-----顺铂的化疗原理-----
内质网的钙流出物可调节顺铂诱导的人宫颈癌细胞凋亡
Calcium efflux from the endoplasmic reticulum regulates cisplatin-induced apoptosis in human cervical cancer HeLa cells
翻译：蓝山
从内质网(ER)到顺铂诱导的细胞凋亡中钙流出物的功能在癌细胞中尚未完全了解。本研究采用western blot分析、流式细胞术、免疫荧光法和3-[4,5-二甲基噻唑-2-yl]-2,5二苯基四唑溴铵法，对暴露于顺铂的人宫颈癌细胞中的钙信号进行研究。在本研究中，顺铂的治疗增加了人宫颈癌HeLa细胞的细胞质和线粒体的游离Ca2+水平，这进一步触发了线粒体介导的和ER应激相关的凋亡通路。特别的是，通过钙螯合剂bis-(o-氨基苯氧基)乙烷、N、N'、N'、N'-四乙酸甲酯的钙离子阻断剂，通过下调钙依赖性蛋白酶、cal疼痛和固有的凋亡蛋白，如caspase- 3、caspase-4和C/EBP同源蛋白(CHOP)，抑制了顺铂诱导的细胞凋亡。此外，使用肌醇三磷酸受体抑制剂，2-氨基乙基二苯基硼酸盐，从ER中抑制钙的流出也产生了类似的效果。这一数据表明，ER中的钙流出物在顺铂诱导的人宫颈癌细胞凋亡中起着重要的作用，它为顺铂的肿瘤细胞杀伤作用和改善顺铂化疗的潜在治疗策略提供了进一步的机制观察。
The function of calcium efflux from the endoplasmic reticulum (ER) in cisplatin-induced apoptosis is not fully understood in cancer cells. The present study used western blot analysis, flow cytometry, immunofluorescence and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay to investigate calcium signaling in human cervical cancer cells exposed to cisplatin. In the present study, treatment with cisplatin increased free Ca2+ levels in the cytoplasm and mitochondria of human cervical cancer HeLa cells, which further triggers the mitochondria-mediated and ER stress-associated apoptosis pathways. Notably, blocking calcium signaling using the calcium chelating agent bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester inhibited cisplatin-induced apoptosis via downregulation of the calcium-dependent proteases, the calpains, and innate apoptosis proteins, such as caspsae-3, caspase-4 and C/EBP homologous protein (CHOP). In addition, use of the inositol triphosphate receptor inhibitor, 2-aminoethyl diphenylborinate, to inhibit calcium efflux from the ER resulted in similar effects. This data indicated that calcium efflux from the ER plays a significant role in cisplatin-induced apoptosis in human cervical cancer HeLa cells, which provides further mechanistic insights into the tumor cell-killing effect of cisplatin and potential therapeutic strategies to improve cisplatin chemotherapy.
http://europepmc.org/articles/PMC4812401
顺铂引起线粒体活性氧反应 这一反应依赖于线粒体氧化还原状态和生物能量功能。
Cisplatin Induces a Mitochondrial-ROS Response That Contributes to Cytotoxicity Depending on Mitochondrial Redox Status and Bioenergetic Functions
翻译：蓝山
顺铂是治疗多种肿瘤的最有效、最广泛使用的抗癌药物之一。
顺铂的细胞毒性作用被认为主要是由细胞核DNA加合物的产生介导的，如果不进行修复，就会导致细胞死亡，这是DNA复制和转录阻断的结果。然而，顺铂引起核DNA (nDNA)损伤的能力不足以解释其高程度的有效性和对正常的后丝分裂组织的毒性作用。几个组织中暴露于顺铂后，在体内观察到氧化损伤，这表明氧化应激在顺铂诱导的剂量限制毒性的发病机制中发挥了作用。然而，顺铂诱导的活性氧（ROS）生成机制及其在正常和癌细胞中对顺铂细胞毒性的贡献仍知之甚少。
通过使用正常和癌细胞的细胞株和芽酵母作为模型系统，我们发现暴露于顺铂引起线粒体依赖的ROS反应，显著提高nDNA损伤引起的细胞毒性效应。ROS生成与顺铂诱导的nDNA损伤的数量无关，而线粒体是蛋白质合成损伤的结果。顺铂引起的线粒体功能障碍在决定其细胞毒性作用方面的作用因细胞而异，取决于线粒体氧化还原状态、线粒体DNA完整性和生物能量功能。因此，通过调控这些细胞参数，我们能够增强顺铂对癌细胞的细胞毒性。
本研究为顺铂诱导的细胞杀伤提供了一种新的机制，并可能导致新的治疗策略的设计，以提高抗癌药物的疗效。
Cisplatin is one of the most effective and widely used anticancer agents for the treatment of several types of tumors. The cytotoxic effect of cisplatin is thought to be mediated primarily by the generation of nuclear DNA adducts, which, if not repaired, cause cell death as a consequence of DNA replication and transcription blockage. However, the ability of cisplatin to induce nuclear DNA (nDNA) damage per se is not sufficient to explain its high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues. Oxidative