修回日期: 2010-06-10
接受日期: 2010-06-15
在线出版日期: 2010-06-18
肿瘤缺氧微环境与肿瘤的发展、转移和患者预后, 以及治疗的效果密切相关, 已经成为癌症研究的热点. 本文介绍了针对肿瘤缺氧微环境所进行的新治疗方法的探索, 包括阻断肿瘤缺氧诱导通路、利用缺氧微环境将无活性的药物前体转化为细胞毒性药物、改变缺氧诱导因子上下游基因的表达等. 最后介绍了作者和研究小组针对肿瘤缺氧微环境的系列研究进展, 包括阻断缺氧诱导因子-1α表达或过表达von Hippel-Lindau蛋白提高免疫治疗、抗新生血管生成治疗、化疗和肝动脉插管治疗恶性肿瘤疗效的研究.
引文著录: 孙学英, 姜宪, 姜洪池. 针对肿瘤缺氧微环境探寻新的治疗方法. 世界华人消化杂志 2010; 18(17): 1741-1746
Revised: June 10, 2010
Accepted: June 15, 2010
Published online: June 18, 2010
Hypoxic microenvironment is closely related to tumorigenesis, progression, metastasis and prognosis and has become a hot topic in cancer research. This article discusses investigations seeking novel therapies targeting the hypoxic microenvironment of tumors, including hypoxic conversion of non-toxic pro-drugs to cytotoxic drugs, and regulation of upstream and downstream genes of hypoxia-inducible factors (HIFs). Additionally, the article reviews our serial studies on tumor hypoxia, including blockade of HIF-1α expression or overexpression of von Hippel-Lindau to enhance the efficacy of immunotherapy, anti-angiogenic therapy, chemotherapy and transarterial embolization to combat malignancies.
- Citation: Sun XY, Jiang X, Jiang HC. Novel therapeutic strategies targeting the hypoxic microenvironment of tumors. Shijie Huaren Xiaohua Zazhi 2010; 18(17): 1741-1746
- URL: https://www.wjgnet.com/1009-3079/full/v18/i17/1741.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v18.i17.1741
绝大多数实体肿瘤内存在缺氧微环境. 肿瘤缺氧微环境影响肿瘤细胞的基因表型, 激活新生血管生成因子包括血管内皮生长因子(vascular endothelial growth factor, VEGF)促进肿瘤新生血管的生成[1], 上调肿瘤细胞能量代谢赖以进行的葡萄糖转运蛋白(glucose transporter, GLUT)和多种糖酵解酶[2,3], 促进肿瘤细胞无氧酵解. 缺氧进一步加剧了肿瘤细胞基因的不稳定性并激活一些肿瘤生存因子, 造成肿瘤对化疗和放疗的耐受[4], 促进了肿瘤的转移[5]. 因此, 肿瘤缺氧微环境与肿瘤的发生发展、预后和转移, 以及治疗的效果密切相关, 而对这一领域的研究已经成为寻找癌症新治疗方法的热点[6,7]. 本文对近年来在肿瘤缺氧微环境研究进展综述如下.
细胞的生长和增殖有赖于充分的氧气和能量供应. 在供氧量正常的组织中, 细胞的能量来源约90%依赖于线粒体的有氧氧化, 仅有10%来源于葡萄糖酵解, 因为后者的能量产生效率较低(仅为有氧氧化的5%). 然而, 肿瘤组织内, 肿瘤细胞失控性生长和增殖消耗大量的营养和氧气, 其内部不能及时、有效的建立新生血管网, 或新生血管网的结构和功能异常, 存在暂时性封闭或"盲端"; 并且通透性较高, 液体外渗至组织间隙导致血流黏滞阻力增加. 这些因素使肿瘤内部血流减少, 氧的供应远少于氧的需求, 肿瘤细胞处于急性(灌注缺乏)或慢性(弥散障碍)缺氧状态. 此外, 恶性肿瘤的自身进程以及抗肿瘤治疗所造成的贫血亦加剧了肿瘤的缺氧, 肿瘤内部缺氧微环境由此产生[8].
