三氧化二砷对胃癌细胞杀伤机制的研究
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摘要
目的
凋亡调控失衡与肿瘤的发生和发展密切相关，诱导肿瘤细胞凋亡已成为肿瘤药物治疗的主要目的与手段。凋亡有两条途径，一条为线粒体途径，另一条为死亡受体途径，多种抗肿瘤药物、射线、活性氧等均通过线粒体途径引起细胞凋亡，在深入研究凋亡调控机制与信号转导途径的过程中，线粒体在细胞凋亡中的作用日益受到人们的关注。
研究表明，过表达Bcl-2能够抑制细胞的凋亡而促进细胞生长。Bcl-2家族是哺乳动物细胞中调节凋亡的重要因子，通过调节线粒体或内质网等细胞内膜结构的通透性，线粒体的通透性改变(permeability transition，PT)是调节凋亡的中心环节，改变与细胞生理功能密切相关的离子或蛋白质因子在细胞内的分布，从而影响细胞的生命活动过程。在凋亡的线粒体途径中，各种凋亡促发因素通过调节线粒体外膜的通透性，释放线粒体膜间隙的促凋亡蛋白质因子如细胞色素C(cytochrome-C)和Smac／DIABLO等进入细胞浆，通过依赖caspase的途径激活细胞凋亡或者释放凋亡诱导因子(apoptosis inducing factor，IF)等通过不依赖caspase的途径引起细胞凋亡。Bcl-2家族中抑制细胞凋亡与促进细胞凋亡蛋白之间的平衡，对其在改变线粒体通透性、调节线粒体蛋白释放起着决定作用，但具体机制还存在许多争论。
对细胞凋亡过程起调节作用的除了非常熟悉的Bcl-2家族外，尚有另外一个家族就是细胞凋亡抑制蛋白(IAPs)家族，凋亡蛋白抑制子(IAP)家族中生存素(Survivin)、X联锁凋亡蛋白抑制子(XIAP)过表达，抑制促凋亡因子对caspase激活及对细胞增殖的影响导致肿瘤细胞对凋亡刺激的抵抗。凋亡与坏死是在形态学上不同的两种细胞死亡形式，凋亡细胞核碎裂形成凋亡小体，细胞膜完整。坏死特点是细胞肿胀，细胞内细胞器裂解，细胞膜不完整。凋亡细胞被巨噬细胞吞噬，坏死细胞则释放细胞内容物引起炎症及组织损害。细胞凋亡的机制研究的非常广泛，而细胞坏死研究较少。
砷剂的抗肿瘤机制主要通过线粒体途径引起细胞凋亡来实现，同时也有引起细胞坏死及自溶，砷剂与巯基结合后，导致MPT开放，线粒体跨膜电位(△ψm)下降或消失，继之呼吸链脱偶联，谷胱甘肽耗竭，活性氧类(reactive oxygen species，ROS)产生及对诱导凋亡非常重要的蛋白酶活化物的释放，包括细胞色素C(Cyto-C)和凋亡诱导因子(AIF)，Cyto-C和AIF均可激活caspase诱导凋亡。Bcl-2的过度表达可抑制砷剂诱导的凋亡相关现象，而通过反义技术使Bcl-2下调可抑制细胞增殖及促进凋亡。砷剂可通过对Bcl-2基因功能的抑制诱导某些肿瘤细胞的凋亡，三氧化二砷(As_2O_3)对肿瘤细胞的杀伤过程中在不同细胞背景下，对Bcl-2、活性氧及凋亡抑制蛋白家族的影响不同。As_2O_3对胃癌细胞的杀伤机制研究很少，具体机制仍不清。本研究以胃癌细胞系MGC803、BCG823、SGC7901为模型，研究As_2O_3对胃癌细胞杀伤过程中线粒体途径相关的活性氧、Bcl-2家族及凋亡抑制蛋白家族中的Survivin在此过程中的作用及在凋亡与坏死两种不同杀伤机制中的差异，进一步明确As_2O_3对胃癌细胞杀伤机制，为在临床中更好应用奠定基础。
材料与方法
1．采用MTT法测定细胞活力，绘制增殖曲线。
2．采用瑞氏-姬姆萨染色，光学显微镜下观察As_2O_3诱导MGC803细胞死亡时的形态学变化。
3．采用流式细胞仪通过PI染色进行细胞周期解析及凋亡判定。
4．采用western blot检测Bcl-2、Survivin及caspase-3蛋白的表达。
5．采用DiOC 6(3)单染法检测线粒体跨膜电位(△ψm)的变化。采用罗丹明和碘化丙啶双染法检测线粒体跨膜电位(△ψm)改变与细胞存活状态的关系。应用流式细胞仪通过2′，7′-二氯乙酰乙酸盐荧光素标记检测细胞内活性氧水平。
6．统计学处理所有数据用3次独立实验的平均值表示，采用学生t检验。
实验结果
1．As_2O_3呈时间、浓度依赖性抑制胃癌MGC803、BCG823、SGC7901细胞的增殖，72h抑制细胞增殖50％的药物浓度(IC_(50))分别为2.8、3.1、10.2uM，MGC803、BCG823、SGC7901细胞的活性氧水平在未用药时活性氧峰值水平分别为20、64.3、57.6，5uM As_2O_3作用24h后三种细胞系活性氧峰值水平分别为100.8、103.3、56.5，在应用5uM的As_2O_3作用24h后，MGC803、BCG823细胞的活性氧水平明显增高，而SGC7901细胞只有少数细胞活性氧增高。2uM及5uM的As_2O_3均引起细胞内的活性氧明显升高，峰荧光强度分别由对照的20升高到为80、70，而25uM的As_2O_3没有引起明显的升高。5uM的As_2O_3作用36h，细胞内活性氧3h开始增高，12h达到高峰值。2、5、25uM As_2O_3作用12及24h后线粒体跨膜电位出现降低，24h的线粒体跨膜电位降低的细胞数分别为8.2％、16.1％、23％，能够呈时间及浓度依赖性降低线粒体跨膜电位。
2．氮乙酰半胱氨酸(NAC)能够明显抑制As_2O_3对MGC803细胞的杀伤作用，联合应用As_2O_3 48h的IC_(50)浓度大于25uM，单独应用时的IC_(50)约为5uM。10mM的NAC与5uM As_2O_3同时应用12h，NAC明显降低细胞内的活性氧水平，对照组为12.4，单独应用As_2O_3组为30.5，而联合应用组为7.2。48h流式细胞仪检测细胞周期，单独应用As_2O_3组亚G_0／G_1期细胞为29.32％，G_2／M期细胞为38.19％，出现了亚二倍体峰及G_2／M期阻滞，联合应用组未见亚二倍体峰及G_2／M期阻滞，细胞周期接近正常对照。
3.5μM As_2O_3作用24h细胞形态学可见细胞核浓聚，染色质非正常分裂及凋亡小体，有明显的M期阻滞。25、50μM As_2O_3作用24h细胞可见明显的核固缩，胞浆空泡变性，未见G_2／M期阻滞。5μM As_2O_3作用24h后细胞出现凋亡细胞由对照的1.2％增加到32.8％。坏死细胞增加到12％。而25μM的As_2O_3作用24h后坏死细胞由5.9％增加到72.2％。而凋亡细胞由对照的1.2％增加到8.1％，结果显示25μM的As_2O_3作用后主要以细胞坏死为主。2、5、25、50uM As_2O_3作用24h，各浓度As_2O_3均能引起线粒体Cyto-C向细胞浆中释放，线粒体内Cyto-C明显减少。
4.5uM的As_2O_3作用MGC803细胞24h，早期凋亡细胞数由1.44增至20.37，坏死细胞由5.7％增加到14.36％，而10uM的BSO与5uM的As_2O_3联合应用组，凋亡细胞由单用As_2O_3的20.37％降至7.44％，坏死细胞由14.36％增加到46.11％。结果提示BSO通过诱导肿瘤细胞坏死而不是凋亡来增加As_2O_3对MGC803细胞的杀伤作用。5uM的ms_2O_3作用24h可激活Procaspase-3后出现17及12KD的活性亚基，10uM的BSO与5uM的As_2O_3联合作用后，Procaspase-3只出现17KD的活性亚基，且表达水平为单用As_2O_3的50％。10mM NAC与BSO同时与5uM的As_2O_3共同作用24h，Caspase-3的表达与对照组没有区别。
5.5μM As_2O_3作用MGC803细胞12-48h均诱导Bcl-2蛋白出现磷酸化，12h可出现，36h Bcl-2蛋白磷酸化达到峰值，48h减弱，25μM的As_2O_3作用24h未见Bcl-2蛋白出现磷酸化，Bcl-2蛋白无明显变化。5μM As_2O_3作用MGC803细胞48h，从24h起可见Caspase-3剪切活化形式17、12 KD的小片段，一直持续到48h，Procaspase-3无明显变化。25μM As_2O_3作用MGC803细胞48h，从24h起下调Procaspase-3，48h接近消失，但未见到活化后形成的小片段。
