过氧化氢通过细胞内超氧化物的产生诱导人TRAIL抗性黑素瘤中的细胞死亡 Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation

　

　

过氧化氢通过细胞内超氧化物的产生诱导人TRAIL抗性黑素瘤中的细胞死亡

Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation

抽象

细胞内活性氧（ROS）如过氧化氢（H2O2）被认为介导由死亡受体配体诱导的细胞凋亡，包括肿瘤坏死因子相关的凋亡诱导配体（TRAIL）。然而，H2O2的作用是有争议的，因为一些证据表明H2O2可作为抗细胞凋亡因子。在这里，我们显示外源施加的H2O2（30-100μM）通过细胞内超氧化物（O2-）生成诱导TRAIL抗性人黑素瘤细胞中的细胞凋亡或坏死。 H2O2诱导凋亡或坏死细胞死亡，取决于所用氧化剂的浓度;低浓度的H2O2优先激活caspase依赖的凋亡途径，而高浓度的H2O2以caspase非依赖性方式诱导凋亡和坏死细胞死亡。

H2O2诱导的细胞死亡与线粒体膜电位崩溃和胱天蛋白酶-3/7激活和ER应激反应增加有关，包括caspase-12和X-box结合蛋白-1（XBP-1）激活。 H2O2甚至在线粒体内诱导细胞内O2-生成，而TRAIL则没有。

超氧化物歧化酶模拟抗氧化剂MnTBaP [Mn（III）四（4-苯甲酸）卟啉氯化物]在相当的浓度下抑制H2O2诱导的O2-产生，凋亡和XBP-1和胱天蛋白酶-12活化。重要的是，H2O2处理在正常原代黑素细胞中引起最小的O2-产生和凋亡。

　

这些数据显示H2O2通过细胞内O2产生诱导内质网相关细胞死亡，并且恶性黑素瘤细胞比正常细胞更易产生氧化细胞死亡。研究结果表明H2O2具有治疗TRAIL耐药黑素瘤的治疗潜力。

　

简介

肿瘤坏死因子相关的凋亡诱导配体（TRAIL）是肿瘤坏死因子细胞因子超家族的成员并且具有同源三聚体结构。由于TRAIL在多种转化细胞和癌细胞中诱导细胞凋亡，但在正常细胞中不诱导凋亡，因此它是癌症预防和治疗的有希望的靶标。然而，越来越多的证据表明，某些癌细胞类型如恶性黑色素瘤，胶质瘤和非小细胞肺癌（NSCLC）细胞对TRAIL诱导的细胞凋亡具有抗性（1）。此外，TRAIL反应性肿瘤获得抗性表型，使得TRAIL治疗无效。克服癌细胞的TRAIL抗性对于有效的TRAIL治疗是必要的，并且迫切需要增强TRAIL有效性的药物（2）。 TRAIL与含有死亡域的受体（DR）结合，例如死亡受体（DR）4 / TRAIL-受体1（TRAIL-R1）和DR5 / TRAIL-R2（3）。这种结合诱导受体的寡聚化和死亡结构域的构象变化，导致形成诱导死亡的信号复合物;随后激活caspase-8。反过来，激活的caspase-8激活效应caspase-3/6/7，后者执行细胞凋亡过程（4,5）。胱天蛋白酶-8的活化也与内在（线粒体）凋亡途径有关。活化的caspase-8可以切割和激活促凋亡的Bcl-2家族分子Bid，后者又激活其他Bcl-2家族分子Bax和Bak，导致它们的寡聚化和线粒体外膜中巨大通道的形成。细胞色素c通过Bax / Bak巨细胞通道释放到胞质溶胶中诱导凋亡小体的组装和胱天蛋白酶-9的活化，导致caspase-3/6/7的活化（4）。最近，各种促细胞凋亡受体激动剂如重组人TRAIL和抗DR4 / DR5的激动剂抗体已经在多种癌细胞类型中进行临床试验，包括恶性黑素瘤和NSCLC细胞。不幸的是，结果显示这些受体激动剂令人失望地只是适度有效（6）。通过内在途径诱导细胞凋亡被认为是常规化学疗法的主要机制，因此是癌症治疗中的关键靶标。然而，临床观察表明，已知凋亡途径的扩增不足以克服癌细胞中的TRAIL抗性。

　

活性氧（ROS）如超氧化物（O2-），过氧化氢（H2O2）和羟基自由基（•OH）是几乎所有需氧生物中正常代谢的产物，也是在异生物暴露后产生的。低生理水平的ROS作为细胞内信号传导的第二信使，是正常细胞功能所必需的，而过量的ROS会对多种大分子造成损害，损害细胞功能，促进细胞死亡（7）。 ROS产生与生物系统解毒氧化剂或修复所导致的损伤的能力之间的不平衡导致氧化应激。几个证据表明细胞内H2O2是DR配体诱导的肿瘤细胞凋亡的介质。黄酮类黄芩素可杀死TNF-α抗性T细胞白血病细胞，并通过增加细胞内H2O2水平使其对TNF-α或TRAIL诱导的细胞凋亡敏感（8）。 LY303511使人神经母细胞瘤细胞对TRAIL敏感，细胞内H2O2的产生在这种作用中发挥作用（9）。另一方面，一些证据表明H2O2在DR配体诱导的细胞凋亡中起抗凋亡因子的作用。低浓度H2O2的持续存在通过灭活前半胱天冬酶-9抑制半胱氨酸蛋白酶介导的Jurkat细胞凋亡（10）。据报道，在人星形细胞瘤细胞中，H2O2以半胱天冬酶依赖性方式产生，并有助于抵抗TRAIL诱导的细胞凋亡（11）。因此，H2O2在DR配体诱导的细胞凋亡中的作用是有争议的。

在这里，我们证明H2O2通过细胞内O2生成诱导TRAIL抗性黑素瘤细胞中的细胞死亡。我们的数据表明细胞内O2介导这些细胞中ER相关的细胞死亡。重要的是，正常的原发性黑素细胞比恶性黑素瘤细胞对氧化细胞死亡的敏感性要小得多。

　

材料和方法

化学品和抗体

试剂来自以下制造商：可溶性重组人TRAIL，Enzo Life Sciences（San Diego，CA）; DATS，Wako Pure Chemicals（日本大阪）; thapsigargin，Sigma-Aldrich（密苏里州圣路易斯市）; z-VAD-氟甲基酮（fmk）（VAD），z-DEVD-fmk（DEVD），z-IETD-fmk（IETD），z-LETD-fmk（LETD）和Mn（III）四（4-苯甲酸）卟啉氯化物（MnTBaP），Calbiochem（La Jolla，CA）。 z-LEVD-fmk（LEVD）和z-ATAD-fmk（ATAD），BioVision（Mountain View，CA）;二氯氢荧光素二乙酸酯（DCFH-DA），二氢乙锭（DHE），MitoSOX™红（MitoSOX）和5,5'，6,6'-四氯-1,1'，3,3'-四乙基 - 苯并咪唑碳酰胺碘化物（JC） -1），Life Technologies Japan（日本东京）。将试剂溶解在二甲基亚砜中，并在使用前用Hanks'平衡盐溶液（HBSS; pH7.4）稀释至终浓度<0.1％。单独的二甲基亚砜浓度为0.1％（载体）在整个研究中没有显示出效果。针对X盒结合蛋白-1（XBP-1）和葡萄糖相关蛋白78（GRP78）的多克隆抗体购自Santa Cruz Biotechnology（Santa Cruz，CA）。所有其他化学品均为分析级。

　

细胞培养

人A2058和SK-MEL-2黑素瘤细胞获自Health Science Research Resource Bank（Osaka，Japan）。人A375黑素瘤细胞获自American Type Culture Collection（Manassas，VA）。将这些细胞在含有5％CO 2的气氛中的含有10％胎牛血清（FBS; JRH Biosciences，Lenexa，KS）的含高葡萄糖的Dulbecco's改良Eagle培养基（DMEM; Sigma-Aldrich）中培养。通过在0.25％胰蛋白酶-EDTA培养基（Life Technologies Japan）中于37℃温育5分钟来收获细胞。从Cascade Biologics（Portland，OR）获得正常人表皮黑素细胞，并在含有5％CO 2的气氛中补充有DermaLife M LifeFactors（Kurabo）的DermaLife Basal培养基（Kurabo，Osaka，Japan）中培养。通过在0.25％胰蛋白酶-EDTA培养基中于37℃温育5分钟收获细胞。