目前尚缺乏直接检测肿瘤或组织内部氧分压的方法, 缺氧的检测仅局限于少数的物理或化学方法. 检测体内缺氧的"金标准"为氧电极极谱法[9], 该技术为将阴极探针置入肿瘤内部, 通过检测氧的离子化过程中产生的电流来测量肿瘤内某个区域的氧分压, 通过计算机实时监控, 可达到每10秒测量1次, 对判断肿瘤预后有一定意义[10,11]. 目前该方法已经应用于临床, 局限性在于他仅能检测肿瘤组织中表浅或一部分区域的氧分压, 由于肿瘤内部难免存在不均一性, 所以他难以准确评估肿瘤整体的缺氧程度; 同时该方法需要消耗检测部位的氧, 因此无法进行重复测量. 由于氧可促进荧光的淬灭, Urano等[12]使用荧光探针检测肿瘤内氧含量, 该方法具有灵敏(可在局部氧分压较低时应用)、高效(多通道同时测量)、可在特定位点重复测量(便于观察对比治疗效果)等优点.
检测肿瘤内缺氧的免疫组织化学方法为, 在外科手术切除肿瘤前将检测外源性缺氧标记物的抗体输入患者体内, 通过肿瘤内的抗原-抗体结合反应, 在显微镜下观察其缺氧部位和程度. 他能反映肿瘤组织内相对氧分压, 但通常只有少数切片能够获得理想的阳性染色, 并且存在样本误差, 使其应用受到限制[13]. 目前, 可供检测肿瘤氧合水平的内源性标志物包括: 低氧诱导因子-1(hypoxia inducible factor-1, HIF-1), 葡萄糖转运蛋白-1(glucose transporter-1, GLUT-1), 碳脱水酶9等, 然而, 他们仅适用于一些特定组织来源的肿瘤(如口腔鳞状细胞癌、宫颈癌、乳腺癌等), 并不适合大多数肿瘤[14,15].
检测肿瘤缺氧的影像学方法包括: 磁共振(magnetic resonance, MR), 正电子发射断层扫描(positron emission tomography, PET), 单光子发射体层显像(single photon emission computerized tomography, SPECT), 此外, 近红外线光谱法(near-infrared spectroscopy, NIRS)及电子顺磁共振光谱法(electron paramagnetic resonance, EPR)正处于实验研究阶段. 传统磁共振成像技术可以监测组织的灌流, 但不能提示组织中氧的水平和分子-基因学改变. 核磁共振波谱法能探测组织内乳酸盐的堆积、ATP的减少及pH值的变化, 但其敏感性和空间分辨率较差[16]. 与磁共振相比, PET检测组织氧含量具有更高的敏感性和准确性. 采用15O2吸入PET检查, 可以评估组织氧合水平、局部氧释放分数以及代谢率等, 已经成为非侵入性检测组织氧水平的"金标准". 由于15O2半衰期较短(小于2 min)及费用昂贵, 限制了该手段在临床中的应用[17]. 临床中常应用一些物质的卤化示踪剂来进行PET扫描, 这些复合物在低氧环境中被还原, 进而与细胞内外的分子共价结合, 用以检测肿瘤内的缺氧状态. 最常用的为18F标记的甲氧甲基硝基咪唑乙醇(18F-FMISO)[18]. 其他如18F-FETNIM[19]、18F-EF5[20]、18F-FAZA[21]、18F-FETA[22]等也有应用的报道, 但前者多局限于头颈部肿瘤的研究, 后者尚处于实验研究阶段.