6.5μM As_2O_3作用MGC803细胞6-36h，结果显示活性氧在贴壁与非贴壁细胞中无明显差别。6h后未贴壁细胞均出现线粒体跨膜电位(△ψm)降低，24h最明显，约55％的细胞线粒体跨膜电位(△ψm)明显降低。而贴壁细胞中未见细胞线粒体跨膜电位(△ψm)的下降。5μM As_2O_3作用24h后分离贴壁与非贴壁细胞，检测Bcl-2、Survivin及Caspase-3蛋白表达，24h非贴壁细胞Bcl-2蛋白出现磷酸化，Survivin表达明显增高，Caspase-3蛋白被活化，该部分细胞为凋亡及G_2／M期细胞，两部分细胞比例接近，而贴壁细胞为G_0／G_1、S及G_2期细胞，Bcl-2蛋白磷酸化水平低于As_2O_3作用24h的水平，Survivin表达接近对照细胞，Caspase-3蛋白未见明显活化。贴壁与非贴壁细胞去除药物后继续培养12、24h，贴壁细胞Bcl-2、Survivin、Caspase-3蛋白表达未见明显变化，非贴壁细胞Bcl-2蛋白磷酸化减弱，Survivin表达在继续培养12h时略降低，24h后明显降低，降至24h分离时的30％。7.500nM Okadaic acid及100nM Staurosporine与G_2／M期细胞作用4h，Okadaicacid能够保持Bcl-2蛋白磷酸化，Staurosporine能够使Bcl-2蛋白脱磷酸化。流式细胞仪检测结果Okadaic acid作用2h、4h时亚二倍体细胞分别为19.51％、40.8％，Staurosporine作用2h、4h时亚二倍体细胞分别为10.61％、11.2％，4h对照细胞的亚二倍体细胞数为15.27％。
8.5uM的As_2O_3从12h起明显上调Survivin蛋白表达，36h达到峰值，上调到对照的2.5倍，48h下降，蛋白表达水平为对照的1.4倍。25 uM的As_2O_3作用72h下调Survivin蛋白表达。Ly294002与As_2O_3共同作用3-24h，各时间点均能抑制As_2O_3对Survivin表达的上调。不同浓度的Ly294002作用12h，呈浓度依赖性抑制As_2O_3对Survivin表达的上调，而对细胞内在的Survivin表达没有影响。且能在24h时增强Bcl-2蛋白磷酸化。25uM Ly294002在24、48h均能增强5uM As_2O_3诱导MGC803细胞凋亡及G_2／M阻滞，As_2O_3单独及联合应用Ly294002 24h的亚二倍体细胞数为8.2％、14.8％，G_2／M期细胞分别为32.38％、56.4％，48h亚二倍体细胞数为15.9％、26.7％，G_2／M期细胞分别为26.72％、42.5％。
讨论
凋亡是机体为维持内环境稳定由基因控制的细胞自主的程序性死亡，与组织器官的发育、机体正常生理活动的维持、衰老及疾病的发生等过程密切相关，对于维持多细胞机体的完整性和自身稳态具有重要意义。线粒体在细胞凋亡的过程中起着重要作用。As_2O_3对早幼粒细胞白血病具有较好的疗效，同时对多种白血病及实体瘤细胞均有杀伤作用，主要机制为诱导肿瘤细胞凋亡。近年来研究表明，不同的凋亡刺激通过活性氧的产生及线粒体跨膜电位的下降引起线粒体膜通透性的改变，导致细胞色素C的释放及Caspase的活化，而引起凋亡的因素如神经酰胺、肿瘤坏死因子、紫外线、蒽环类抗肿瘤药等被证实通过活性氧产生引起凋亡，As_2O_3作为新的凋亡诱导剂主要通过活性氧的产生，尤其是过氧化氢(H2O2)，诱导肿瘤细胞凋亡，也有文献报道肿瘤细胞对As_2O_3的敏感性与细胞内在固有活性氧水平有关。Bcl-2蛋白家族在线粒体凋亡途径中发挥重要作用，As_2O_3通过下调Bcl-2蛋白表达、诱导自身或相互间形成二聚体、Bcl-2磷酸化、蛋白水解及线粒体膜转位等方式，参与PTP的形成、开放及MPT的调节，研究发现Bcl-2蛋白能够抑制脂质过氧化引起的细胞凋亡，Bcl-2蛋白参与As_2O_3对肿瘤细胞的杀伤。As_2O_3对肿瘤细胞的杀伤与活性氧、线粒体跨膜电位及与凋亡蛋白之间的关系仍有争议，本研究结果显示As_2O_3对三种胃癌细胞系均有杀伤作用，但对As_2O_3的敏感性不同，在细胞内在活性氧上反映不出敏感性差异，而与As_2O_3作用后活性氧的升高水平有关，结果显示，应用NAC能够明显抑制As_2O_3对肿瘤细胞的杀伤作用，NAC能够抑制细胞内活性氧的产生及As_2O_3诱导胃癌MGC803细胞的G_2／M期阻滞和凋亡，由此可见As_2O_3诱导胃癌MGC803细胞的G_2／M期阻滞和凋亡需要活性氧的参与。但在5μM As_2O_3作用6-36h的不同时间点，结果显示活性氧在贴壁与非贴壁细胞中无明显差别。As_2O_3诱导胃癌MGC803细胞的凋亡需要活性氧的产生，活性氧的产生对于低浓度As_2O_3诱导胃癌MGC803细胞凋亡是必需但不是充分条件，还有另外机制参与。25μM的As_2O_3作用后主要以细胞膜完整性破坏为表现的细胞坏死为主。我们的结果显示，高浓度As_2O_3引起细胞坏死为主，不引起细胞周期阻滞及凋亡，低浓度As_2O_3引起细胞周期阻滞及细胞凋亡为主，同时伴有少数细胞坏死。BSO通过不依赖Caspase-3的细胞坏死途径来增强As_2O_3对MGC803细胞的杀伤。As_2O_3诱导MGC803细胞G_2／M期阻滞和凋亡过程中出现Bcl-2磷酸化而未见下调，通过对Bcl-2磷酸化的调节证明Bcl-2磷酸化促进As_2O_3诱导MGC803凋亡。Survivin表达的上调作为肿瘤细胞通过G_2／M检测点的促进因素，使细胞对As_2O_3诱导的凋亡产生抵抗，使肿瘤细胞耐药，Ly294002抑制As_2O_3对胃癌细胞Survivin表达的上调。
As_2O_3对肿瘤细胞的杀伤需要活性氧的参与，但单纯的活性氧增高不能引起细胞凋亡，还需要其他因素共同参与，低浓度As_2O_3选择性诱导胃癌MGC803细胞G_2／M期阻滞及凋亡与Bcl-2蛋白磷酸化丧失其抗凋亡作用有关。Survivin表达的上调为肿瘤细胞获得性耐药增加对化疗的抵抗，下调survivin蛋白的表达增加As_2O_3的敏感性，提示survivin基因有望成为肿瘤治疗的理想靶标。
结论
As_2O_3对胃癌细胞的杀伤需要活性氧的参与，肿瘤细胞对As_2O_3的敏感性与As_2O_3作用后活性氧的升高相关。线粒体途径是As_2O_3诱导凋亡与坏死的共同通路，BSO增强低浓度As_2O_3对胃癌细胞的杀伤是通过改As_2O_3对胃癌细胞的杀伤模式，由凋亡转变为坏死来完成。G_2／M期肿瘤细胞对As_2O_3更敏感可能与G_2／M期细胞Bcl-2蛋白磷酸化，使其丧失其抗凋亡作用有关。As_2O_3作用后Survivin表达的上调为肿瘤细胞获得性耐药，Survivin表达的上调抵抗As_2O_3诱导的肿瘤细胞凋亡，下调survivin蛋白的表达增加As_2O_3的敏感性。
Apoptosis is a cell-autonomous programmed cell death mechanism that is utilized extensively during development, tissue homeostasis and maintenance of the immune system in adult organisms. These are two pathways for apoptosis are commonly referred to as the intrinsic and extrinsic pathways. Multiple signals converge on mitochondria, including DNA damage, microtubule disruption, and growth-factor deprivation. The intrinsic pathway centers on mitochondria as initiators of cell death seem to be very important for anticancer drugs.
