不依赖于p53的表达。4.奥沙利铂和ERK抑制剂可以协同诱导肠癌细胞的凋亡,并且不依赖于p53的表达。将LoVo细胞和p53表达明显减弱的LoVo p53-/-细胞克隆,分别分为三组:对照无处理组,奥沙利铂单纯处理组,奥沙利铂和PD98059联合处理组。结果提示在三个处理组中,LoVo p53-/-细胞克隆与对照细胞组凋亡细胞数无明显差别。5.抑制PUMA的表达可以减少奥沙利铂和ERK抑制剂协同诱导的肠癌细胞的凋亡。采用稳定转染pcDNA3.1-的LoVo细胞及LoVo PUMA_AS细胞,分别分为三组:对照无处理组,奥沙利铂单纯处理组,奥沙利铂和PD98059联合处理组。结果表明:LoVo PUMA_AS细胞中奥沙利铂单纯处理及奥沙利铂和PD98059联合处理后细胞凋亡均较对照细胞明显减少,差别有显著性意义。6.过氧化氢能够诱导PUMA启动子的活性,从而诱导PUMA的表达。LoVo细胞转染0.5μg-336/+157 PUMA-Luc报告基因质粒,同时转入0.1μgpSVβ-Galactosidase质粒做为对照。24h后分别给予0.04,0.24,0.64mmol/L过氧化氢刺激肠癌LoVo细胞15min,24h后检测荧光素酶报告基因及pSVβ-Galactosidase报告基因活性。结果提示过氧化氢能够诱导PUMA启动子的活性。7.过氧化氢能够通过增强p53活性而转录激活PUMA启动子,Sp1可以增强这种转录激活作用。肠癌LoVo细胞分别转染-336/+157 PUMA-Luc、-36/+157PUMA-Luc及-336/157△-126/-25 PUMA-Luc三种报告基因质粒及对照质粒,24h后给予过氧化氢0.64mmol/L处理15min,24h后检测荧光素酶报告基因及pSVβ-Galactosidase报告基因活性。结果提示过氧化氢诱导PUMA启动子的活性是通过p53转录因子的调控,这一过程需要Sp1转录因子的辅助作用。肠癌LoVo细胞分为对照无处理组,过氧化氢单纯处理组,过氧化氢及PFT-α联合处理组,过氧化氢及普卡霉素联合处理组。给予0.64mmol/L过氧化氢处理15min前1h给予PFT-α或普卡霉素,24h后蛋白印迹法检测Sp1和p53蛋白的表达,结果提示普卡霉素可以抑制过氧化氢诱导的Sp1的表达,而对p53的表达无影响,而PFT-α可以抑制过氧化氢诱导的p53的表达,而对Sp1的表达无影响。8.普卡霉素和PFT-α可以协同抑制过氧化氢诱导的PUMA的表达及启动子活性。肠癌LoVo细胞分别转染-336/+157 PUMA-Luc、-36/+157PUMA-Luc及-336/+157△-126/-25三种报告基因质粒及对照质粒,24h后给予过氧化氢0.64mmol/L处理15min,给予过氧化氢前1h分别给予PFT-α或普卡霉素,24h后蛋白印迹法检测PUMA蛋白的表达。结果提示普卡霉素及PFT-α具有协同抑制PUMA表达的作用。同时检测荧光素酶报告基因活性,提示二者联合使用对过氧化氢诱导的PUMA启动子活性较单独使用具有更强的抑制作用。9.Sp1参与过氧化氢通过诱导PUMA表达引起的肠癌LoVo细胞的凋亡。从上述结果可以看到,Sp1参与过氧化氢对PUMA表达的调节作用,为探讨这种调节作用对PUMA表达介导的肠癌细胞凋亡作用的影响,采用普卡霉素和PFT-α抑制Sp1及p53的表达,利用Hoechst 33258染色检测肠癌LoVo细胞的凋亡。结果提示普卡霉素和PFT-α联合应用对过氧化氢诱导的肠癌细胞凋亡的抑制作用增强。二者联合应用后procaspase-3,9的表达增加,而procaspase-8表达无变化,提示普卡霉素和PFT-α联合应用是通过线粒体途径发挥作用,从而进一步证明了其对PUMA表达的调控作用。结论1.MAPK信号通路中重要成员ERK1/2参与奥沙利铂对PUMA的调节作用,这种调节作用可以不依赖于p53的表达;2.尽管JNK1/2也被奥沙利铂激活,但并不参与其对PUMA的调节作用;3.抑制ERK1/2的磷酸化激活可以协同加强奥沙利铂诱导PUMA的表达;4.奥沙利铂和ERK1/2的磷酸化失活可以通过协同诱导PUMA的表达而诱导肠癌LoVo细胞的凋亡;5.在过氧化氢诱导PUMA表达的过程中,转录因子Sp1参与p53对PUMA表达的调控,从而诱导肠癌细胞的凋亡,二者具有协同作用,其单独调控作用不明显。更多还原

【Abstract】 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/+157△-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.更多还原