缺氧诱导因子(hypoxia-inducible factor, HIF)是肿瘤缺氧反应中关键性因子, 也是调节肿瘤新生血管生成、能量代谢、细胞增殖、浸润和转移等相关基因的上游转录调节蛋白[23,24]. 肿瘤细胞通过激活HIF信号通路并利用缺氧诱导反应, 导致肿瘤恶性程度的增加和肿瘤生物学行为的改变. 到目前为止, 已经证明的受HIF调节和控制的基因超过100余种, 包括血管生成因子(VEGF, ANG-1, ANG-2, VEGFR, IGF-2), 能量代谢相关因子(ALDA, GLUT-1, GLUT-3, LDHA, PFK1, ENO1), 细胞增殖相关因子(cyclin D1)等[7]. 这些基因都是影响肿瘤生存和生长的关键要素[2,23], 决定着肿瘤发生、生长、转移和对放化疗耐受[25]. 因此, 下调HIF的基因表达、或促进他的降解、抑制他的调节功能等, 可以阻断肿瘤生长所必需的缺氧诱导反应, 达到抑制肿瘤生长的效果[7].
HIF是由α和β亚单位组成的异构二聚体, 在低氧条件下HIF与靶基因的缺氧反应元件(HRE)结合而调节上述基因的表达[26]. 在有氧条件下, HIFα亚单位内特异的氨基酸残基转录后羟基化, 与VHL蛋白的E3泛素连接酶复合体相互反应, 被蛋白酶降解, 而失去活性[27]. HIF-1α是最早被认识的HIF异构体[28], 在寻找HIF家族新成员或与HIF-1β(ARNT)相互反应的亚单位时发现了HIF-2α和HIF-3α. HIF-3α与其他二者关系较疏, 在按一定的顺序组合时他编码一种能拮抗HRE依赖性基因表达的多肽. 然而, HIF-1α和HIF-2α密切相关, 均能活化HRE依赖性基因的转录[29]. HIF-2α(也称为EPAS1)与HIF-1α有48%的同源氨基酸序列, 分子结构相似, 包含bHLH、PAS和ODD结构. HIF-2α与HIF-1α在缺氧环境下的稳定以及转录激活方式很相似, 都能与ARNT形成异构二聚体并调节基因的表达[7]. 但是其不同之处正在不断被人们发现[26]. 比如HIF-1α在缺氧环境中的稳定是短暂的, 在持续缺氧情况下HIF-1α的含量会逐渐减少直至消失. 相反, HIF-2α的含量则在缺氧的一段时期内持续增加, 缺氧诱导基因如VEGF在缺氧早期主要由HIF-1α激活, 而在之后的时间内主要由HIF-2α激活[30]. HIF-1α与HIF-2α在很多人类肿瘤组织中表达, 但二者的表达有一定差异[31]. 在肿瘤组织以及非肿瘤组织中氧分压较高的条件下, HIF-2α均比HIF-1α更能稳定表达[26]. 另外, 与HIF-1α不同, HIF-2α仅能在特定细胞内表达, 包括肝细胞[32]. 这些不同的特点决定HIF-1α和HIF-2α在肿瘤缺氧诱导反应中具有不同的功能. 在肝细胞肝癌中, 尽管二者都呈阳性表达, HIF-2α与患者的预后关系更加密切[31,33].
当组织内氧浓度<1%(PO2<7 mmHg)时, 能抑制细胞增殖、促进其分化并诱导细胞发生坏死和凋亡. 另一方面, 缺氧作为应激因素, 使肿瘤内部分细胞发生适应性改变, 他们通过改变相关基因(HIF-1α、AP-1、NF-κB)的表达获得更有侵袭性的表型, 细胞的侵袭、增殖和抵御凋亡的能力增加, 从而促使其更易发生局部、远处转移及对治疗的抵抗[8]. 缺氧同样会促进肿瘤新生血管生成. 其机制为缺氧能够上调多种促血管生成因子如VEGF、血小板源性生长因子-B(platelet-derived growth factor-B, PDGF-B)、TGF-β、胰岛素样生长因子-2及表皮生长因子(epidermal growth factor, EGF)等, 同时下调血管生成抑制因子如angiostatin、endostatin、16 kDa催乳素及白血病抑制因子等. 研究表明, 缺氧能活化VEGF基因转录并增加VEGF mRNA的稳定性[34]. 此外, 缺氧还能通过上调MMP-9[5]、下调细胞间黏附分子[35]的表达来促进肿瘤的转移. 当组织内氧浓度<0.1%(PO2<0.7 mmHg)时, 肿瘤细胞基因组变得不稳定, 选择压力增加, 促进细胞的恶性筛选. 缺氧导致肿瘤细胞遗传不稳定性的机制为促使双微体融合并重组于脆性位点上[36]; 增加细胞内核酸内切酶的活性. 为适应这种极度缺氧的环境, 肿瘤细胞的侵袭性增加, 进展迅速, 又加剧了肿瘤的缺氧, 如此形成恶性循环.