Bc1-2 family proteins are the most prominent of the intrinsic pathway in regulating cell apoptosis, Mitochondria is central to the apoptosis activities pathway in many physiological and pathological conditions. The mitochondrial inner transmembrane potential (△(?)m) collapses in apoptosis, indicating the opening of a large conductance channel known as the mitochondrial PT pore (PTP). Apoptosis stimuli cause these organelles to release cytochrome c (cyt c) and other apoptotic proteins into cytosol. In the cytosol, cyt-e binds the caspase-activating protein, apoptotic protease-activating factorl (Apaf 1), triggering its oligomerization into a hepatmeric complex that binds procaspase-9, forming a multiprotein structure known as the "apoptosome." Physical binding of Apafl to procaspase-9 is mediated by their caspase recruitment domains (CARDs), through homotypic CARD-CARD binding. Activation of apoptosome associated cell death protease caspase-9 then initiates a proteolytic cascade, where activated caspase-9 cleaves and activates downstream effectors proteases, such as procaspase-3. The Bc1-2 family of pro- and antiapoptotic proteins constitutes a critical control point for apoptosis, and the family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, involving dimerization, phosphorylation, proteolytic cleavage and mitochondrial translocation. The mitochondrial inner transmembrane potential (△(?)m) collapses in apoptosis, indicating the opening of a large conductance channel known as the mitochondrial PT pore (PTP). But the mechanisms in anti-apoptosis are conversal.
The intrinsic and extrinsic pathways for apoptosis converge on downstream effectors caspases. Certain effectors caspases are targets of suppression by an endogenous family of anti apoptotic proteins called inhibitor of apoptosis proteins (IAPs). The human genome encodes 8 lAP family members Survivin, X-linked inhibitor of apoptosis (X-IAP) and so on. Pathologic over expression of lAPs has been documented in cancer and leukemia. The functional importance of IAPs for apoptosis suppression in cancers has been documented by antisense experiments, in which knocking-down expression of Survivin, X-IAP or others induced apoptosis of tumor cell lines in culture or sensitized to apoptosis induced by anticancer drugs.
Gastric cancer is one of the most common cancers in China. Conventional chemotherapy to most patients with advanced stage is usually not satisfactory. Therefore, discovery of new drugs for the treatment of gastric cancer is urgent.
As_2O_3 is an active ingredient of a traditional Chinese medicine (TCM) has shown substantial efficacy in treating both newly diagnosed and relapsed patients with acute promyelocytic leukemia (APL) with few adverse effects and only minimal myelosuppression As_2O_3 has been shown to be an effective inducer of apoptosis in some solid cancer cells, such as gastric cancer, esophageal, prostate and ovarian carcinoma, neuroblastoma, hepatocarcinoma as well as head and neck cancers. Reports showed that As_2O_3 was also effective in inhibiting the growth of these cancers and which has been shown to be an effective inducer of apoptosis in some solid cancer cells. But the mechanism by which arsenic kills cancer cells remains unclear. Some reports have shown that arsenic-induced apoptosis is caused by a direct effect on the mitochondrial permeability transition pore, resulting in loss of the mitochondrial transmembrane potential. Other studies have demonstrated that arsenic compounds can disrupt mitosis, ROS, Bc1-2 family proteins, inhibitor of apoptosis proteins (lAPs) and therefore induce apoptosis in a variety of cell systems Because arsenic affects so many cellular and physiological pathways, a wide variety of malignancies, including both hematologic cancer and solid tumors derived from several tissue types, may be susceptible to therapy with arsenic trioxide. These multiple actions of arsenic trioxide also highlight the need for additional mechanistic studies to determine which actions mediate the diverse biological effects of this agent.
In the present study, we will investigate the mechanisms of As_2O_3, mainly the role of Bc1-2, ROS and Survivin in As_2O_3 induced in human gastric cancer cell death.
Materials and Methods
1. Growth inhibition assay in vitro growth inhibition effect of As_2O_3 on gastric cancer cells was determined by MTT assay.
2. Cell morphology and mitotic analysis ware performed by cytospin preparation with Wright-Giemsa stain. The slides were viewed and photographed under a light microscope.
3. Flow cytometric analysis of mitochondrial transmembrane potential (△(?)m), membrane integrity, ROS, and Cell cycle.
4. Expression of Bcl-2、Bax、Survivin、Smac/DIABLO and Caspase-3 were analyzed by Western blot.
5. Statistical Analysis. All assays were set up in triplicate experiments, the results were showed as the mean±SD. Statistical analysis was determined by Student's t test.
Results
1. As_2O_3 inhibited human gastric cancer cells growth through different mechanisms. The MGC803, BCG823, SGC7901 gastric cancer cell lines exhibited susceptibility to As_2O_3 and showed in time and dose dependent manner, The IC_(50) value for As_2O_3 in MGC803, BCG823, SGC7901 gastric cancer cells was about 2.8,3.1,10.2uM. MGC803, BCG823 was more susceptibility to As203 than SGC7901, the susceptibility was associated with the production of ROS but not with the internal ROS level. After treatment with SuM for 24h, the peak level of ROS was 100.8、103.3、56.5 respectively. The ROS in SGC7901 did not increase. Low concentrations of As_2O_3 can induce ROS production and reach peak level at 12h, and which also induce lose MPT, after treatment with 2, 5, 25uMAs_2O_3 for12-24h, the cells with lower MPT were 8.2%, 16.1%, 23% and in time and dose dependent manner.
2. NAC can relieve the inhibition of growth induced by As203 and in combined with NAC the IC50 (48h) is more than 25uM, six times than As_2O_3 alone. NAC can decrease the ROS level induced by As_2O_3, the apoptosis and cell cycle was recovered. The ROS level was decreased to 7.2. The cell cycle was similar to the control.