荧光显微镜测定细胞死亡率

如前所述（12）通过进行荧光显微镜评估总细胞死亡。简而言之，将细胞（1×10 4个细胞）置于8室盖玻片（Asahi Glass Co.，Tokyo，Japan）上，并在37℃，5％CO 2的气氛中用待测试的试剂处理24小时。。然后使用商业上可获得的试剂盒（LIVE /DEAD®Viability/ Cytotoxicity kit; Life Technologies）分别用4μM的钙黄绿素-AM和溴化乙锭同型二聚体-1（EthD-1）对细胞进行染色，以分别标记活细胞和死细胞。日本）根据制造商的说明。使用荧光显微镜（IX71倒置显微镜，Olympus，Tokyo，Japan）获得图像，并使用LuminaVision软件（Mitani Corporation，Fukui，Japan）进行分析。

　

凋亡细胞死亡的测定

如前所述（12）定量评估凋亡细胞死亡。简而言之，将用24孔板（2×10 5细胞/孔）铺板的细胞用TRAIL和待测试的试剂单独或一起在含有10％FBS（FBS / DMEM）的DMEM中处理20小时。随后，使用市售试剂盒（Annexin V FITC Apoptosis Detection Kit I; BD Biosciences，San Jose，CA），用FITC-缀合的膜联蛋白V和PI对细胞染色。使用CellQuest软件（BD Biosciences）在FACSCalibur流式细胞仪（BD Biosciences）中分析染色的细胞。评估了四个细胞亚群：活细胞（膜联蛋白V- / PI-）;早期凋亡细胞（Annexin V + / PI-）;晚期凋亡细胞（Annexin V + / PI +）;和坏死/受损细胞（膜联蛋白V- / PI +）。膜联蛋白V +细胞被认为是凋亡细胞。

测量线粒体膜电位（ΔΨm）去极化和caspase-3/7激活

通过先前描述的方法（12）同时测量ΔΨm去极化和胱天蛋白酶-3/7活化。简言之，将用24孔板（2×10 5细胞/孔）铺板的细胞用待在FBS / DMEM中测试的试剂处理24小时，用双传感器MitoCasp（Cell Technology Inc.，Mountain View，CA）染色。），并使用CellQuest软件分析其在FACSCalibur中的caspase-3/7活性和ΔΨm。还通过前述方法（13）使用亲脂性阳离子JC-1测量ΔΨm的变化。

测量胱天蛋白酶-12活化

如前所述（12），使用与FITC缀合的半胱天冬酶-12抑制剂ATAD检测活细胞中活化的胱天蛋白酶-12。该化合物与活性caspase-12结合，但不与无活性的caspase-12结合。根据制造商的方案，使用CaspGLOW荧光素Caspase-12染色试剂盒（BioVision），用FITC-ATAD在37℃下将细胞（2×10 5个细胞/ ml）染色30分钟。使用FACSCalibur的FL-1通道测定荧光，并使用CellQuest软件进行分析。

　

测量细胞内ROS

使用氧化敏感性DHE和DCFH-DA通过流式细胞术通过先前描述的方法测量细胞内ROS的产生（14）。简而言之，将待重悬于HBSS中的细胞（4×105 /500μl）用待测试的试剂处理，并在37℃下孵育不同的时间段，然后在37℃下用5μMDHE或DCFH-DA孵育15分钟。。洗涤细胞，在冰上重悬于HBSS中并在4℃下离心。分别使用FACSCalibur的FL-1和FL-2通道测量绿色荧光（DCFH-DA）和红色荧光（DHE），并使用CellQuest软件进行分析。如前所述（14），使用线粒体靶向探针MitoSOX Red测量线粒体O2-产生。简而言之，将悬浮在HBSS中的细胞（4×105 /500μl）用待测试的试剂处理，并在37℃下孵育不同的时间段，然后在5℃下与5μMMitoSOXRed在37℃下孵育15分钟。洗涤细胞，在冰上重悬于HBSS中，并在4℃下离心。使用FACSCalibur的FL-2通道测量红色荧光，并通过CellQuest软件分析。

荧光显微镜检测细胞间ROS

将细胞（1×104）铺在8室盖玻片上，并用待测试的试剂在37℃，含5％CO 2的气氛中处理30分钟。除去培养基后，用4μM的DCFH-DA和DHE染色细胞，分别标记产生H 2 O 2和O 2 - 的细胞。用荧光显微镜获得图像并使用LuminaVision软件分析。

蛋白质印迹分析

通过前述方法（12）进行蛋白质印迹分析。将6孔板中的细胞（1×10 6细胞/ ml /孔）用待测试的试剂在37℃处理24小时，洗涤并用SDS-样品缓冲液裂解。将全细胞裂解物进行SDS-PAGE并转移至PVDF膜（Nippon Millipore，Tokyo，Japan）。用BlockAce（Dainippon Sumitomo Pharma，Osaka，Japan）在室温下封闭膜60分钟后，使用特异性抗体检测膜上的GRP78和XBP-1蛋白。使用ECL Prime Western Blotting Reagent（GE Healthcare Japan，Tokyo，Japan）检测抗体 - 抗原复合物。为了验证相等的加载，用抗β-肌动蛋白或GAPDH的抗体重新探测膜。

结果

H2O2诱导人TRAIL抗性黑素瘤细胞中的细胞死亡

首先，我们检查了外源应用H2O2对黑素瘤细胞存活的影响。在用不同浓度的H 2 O 2处理后，用钙黄绿素-AM和EthD-1对A375细胞染色，并进行荧光显微镜分析。活细胞用钙黄绿素-AM染成绿色，而具有受损细胞膜的死细胞用EthD-1染成红色。如图1A所示，用100μMH2 O 2处理24小时导致相当大的细胞死亡，而用100ng / ml TRAIL处理具有边际效应。类似地，A2058细胞和SK-MEL-2细胞被H2O2杀死，但不被TRAIL杀死（图1B和C）。此外，在用TRAIL和H 2 O 2处理的细胞中观察到比用单独的任一种药剂处理的细胞更明显的细胞死亡。通过使用膜联蛋白V / PI染色的细胞凋亡测量证实了H2O2对TRAIL抗性黑素瘤细胞的细胞毒性作用。 H2O2处理导致A375细胞中细胞凋亡的剂量和时间依赖性增加（图1D和E）。用H2O2处理高达30μM的24小时导致细胞凋亡（膜联蛋白V +细胞）仅有适度（最大15％）增加，并且在72小时后观察到中度（35％）细胞凋亡。用100μMH2 O 2处理24小时引起中度凋亡，并且70-90％的细胞在72小时经历凋亡。虽然30μMH2O2主要引起细胞凋亡，但对坏死的诱导最小，100μMH2O2似乎也引起坏死，因为不仅膜联蛋白V +而且膜联蛋白V- / PI +细胞也增加。根据膜联蛋白V- / PI +细胞的基础水平，在不同实验中效果差异很大。水平变化范围为1.6-9.2％，当水平相对较高时，坏死细胞增加至25％。这些数据表明，在某些情况下，这些细胞会自发地发生坏死，并且高浓度的H2O2会促进这一过程。与荧光显微镜分析一致，单独TRAIL在72小时内引起最小的细胞凋亡。然而，在用TRAIL加30μM H2 O 2处理的细胞中观察到更高的细胞凋亡幅度，但是没有观察到TRAIL加100μM H2 O 2的细胞（图1F）。在所有测试的细胞系中H2O2以剂量依赖性方式诱导凋亡，而在一些但不是所有细胞系中观察到H2O2的TRAIL诱导的细胞凋亡的显着扩增（数据未显示）。在低浓度下H2O2的浓度比在高浓度下的H2O2更明显。这些数据显示H2O2处理诱导人TRAIL抗性黑素瘤细胞中的细胞死亡。随后，我们使用A375细胞作为模型细胞系统更详细地研究了H2O2诱导的细胞死亡。

图1

H2O2诱导TRAIL抗性人黑素瘤细胞中的细胞死亡。（A）人A375，（B）A2058和（C）SK-MEL-2黑素瘤细胞用30μMH2 O 2和100ng / ml TRAIL单独或组合处理24小时。除去培养基后，用钙黄绿素-AM和溴化乙锭同二聚体-1（EthD-1）对细胞进行染色，以分别标记活细胞（绿色）和具有受损细胞膜（红色）的死细胞。使用荧光显微镜（×100）获得图像。显示的结果代表4个独立实验。（D-F）将A375细胞用30或100μMH2O2和100ng / ml TRAIL单独或组合处理（D）24或（E，F）72小时，用FITC-膜联蛋白V / PI染色并通过流式细胞术。膜联蛋白V +细胞被认为是凋亡细胞。数据代表4至8个独立实验的平均值±SE。 * P <0.05; ** P <0.01; *** P <0.001。