现已证实, 缺氧是实体肿瘤对放疗、化疗产生耐受性的重要原因之一. 缺氧能诱导肿瘤多耐药基因(multidrug resistence-1, MDR-1)的表达, 增加肿瘤细胞对化疗药物的抵抗性[37]. 缺氧能改变肿瘤细胞的周期, 使多数肿瘤细胞停滞于G1期, 对化疗药物不敏感; 缺氧还可减少肿瘤细胞内拓扑异构酶Ⅱ的表达, 造成拓扑异构酶代谢的化疗药物的耐药[38]. 如前所述, 肿瘤内不能及时、有效的建立新生血管网, 或新生血管网的结构和功能异常, 导致肿瘤细胞与化疗药物接触的机会减少. 同时, 缺氧导致肿瘤细胞产生凋亡抑制, 而诱导细胞凋亡是化疗药物发挥作用的重要机制之一, 因此缺氧能增加肿瘤细胞的化疗耐药. Williams等比较了HIF-1β缺陷型与正常野生型肿瘤的放疗反应性, 发现前者对放疗的敏感性明显增加, 因此他们总结缺氧增加肿瘤放疗耐受的机制为: 自由基造成肿瘤细胞DNA损伤的"固定化"; 缺氧/HIF-1诱导多种基因表达从而造成细胞对放疗的耐受[39].
多数实体肿瘤内部存在缺氧微环境和HIF及其相关下游基因的表达, 并且与预后密切相关[40]. 一些学者针对肿瘤的上述生物学特征, 对肿瘤的治疗进行了有益探索, 包括阻断肿瘤的缺氧诱导通路、利用缺氧微环境将无活性的药物前体转化为细胞毒性药物、改变HIF的上、下游基因的表达等.
Yang等将酪氨酸激酶抑制剂PTK787与缺氧联合治疗大鼠原位肝癌, 发现与单独缺氧相比, 联合治疗通过抑制肿瘤细胞增殖、迁移、诱导肿瘤细胞坏死和凋亡以及抑制内皮细胞运动和血管生成, 显著缩小了肿瘤体积、延长了大鼠的生存期[41]. Zhao等采用RNA干扰技术减少视网膜色素上皮细胞内HIF-1α的表达, 发现能有效抑制共培养的脉络膜微血管内皮细胞的增殖、迁移和血管形成[42]. 蒽醌AQ4N是需缺氧环境活化的药物前体, 一期临床试验显示出良好的耐受性[43]. 他对多种移植瘤内的低氧细胞均有抑制作用, 他在体内经双电子还原途径转化为AQ4, 后者为DNA嵌入剂/拓扑异构酶Ⅱ抑制剂, 作用于分裂期细胞, 但由于其半衰期较长, 对低氧环境中的细胞亦可能有作用[44]. 其他需缺氧环境活化的药物前体包括SN23862、indoloquinones和cobalt(Ⅲ)复合物等[23]. Mxil是c-myc的拮抗剂, 为HIF的下游基因, 通过shRNA沉默Mxil基因能抑制裸鼠786-0肿瘤的生长, 并且主要是通过抑制细胞增殖来实现的[45].