3.5μM As_2O_3 can induced typical apoptosis appearance, mitosis arrest and apoptosis body. But 25μM As_2O_3 did not induced cell cycle arrest and no apoptosis body, cells show swelling in cytosol. All different concentration As203 released Cyto-C from mitochondrial to Cytosol. Treatment with 5μM As_2O_3 24h induced apoptotic cell froml.2% to 32.8%, necrotic cells reached to 12%. But treatment with 25μM As_2O_3 for 24h induced necrotic cells from 5.9% to72.2%.
4. After treatment with 5uM As_2O_3 for 24h, the apoptotic cells increase to 20.37% and necrosis cells increase from 5.7%to 14.36%. 5uM As203 combined with 10uM BSO can switch cell from apoptosis to necrosis and the necrotic cell was about 46.11%. As203 combined with BSO killed MGC803 cells through caspase-3 independent manner, 12KD active subunit ofprcaspase-3 disappear and 17KD active subunit was in a half.
5.5μM As_2O_3 can induce Bc1-2 protein phosphorylation from 12 and reached peak level at 36h and decreased after 48h, treatment with 5μM As_2O_3 results in Caspase-3 active and be cleaved into small parts, but treatment with 25μM As_2O_3 doesn't induce Bc1-2 protein phosphorylation and Pro-Caspase-3 was actived. The level of Pro-Caspase-3 decrease.
6. After treatment with 5μM As_2O_3 for 6-36h, we separate the attaching and floating cell by lightly shaking the culture bottles, the floating cells show the decrease of△(?)m and which was not seen in the attaching cells, the floating cells display Bc1-2 phosphorylation, up regulation of survivin and caspase-3 was actived. The attaching cells show nothing, after washed with culture media 3 times, the floating and attaching cells were continue cultured for future 12 and 24h,the floating cells show that most of them appear apoptosis, Bcl-2 protein de-phosphorylation and the expression of survivin protein decrease, the attaching cell display small part apoptosis followed the decrease of the cells in G_2 phase, but Bc1-2 phosphorylation, up regulation of survivin and active caspase-3 was not induced. The level of Bcl-2, survivin and caspase-3 protein show no change.
7.500nM Okadaic acid can keep Bcl-2 protein phosphorylation than the control in the floating cells for 4h. 100nM Staurosporine can promote Bcl-2 protein de-phosphorylation than the control in the floating cells for 4h.the cells treatment with 500nM Okadaic acid showed more apoptosis than the cells treatment with Staurosporine, the apoptotic cells in treatment with Okadaic acid 2 and 4h was 19.51%, 40.8% and in that with Staurosporine was 10.61%, 11.2% respectively for 2 and 4h.
8.5uM As_2O_3 can increase the expression of Survivin protein from12h and reach the peak after treatment with As203 36h ,the expression of Survivin protein is 2.5 times higher than that of the control and 1.4 times in 48h, 25 uM As203 decrease the expression of surviving, Ly294002, an inhibiter of PI3 Kinase inhibiter can inhibit the up-regulation of survivin induced by As_2O_3 which enhanced the apoptosis and G_2/M phase arrest, he sub G0/GI phase cells was increased from 8.2% to 14.8% in 24h and from 15.9%to 26.7% in 48h, the cells in G_2/M phase were 32.38% to 56.4% in 24h and from 26.72% to 42.5% in 48h.
Discussion
Cell death can occur by either of two distinct mechanisms, apoptosis or necrosis. Apoptosis is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Necrosis is the pathological process which occurs when cells are exposed to a serious physical or chemical insult. Necrotic cell death is often associated with extensive tissue damage resulting in an intense inflammatory response. In the last few years, interest in apoptosis has increased greatly. Great progress has been made in the understanding of the basic mechanisms of apoptosis and the gene products involved. This genetic program is vital for normal development, for maintenance of tissue homeostasis, and for an effective immune system. Not surprisingly therefore, its disturbance is implicated in numerous pathological conditions, ranging from degenerative disorders to autoimmunity and cancer. Mitochondria are pivotal in controlling cell life and death. Reduced mitochondrial membrane potential (△(?)m) is considered as an initial and irreversible step towards apoptosis. The PTP is a multiprotein complex spanning the inner and outer mitochondrial membranes whose activation results in dissipation of the mitochondrial inner membrane potential, and eventual disruption of the outer mitochondrial membrane. In most cases, Bcl-2 family proteins are postulated to play a central role by controlling the permeabilization of mitochondrial membranes, either as pore forming sub-units or as regulators of intrinsic mitochondrial channels. Anti-apoptotic members of Bcl-2 family (e.g. Bcl-2 and Bcl-xL) act primarily to preserve mitochondrial integrity by suppressing the release of cytochrome c and Smac/DIABLO. Mitochondrial efflux of Cyt-C, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bc1-2 over expressing cells. Thus, in addition to modulating apoptosis- associated mitochondrial cytochrome c release.
Arsentie has potential as an effective chemotherapeutic agent in treatment of a variety of cancers, but arsenties's chemotherapeutic mechanism is poorly understood. In many countries, As203 is approved for the treatment of APL, which is most often characterized by the presence of the oncogenic fusion protein PML-RARa. it is thought that arsentie exerts its proapoptotic and differentiate effects on these cells by inducing PML-RARo degradation and downregulation Bcl-2 protein. However, degradation of the oncoprotein is not the sole mechanism of arsenite action, because arsenite does induce apoptosis in cell lines that lack the fusion protein. Therefore, arsenite may be useful in the treatment of cancers other than APL. Recently, studies were carried out in the treatment of solid tumors. Now, the molecular mechanism of action by which As203 induces cell death remains poorly understood. In terms of a mechanism for the effect of on APL cells, it has been suggested based on experiments, with NB4 cells of APL that arsenic caused apoptosis directly through down-regulation of Bcl-2, some investigator reported that Bcl-2 was not down-regulated in arsenic trioxide-treated HL-60 cells. Our results showed that low concentration 5μM As_2O_3 induced Bcl-2 phosphorylation from 12 to 48h and reached peak level at 36h and the level of Bc1-2 protein did not change, procaspase-3 was cleavage into small molecular parts from 24h, high concentration 25μM As_2O_3 did not induced Bcl-2 protein phosphorylation and activated Caspase 3, mainly increased Rh123~+ PI~+ necrotic cells. As_2O_3 can induce ROS production and associated with the sensitivity to As_2O_3. BSO can switch cell from apoptosis to necrosis. AS_2O_3 combined with BSO killed MGC803 cells through caspase-3 independent manner, Okadaic acid can keep Bcl-2 protein phosphorylation, cells are more susceptible to a death signal during G2/M and that characteristic can be attributed to phosphorylation of Bcl-2.Staurosporine can promote Bc1-2 protein de-phosphorylation and inhibit apoptosis. Ly294002, an inhibiter of PI3 Kinase inhibiter can inhibit the up-regulation of survivin induced by As203 enhanced the apoptosis and G2/M phase arrest, this result is consistent with us. Bc1-2 phosphorylation may lose it anti-apoptosis function and activated Caspase-3 results in MGC803 cells apoptosis.
Conclusion
In summary, the present results indicate that ROS production play an important role in As_2O_3 killed human gastric cancer cell death, As203 killed human gastric cancer cell death via apoptosis and necrosis, Both apoptosis and necrosis are share the same mitochondrial pathway. Cells are more susceptible to As203 during G2/M and that characteristic can be attributed to phosphorylation of Bcl-2, As_2O_3 can increase the expression of Survivin protein may confer drug resistance through promoting cells through the mitosis. Inhibition the up-regulation of the Survivin expression sensitized gastric cancer cells to apoptosis induced by As203, so survivin may be a target for cancer therapy.
