NOX-1基因及其在恶性肿瘤中的作用和分子机制

徐国利(综述),范跃祖(审校)

同济大学附属同济医院普外科,上海 200065

NADPH氧化酶-1(NADPH oxidase-1,NOX-1)是NADPH氧化酶(nicotinamide adenine dinucleo-tide phosphate oxidase,NADPH oxidase,NOX)家族成员之一。NOX是细胞内一组具有氧化活性的蛋白[1],是由膜亚基gp91phox(即NOX-2)和跨膜亚基p22phox,胞浆亚基p47phox、p67phox、p40phox和小分子GTPase结合蛋白Rac 等组成的酶复合体[2-4]。还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)即还原型辅酶Ⅱ,在很多生物体化学反应中起着递氢体的作用,对机体具有重要意义。

1 NOX-1的结构及基因定位

研究发现,在不同种类的细胞中存在着一系列NADPH氧化酶催化亚基的同源氧化酶。NOX-1,亦即非吞噬细胞氧化酶1(Non-phagocytic cell oxidase 1),是哺乳动物中第一个被确认为NADPH氧化酶膜亚基gp91phox同源物的氧化酶[5];含有564 个氨基酸,与其同系物NOX-2(即膜亚基gp91phox)、NOX-3、NOX-4、NOX-5、DUOX-1和DUOX-2被称为NOX家族。该家族蛋白分布于几乎所有的器官、组织和细胞,其主要生物学功能是产生活性氧类(reactive oxygen species,ROS)。

人类NOX-1蛋白调控NOX-1基因,又名MOX-1(mitogenic oxidase-1,有丝分裂氧化酶-1)基因或NOH-1(NADPH oxidase homologue-1,NADPH 氧化酶同源物-1)基因[6-8];该基因位于X 染色体q22。NOX-1基因至少有3种剪切变构体(splice variants),即NOX1α(外显子1～13)、NOX1β(外显子1～10,12,13)、NOX1γ(外显子1～5,14);选择性地剪接NOX-1外显子11,不能编码蛋白产生超氧化物[9]。

2 NOX-1的表达分布及生理学功能

2.1 NOX-1的表达分布

正常情况下,NOX-1在结肠上皮细胞内有丰富表达[10],不仅调节组织增生和细胞分化相关的信号转导,还参与机体宿主防御[11]。NOX-1在血管平滑肌细胞、内皮细胞、破骨细胞、周细胞、肺上皮细胞以及子宫、胎盘、前列腺等细胞和组织中低表达,在前列腺癌、结直肠癌、胃腺癌等组织或细胞中高表达[12]。在前期试验中,我们还发现在胆囊癌相关成纤维细胞中NOX-1表达增高。

2.2 NOX-1的生理学功能

NOX-1等NOX家族蛋白的主要生物学功能是产生ROS[13]。ROS是一类在需氧代谢和有氧环境中形成、在分子组成上含氧,且比氧自身有更高化学活性的物质的总称[14]。ROS是氧衍生的小分子物质,包括超氧化物、羟自由基、过氧化氢和一些既是氧化因子又能被轻易转换成自由基的物质如次氯酸、臭氧及单态氧等[15]。机体内ROS有许多来源,其主要生成途径包括环氧化酶、细胞色素p450、内皮一氧化氮合酶(nitric oxide synthase,NOS)、脂肪氧化酶、线粒体呼吸、NADPH 氧化酶NOX家族、黄嘌呤氧化酶(xanthine oxidase,XO) 和髓过氧化物酶(MPO)等[16]。其中,吞噬细胞NADPH氧化酶是最早被确认的产生ROS的体系,它在正常情况下处于静息状态,而在各种刺激因子作用下活化后参与了机体的宿主防御反应[17]。

不同于吞噬细胞NADPH氧化酶,NOX家族在正常时保持一定活性,产生胞内胞外ROS,通过此途径产生的ROS并不主要起细胞防御功能,而是参与细胞分化、增殖、凋亡(细胞内源性ROS)及细胞间信号通路的调控(细胞外源性ROS)等[18],起到信号分子和基因表达开关的作用,从而参与了机体多数器官生理功能的调控。当受到胞外刺激因子的刺激时,NOX家族蛋白则过表达,产生更高浓度的ROS,诱导人体疾病的发生、发展[19]。

可见,适量的ROS可通过参与细胞生长、分化、凋亡信号和酶活性的调控,通过刺激炎性因子产生参与炎症反应,清除病原微生物和外源物质从而保护机体;而过量的ROS则造成细胞发展异常,引起各种病理生理改变,甚至触发细胞死亡从而对机体造成负面影响[20]。NOX源性ROS 牵涉到的疾病有慢性肉芽肿[21-22]、阿尔茨海默病[23]、胃肠道炎症[24]、高血压[25]、动脉粥样硬化[26]、肿瘤、甲状腺功能减退[27]、囊肿性纤维化[28]、类风湿性关节炎和糖尿病等[1,24,29-30]。

3 NOX-1与人类恶性肿瘤

肿瘤的发生常伴有多种基因的异常表达。NOX家族基因的过度表达是许多肿瘤细胞的特征之一。目前报道,与NOX-1相关的肿瘤包括结肠癌、胃癌、前列腺癌等。

研究报道,结肠癌NOX-1表达并不比正常结肠组织高[31],NOX-1表达水平与肿瘤恶性程度无明显相关[32-33],说明在结肠组织中NOX-1主要是调节细胞分化,而不是肿瘤生成;而另一项研究的结果则与此相反,NOX-1 mRNA在结肠癌中表达水平与原癌基因K-Ras激活变异及肿瘤表型相关[34]。从而推测,在结肠癌的发生发展过程中,NOX-1可能主要在早期起促进作用,而在肿瘤进展阶段表达反而降低[32]。