damage has been observed in vivo following exposure to cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced dose-limiting toxicities. However, the mechanism of cisplatin-induced generation of ROS and their contribution to cisplatin cytotoxicity in normal and cancer cells is still poorly understood. By employing a panel of normal and cancer cell lines and the budding yeast Saccharomyces cerevisiae as model system, we show that exposure to cisplatin induces a mitochondrial-dependent ROS response that significantly enhances the cytotoxic effect caused by nDNA damage. ROS generation is independent of the amount of cisplatin-induced nDNA damage and occurs in mitochondria as a consequence of protein synthesis impairment. The contribution of cisplatin-induced mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function. Thus, by manipulating these cellular parameters, we were able to enhance cisplatin cytotoxicity in cancer cells. This study provides a new mechanistic insight into cisplatin-induced cell killing and may lead to the design of novel therapeutic strategies to improve anticancer drug efficacy.
Rossella Marullo, Erica Werner, Natalya Degtyareva, Bryn Moore, Giuseppe Altavilla, Suresh S. Ramalingam, Paul W. Doetsch
Published: November 19, 2013https://doi.org/10.1371/journal.pone.0081162
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081162
顺铂诱导内质网应激和独立于细胞核的凋亡信号
Cisplatin induces endoplasmic reticulum stress and nucleus independent apoptotic signaling.
翻译：蓝山
DNA损伤被认为是顺铂的抗增殖作用的主要原因，顺铂是抗癌治疗的基石。然而，顺铂可以被期望与除DNA以外的亲核物质反应。我们在这里证明，顺铂诱导的细胞凋亡信号可能独立于DNA损伤。顺铂诱导的caspase-3在细胞质中激活需要钙和钙依赖的蛋白酶calpain的活性。众所周知，calpain活化可能与内质网(ER)应激有关，提示ER是顺铂的细胞质目标。与这个假说相一致，顺铂诱导了在细胞质内和完整的细胞中，特定于ER的caspase-12的激活。顺铂还诱导Grp78/BiP的表达增加，这是ER应激的另一个标记。相比之下，破坏DNA的拓扑异构酶II抑制剂etoposide并没有诱导细胞凋亡信号，也没有在完整细胞中产生ER应激。我们由此确定了顺铂的一种新的作用机制。
研究结果对了解耐药机理和独特的效率有一定的指导意义。
DNA damage is believed to be the main cause of the antiproliferative effect of cisplatin, a cornerstone agent in anticancer therapy. However, cisplatin can be expected to react also with nucleophiles other than DNA. Using enucleated cells (cytoplasts) we demonstrate here that cisplatin-induced apoptotic signaling may occur independently of DNA damage. Cisplatin-induced caspase-3 activation in cytoplasts required calcium and the activity of the calcium-dependent protease calpain. It is known that calpain activation may be associated with endoplasmic reticulum (ER) stress, suggesting that the ER is a cytosolic target of cisplatin. Consistent with this hypothesis, cisplatin induced calpain-dependent activation of the ER-specific caspase-12 in cytoplasts as well as in intact cells. Cisplatin also induced increased expression of Grp78/BiP, another marker of ER stress. By contrast, the DNA-damaging topoisomerase II inhibitor etoposide did not induce apoptotic signaling in cytoplasts nor ER stress in intact cells. We have thus identified a novel mechanism of action of cisplatin. The results have implications for the understanding of resistance mechanisms as well as the unique efficiency of this drug.
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J Biol Chem. 2003 Mar 14;278(11):9100-6. Epub 2002 Dec 31.
https://www.ncbi.nlm.nih.gov/pubmed/12509415/