凋亡调控失衡与肿瘤的发生和发展密切相关，诱导肿瘤细胞凋亡已成为肿瘤药物治疗的主要目的与手段。凋亡有两条途径，一条为线粒体途径，另一条为死亡受体途径，多种抗肿瘤药物、射线、活性氧等均通过线粒体途径引起细胞凋亡，在深入研究凋亡调控机制与信号转导途径的过程中，线粒体在细胞凋亡中的作用日益受到人们的关注。
研究表明，过表达Bcl-2能够抑制细胞的凋亡而促进细胞生长。Bcl-2家族是哺乳动物细胞中调节凋亡的重要因子，通过调节线粒体或内质网等细胞内膜结构的通透性，线粒体的通透性改变(permeability transition，PT)是调节凋亡的中心环节，改变与细胞生理功能密切相关的离子或蛋白质因子在细胞内的分布，从而影响细胞的生命活动过程。在凋亡的线粒体途径中，各种凋亡促发因素通过调节线粒体外膜的通透性，释放线粒体膜间隙的促凋亡蛋白质因子如细胞色素C(cytochrome-C)和Smac／DIABLO等进入细胞浆，通过依赖caspase的途径激活细胞凋亡或者释放凋亡诱导因子(apoptosis inducing factor，IF)等通过不依赖caspase的途径引起细胞凋亡。Bcl-2家族中抑制细胞凋亡与促进细胞凋亡蛋白之间的平衡，对其在改变线粒体通透性、调节线粒体蛋白释放起着决定作用，但具体机制还存在许多争论。
对细胞凋亡过程起调节作用的除了非常熟悉的Bcl-2家族外，尚有另外一个家族就是细胞凋亡抑制蛋白(IAPs)家族，凋亡蛋白抑制子(IAP)家族中生存素(Survivin)、X联锁凋亡蛋白抑制子(XIAP)过表达，抑制促凋亡因子对caspase激活及对细胞增殖的影响导致肿瘤细胞对凋亡刺激的抵抗。凋亡与坏死是在形态学上不同的两种细胞死亡形式，凋亡细胞核碎裂形成凋亡小体，细胞膜完整。坏死特点是细胞肿胀，细胞内细胞器裂解，细胞膜不完整。凋亡细胞被巨噬细胞吞噬，坏死细胞则释放细胞内容物引起炎症及组织损害。细胞凋亡的机制研究的非常广泛，而细胞坏死研究较少。
砷剂的抗肿瘤机制主要通过线粒体途径引起细胞凋亡来实现，同时也有引起细胞坏死及自溶，砷剂与巯基结合后，导致MPT开放，线粒体跨膜电位(△ψm)下降或消失，继之呼吸链脱偶联，谷胱甘肽耗竭，活性氧类(reactive oxygen species，ROS)产生及对诱导凋亡非常重要的蛋白酶活化物的释放，包括细胞色素C(Cyto-C)和凋亡诱导因子(AIF)，Cyto-C和AIF均可激活caspase诱导凋亡。Bcl-2的过度表达可抑制砷剂诱导的凋亡相关现象，而通过反义技术使Bcl-2下调可抑制细胞增殖及促进凋亡。砷剂可通过对Bcl-2基因功能的抑制诱导某些肿瘤细胞的凋亡，三氧化二砷(As_2O_3)对肿瘤细胞的杀伤过程中在不同细胞背景下，对Bcl-2、活性氧及凋亡抑制蛋白家族的影响不同。As_2O_3对胃癌细胞的杀伤机制研究很少，具体机制仍不清。本研究以胃癌细胞系MGC803、BCG823、SGC7901为模型，研究As_2O_3对胃癌细胞杀伤过程中线粒体途径相关的活性氧、Bcl-2家族及凋亡抑制蛋白家族中的Survivin在此过程中的作用及在凋亡与坏死两种不同杀伤机制中的差异，进一步明确As_2O_3对胃癌细胞杀伤机制，为在临床中更好应用奠定基础。
材料与方法
1．采用MTT法测定细胞活力，绘制增殖曲线。
2．采用瑞氏-姬姆萨染色，光学显微镜下观察As_2O_3诱导MGC803细胞死亡时的形态学变化。
3．采用流式细胞仪通过PI染色进行细胞周期解析及凋亡判定。
4．采用western blot检测Bcl-2、Survivin及caspase-3蛋白的表达。
5．采用DiOC 6(3)单染法检测线粒体跨膜电位(△ψm)的变化。采用罗丹明和碘化丙啶双染法检测线粒体跨膜电位(△ψm)改变与细胞存活状态的关系。应用流式细胞仪通过2′，7′-二氯乙酰乙酸盐荧光素标记检测细胞内活性氧水平。
6．统计学处理所有数据用3次独立实验的平均值表示，采用学生t检验。
实验结果
1．As_2O_3呈时间、浓度依赖性抑制胃癌MGC803、BCG823、SGC7901细胞的增殖，72h抑制细胞增殖50％的药物浓度(IC_(50))分别为2.8、3.1、10.2uM，MGC803、BCG823、SGC7901细胞的活性氧水平在未用药时活性氧峰值水平分别为20、64.3、57.6，5uM As_2O_3作用24h后三种细胞系活性氧峰值水平分别为100.8、103.3、56.5，在应用5uM的As_2O_3作用24h后，MGC803、BCG823细胞的活性氧水平明显增高，而SGC7901细胞只有少数细胞活性氧增高。2uM及5uM的As_2O_3均引起细胞内的活性氧明显升高，峰荧光强度分别由对照的20升高到为80、70，而25uM的As_2O_3没有引起明显的升高。5uM的As_2O_3作用36h，细胞内活性氧3h开始增高，12h达到高峰值。2、5、25uM As_2O_3作用12及24h后线粒体跨膜电位出现降低，24h的线粒体跨膜电位降低的细胞数分别为8.2％、16.1％、23％，能够呈时间及浓度依赖性降低线粒体跨膜电位。
2．氮乙酰半胱氨酸(NAC)能够明显抑制As_2O_3对MGC803细胞的杀伤作用，联合应用As_2O_3 48h的IC_(50)浓度大于25uM，单独应用时的IC_(50)约为5uM。10mM的NAC与5uM As_2O_3同时应用12h，NAC明显降低细胞内的活性氧水平，对照组为12.4，单独应用As_2O_3组为30.5，而联合应用组为7.2。48h流式细胞仪检测细胞周期，单独应用As_2O_3组亚G_0／G_1期细胞为29.32％，G_2／M期细胞为38.19％，出现了亚二倍体峰及G_2／M期阻滞，联合应用组未见亚二倍体峰及G_2／M期阻滞，细胞周期接近正常对照。
3.5μM As_2O_3作用24h细胞形态学可见细胞核浓聚，染色质非正常分裂及凋亡小体，有明显的M期阻滞。25、50μM As_2O_3作用24h细胞可见明显的核固缩，胞浆空泡变性，未见G_2／M期阻滞。5μM As_2O_3作用24h后细胞出现凋亡细胞由对照的1.2％增加到32.8％。坏死细胞增加到12％。而25μM的As_2O_3作用24h后坏死细胞由5.9％增加到72.2％。而凋亡细胞由对照的1.2％增加到8.1％，结果显示25μM的As_2O_3作用后主要以细胞坏死为主。2、5、25、50uM As_2O_3作用24h，各浓度As_2O_3均能引起线粒体Cyto-C向细胞浆中释放，线粒体内Cyto-C明显减少。
4.5uM的As_2O_3作用MGC803细胞24h，早期凋亡细胞数由1.44增至20.37，坏死细胞由5.7％增加到14.36％，而10uM的BSO与5uM的As_2O_3联合应用组，凋亡细胞由单用As_2O_3的20.37％降至7.44％，坏死细胞由14.36％增加到46.11％。结果提示BSO通过诱导肿瘤细胞坏死而不是凋亡来增加As_2O_3对MGC803细胞的杀伤作用。5uM的ms_2O_3作用24h可激活Procaspase-3后出现17及12KD的活性亚基，10uM的BSO与5uM的As_2O_3联合作用后，Procaspase-3只出现17KD的活性亚基，且表达水平为单用As_2O_3的50％。10mM NAC与BSO同时与5uM的As_2O_3共同作用24h，Caspase-3的表达与对照组没有区别。
5.5μM As_2O_3作用MGC803细胞12-48h均诱导Bcl-2蛋白出现磷酸化，12h可出现，36h Bcl-2蛋白磷酸化达到峰值，48h减弱，25μM的As_2O_3作用24h未见Bcl-2蛋白出现磷酸化，Bcl-2蛋白无明显变化。5μM As_2O_3作用MGC803细胞48h，从24h起可见Caspase-3剪切活化形式17、12 KD的小片段，一直持续到48h，Procaspase-3无明显变化。25μM As_2O_3作用MGC803细胞48h，从24h起下调Procaspase-3，48h接近消失，但未见到活化后形成的小片段。