H2O2以Caspase(半胱天冬酶)依赖性或非依赖性方式诱导黑素瘤细胞死亡，这取决于所应用的浓度

为了解H2O2诱导的细胞死亡的潜在机制，我们检查了细胞死亡是否依赖于caspase。使用半胱天冬酶特异性抑制剂的分析显示，H2O2诱导的细胞凋亡表现出对给定半胱天冬酶抑制剂的区别敏感性，这取决于所施用的氧化剂的浓度。一般的半胱天冬酶抑制剂VAD显着抑制由30μMH2O2诱导的细胞凋亡（最大抑制63％），而半胱天冬酶-3/7抑制剂DEVD将其降低40％（图2A）。引人注目的是，caspase-12抑制剂ATAD与VAD一样有效，并且在抑制细胞凋亡方面明显比DEVD更有效。相反，caspase-4抑制剂LEVD增强而非抑制细胞凋亡。有趣的是，所有这些抑制剂都能最低限度地抑制100μM H2O2诱导的细胞凋亡（图2B）。我们进一步检查了H2O2对ΔΨm和caspase-3/7活化的影响，因为这些是内在细胞凋亡途径的标志。使用荧光ΔΨm敏感染料或半胱天冬酶-3 / 7特异性探针的流式细胞术分析显示H2O2以剂量依赖性方式诱导ΔΨm去极化和胱天蛋白酶-3/7活化（图2C和D）。这些数据显示H2O2刺激多种死亡途径，包括内在的凋亡和不依赖于半胱天冬酶的凋亡和坏死的死亡途径，这取决于所应用的浓度。

图2

H2O2以半胱天冬酶依赖性或非依赖性方式诱导黑素瘤细胞凋亡，这取决于所应用的浓度。在存在或不存在30μM的z-DEVD-fmk（DEVD，特异于caspase-3/7），z-ATAD的情况下，用（A）30或（B）100μMH2O2处理A375细胞24小时。 -fmk（ATAD，特异于caspase-12），z-LEVD-fmk（LEVD，特异于caspase-4）和z-VAD-fmk（VAD，一般半胱天冬酶抑制剂）24小时和凋亡细胞通过进行膜联蛋白测量V / PI染色。数据代表5至7个独立实验的平均值±SE。 ** P <0.01; *** P <0.001。（C和D）将A375细胞用30或100μMH2 O 2处理24小时。使用双传感器MitoCasp通过流式细胞术测量（C）线粒体膜电位（ΔΨm）和（D）胱天蛋白酶-3/7活化的损失。数据代表来自4个独立实验的平均值±SE。 * P <0.05; ** P <0.01; *** P <0.001。

　

H2O2诱导细胞内超氧阴离子（O2-）产生，其介导人TRAIL抗性黑素瘤细胞中的细胞凋亡

为了阐明ROS在H2O2诱导的细胞死亡中的潜在作用，我们分析了使用氧化敏感染料DCF或DHE处理H2O2后细胞内ROS的产生。 DCF可以与多种氧化剂如H2O2，过氧亚硝酸盐（ONOO-）和·OH反应，但DCF荧光的增加可以被认为主要代表增加的H2O2水平，因为H2O2在这些DCF反应氧化剂中是最稳定的。另一方面，DHE经历双电子氧化以形成DNA结合的溴化乙锭;反应由O2-介导，但不由H2O2或ONOO-介导。因此，DCFH-DA和DHE已广泛用于评估各种啮齿动物和人类非转化和转化细胞中细胞内H2O2和O2-的产生（15-18）。用H2O2处理A375细胞30分钟，并通过进行荧光显微镜分析其DCF或DHE荧光。如图3A所示，在TRAIL处理的细胞中仅观察到DCF（绿色）荧光的适度增加，而不是DHE（红色）荧光。 DCF荧光的这种增加是短暂的并且在1小时内下降至低于基础水平。另一方面，出乎意料的是，H2O2处理不仅增加了DCF荧光而且增加了DHE荧光，表明细胞内O2-的产生。通过流式细胞术分析证实了这种氧化反应。最初在1小时观察到DHE信号的增加（图3B），并且持续至少4小时;超氧化物歧化酶（SOD）模拟抗氧化剂MnTBaP完全消除了信号（图3C）。

图3

H2O2诱导细胞内O2-产生，其介导人TRAIL抗性黑素瘤细胞中的细胞凋亡。（A）用100μM H2 O 2或100ng / ml TRAIL处理A375细胞30分钟。除去培养基后，将细胞染色DCFH-DA（绿色）和DHE（红色）以分别检测细胞内H 2 O 2和O 2 - 。用荧光显微镜（×100;比例尺，100μm）获得图像。显示的结果代表3个独立实验。（B）用100μM H2 O 2处理A375细胞60分钟，并使用DHE和DCFH-DA通过流式细胞术测量细胞内ROS产生。数据代表来自3个独立实验的平均值±SE。 *** P <0.001。（C）在存在或不存在30μM MnTBaP的情况下，用100μM H2 O 2处理A375细胞4小时，并通过流式细胞术测量细胞内O 2 - 产生。数据代表来自7个独立实验的平均值±SE。 ** P <0.01。（D）用100μM H2 O 2处理A375细胞60分钟，并使用线粒体靶向O 2 - 探针MitoSOX Red通过流式细胞术测量线粒体O 2产生。数据代表来自7个独立实验的平均值±SE。 * P <0.05。（E）在存在或不存在30μMMnTBaP的情况下，用100μM H2 O 2处理A375细胞4小时，并通过流式细胞术测量线粒体O 2 - 产生。数据代表来自6个独立实验的平均值±SE。 ** P <0.01; *** P <0.001。（F）在存在或不存在30μM MnTBaP的情况下，用30或100μM H2 O 2处理A375细胞24小时，用膜联蛋白V / PI染色，并通过流式细胞术分析凋亡细胞死亡。数据代表来自5个独立实验的平均值±SE。 * P <0.05; ** P <0.01。

因为线粒体是生理条件下ROS产生的主要部位，我们检查了这种细胞器在H2O2诱导的O2产生中的作用。 MitoSOX定位于线粒体，并作为荧光探针用于选择性检测细胞器中的O2-（15,19）。如图3D所示，最初在处理后1小时观察到MitoSOX信号的显着增加，并且增加持续至少4小时;再次，MnTBaP完全消除了这种效应（图3E）。在A2058细胞中也观察到类似的效果（数据未显示）。为了解细胞内O2-在H2O2诱导的细胞死亡中的作用，我们检测了MnTBaP处理对细胞死亡的影响。抗氧化剂以剂量依赖性方式阻断细胞凋亡（图3F）。除细胞凋亡外，100μM H2O2诱导的坏死细胞死亡也减少。相反，过氧化氢酶对细胞死亡没有影响（数据未显示）。这些结果显示H2O2而非TRAIL诱导细胞内O2-的产生，包括在TRAIL抗性黑素瘤细胞中的线粒体内部，并且O2-介导细胞凋亡。

H2O2诱导ER应激反应，同时清除O2-抑制它们

Caspase-12普遍表达并定位于ER膜。它被ER应激特异性激活，在应激诱导的细胞凋亡中发挥关键作用（22-25）。因此，获得的数据表明ER应激包括胱天蛋白酶-12活化在氧化细胞死亡中的可能作用。为了测试这种观点，我们检查了H2O2是否调节半胱天蛋白酶-12活化。使用FITC-ATAD的荧光分析显示，在有效诱导细胞凋亡的浓度下，H 2 O 2以剂量依赖性方式诱导半胱天蛋白酶-12活化（图4A和B）。此外，MnTBaP处理阻断了H2O2诱导的半胱天蛋白酶-12活化。用MnTBaP（100μM）处理几乎完全消除了30μMH2 O 2的作用并且将100μMH2 O 2的作用降低了50％（图4C）。这些数据显示清除O2-抑制H2O2诱导的细胞死亡和半胱天蛋白酶-12活化。

