摘要
硒是具有抗癌作用的人体必需微量元素之一,大量证据表明,超营养剂量的硒能够诱导前列腺癌、肝癌、结直肠癌、肺癌、乳腺癌、白血病等多种肿瘤细胞的凋亡。在本实验室的前期工作中已经发现,20μM亚硒酸钠(sodium selenite)能够诱导人急性早幼粒细胞白血病(acute promyelocytic leukemia, APL)细胞株NB4发生显著凋亡,活性氧(active oxygen species, ROS)是关键的上游调控因素。亚硒酸钠刺激NB4细胞迅速产生大量活性氧,通过活性氧启动线粒体凋亡通路与内质网应激(endoplasmic reticulum stress, ER stress)凋亡通路。为了详细阐明NB4细胞在亚硒酸钠诱导凋亡的过程中发生的分子生物学变化,我们仍然需要进行更深入的探索。
本研究在前期工作的基础上,进一步探讨了在亚硒酸钠诱导NB4细胞凋亡过程中,一些受到活性氧调节的重要分子的变化情况、调节通路及其对凋亡的意义,重点对关键抗氧化酶MnSOD(manganese superoxide dismutase)进行了研究。经Western bolt发现,亚硒酸钠在诱导NB4细胞凋亡的同时引起了MnSOD的显著上调,活性氧清除剂MnTMPyP完全阻断该上调效应,提示活性氧是其上游调控因素。经不同活性氧探针检测发现,亚硒酸钠作用后诱导两种细胞内活性氧超氧自由基(superoxide radical)与过氧化氢(hydrogen peroxide)迅速而显著的升高,其中超氧自由基可能对于MnSOD的上调起主要作用。就具体通路而言,蛋白激酶ERK(extracellular signal-regulated kinase)与转录因子p53是活性氧介导MnSOD上调的关键因素。cDNA测序结果证明本研究使用的NB4细胞表达野生型的p53。经Western blot检测核蛋白提取物和免疫荧光染色发现,亚硒酸钠诱导细胞产生活性氧后,活性氧促使ERK2从胞质转位至核内,磷酸化p53的关键位点Ser15。免疫共沉淀结果显示,ERK2在核内通过直接作用的方式磷酸化p53。采用该方法还发现,在未经亚硒酸钠作用的NB4细胞的核内,p53与其抑制性蛋白MDM2(mouse double minute 2)处于结合状态,而经亚硒酸钠处理后,核内p53被ERK磷酸化,从而与MDM2解离而被激活。采取p53选择性抑制剂Pifithrin-α(PFT)抑制其转录活性,或通过siRNA干扰降低其表达,均能显著抑制亚硒酸钠诱导的MnSOD上调,提示p53作为MnSOD的上游转录因子诱导了其表达。对于p53缺失或突变的白血病细胞株HL-60或U937,亚硒酸钠不能诱导MnSOD上调,进一步证明了p53的重要作用。通过上述研究阐明了亚硒酸钠诱导NB4细胞凋亡过程中,细胞内存在一条ROS-ERK-p53-MnSOD信号通路,该通路可能是NB4细胞在面临氧化应激时启动的重要抗氧化机制。
前期研究已经发现,在未经亚硒酸钠处理的NB4细胞中存在活跃的自噬作用,亚硒酸钠处理后,在诱导凋亡的同时伴有自噬的减弱。本研究在此基础上进行了进一步探讨,发现p53的激活除了诱导MnSOD上调,还是亚硒酸钠促进NB4细胞由自噬转向凋亡的关键因素。Western blot和免疫共沉淀结果显示,亚硒酸钠通过蛋白激酶ERK和p38MAPK引起p53关键位点Ser15的磷酸化,进而导致p53与其抑制性蛋白MDM2解离并被激活。借助免疫荧光染色与免疫共沉淀发现,在此过程中核仁蛋白B23从核仁转位至核质,与MDM2共定位并发生相互作用,从而起到稳定p53的作用。采用p53抑制剂PFT抑制其活性或通过siRNA干扰降低其表达,均能显著逆转亚硒酸钠诱导的凋亡、caspases激活以及自噬标志分子Beclin-1和LC-3的表达下调,表明激活后的p53一方面促进凋亡的进行,另一方面抑制自噬,从而在NB4细胞由自噬转向凋亡的过程中扮演关键角色。
本研究还发现,在亚硒酸钠诱导NB4细胞凋亡后期,蛋白激酶PKCalpha(protein kinase Calpha)在蛋白水平与磷酸化水平均显著下调,其下调同样由活性氧所介导。采用PKCalpha抑制剂与siRNA干扰发现,PKCalpha在NB4细胞中发挥抗凋亡作用,并且该作用建立在其对蛋白激酶ERK与Akt调节的基础之上,活性氧探针检测发现,PKCalpha还具有抑制活性氧产生的作用。此外,活性氧还激活了蛋白水解酶caspase-3,诱导磷酸酶PP2Ac的表达,在凋亡后期,活性氧通过caspase-3与PP2Ac分别在蛋白水平与磷酸化水平下调PKCalpha,从而加速了凋亡的进行。因此,下调抗凋亡激酶PKCalpha可能是亚硒酸钠在凋亡后期维持并促进凋亡的重要机制之一。
细胞色素C氧化酶亚基IV(cytochrome c oxidase subunit IV, COX IV)是线粒体呼吸链的重要组分,在亚硒酸钠诱导NB4细胞凋亡后期,该蛋白同样受到活性氧的显著下调,RT-PCR显示其mRNA水平没有明显变化,提示非转录机制参与其下调。进一步研究发现,活性氧通过激活caspase-3介导COX IV的下调。采用shRNA表达载体抑制COX IV的表达,能够显著增强亚硒酸钠诱导的NB4细胞凋亡,表明亚硒酸钠可能通过ROS-caspase-3-COX IV这一途径损伤线粒体呼吸链,加速凋亡的进行。
Selenium is one of the essential trace elements with anti-tumor properties. Large amount of evidence indicates that supranutritional selenium is able to induce apoptosis in diverse tumor cell lines, including prostatic cancer, hepatoma, colorectal cancer, lung cancer, breast cancer and leukemia. In the previous study of our group, we found 20μM of sodium selenite induced pronounced apoptosis in NB4 cells derived from human acute promyelocytic leukemia (APL), while reactive oxygen species (ROS) played an upstream and critical role. Under selenite exposure, NB4 cells produced considerable ROS, by which mitochondrial apoptosis pathway and endoplasmic reticulum stress (ER stress) pathway were initiated. However, the detailed molecular alterations in selenite-induced apoptosis still require further investigations.