6.5μM As_2O_3作用MGC803细胞6-36h，结果显示活性氧在贴壁与非贴壁细胞中无明显差别。6h后未贴壁细胞均出现线粒体跨膜电位(△ψm)降低，24h最明显，约55％的细胞线粒体跨膜电位(△ψm)明显降低。而贴壁细胞中未见细胞线粒体跨膜电位(△ψm)的下降。5μM As_2O_3作用24h后分离贴壁与非贴壁细胞，检测Bcl-2、Survivin及Caspase-3蛋白表达，24h非贴壁细胞Bcl-2蛋白出现磷酸化，Survivin表达明显增高，Caspase-3蛋白被活化，该部分细胞为凋亡及G_2／M期细胞，两部分细胞比例接近，而贴壁细胞为G_0／G_1、S及G_2期细胞，Bcl-2蛋白磷酸化水平低于As_2O_3作用24h的水平，Survivin表达接近对照细胞，Caspase-3蛋白未见明显活化。贴壁与非贴壁细胞去除药物后继续培养12、24h，贴壁细胞Bcl-2、Survivin、Caspase-3蛋白表达未见明显变化，非贴壁细胞Bcl-2蛋白磷酸化减弱，Survivin表达在继续培养12h时略降低，24h后明显降低，降至24h分离时的30％。7.500nM Okadaic acid及100nM Staurosporine与G_2／M期细胞作用4h，Okadaicacid能够保持Bcl-2蛋白磷酸化，Staurosporine能够使Bcl-2蛋白脱磷酸化。流式细胞仪检测结果Okadaic acid作用2h、4h时亚二倍体细胞分别为19.51％、40.8％，Staurosporine作用2h、4h时亚二倍体细胞分别为10.61％、11.2％，4h对照细胞的亚二倍体细胞数为15.27％。
8.5uM的As_2O_3从12h起明显上调Survivin蛋白表达，36h达到峰值，上调到对照的2.5倍，48h下降，蛋白表达水平为对照的1.4倍。25 uM的As_2O_3作用72h下调Survivin蛋白表达。Ly294002与As_2O_3共同作用3-24h，各时间点均能抑制As_2O_3对Survivin表达的上调。不同浓度的Ly294002作用12h，呈浓度依赖性抑制As_2O_3对Survivin表达的上调，而对细胞内在的Survivin表达没有影响。且能在24h时增强Bcl-2蛋白磷酸化。25uM Ly294002在24、48h均能增强5uM As_2O_3诱导MGC803细胞凋亡及G_2／M阻滞，As_2O_3单独及联合应用Ly294002 24h的亚二倍体细胞数为8.2％、14.8％，G_2／M期细胞分别为32.38％、56.4％，48h亚二倍体细胞数为15.9％、26.7％，G_2／M期细胞分别为26.72％、42.5％。
讨论
凋亡是机体为维持内环境稳定由基因控制的细胞自主的程序性死亡，与组织器官的发育、机体正常生理活动的维持、衰老及疾病的发生等过程密切相关，对于维持多细胞机体的完整性和自身稳态具有重要意义。线粒体在细胞凋亡的过程中起着重要作用。As_2O_3对早幼粒细胞白血病具有较好的疗效，同时对多种白血病及实体瘤细胞均有杀伤作用，主要机制为诱导肿瘤细胞凋亡。近年来研究表明，不同的凋亡刺激通过活性氧的产生及线粒体跨膜电位的下降引起线粒体膜通透性的改变，导致细胞色素C的释放及Caspase的活化，而引起凋亡的因素如神经酰胺、肿瘤坏死因子、紫外线、蒽环类抗肿瘤药等被证实通过活性氧产生引起凋亡，As_2O_3作为新的凋亡诱导剂主要通过活性氧的产生，尤其是过氧化氢(H2O2)，诱导肿瘤细胞凋亡，也有文献报道肿瘤细胞对As_2O_3的敏感性与细胞内在固有活性氧水平有关。Bcl-2蛋白家族在线粒体凋亡途径中发挥重要作用，As_2O_3通过下调Bcl-2蛋白表达、诱导自身或相互间形成二聚体、Bcl-2磷酸化、蛋白水解及线粒体膜转位等方式，参与PTP的形成、开放及MPT的调节，研究发现Bcl-2蛋白能够抑制脂质过氧化引起的细胞凋亡，Bcl-2蛋白参与As_2O_3对肿瘤细胞的杀伤。As_2O_3对肿瘤细胞的杀伤与活性氧、线粒体跨膜电位及与凋亡蛋白之间的关系仍有争议，本研究结果显示As_2O_3对三种胃癌细胞系均有杀伤作用，但对As_2O_3的敏感性不同，在细胞内在活性氧上反映不出敏感性差异，而与As_2O_3作用后活性氧的升高水平有关，结果显示，应用NAC能够明显抑制As_2O_3对肿瘤细胞的杀伤作用，NAC能够抑制细胞内活性氧的产生及As_2O_3诱导胃癌MGC803细胞的G_2／M期阻滞和凋亡，由此可见As_2O_3诱导胃癌MGC803细胞的G_2／M期阻滞和凋亡需要活性氧的参与。但在5μM As_2O_3作用6-36h的不同时间点，结果显示活性氧在贴壁与非贴壁细胞中无明显差别。As_2O_3诱导胃癌MGC803细胞的凋亡需要活性氧的产生，活性氧的产生对于低浓度As_2O_3诱导胃癌MGC803细胞凋亡是必需但不是充分条件，还有另外机制参与。25μM的As_2O_3作用后主要以细胞膜完整性破坏为表现的细胞坏死为主。我们的结果显示，高浓度As_2O_3引起细胞坏死为主，不引起细胞周期阻滞及凋亡，低浓度As_2O_3引起细胞周期阻滞及细胞凋亡为主，同时伴有少数细胞坏死。BSO通过不依赖Caspase-3的细胞坏死途径来增强As_2O_3对MGC803细胞的杀伤。As_2O_3诱导MGC803细胞G_2／M期阻滞和凋亡过程中出现Bcl-2磷酸化而未见下调，通过对Bcl-2磷酸化的调节证明Bcl-2磷酸化促进As_2O_3诱导MGC803凋亡。Survivin表达的上调作为肿瘤细胞通过G_2／M检测点的促进因素，使细胞对As_2O_3诱导的凋亡产生抵抗，使肿瘤细胞耐药，Ly294002抑制As_2O_3对胃癌细胞Survivin表达的上调。
As_2O_3对肿瘤细胞的杀伤需要活性氧的参与，但单纯的活性氧增高不能引起细胞凋亡，还需要其他因素共同参与，低浓度As_2O_3选择性诱导胃癌MGC803细胞G_2／M期阻滞及凋亡与Bcl-2蛋白磷酸化丧失其抗凋亡作用有关。Survivin表达的上调为肿瘤细胞获得性耐药增加对化疗的抵抗，下调survivin蛋白的表达增加As_2O_3的敏感性，提示survivin基因有望成为肿瘤治疗的理想靶标。
结论
As_2O_3对胃癌细胞的杀伤需要活性氧的参与，肿瘤细胞对As_2O_3的敏感性与As_2O_3作用后活性氧的升高相关。线粒体途径是As_2O_3诱导凋亡与坏死的共同通路，BSO增强低浓度As_2O_3对胃癌细胞的杀伤是通过改As_2O_3对胃癌细胞的杀伤模式，由凋亡转变为坏死来完成。G_2／M期肿瘤细胞对As_2O_3更敏感可能与G_2／M期细胞Bcl-2蛋白磷酸化，使其丧失其抗凋亡作用有关。As_2O_3作用后Survivin表达的上调为肿瘤细胞获得性耐药，Survivin表达的上调抵抗As_2O_3诱导的肿瘤细胞凋亡，下调survivin蛋白的表达增加As_2O_3的敏感性。
Apoptosis is a cell-autonomous programmed cell death mechanism that is utilized extensively during development, tissue homeostasis and maintenance of the immune system in adult organisms. These are two pathways for apoptosis are commonly referred to as the intrinsic and extrinsic pathways. Multiple signals converge on mitochondria, including DNA damage, microtubule disruption, and growth-factor deprivation. The intrinsic pathway centers on mitochondria as initiators of cell death seem to be very important for anticancer drugs.