图4

H2O2诱导ER应激反应和清除O2-抑制它们。（A和B）将A375细胞用30或100μMH2 O 2处理24小时，通过流式细胞术测量细胞可渗透底物FITC-ATAD-fmk的转化来评估半胱天蛋白酶-12的功能活化。图A显示了典型的直方图。图B中显示的数据代表来自4个独立实验的平均值±SE。 ** P <0.01; *** P <0.001。（C）在存在或不存在30μM MnTBaP的情况下，用30或100μMH2 O 2处理A375细胞24小时，并通过流式细胞术评估胱天蛋白酶-12活化。数据代表来自4个独立实验的平均值±SE。 * P <0.05。（D和E）A375细胞单独或组合用100μM H2 O 2和30μM MnTBaP处理。 Thapsigargin（Tg，1μM）作为ER应激反应的阳性对照。然后洗涤细胞，用SDS-样品缓冲液裂解并通过蛋白质印迹分析用特异性抗体分析GRP78和XBP-1含量。为了验证相等的加载，用抗β-肌动蛋白抗体重新探测印迹。数据代表3个独立实验。

为了获得诱导ER应激的进一步证据，我们在H2O2处理后评估了2个未折叠蛋白反应（UPR）蛋白GRP78和XBP-1的水平。 Western印迹分析显示用阳性对照毒胡萝卜素处理显着上调GRP78的表达24小时，而H2O2处理则没有（图4D）。另一方面，H2O2增加了XBP-1的表达，尽管在不同的实验中程度变化很大。 XBP-1的活性剪接形式（XBP-1s）和无活性未剪接形式（XBP-1u）都增加，并且这些效果被MnTBaP处理完全抑制（图4E）。总的来说，这些数据表明H2O2诱导ER应激反应，清除O2-抑制它们。

H2O2在原代黑素细胞中诱导最小的细胞凋亡和O2-产生

我们检查了H2O2对原发性正常黑素细胞的细胞毒作用。荧光显微镜分析显示，用100ng / ml TRAIL和100μMH2O2单独或组合处理24小时导致正常黑素细胞中的细胞死亡（数据未显示）和细胞凋亡（图5A）最小化。此外，在4小时H 2 O 2处理后仅观察到最小的细胞内和线粒体O 2 - 产生（图5B和C）。这些数据表明黑素细胞对H2O2诱导的细胞死亡和O2-产生具有抗性。

　

图5

H2O2在正常原代黑素细胞中诱导最小的细胞凋亡和O2-产生。（A）用30或100μMH2 O 2处理黑素细胞，用膜联蛋白V / PI染色，并通过流式细胞术分析凋亡细胞死亡。数据代表4个独立实验。将装载有（B）DHE或（C）MitoSOX Red的黑素细胞用100μMH2 O 2处理4小时，并通过流式细胞术分析荧光。数据代表来自4个独立实验的平均值±SE。 NS，不重要。

讨论

在本研究中，我们研究了H2O2在TRAIL诱导的细胞凋亡中的可能作用。 TRAIL在人TRAIL抗性黑素瘤细胞中不诱导或仅诱导细胞内H 2 O 2水平的边际增加。另一方面，以相对低的浓度（30-100μM）外源施加的H 2 O 2基本上杀死了这些细胞。此外，在某些情况下，当组合施用H2O2和TRAIL时，观察到细胞凋亡的协同诱导。总之，这些数据表明H2O2是调节剂而不是TRAIL细胞毒性作用的主要介质。有趣的是，用低浓度的H2O2比用高浓度的H2O2更清楚地观察到协同作用，这表明随着浓度的增加，除了其内在机制之外，H2O2还刺激至少部分与TRAIL共享的凋亡途径。 H2O2诱导细胞凋亡或坏死细胞死亡，这取决于所用氧化剂的浓度。内在的线粒体途径被认为是细胞凋亡的主要机制。与此观点一致，低浓度H2O2诱导的细胞死亡是半胱天冬酶依赖性的，并且与线粒体膜电位（ΔΨm）塌陷和半胱天蛋白酶-3/7活化增加有关。然而，caspase-3/7的抑制仅部分阻断了细胞凋亡。这些数据表明虽然内在线粒体途径确实在诱导细胞凋亡中发挥作用，但另一种半胱天冬酶级联也可能参与这种半胱天冬酶依赖性细胞凋亡。

ER可以通过独立于内在（线粒体）和外在（死亡受体）途径的途径启动细胞死亡。 ER相关细胞死亡被认为是由caspase-12介导的（22-26）。多种细胞条件如葡萄糖剥夺，缺氧，钙稳态紊乱和过量的ROS可引起ER应激，其特征在于未折叠蛋白的积累。 ER应激激活适应性UPR，其由于蛋白质合成抑制，伴侣蛋白上调和蛋白质降解的增加而保护细胞。如果UPR激活不能缓解ER应激，则细胞经历ER介导的细胞凋亡（22-26）。在ER应激后，分子伴侣分子GRP78从跨膜蛋白解离，例如需要酶1α（IRE1α）和激活转录因子6（ATF6）的肌醇。游离的ATF6易位至激活的高尔基体。活性ATF6进而进入细胞核并启动转录因子XBP-1的表达。活化的IRE1α剪接转录的XBP-1 mRNA以允许翻译成熟的XBP-1蛋白，其充当转录因子并介导参与ER功能的众多基因的转录上调（20,21,23）。我们的数据显示H2O2诱导ER应激，如显示的胱天蛋白酶-12活化，并上调成熟XBP-1蛋白的表达。此外，caspase-12的抑制强烈阻断了H2O2诱导的细胞凋亡。总的来说，我们的数据表明ER介导的涉及caspase-12的凋亡途径在H2O2诱导的细胞凋亡中起关键作用。

有趣的是，虽然在包括小鼠，大鼠，兔和牛在内的各种哺乳动物细胞中已经报道了在细胞凋亡期间诱导ER应激后caspase-12的激活（26），但caspase-12在ER介导的人细胞凋亡中的作用是一个有争议的问题。这可能是因为人类caspase-12基因含有几个阻断其表达的突变（27）。尽管如此，越来越多的证据表明，caspase-12样蛋白存在并在人类细胞中被由不同原因激活，包括H2O2，顺铂，tetrocarcin A和体温过高（12,28-33）诱导的ER应激。

最近，对ER应激的适应被认为是恶性肿瘤和癌症细胞治疗抗性的关键驱动因素，包括恶性黑色素瘤细胞，GRP78在这种适应中发挥关键作用（34,35）。 GRP78表达与肿瘤发展和生长有关，并且与化学治疗药物如顺铂和阿霉素的耐药性相关（34-36）。在该研究中，毒胡萝卜素显着增加GRP78表达，而H2O2降低黑素瘤细胞中GRP78的表达;这些细胞被H2O2杀死，但不是毒胡萝卜素。另一方面，在毒胡萝卜素敏感的Jurkat白血病细胞中GRP78表达最低程度增加（Inoue和Suzuki，未发表的数据）。已显示GRP78通过抑制半胱天蛋白酶-4或胱天蛋白酶-7活性发挥其抗凋亡功能（36）。然而，caspase-4似乎负向调节H2O2诱导的细胞凋亡而不是正向的调节，因为酶活性的抑制显着增强细胞凋亡。鉴于caspase-4和caspase-12之间的结构相似性，GRP78也可能靶向caspase-12以抵消ER介导的细胞凋亡。

ROS水平受抗氧化防御系统控制，包括抗氧化酶，含锰或铜锌的超氧化物歧化酶，催化O2-歧化为H2O2，过氧化氢酶和谷胱甘肽过氧化物酶，降解H2O2。我们的数据显示这些酶对H2O2诱导的细胞死亡没有影响。 H2O2是一种可扩散的分子，很容易通过细胞膜转运到细胞外空间。因此，通过过氧化氢酶清除细胞外H 2 O 2可能最终导致细胞内H 2 O 2水平的降低。因此，过氧化氢酶抑制H2O2诱导的细胞死亡的无效性表明H2O2原位在细胞死亡中起次要作用。 MnTBaP增强H2O2诱导的细胞死亡的无效性支持这一观点，因为MnTBaP增加细胞内H2O2水平。相反，我们的数据显示O2-是H2O2诱导的细胞死亡的关键介质。 H2O2在有效诱导细胞死亡的浓度下诱导持续的细胞内O2-产生。与线粒体作为细胞凋亡过程中最常见的ROS来源的作用一致，H2O2诱导了大量的线粒体O2• - 生成。此外，MnTBaP，一种可透过细胞的SOD模拟物，减少H2O2诱导的线粒体O2-生成和细胞死亡。此外，MnTBaP阻断H2O2诱导的ER应激反应，如caspase-12和XBP-1激活。总的来说，这些数据表明超氧阴离子（O2-）最可能源自线粒体介导ER介导的细胞凋亡，从而促进H2O2诱导的细胞死亡。