Based on our previous work, we further explored the alterations of some ROS-regulated important molecules, as well as their regulation pathways and the influences on selenite-induced apoptosis. Here we discovered by Western blot that the expression of manganese superoxide dismutase (MnSOD) was dramatically elevated in selenite-induced apoptosis of NB4 cells. ROS scavagener MnTMPyP completely abrogated such upregulation, suggesting ROS acted as the upstream regulator. ROS detection revealed that two intracelluler ROS, superoxide radicals and hydrogen peroxide were immediately generated after selenite exposure, in which superoxide radicals might be critical for elevation of MnSOD. As to the detailed mechanisms, we elucidated that ERK and p53 were closely involved. cDNA sequencing validated that NB4 cells used in the present study expressed wild-type p53. Western blot of the nuclear fractions and immunofluorescence staining indicated that after production of ROS induced by selenite, ERK2 was driven by ROS to be translocated from the cytoplasm into the nucleus, and subsequently phosphorylated p53 at the vital site Ser15. Immunoprecipitation results demonstrated ERK2 phosphorylated p53 by a direct bingding, leading to activation of p53 by dissociation from its inhibitory protein mouse double minute 2 (MDM2). Inhibition of p53 transactivation ability by its selective inhibitor Pifithrin-a (PFT) or suppression of its expression by siRNA abolished selenite-induced upregulation of MnSOD, indicating p53 mediated elevated expression of MnSOD as its upstream transcription factor. Additionally, in leukemia cell lines with null or mutant p53 such as HL-60 and U937, selenite could not induce upregulation of MnSOD, further corroborating the critical role of p53 in this modulation. We speculate a ROS-ERK-p53-MnSOD pathway exists in NB4 cells when apoptosis is induced by selenite. This is probably an important mechanism by which NB4 cells counteract oxidative stress induced by selenite.
It was revealed by our previous research that selenite repressed autophagy accompanied by the induction of apoptosis in NB4 cells. In the present study, we further explored the mechanisms underlying the switch of NB4 cells from autophagy to apoptosis, and elucidated p53 played a key role. Western blot indicated selenite induced phosphorylation of p53 at the vital site ser15 via ERK and p38MAPK. Immunofluorescence staining and immunoprecipitation showed p53 dissociated with MDM2, while the nucleolar protein B23 transferred from the nucleolus to the nucleoplasm and associated with MDM2, probably stabilizing p53. p53 inhibitor PFT and siRNA interference notably reversed selenite-induced apoptosis, caspases activation, and decrease of autophagic protein Beclin-1 and LC-3, manifesting that active p53 mediated induction of apoptosis and inhibition of autophagy, thus switching NB4 cells from autophagy to apoptosis.
We also revealed that protein kinase Calpha (PKCalpha) was dramatically reduced at later stage of selenite-induced apoptosis, which was mediated by ROS. Besides, by means of PKCalpha inhibitor and siRNA interference, we confirmed that PKCalpha played an anti-apoptotic role through its effects on ERK 1/2 and Akt. ROS detection indicated PKCalpha also inhibited generation of intracellular ROS. At later stage, PKCalpha was downregulated at protein level and phosphorylation level by caspase-3 and PP2Ac respectively under the regulation of ROS. In summary, we speculate that PKCalpha functions to counteract apoptosis in NB4 cells, and its downregulation seems an important mechanism maintaining and promoting apoptosis induced be selenite, especially at later stage.
COX IV (cytochrome c oxidase subunit IV) is an essential component of mitochondrial respiratory chain. At later stage of selenite-induced apoptosis of NB4 cells, COX IV was remarkably downregulated at protein level without evident alteration in mRNA level, suggesting this downregulation was independent of transcriptional regulation. Further investigation illuminated that ROS mediated COX IV downregulation through caspase-3 activation. COX IV inhibition by shRNA expression vectors markedly enhanced selenite-induced apoptosis of NB4 cells, indicating selenite probably damaged the integrity and function of mitochondrial respiratory chain through the ROS-caspase-3-COX IV pathway, thus accelerating the proceeding of apoptosis.
引文
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