作者和研究小组自2000年始, 针对肿瘤的缺氧微环境进行了系列研究. 采用反义基因技术阻断HIF-1α的表达能增强B7-1免疫基因治疗对EL-4淋巴瘤的疗效. 其机制为下调VEGF的表达, 减少肿瘤的MVD, 同时诱导NK细胞对肿瘤细胞的杀伤作用, 增强小鼠的T细胞免疫功能[46]. von Hippel-Lindau蛋白(pVHL)与HIF的α亚单位结合, 调节其泛素化, 促进HIF-1α的降解, 肿瘤内VHL功能的缺失能导致HIF-1α过表达, 并造成血管生成增多[47]. 我们构建编码VHL表达载体, 并与反义HIF-1α的表达载体联合, 显著减少了肿瘤内血管密度, 促进肿瘤细胞的凋亡, 下调HIF-1α和VEGF的表达, 达到了根除肿瘤的效果[48,49]. 最近发现阻断肿瘤HIF-1的表达可以提高抗肿瘤新生血管生成治疗的疗效[50].
近年来, 针对肝癌对化疗极其不敏感的特点, 针对肿瘤HIF探索提高肝癌化疗的新途径. 作为目前最有效的化疗药物之一, 多柔比星仅对4%-10.5%肝癌患者有效[51]. 研究发现反义HIF基因治疗提高了多柔比星治疗肝癌的效果, 抑制了新生血管生成和细胞增殖, 促进了肿瘤细胞的凋亡[52]. 肝动脉插管化疗(transcatheter arterial chemoembolization, TAE)是治疗肝癌的主要非手术方法之一. 但是国内外多项大宗病例分析发现TAE治疗肝癌的效果短暂, 主要原因在于栓塞后肿瘤很快形成新的侧支循环[53,54]. TAE治疗后残存肿瘤及周围肝脏组织中HIF-1α和VEGF的表达明显上调. 因此我们联合TAE和针对HIF的基因治疗弥补了TAE的缺点, 肿瘤体积明显小于TAE单独治疗的肿瘤. 研究中发现, 阻断HIF的表达可以下调VEGF、GLUT、LDHA和PCNA的表达[55].
实体肿瘤普遍存在缺氧微环境, 缺氧对肿瘤的生长、进展发挥十分重要且复杂的作用, 目前尚缺乏简单、有效评估肿瘤缺氧程度的手段, 肿瘤缺氧信号通路是一个受多因素调控的复杂网络, 如何阻断该信号通路, 并通过该通路寻找新的治疗策略、提高癌症治疗效果, 是将来努力的方向.
绝大多数实体肿瘤内存在缺氧微环境. 肿瘤缺氧微环境影响肿瘤细胞的基因表型, 激活新生血管生成因子包括血管内皮生长因子(VEGF)促进肿瘤新生血管的生成, 上调肿瘤细胞能量代谢赖以进行的葡萄糖转运蛋白(GLUT)和多种糖酵解酶, 促进肿瘤细胞无氧酵解. 缺氧进一步加剧了肿瘤细胞基因的不稳定性并激活一些肿瘤生存因子, 造成肿瘤对化疗和放疗的耐受, 促进了肿瘤的转移.
陈积圣, 教授, 中山大学孙逸仙纪念医院肝胆外科.
肿瘤缺氧微环境与肿瘤的发生发展、预后和转移, 以及治疗的效果密切相关, 而对这一领域的研究已经成为寻找癌症新治疗方法的热点.
Urano等使用荧光探针检测肿瘤内氧含量, 该方法具有灵敏(可在局部氧分压较低时应用)、高效(多通道同时测量)、可在特定位点重复测量(便于观察对比治疗效果)等优点.
本文选题先进, 针对肿瘤缺氧微环境的病理生理特点, 探索肿瘤新的治疗方式, 具有新颖性和科学性, 前沿性.
编辑: 李军亮 电编: 吴鹏朕
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