　

Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation

Abstract

Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) are thought to mediate apoptosis induced by death receptor ligands, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the role of H2O2 is controversial, since some evidence suggests that H2O2 acts as an anti-apoptotic factor. Here, we show that exogenously applied H2O2 (30‑100 µM) induces cell death in TRAIL-resistant human melanoma cells via intracellular superoxide (O2-) generation. H2O2 induced apoptotic or necrotic cell death, depending on the concentration of the oxidant applied; low concentrations of H2O2 preferentially activated the caspase-dependent apoptotic pathway, while high concentrations of H2O2 induced apoptotic and necrotic cell death in a caspase-independent manner. The H2O2-induced cell death was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation and ER stress responses including caspase-12 and X-box-binding protein-1 (XBP-1) activation. H2O2 induced intracellular O2- generation even within the mitochondria, while TRAIL did not. The superoxide dismutase mimetic antioxidant MnTBaP [Mn (III) tetrakis (4-benzonic acid) porphyrin chloride] inhibited the H2O2‑induced O2- generation, apoptosis and XBP-1 and caspase-12 activation at comparable concentrations. Importantly, H2O2 treatment caused minimal O2- generation and apoptosis in normal primary melanocytes. These data show that H2O2 induces endoplasmic reticulum-associated cell death via intracellular O2- generation and that malignant melanoma cells are more susceptible than normal cells to this oxidative cell death. The findings suggest that H2O2 has therapeutic potential in the treatment of TRAIL‑resistant melanoma.

　

Introduction

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor cytokine superfamily and has a homotrimeric structure. Since TRAIL induces apoptosis in a variety of transformed and cancer cells, but not in normal cells, it is a promising target for cancer prevention and treatment. However, growing evidence suggests that some cancer cell types such as malignant melanoma, glioma and non-small cell lung cancer (NSCLC) cells are resistant to TRAIL-induced apoptosis (1). Moreover, TRAIL-responsive tumors acquire a resistant phenotype that renders TRAIL therapy ineffective. Overcoming the TRAIL resistance of cancer cells is necessary for effective TRAIL therapy and drugs potentiating TRAIL effectiveness are urgently required (2). TRAIL binds to receptors (DRs) that contain death domains such as death receptor (DR) 4/TRAIL-receptor 1 (TRAIL-R1) and DR5/TRAIL-R2 (3). This binding induces oligomerization of the receptors and conformational changes in the death domains, resulting in the formation of a death-inducing signaling complex; and subsequent activation of caspase-8. In turn, activated caspase-8 activates the effector caspase-3/6/7, which executes the apoptotic process (4,5). The activation of caspase-8 is also linked to the intrinsic (mitochondrial) apoptotic pathway. Activated caspase-8 can cleave and activate the pro-apoptotic Bcl-2-family molecule Bid, which in turn activates other Bcl-2-family molecules, Bax and Bak, resulting in their oligomerization and the formation of megachannels in the outer mitochondrial membrane. The release of cytochrome c through the Bax/Bak megachannels into the cytosol induces the assembly of apoptosome and the activation of caspase-9, resulting in the activation of caspase-3/6/7 (4). Recently, various pro-apoptotic receptor agonists such as recombinant human TRAIL and agonistic antibodies against DR4/DR5 have been subjected to clinical trials in a variety of cancer cell types, including malignant melanoma and NSCLC cells. Unfortunately, the results showed that these receptor agonists were disappointedly only modestly effective (6). Induction of apoptosis by the intrinsic pathway is considered to be the major mechanism of conventional chemotherapy and is therefore a critical target in cancer treatment. However, clinical observations suggest that amplification of the known apoptotic pathways is not sufficient for overcoming TRAIL resistance in cancer cells.

Reactive oxygen species (ROS) such as superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH) are products of normal metabolism in virtually all aerobic organisms and are also produced following xenobiotic exposure. Low physiological levels of ROS function as second messengers in intracellular signaling and are required for normal cell function, while excessive ROS cause damage to multiple macromolecules, impair cell function, and promote cell death (7). Imbalance between ROS production and the ability of a biological system to detoxify oxidants or to repair the resulting damage leads to oxidative stress. Several lines of evidence suggest that intracellular H2O2 is a mediator of DR ligand-induced apoptosis in tumor cells. The flavonoid wogonin kills TNF-α-resistant T-cell leukemia cells and sensitizes them to TNF-α- or TRAIL-induced apoptosis by increasing intracellular H2O2 levels (8). LY303511 sensitizes human neuroblastoma cells to TRAIL, and intracellular H2O2 generation plays a role in this effect (9). On the other hand, some evidence suggests that H2O2 acts as an anti-apoptotic factor in DR ligand-induced apoptosis. The continuous presence of low concentrations of H2O2 inhibits caspase-mediated apoptosis in Jurkat cells by inactivating pro-caspase-9 (10). It was reported that in human astrocytoma cells, H2O2 is generated in a caspase-dependent manner and contributes to resistance to TRAIL-induced apoptosis (11). Thus, the role of H2O2 in DR ligand-induced apoptosis is controversial.

Here we demonstrate that H2O2 induces cell death in TRAIL-resistant melanoma cells via intracellular O2− generation. Our data suggest that the intracellular O2− mediates ER-associated cell death in these cells. Importantly, normal primary melanocytes were much lesser sensitive than malignant melanoma cells to oxidative cell death.

Materials and methods

Chemicals and antibodies

Reagents were obtained from the following manufacturers: soluble recombinant human TRAIL, Enzo Life Sciences (San Diego, CA); DATS, Wako Pure Chemicals (Osaka, Japan); thapsigargin, Sigma-Aldrich (St. Louis, MO); z-VAD-fluoromethylketone (fmk) (VAD), z-DEVD-fmk (DEVD), z-IETD-fmk (IETD), z-LETD-fmk (LETD) and Mn (III) tetrakis (4-benzonic acid) porphyrin chloride (MnTBaP), Calbiochem (La Jolla, CA). z-LEVD-fmk (LEVD) and z-ATAD-fmk (ATAD), BioVision (Mountain View, CA); dichlorohydrofluorescein diacetate (DCFH-DA), dihydroethidium (DHE), MitoSOX™ Red (MitoSOX) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benz-imidazolocarbocyamine iodide (JC-1), Life Technologies Japan (Tokyo, Japan). Reagents were dissolved in dimethylsulfoxide and diluted with Hanks’ balanced salt solution (HBSS; pH 7.4) to a final concentration of <0.1% before use. Dimethylsulfoxide alone at a concentration of 0.1% (vehicle) showed no effects throughout this study. Polyclonal antibodies against X-box-binding protein-1 (XBP-1) and glucose-related protein 78 (GRP78) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All the other chemicals were of analytical grade.

Cell culture

Human A2058 and SK-MEL-2 melanoma cells were obtained from Health Science Research Resource Bank (Osaka, Japan). Human A375 melanoma cells were obtained from American Type Culture Collection (Manassas, VA). These cells were cultured in high glucose-containing Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS) in a 5% CO2-containing atmosphere. The cells were harvested by incubation in 0.25% trypsin-EDTA medium (Life Technologies Japan) for 5 min at 37°C. Normal human epidermal melanocytes were obtained from Cascade Biologics (Portland, OR) and cultured in DermaLife Basal medium (Kurabo, Osaka, Japan) supplemented with DermaLife M LifeFactors (Kurabo) in a 5% CO2-containing atmosphere. The cells were harvested by incubation in 0.25% trypsin-EDTA medium for 5 min at 37°C.

Determination of cell death by fluorescence microscopy

The overall cell death was evaluated by performing fluorescence microscopy as previously described (12). Briefly, the cells (1×104 cells) were placed on 8-chamber coverslips (Asahi Glass Co., Tokyo, Japan) and treated with the agents to be tested for 24 h at 37°C in a 5% CO2-containing atmosphere. The cells were then stained with 4 μM each of calcein-AM and ethidium bromide homodimer-1 (EthD-1) to label live and dead cells, respectively, using a commercially available kit (LIVE/DEAD® Viability/Cytotoxicity kit; Life Technologies Japan) according to the manufacturer’s instructions. Images were obtained using a fluorescence microscope (IX71 inverted microscope, Olympus, Tokyo, Japan) and analyzed using the LuminaVision software (Mitani Corporation, Fukui, Japan).