NOX-1同样与胃癌相关。据报道,NOX-1在人胃正常上皮黏膜细胞、慢性萎缩性胃炎、胃腺瘤及胃癌周围组织中表达水平较低,在胃的肠型及弥漫型腺癌包括印戒细胞癌中高表达[35]。研究表明,幽门螺杆菌脂多糖通过TLR4(Toll-like receptor 4)信号途径激活NOX-1源性ROS[36],由于幽门螺杆菌感染与胃腺癌密切相关[37],故推测NOX-1可能通过上调炎症反应和氧自由基活性促进胃癌发生;siRNA沉默NOX-1基因可抑制胃癌细胞生长[9]。

在前列腺癌,NOX-1高表达且能介导产生血管内皮生长因子(vascular endothelial growth factor,VEGF),但具体机制尚不清楚[38]。肿瘤相关成纤维细胞(carcinoma-associated fibroblast,CAF)可通过促炎信号利用ROS介导前列腺癌上皮间质转化(epithelial mesenchymal transition,EMT)从而影响肿瘤生成[39]。

另有报道,NOX-1 可通过上调表皮生长因子(epidermal growth factor,EGF)受体调控自分泌肝癌细胞体外生长[40]。

4 NOX-1在肿瘤中的作用和分子机制

NOX-1在肿瘤中的作用与癌基因Ras激活变异相关。研究表明,正常情况下,生长因子(growth factor,GF)激活受体酪氨酸激酶(receptor tyrosine kinase,TRK),通过Ras-Raf-有丝分裂原激活蛋白激酶(mitogen active kinase,MEK)-细胞外信号调节蛋白激酶(extracellar regulated kinase,ERK)-GATA结合蛋白(GATA binding protein,GATA-6)通路诱导NOX-1表达并产生H2O2;在病理条件下,癌基因Ras(Ras V12)持续刺激NOX-1过表达并产生大量ROS[41]。K-Ras转化NRK细胞(K-Ras V12-transformed normal rat kidney cells)NOX-1 mRNA表达上调,EGF刺激细胞也可达到相同的效果[42]。NOX-1 siRNA可阻断K-Ras/NRK细胞锚定非依赖生长,逆转形态学改变,抑制其裸鼠移植瘤生长[42]。但NOX-1本身并不具备转化能力[18]。在乳头状瘤病毒E6/E7转化的人角质细胞体外实验中可观察到,ERK激活和NOX-1表达上调[43]。

NOX源性ROS在肿瘤形成的各个阶段均发挥重要作用,ROS过表达长期以来都被认为是癌发生发展的高危因子。ROS能使细胞内主要成分如DNA、蛋白质和脂质受到严重的氧化损伤;其中,DNA受损是最常见的一种类型,主要有嘌呤和嘧啶碱基、DNA-蛋白质交联的改变以及寡核苷酸链和碱基位点的断裂等[44]。因此,参与肿瘤早期阶段基因突变和DNA异常修饰而引起的DNA损伤,是NOX源性ROS的重要作用机制。

在肿瘤的发展阶段,高表达NOX-1所产生的ROS不仅干扰细胞形态,而且促进肿瘤细胞的增殖、迁徙、转移,抑制凋亡等[20]。ROS作为信号分子,通过调节ERK1/2通路及核转录因子(nuclear factor-κB,NF-κB)活化促进细胞增殖和分化[45]。NOX-1源性ROS激活的NF-κB还能介导炎症细胞因子及给肠道细胞提供强力的抗凋亡信号从而促进结肠癌发展[46]。通过TLR4信号上调NF-κB介导的NOX-1转录,增加ROS,可加速结肠癌细胞粘附[47]。ROS还可通过增加细胞迁移速率而促进肿瘤细胞侵袭、转移[48]。另外,丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号转导通路在细胞增殖、分化、凋亡等生物学反应中亦具有至关重要的作用,如NOX源性ROS可通过激活MAPKs通路促进细胞进展[49-50]。ROS通过累积缺氧诱导因子1α(hypoxia-inducible factor-1 alpha,HIF-1α)转录子(HIF-1α transcription factor)和CXCL12趋化因子促进成纤维细胞转化为具有高迁徙力的肌成纤维细胞(MF),进而加速肿瘤播散[51]。NOX-1源性ROS还影响基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)和VEGF产生[41],而这两者与多种肿瘤的增殖、侵袭、转移尤其是血管/淋巴管生成密切相关,从而促进肿瘤细胞的增殖和肿瘤血管生成[6],最终促进肿瘤发生。

5 结语

如上所述,NOX(包括NOX-1)源性ROS的作用主要依赖于其信号转导,但研究主要集中在体外细胞试验而体内试验研究报道极少。在体内环境中,与肿瘤进展相关的不仅是癌细胞本身、癌基因和肿瘤抑制剂,还包含基质细胞构成的肿瘤微环境。目前,除了NOX-1生物学功能作用如NOX-1对肿瘤发生发展的作用、建立基因敲除/上调NOX-1表达和活性小鼠模型、NOX-1转录激活造成ROS过表达及其分子机制有待进一步研究外,临床验证NOX-1在胆囊癌细胞和间质标记阳性的间质细胞中的表达,并探讨与胆囊癌临床病理参数与患者预后的相关性,显得非常重要;但迄今未见相关报道。可以冀望,通过探讨NOX-1在胆囊癌等恶性肿瘤细胞和间质标记阳性间质细胞中表达及其临床意义,有可能寻找到胆囊癌诊断、治疗、预后判断的新靶点。

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