Bc1-2 family proteins are the most prominent of the intrinsic pathway in regulating cell apoptosis, Mitochondria is central to the apoptosis activities pathway in many physiological and pathological conditions. The mitochondrial inner transmembrane potential (△(?)m) collapses in apoptosis, indicating the opening of a large conductance channel known as the mitochondrial PT pore (PTP). Apoptosis stimuli cause these organelles to release cytochrome c (cyt c) and other apoptotic proteins into cytosol. In the cytosol, cyt-e binds the caspase-activating protein, apoptotic protease-activating factorl (Apaf 1), triggering its oligomerization into a hepatmeric complex that binds procaspase-9, forming a multiprotein structure known as the "apoptosome." Physical binding of Apafl to procaspase-9 is mediated by their caspase recruitment domains (CARDs), through homotypic CARD-CARD binding. Activation of apoptosome associated cell death protease caspase-9 then initiates a proteolytic cascade, where activated caspase-9 cleaves and activates downstream effectors proteases, such as procaspase-3. The Bc1-2 family of pro- and antiapoptotic proteins constitutes a critical control point for apoptosis, and the family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, involving dimerization, phosphorylation, proteolytic cleavage and mitochondrial translocation. The mitochondrial inner transmembrane potential (△(?)m) collapses in apoptosis, indicating the opening of a large conductance channel known as the mitochondrial PT pore (PTP). But the mechanisms in anti-apoptosis are conversal.
The intrinsic and extrinsic pathways for apoptosis converge on downstream effectors caspases. Certain effectors caspases are targets of suppression by an endogenous family of anti apoptotic proteins called inhibitor of apoptosis proteins (IAPs). The human genome encodes 8 lAP family members Survivin, X-linked inhibitor of apoptosis (X-IAP) and so on. Pathologic over expression of lAPs has been documented in cancer and leukemia. The functional importance of IAPs for apoptosis suppression in cancers has been documented by antisense experiments, in which knocking-down expression of Survivin, X-IAP or others induced apoptosis of tumor cell lines in culture or sensitized to apoptosis induced by anticancer drugs.
Gastric cancer is one of the most common cancers in China. Conventional chemotherapy to most patients with advanced stage is usually not satisfactory. Therefore, discovery of new drugs for the treatment of gastric cancer is urgent.
As_2O_3 is an active ingredient of a traditional Chinese medicine (TCM) has shown substantial efficacy in treating both newly diagnosed and relapsed patients with acute promyelocytic leukemia (APL) with few adverse effects and only minimal myelosuppression As_2O_3 has been shown to be an effective inducer of apoptosis in some solid cancer cells, such as gastric cancer, esophageal, prostate and ovarian carcinoma, neuroblastoma, hepatocarcinoma as well as head and neck cancers. Reports showed that As_2O_3 was also effective in inhibiting the growth of these cancers and which has been shown to be an effective inducer of apoptosis in some solid cancer cells. But the mechanism by which arsenic kills cancer cells remains unclear. Some reports have shown that arsenic-induced apoptosis is caused by a direct effect on the mitochondrial permeability transition pore, resulting in loss of the mitochondrial transmembrane potential. Other studies have demonstrated that arsenic compounds can disrupt mitosis, ROS, Bc1-2 family proteins, inhibitor of apoptosis proteins (lAPs) and therefore induce apoptosis in a variety of cell systems Because arsenic affects so many cellular and physiological pathways, a wide variety of malignancies, including both hematologic cancer and solid tumors derived from several tissue types, may be susceptible to therapy with arsenic trioxide. These multiple actions of arsenic trioxide also highlight the need for additional mechanistic studies to determine which actions mediate the diverse biological effects of this agent.
In the present study, we will investigate the mechanisms of As_2O_3, mainly the role of Bc1-2, ROS and Survivin in As_2O_3 induced in human gastric cancer cell death.
Materials and Methods
1. Growth inhibition assay in vitro growth inhibition effect of As_2O_3 on gastric cancer cells was determined by MTT assay.
2. Cell morphology and mitotic analysis ware performed by cytospin preparation with Wright-Giemsa stain. The slides were viewed and photographed under a light microscope.
3. Flow cytometric analysis of mitochondrial transmembrane potential (△(?)m), membrane integrity, ROS, and Cell cycle.
4. Expression of Bcl-2、Bax、Survivin、Smac/DIABLO and Caspase-3 were analyzed by Western blot.
5. Statistical Analysis. All assays were set up in triplicate experiments, the results were showed as the mean±SD. Statistical analysis was determined by Student's t test.
Results
1. As_2O_3 inhibited human gastric cancer cells growth through different mechanisms. The MGC803, BCG823, SGC7901 gastric cancer cell lines exhibited susceptibility to As_2O_3 and showed in time and dose dependent manner, The IC_(50) value for As_2O_3 in MGC803, BCG823, SGC7901 gastric cancer cells was about 2.8,3.1,10.2uM. MGC803, BCG823 was more susceptibility to As203 than SGC7901, the susceptibility was associated with the production of ROS but not with the internal ROS level. After treatment with SuM for 24h, the peak level of ROS was 100.8、103.3、56.5 respectively. The ROS in SGC7901 did not increase. Low concentrations of As_2O_3 can induce ROS production and reach peak level at 12h, and which also induce lose MPT, after treatment with 2, 5, 25uMAs_2O_3 for12-24h, the cells with lower MPT were 8.2%, 16.1%, 23% and in time and dose dependent manner.
2. NAC can relieve the inhibition of growth induced by As203 and in combined with NAC the IC50 (48h) is more than 25uM, six times than As_2O_3 alone. NAC can decrease the ROS level induced by As_2O_3, the apoptosis and cell cycle was recovered. The ROS level was decreased to 7.2. The cell cycle was similar to the control.