Determination of apoptotic cell death

Apoptotic cell death was quantitatively assessed as previously described (12). Briefly, the cells plated in 24-well plates (2×105 cells/well) were treated with TRAIL and the agents to be tested alone or together for 20 h in DMEM containing 10% FBS (FBS/DMEM). Subsequently, the cells were stained with FITC-conjugated Annexin V and PI using a commercially available kit (Annexin V FITC Apoptosis Detection Kit I; BD Biosciences, San Jose, CA). The stained cells were analyzed in a FACSCalibur flow cytometer (BD Biosciences) using the CellQuest software (BD Biosciences). Four cellular subpopulations were evaluated: viable cells (Annexin V−/PI−); early apoptotic cells (Annexin V+/PI−); late apoptotic cells (Annexin V+/PI+); and necrotic/damaged cells (Annexin V−/PI+). Annexin V+ cells were considered to be apoptotic cells.

Measurements of mitochondrial membrane potential (ΔΨm) depolarization and caspase-3/7 activation

ΔΨm depolarization and caspase-3/7 activation were simultaneously measured by a previously described method (12). Briefly, the cells plated in 24-well plates (2×105 cells/well) were treated with the agents to be tested in FBS/DMEM for 24 h, stained with the dual sensor MitoCasp (Cell Technology Inc., Mountain View, CA), and analyzed for their caspase-3/7 activity and ΔΨm in the FACSCalibur using the CellQuest software. Changes in ΔΨm were also measured using the lipophilic cation JC-1 by a previously described method (13).

Measurement of caspase-12 activation

Activated caspase-12 in living cells was detected using the caspase-12 inhibitor ATAD conjugated to FITC as previously described (12). This compound binds to active caspase-12, but not to inactive caspase-12. The cells (2×105 cells/ml) were stained with FITC-ATAD for 30 min at 37°C using a CaspGLOW Fluorescein Caspase-12 Staining Kit (BioVision) according to the manufacturer’s protocol. Fluorescence was determined using the FL-1 channel of the FACSCalibur and analyzed using the CellQuest software.

Measurement of intracellular ROS

The production of intracellular ROS was measured using the oxidation sensitive DHE and DCFH-DA by flow cytometry by a previously described method (14). Briefly, cells (4×105/500 μl) resuspended in HBSS were treated with the agents to be tested and incubated at 37°C for various time periods, then incubated with 5 μM DHE or DCFH-DA for 15 min at 37°C. The cells were washed, resuspended in HBSS on ice and centrifuged at 4°C. The green fluorescence (DCFH-DA) and red fluorescence (DHE) were measured using the FL-1 and FL-2 channels of the FACSCalibur, respectively, and analyzed using the CellQuest software. Mitochondrial O2− generation was measured using the mitochondria-targeting probe MitoSOX Red as previously described (14). Briefly, the cells (4×105/500 μl) suspended in HBSS were treated with the agents to be tested and incubated at 37°C for various time periods, then incubated with 5 μM MitoSOX Red for 15 min at 37°C. The cells were washed, resuspended in HBSS on ice, and centrifuged at 4°C. The red fluorescence was measured using the FL-2 channels of the FACSCalibur and analyzed by CellQuest software.

Detection of intercellular ROS by fluorescent microscopy

The cells (1×104) were plated on 8-chamber cover glasses and treated with the agents to be tested for 30 min at 37°C in a 5% CO2 containing atmosphere. After removal of the medium, the cells were stained with 4 μM each of DCFH-DA and DHE to label cells producing H2O2 and O2−, respectively. Images were obtained with the fluorescence microscope and analyzed using LuminaVision software.

Western blot analysis

Western blot analysis was carried out by the previously described method (12). The cells in 6-well plates (1×106 cells/ml/well) were treated with the agents to be tested for 24 h at 37°C, washed and lysed with SDS-sample buffer. The whole cell lysates were subjected to SDS-PAGE and transferred to PVDF membranes (Nippon Millipore, Tokyo, Japan). After blocking the membranes with BlockAce (Dainippon Sumitomo Pharma, Osaka, Japan) at room temperature for 60 min, GRP78 and XBP-1 proteins on the membranes were detected using specific antibodies. Antibody-antigen complexes were detected using the ECL Prime Western Blotting Reagent (GE Healthcare Japan, Tokyo, Japan). To verify equal loading, the membranes were re-probed with an antibody against β-actin or GAPDH.

Results

H2O2 induces cell death in human TRAIL-resistant melanoma cells

First, we examined the effect of exogenously applied H2O2 on melanoma cell survival. After treatment with H2O2 at varying concentrations, A375 cells were stained with calcein-AM and EthD-1 and subjected to fluorescence microscopic analysis. Live cells were stained green with calcein-AM, while dead cells with compromised cell membranes were stained red with EthD-1. As shown in Fig. 1A, treatment with 100 μM H2O2 for 24 h resulted in considerable cell death, while treatment with 100 ng/ml TRAIL had a marginal effect. Similarly, A2058 cells and SK-MEL-2 cells were killed by H2O2, but not by TRAIL (Fig. 1B and C). In addition, more pronounced cell death was observed in the cells treated with TRAIL and H2O2 than in the cells treated with either of the agents alone. The cytotoxic effects of H2O2 on TRAIL-resistant melanoma cells were confirmed by apoptosis measurements using Annexin V/PI staining. H2O2 treatment resulted in a dose- and time-dependent increase in apoptosis in the A375 cells (Fig. 1D and E). Treatment with H2O2 up to 30 μM for 24 h resulted in only a modest (maximum 15%) increase in apoptosis (Annexin V+ cells) and a moderate (35%) apoptosis was observed after 72 h. Treatment with 100 μM H2O2 for 24 h caused a moderate apoptosis and 70–90% of the cells underwent apoptosis at 72 h. While 30 μM H2O2 primarily caused apoptosis with minimal induction of necrosis, 100 μM H2O2 appeared to also cause necrosis, since not only Annexin V+ but also Annexin V−/PI+ cells were increased. The effect varied considerably in different experiments depending on the basal level of Annexin V−/PI+ cells. The level varied ranging from 1.6–9.2% and when the level was relatively high, necrotic cells were increased up to 25%. These data suggest that under certain circumstances, these cells spontaneously undergo necrosis and that high concentrations of H2O2 promotes this process. Consistent with the analysis by fluorescent microscopy, TRAIL alone caused minimal apoptosis for 72 h. However, higher amplitude of apoptosis was observed in the cells treated with TRAIL plus 30 μM H2O2, but not TRAIL plus 100 μM H2O2 (Fig. 1F). H2O2 induced apoptosis in a dose-dependent manner in all the cell lines tested while substantial amplification of TRAIL-induced apoptosis by H2O2 was observed in some but not all cell lines (data not shown). The amplification was more pronounced with H2O2 at low concentrations than with H2O2 at high concentrations. These data show that H2O2 treatment induces cell death in human TRAIL-resistant melanoma cells. Subsequently, we investigated the H2O2-induced cell death in greater detail using A375 cells as a model cell system.



Figure 1

H2O2 induces cell death in TRAIL-resistant human melanoma cells. (A) Human A375, (B) A2058 and (C) SK-MEL-2 melanoma cells were treated with 30 μM H2O2 and 100 ng/ml TRAIL alone or in combination for 24 h. After removal of the medium, the cells were stained with calcein-AM and ethidium bromide homodimer-1 (EthD-1) to label live cells (green) and dead cells with compromised cell membranes (red), respectively. Images were obtained using a fluorescence microscope (×100). The results shown are representative of 4 independent experiments. (D–F) A375 cells were treated with 30 or 100 μM H2O2 and 100 ng/ml TRAIL alone or in combination for (D) 24 or (E,F) 72 h, stained with FITC-Annexin V/PI and analyzed by flow cytometry. Annexin V+ cells were considered to be apoptotic cells. The data represent means ± SE from 4 to 8 independent experiments. *p<0.05; **p<0.01; ***p<0.001.

H2O2 induces melanoma cell death in a caspase-dependent or -independent manner, depending on the concentration applied

To understand the mechanisms underlying the H2O2-induced cell death, we examined whether or not the cell death was caspase-dependent. Analysis using caspase-specific inhibitors revealed that H2O2-induced apoptosis exhibited discriminate sensitivities to the given caspase inhibitor depending on the concentration of the oxidant applied. The general caspase inhibitor VAD pronouncedly inhibited the apoptosis induced by 30 μM H2O2 (maximum of 63% inhibition), while the caspase-3/7 inhibitor DEVD reduced it by 40% (Fig. 2A). Strikingly, the caspase-12 inhibitor ATAD was as effective as VAD and was significantly more effective than DEVD at inhibiting apoptosis. By contrast, the caspase-4 inhibitor LEVD enhanced rather than suppressed apoptosis. Interestingly, all these inhibitors minimally inhibited the apoptosis induced by 100 μM H2O2 (Fig. 2B). We further examined the effects of H2O2 on the ΔΨm and caspase-3/7 activation, as these are hallmarks of the intrinsic apoptotic pathway. Flow cytometric analysis using the fluorescent ΔΨm-sensitive dye or the caspase-3/7-specific probe revealed that H2O2 induced ΔΨm depolarization and caspase-3/7 activation in a dose-dependent manner (Fig. 2C and D). These data show that H2O2 stimulates multiple death pathways including intrinsic apoptotic and caspase-independent apoptotic and necrotic death pathways, depending on the concentration applied.



Figure 2

H2O2 induces melanoma cell apoptosis in a caspase-dependent or -independent manner, depending on the concentration applied. A375 cells were treated with (A) 30 or (B) 100 μM H2O2 for 24 h, in the presence or absence of 30 μM each of z-DEVD-fmk (DEVD, specific for caspase-3/7), z-ATAD-fmk (ATAD, specific for caspase-12), z-LEVD-fmk (LEVD, specific for caspase-4) and z-VAD-fmk (VAD, general caspase inhibitor) for 24 h and apoptotic cells were measured by performing Annexin V/PI staining. The data represent means ± SE from 5 to 7 independent experiments. **p<0.01; ***p<0.001. (C and D) A375 cells were treated with 30 or 100 μM H2O2 for 24 h. Loss of (C) mitochondrial membrane potential (ΔΨm) and (D) caspase-3/7 activation were measured using the dual sensor MitoCasp by flow cytometry. The data represent means ± SE from 4 independent experiments. *p<0.05; **p<0.01; ***p<0.001.

H2O2 induces intracellular O2− generation, which mediates apoptosis in human TRAIL-resistant melanoma cells

To elucidate the potential role of ROS in the H2O2-induced cell death, we analyzed the generation of intracellular ROS after H2O2 treatment using the oxidation-sensitive dye DCF or DHE. DCF can react with multiple oxidants such as H2O2, peroxynitrite (ONOO−) and •OH, but an increase in DCF fluorescence can be considered to primarily represent increased H2O2 level, since H2O2 is the most stable among these DCF-reacting oxidants. On the other hand, DHE undergoes two-electron-oxidation to form DNA-binding ethidium bromide; the reaction is mediated by O2−, but not by H2O2 or ONOO−. Consequently, DCFH-DA and DHE have been widely used to assess the generation of intracellular H2O2 and O2−, respectively, in various rodent and human non-transformed and transformed cells (15–18). A375 cells were treated with H2O2 for 30 min and analyzed for their DCF or DHE fluorescence by performing fluorescence microscopy. As shown in Fig. 3A, only a modest increase in DCF (green) fluorescence, but not DHE (red) fluorescence, was observed in TRAIL-treated cells. This increase in DCF fluorescence was transient and declined to below the basal level within 1 h. On the other hand, unexpectedly, H2O2 treatment increased not only DCF fluorescence but also DHE fluorescence, indicating the production of intracellular O2−. This oxidative response was confirmed by flow cytometric analysis. The increase in the DHE signal was initially observed at 1 h (Fig. 3B), and it lasted for at least 4 h; the signal was completely abolished by superoxide dismutase (SOD) mimetic antioxidant MnTBaP (Fig. 3C).



Figure 3

H2O2 induces intracellular O2− generation, which mediates apoptosis in human TRAIL-resistant melanoma cells. (A) A375 cells were treated with 100 μM H2O2 or 100 ng/ml TRAIL for 30 min. After removal of the medium, the cells were stained DCFH-DA (green) and DHE (red) to detect intracellular H2O2 and O2−, respectively. Images were obtained with a fluorescence microscope (×100; scale bar, 100 μm). The results shown are representative of 3 independent experiments. (B) A375 cells were treated with 100 μM H2O2 for 60 min and intracellular ROS production was measured using DHE and DCFH-DA by flow cytometry. The data represent means ± SE from 3 independent experiments. ***p<0.001. (C) A375 cells were treated with 100 μM H2O2 for 4 h in the presence or absence of 30 μM MnTBaP and intracellular O2− generation was measured by flow cytometry. The data represent means ± SE from 7 independent experiments. **p<0.01. (D) A375 cells were treated with 100 μM H2O2 for 60 min and mitochondrial O2− production was measured using the mitochondria-targeting O2− probe MitoSOX Red by flow cytometry. The data represent means ± SE from 7 independent experiments. *p<0.05. (E) A375 cells were treated with 100 μM H2O2 for 4 h in the presence or absence of 30 μM MnTBaP and mitochondrial O2− generation was measured by flow cytometry. The data represent means ± SE from 6 independent experiments. **p<0.01; ***p<0.001. (F) A375 cells were treated with 30 or 100 μM H2O2 for 24 h in the presence or absence of 30 μM MnTBaP, stained with Annexin V/PI and analyzed for apoptotic cell death by flow cytometry. The data represent means ± SE from 5 independent experiments. *p<0.05; **p<0.01.

Because mitochondria are the major site of ROS generation under physiological conditions, we examined the role of this organelle in H2O2-induced O2− generation. MitoSOX localizes to the mitochondria and serves as a fluoroprobe for selective detection of O2− in the organelle (15,19). As shown in Fig. 3D, a substantial increase in the MitoSOX signal was initially observed at 1 h after treatment and the increase lasted for at least 4 h; again, MnTBaP completely abolished the effect (Fig. 3E). Similar effects were also observed in A2058 cells (data not shown). To understand the role of intracellular O2− in H2O2-induced cell death, we examined the effect of MnTBaP treatment on cell death. The antioxidant blocked apoptosis in a dose-dependent manner (Fig. 3F). In addition to apoptosis, necrotic cell death induced by 100 μM H2O2 was also reduced. On the contrary, catalase had no effect on the cell death (data not shown). These results show that H2O2, but not TRAIL, induces the generation of intracellular O2−, including inside the mitochondria in TRAIL-resistant melanoma cells and that the O2− mediates apoptosis.

H2O2 induces ER stress responses while scavenging of O2− inhibits them

Caspase-12 is ubiquitously expressed and is localized to the ER membrane. It is specifically activated by ER stress to play a key role in the stress-induced apoptosis (22–25). Consequently, the data obtained suggested the possible role of ER stress including caspase-12 activation in oxidative cell death. To test this view, we examined whether H2O2 modulates caspase-12 activation. Fluorometric analysis using FITC-ATAD revealed that H2O2 induced caspase-12 activation in a dose-dependent manner at concentrations that effectively induced apoptosis (Fig. 4A and B). Furthermore, MnTBaP treatment blocked H2O2-induced caspase-12 activation. Treatment with MnTBaP (100 μM) almost completely abolished the effect of 30 μM H2O2 and reduced the effect of 100 μM H2O2 by 50% (Fig. 4C). These data show that scavenging of O2− inhibits H2O2-induced cell death and caspase-12 activation.



Figure 4

H2O2 induces ER stress responses and scavenging of O2− inhibits them. (A and B) A375 cells were treated with 30 or 100 μM H2O2 for 24 h and functional activation of caspase-12 was assessed by measuring the conversion of a cell-permeable substrate, FITC-ATAD-fmk by flow cytometry. Panel A shows typical histogram. The data shown in panel B represent means ± SE from 4 independent experiments. **p<0.01; ***p<0.001. (C) A375 cells were treated with 30 or 100 μM H2O2 for 24 h in the presence or absence of 30 μM MnTBaP and caspase-12 activation was assessed by flow cytometry. The data represent means ± SE from 4 independent experiments. *p<0.05. (D and E) A375 cells were treated with 100 μM H2O2 and 30 μM MnTBaP alone or in combination. Thapsigargin (Tg, 1 μM) served as a positive control of ER stress response. The cells were then washed, lysed with SDS-sample buffer and analyzed for GRP78 and XBP-1 content by western blot analysis with specific antibodies. To verify equal loading, the blots were re-probed with an anti-β-actin antibody. The data are representative of 3 independent experiments.