3.5μM As_2O_3 can induced typical apoptosis appearance, mitosis arrest and apoptosis body. But 25μM As_2O_3 did not induced cell cycle arrest and no apoptosis body, cells show swelling in cytosol. All different concentration As203 released Cyto-C from mitochondrial to Cytosol. Treatment with 5μM As_2O_3 24h induced apoptotic cell froml.2% to 32.8%, necrotic cells reached to 12%. But treatment with 25μM As_2O_3 for 24h induced necrotic cells from 5.9% to72.2%.
4. After treatment with 5uM As_2O_3 for 24h, the apoptotic cells increase to 20.37% and necrosis cells increase from 5.7%to 14.36%. 5uM As203 combined with 10uM BSO can switch cell from apoptosis to necrosis and the necrotic cell was about 46.11%. As203 combined with BSO killed MGC803 cells through caspase-3 independent manner, 12KD active subunit ofprcaspase-3 disappear and 17KD active subunit was in a half.
5.5μM As_2O_3 can induce Bc1-2 protein phosphorylation from 12 and reached peak level at 36h and decreased after 48h, treatment with 5μM As_2O_3 results in Caspase-3 active and be cleaved into small parts, but treatment with 25μM As_2O_3 doesn't induce Bc1-2 protein phosphorylation and Pro-Caspase-3 was actived. The level of Pro-Caspase-3 decrease.
6. After treatment with 5μM As_2O_3 for 6-36h, we separate the attaching and floating cell by lightly shaking the culture bottles, the floating cells show the decrease of△(?)m and which was not seen in the attaching cells, the floating cells display Bc1-2 phosphorylation, up regulation of survivin and caspase-3 was actived. The attaching cells show nothing, after washed with culture media 3 times, the floating and attaching cells were continue cultured for future 12 and 24h,the floating cells show that most of them appear apoptosis, Bcl-2 protein de-phosphorylation and the expression of survivin protein decrease, the attaching cell display small part apoptosis followed the decrease of the cells in G_2 phase, but Bc1-2 phosphorylation, up regulation of survivin and active caspase-3 was not induced. The level of Bcl-2, survivin and caspase-3 protein show no change.
7.500nM Okadaic acid can keep Bcl-2 protein phosphorylation than the control in the floating cells for 4h. 100nM Staurosporine can promote Bcl-2 protein de-phosphorylation than the control in the floating cells for 4h.the cells treatment with 500nM Okadaic acid showed more apoptosis than the cells treatment with Staurosporine, the apoptotic cells in treatment with Okadaic acid 2 and 4h was 19.51%, 40.8% and in that with Staurosporine was 10.61%, 11.2% respectively for 2 and 4h.
8.5uM As_2O_3 can increase the expression of Survivin protein from12h and reach the peak after treatment with As203 36h ,the expression of Survivin protein is 2.5 times higher than that of the control and 1.4 times in 48h, 25 uM As203 decrease the expression of surviving, Ly294002, an inhibiter of PI3 Kinase inhibiter can inhibit the up-regulation of survivin induced by As_2O_3 which enhanced the apoptosis and G_2/M phase arrest, he sub G0/GI phase cells was increased from 8.2% to 14.8% in 24h and from 15.9%to 26.7% in 48h, the cells in G_2/M phase were 32.38% to 56.4% in 24h and from 26.72% to 42.5% in 48h.
Discussion
Cell death can occur by either of two distinct mechanisms, apoptosis or necrosis. Apoptosis is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Necrosis is the pathological process which occurs when cells are exposed to a serious physical or chemical insult. Necrotic cell death is often associated with extensive tissue damage resulting in an intense inflammatory response. In the last few years, interest in apoptosis has increased greatly. Great progress has been made in the understanding of the basic mechanisms of apoptosis and the gene products involved. This genetic program is vital for normal development, for maintenance of tissue homeostasis, and for an effective immune system. Not surprisingly therefore, its disturbance is implicated in numerous pathological conditions, ranging from degenerative disorders to autoimmunity and cancer. Mitochondria are pivotal in controlling cell life and death. Reduced mitochondrial membrane potential (△(?)m) is considered as an initial and irreversible step towards apoptosis. The PTP is a multiprotein complex spanning the inner and outer mitochondrial membranes whose activation results in dissipation of the mitochondrial inner membrane potential, and eventual disruption of the outer mitochondrial membrane. In most cases, Bcl-2 family proteins are postulated to play a central role by controlling the permeabilization of mitochondrial membranes, either as pore forming sub-units or as regulators of intrinsic mitochondrial channels. Anti-apoptotic members of Bcl-2 family (e.g. Bcl-2 and Bcl-xL) act primarily to preserve mitochondrial integrity by suppressing the release of cytochrome c and Smac/DIABLO. Mitochondrial efflux of Cyt-C, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bc1-2 over expressing cells. Thus, in addition to modulating apoptosis- associated mitochondrial cytochrome c release.
Arsentie has potential as an effective chemotherapeutic agent in treatment of a variety of cancers, but arsenties's chemotherapeutic mechanism is poorly understood. In many countries, As203 is approved for the treatment of APL, which is most often characterized by the presence of the oncogenic fusion protein PML-RARa. it is thought that arsentie exerts its proapoptotic and differentiate effects on these cells by inducing PML-RARo degradation and downregulation Bcl-2 protein. However, degradation of the oncoprotein is not the sole mechanism of arsenite action, because arsenite does induce apoptosis in cell lines that lack the fusion protein. Therefore, arsenite may be useful in the treatment of cancers other than APL. Recently, studies were carried out in the treatment of solid tumors. Now, the molecular mechanism of action by which As203 induces cell death remains poorly understood. In terms of a mechanism for the effect of on APL cells, it has been suggested based on experiments, with NB4 cells of APL that arsenic caused apoptosis directly through down-regulation of Bcl-2, some investigator reported that Bcl-2 was not down-regulated in arsenic trioxide-treated HL-60 cells. Our results showed that low concentration 5μM As_2O_3 induced Bcl-2 phosphorylation from 12 to 48h and reached peak level at 36h and the level of Bc1-2 protein did not change, procaspase-3 was cleavage into small molecular parts from 24h, high concentration 25μM As_2O_3 did not induced Bcl-2 protein phosphorylation and activated Caspase 3, mainly increased Rh123~+ PI~+ necrotic cells. As_2O_3 can induce ROS production and associated with the sensitivity to As_2O_3. BSO can switch cell from apoptosis to necrosis. AS_2O_3 combined with BSO killed MGC803 cells through caspase-3 independent manner, Okadaic acid can keep Bcl-2 protein phosphorylation, cells are more susceptible to a death signal during G2/M and that characteristic can be attributed to phosphorylation of Bcl-2.Staurosporine can promote Bc1-2 protein de-phosphorylation and inhibit apoptosis. Ly294002, an inhibiter of PI3 Kinase inhibiter can inhibit the up-regulation of survivin induced by As203 enhanced the apoptosis and G2/M phase arrest, this result is consistent with us. Bc1-2 phosphorylation may lose it anti-apoptosis function and activated Caspase-3 results in MGC803 cells apoptosis.
Conclusion
In summary, the present results indicate that ROS production play an important role in As_2O_3 killed human gastric cancer cell death, As203 killed human gastric cancer cell death via apoptosis and necrosis, Both apoptosis and necrosis are share the same mitochondrial pathway. Cells are more susceptible to As203 during G2/M and that characteristic can be attributed to phosphorylation of Bcl-2, As_2O_3 can increase the expression of Survivin protein may confer drug resistance through promoting cells through the mitosis. Inhibition the up-regulation of the Survivin expression sensitized gastric cancer cells to apoptosis induced by As203, so survivin may be a target for cancer therapy.
引文
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