To obtain further evidence for the induction of ER stress, we assessed the levels of 2 unfolded protein response (UPR) proteins GRP78 and XBP-1, after H2O2 treatment. Western blot analysis showed that treatment with the positive control thapsigargin considerably upregulated the expression of GRP78 for 24 h, while H2O2 treatment did not (Fig. 4D). On the other hand, H2O2 increased the expression of XBP-1, although the degree varied considerably in different experiments. Both the active spliced form (XBP-1s) and inactive unspliced form (XBP-1u) of XBP-1 were increased, and these effects were totally inhibited by MnTBaP treatment (Fig. 4E). Collectively, these data show that H2O2 induces ER stress responses and that scavenging of O2− inhibits them.

H2O2 induces minimal apoptosis and O2− generation in primary melanocytes

We examined the cytotoxic effect of H2O2 on primary normal melanocytes. Fluorescence microscopic analysis revealed that treatment with 100 ng/ml TRAIL and 100 μM H2O2 alone or in combination for 24 h resulted in minimal cell death (data not shown) and apoptosis (Fig. 5A) in normal melanocytes. In addition, only minimal intracellular and mitochondrial O2− generation was observed after 4-h H2O2 treatment (Fig. 5B and C). These data indicate that melanocytes are resistant to H2O2-induced cell death and O2− generation.



Figure 5

H2O2 induces minimal apoptosis and O2− generation in normal primary melanocytes. (A) Melanocytes were treated with 30 or 100 μM H2O2, stained with Annexin V/PI, and analyzed for apoptotic cell death by flow cytometry. The data are representative of 4 independent experiments. Melanocytes loaded with (B) DHE or (C) MitoSOX Red were treated with 100 μM H2O2 for 4 h and analyzed for fluorescence by flow cytometry. The data represent means ± SE from 4 independent experiments. NS, not significant.

Discussion

In the present study, we investigated the possible role of H2O2 in TRAIL-induced apoptosis. TRAIL induced no or only a marginal increase in intracellular H2O2 levels in human TRAIL-resistant melanoma cells. On the other hand, exogenously applied H2O2 at relatively low concentrations (30–100 μM) substantially killed these cells. In addition, under certain circumstances, a synergistic induction of apoptosis was observed when H2O2 and TRAIL were applied in combination. Collectively, these data indicate that H2O2 is a modulator rather than primary mediator of the cytotoxic effect of TRAIL. Interestingly, the synergism was more clearly observed with low concentrations of H2O2 than with high concentrations of H2O2, suggesting that as the concentration increases, in addition to its intrinsic mechanism, H2O2 also stimulates apoptotic pathways that are at least partially shared with TRAIL. H2O2 induced apoptotic or necrotic cell death, depending on the concentration of the oxidant applied. The intrinsic mitochondrial pathway is considered to be the major mechanism of apoptosis. Consistent with this view, the cell death induced by low concentrations of H2O2 was caspase-dependent and was associated with increased ΔΨm collapse and caspase-3/7 activation. However, inhibition of caspase-3/7 only partially blocked apoptosis. These data suggest that while the intrinsic mitochondrial pathway does play a role in inducing apoptosis, another caspase cascade may also be involved in this caspase-dependent apoptosis.

ER can initiate cell death through a pathway that is independent of intrinsic (mitochondria) and extrinsic (death receptor) pathways. ER-associated cell death is thought to be mediated by caspase-12 (22–26). A variety of cellular conditions such as glucose deprivation, hypoxia, disturbance of calcium homeostasis and excess ROS can cause ER stress, which is characterized by the accumulation of unfolded proteins. ER stress activates the adaptive UPR, which protects cells owing to protein synthesis inhibition, chaperone protein upregulation and an increase in protein degradation. If UPR activation is not able to relieve ER stress, the cells undergo ER-mediated apoptosis (22–26). Upon ER stress, the chaperone molecule GRP78 dissociates from the transmembrane proteins, such as inositol requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6). The free ATF6 translocates to the Golgi apparatus where it is activated. The active ATF6 in turn enters the nucleus and initiates the expression of the transcription factor XBP-1. Activated IRE1α splices the transcribed XBP-1 mRNA to allow translation of the mature XBP-1 protein, which acts as a transcription factor and mediates the transcriptional upregulation of numerous genes involved in ER function (20,21,23). Our data showed that H2O2 induced ER stress, as shown by caspase-12 activation and upregulated the expression of the mature XBP-1 protein. Furthermore, inhibition of caspase-12 strongly blocked the H2O2-induced apoptosis. Collectively, our data suggest that the ER-mediated apoptotic pathway involving caspase-12 plays a key role in H2O2-induced apoptosis.

Interestingly, while activation of caspase-12 following the induction of ER stress during apoptosis has been reported in various mammalian cells including mouse, rat, rabbit and cow (26), the role of caspase-12 in ER-mediated apoptosis of human cells is a matter of debate. This might be because the human caspase-12 gene contains several mutations that block its expression (27). Nevertheless, an increasing body of evidence suggests that a caspase-12-like protein exists and is activated in human cells following the induction of ER stress by divergent causes, including H2O2, cisplatin, tetrocarcin A and hyperthermia (12,28–33).

Recently, adaptation to ER stress was suggested to be a key driver of malignancy and resistance to therapy in cancer cells, including malignant melanoma cells, with GRP78 playing a key role in this adaptation (34,35). GRP78 expression is associated with tumor development and growth and is correlated with resistance to chemotherapeutic drugs such as cisplatin and adriamycin (34–36). In this study, thapsigargin substantially increased GRP78 expression, while H2O2 decreased GRP78 expression in melanoma cells; these cells were killed by H2O2, but not thapsigargin. On the other hand, GRP78 expression was minimally increased in thapsigargin-sensitive Jurkat leukemia cells (Inoue and Suzuki, unpublished data). GRP78 has been shown to exert its anti-apoptotic function by inhibiting caspase-4 or caspase-7 activity (36). However, caspase-4 appears to regulate H2O2-induced apoptosis negatively rather than positively, as inhibition of the enzymatic activity significantly enhanced the apoptosis. Given the structural similarity between caspase-4 and caspase-12, it is possible that GRP78 may also target caspase-12 to counteract ER-mediated apoptosis.

ROS levels are controlled by the antioxidant defense system, including the antioxidant enzymes manganese- or copper-zinc-containing superoxide dismutase, which catalyze the dismutation of O2− into H2O2, and catalase and glutathione peroxidase, which degrade H2O2. Our data showed that these enzymes had no effects on H2O2-induced cell death. H2O2 is a diffusible molecule that is readily transported across the cell membrane to the extracellular space. Consequently, scavenging of extracellular H2O2 by catalase may eventually result in a decrease in the intracellular H2O2 level. Therefore, the ineffectiveness of catalase to suppress H2O2-induced cell death suggests that H2O2in situ plays a minor role in the cell death. The ineffectiveness of MnTBaP to enhance H2O2-induced cell death supports this view, since MnTBaP increased intracellular H2O2 levels. Instead, our data showed that O2− is a key mediator in H2O2-induced cell death. H2O2 induced persistent intracellular O2− generation at concentrations that effectively induced cell death. Consistent with the role of mitochondria as the most common source of ROS during apoptosis, H2O2 induced substantial mitochondrial O2•− generation. Moreover, MnTBaP, a cell permeable SOD mimetic, reduced the H2O2-induced mitochondrial O2− generation and cell death, In addition, MnTBaP blocked H2O2-induced ER stress responses such as caspase-12 and XBP-1 activation. Collectively, these data suggest O2− most likely derived from the mitochondria mediates ER-mediated apoptosis, thereby promoting H2O2-induced cell death.

In conclusion, we have demonstrated for the first time that H2O2 induces cell death in TRAIL-resistant human melanoma cells via intracellular O2− generation. Further studies on the mechanisms by which H2O2 induces this O2− generation are under way. Since melanoma cells are much more susceptible to oxidative cell death than normal primary melanocytes, H2O2 has therapeutic potential in the treatment of malignant melanoma.

Acknowledgements

We thank Dr M. Murai for her technical assistance. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (KAKENHI 23591631; to Y.S.) and Grants-in-Aid from Nihon University (to Y.S.).

　

Affiliations: Division of Molecular Cell Immunology and Allergology, Department of Biomedical Sciences, Nihon University School of Medicine, Tokyo, Japan, Department of Dermatology, Nihon University Surugadai Hospital, Tokyo, Japan

Authors: Mizuki Tochigi Toshio Inoue Miki Suzuki-Karasaki Toyoko Ochiai Chisei Ra Yoshihiro Suzuki-Karasaki

Published online on: January 10, 2013     https://doi.org/10.3892/ijo.2013.1769

Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation https://www.spandidos-publications.com/10.3892/ijo.2013.1769

　