肝动脉断流对肝癌侵袭转移潜能的影响及其机理和干预研究
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摘要
“去血供疗法”是临床上针对不可切除肝癌常用的治疗手段,主要包括肝动脉结扎(Hepatic artery ligation, HAL),经肝动脉栓塞(Transarterial embolization, TAE)和经肝动脉化疗栓塞(Transarterial chemoembolization, TACE)等。其中TACE是目前中晚期肝癌的标准治疗手段。另外,近年成为终末期肝癌标准疗法的抗血管生成治疗(如Sorafinib和Sunitinib等)在一定程度上也属于“药物性去血供”的范畴。值得注意的是,虽然“去血供治疗”疗效被多数临床随机对照研究或荟萃分析证实,但其也存在以下不确定性：①治疗不彻底,残留少数肿瘤细胞,成为日后肝癌复发、转移的来源。②疗效短暂,仅能延缓而不是阻断肝癌发展,病人中位生存期最多延长至16个月。③少临床观察发现治疗本身促进残癌细胞的侵袭、转移,甚至导致病人生存期缩短。④多中心临床随机对照分析提示术前TAE／TACE不能延长可切除肝癌病人的生存期,可能与“去血供”后肿瘤肺转移增加有关[9]。
基于上述发现,本实验拟研究“去血供治疗”对残余肝癌侵袭、转移潜能的影响,分析其分子机制及可干预靶点,为进一步提高“去血供”疗效提供理论依据。
由于获取“去血供治疗”后临床肝癌样本较为困难,开展“去血供”后残余肝癌生物学特性的研究多通过动物实验来完成,主要选用动物源性肿瘤,不具有人源性背景；且一般采用经血管直接注入癌细胞的“实验性转移模式(Experimental metastatis)”,不能完整反映肝癌侵袭、转移的全过程。本实验用较高转移潜能的人肝癌细胞(MHCC97)及较低转移潜能的HepG2或Hep3B细胞行裸鼠原位种植,继以HAL模拟临床“去血供”,为开展相关研究提供了合适的体内实验平台。
该动物模型特征如下：人肝癌裸鼠原位种植成瘤率几近100%(56／56)；较高转移潜能的MHCC97(或带红色荧光标记的MHCC97-R)移植瘤自发肺转移率达40%以上[17]；而低转移潜能的HepG2(或带红色荧光标记的HepG2-R)或Hep3B(或带红色荧光标记的Hep3B-R)移植瘤腹腔转移率也超过50%。通过分析肝脏移植瘤生长曲线、瘤内血供(彩色多普勒超声)及侧枝循环建立时间,选定原位接种肝癌术后2周为HAL手术时机。HAL手术成功率达到93.3%(28／30)HAL后,移植瘤内缺氧细胞(Pimonidazole阳性染色)及组织缺氧诱导因子-1α(Hypoxia-induced factor-1α, HIF-1α)蛋白水平显著增加(P<0.05),证实“去血供”有效。
考虑到“去血供治疗”主要的生物学效应是诱发或加重瘤内缺氧,为进一步分析缺氧对肿瘤细胞生物学特性的影响,本研究用氯化钴(CoCl2)诱导肝癌细胞缺氧建立了体外模型。通过分析其量效性,选定100μmol/L作为CoCl2诱导肝癌细胞缺氧的有效浓度；同时对比其时效性,发现处理肝癌细胞12h后,细胞内HIF-1α蛋白表达即增加,48h-72h达到高峰。因此以48h-72h作为体外实验的有效时间段。
利用上述HAL模型,本研究观察“去血供治疗”对肝癌原发瘤生长和残癌细胞侵袭、转移潜能的影响。
结果发现,(1)HAL后肿瘤组织坏死增加,残癌细胞增殖抑制,原发瘤体积缩小——MHCC97-R移植瘤体积：HAL组2.0±0.2 cm3 vs.假手术组4.0±0.6cm3(P<0.01)；HepG2-R移植瘤体积：HAL组2.8±0.2 cm3 vs.假手术组4.6±0.9mm3(P<0.05)。(2)但残癌细胞的局部侵袭和远处转移明显增加,表现为：①肝脏原发瘤组织学显示,HAL导致肿瘤包膜破坏、残癌细胞局部侵袭,微血管侵犯和癌栓形成。②体视荧光显微镜显示,HAL增加肝癌移植瘤肝内、外转移(MHCC97-R:肝内播散+腹膜种植+肺转移；HepG2-R:肝内播散+腹膜种植)。③荷瘤裸鼠肺组织连续切片发现,MHCC97-R肝癌细胞肺转移率和转移灶数量也在HAL后显著增加(HAL组83.3%[10／12]与66.1±15.6个／只vs.假手术组33.3%[4／12]与49.3±6.8个／只,P<0.05)。(3)生存分析发现,HAL不能延长动物生存时间——MHCC97-R:HAL组56.0±4.6天vs.假手术组60.7±5.8天(P>0.05)：HepG2-R:HAL组54.3±4.5天vs.假手术组60.7±5.8天(P>0.05)。类似结果也见于体外实验,进一步证实“去血供”促癌效应与缺氧的相关性：100μmol/L CoCl2诱导缺氧显著抑制肝癌细胞增殖,但提高了细胞运动和侵袭潜能：(1)缺氧培养48h后,肝癌细胞迁移距离较常氧组增加：MHCC97细胞：缺氧组472.5±87.2μm vs.常氧组346.8±65.6μm(P=0.022)；HepG2细胞：缺氧组474.9±65.4μm vs.常氧组388.3±67.7μm(P=0.041)。(2)缺氧培养72h后,肝癌细胞侵袭数目也较常氧组增加(个／200x视野)：MHCC97:缺氧组75.8±12.7 vs.常氧组43.3±4.9(P=0.003)；HepG2:缺氧组52.0±4.6 vs.常氧组26.7±5.5(P<0.0001)。
结果证实,“去血供治疗”虽然抑制肝癌原发瘤生长,但促进了残癌细胞的侵袭、转移,与缺氧的生物学效应有关。
缺氧是“去血供治疗”的主要抗癌机制,但也有上述促癌效应。近年来缺氧增加肿瘤细胞侵袭、转移潜能及诱导放、化疗抵抗等均被广泛证实,并以促进肿瘤血管新生最为重要。然而瘤内缺氧具有波动性,新生肿瘤血管及侧枝循环反过来缓解缺氧,也减弱了其促癌效应。本研究对HAL后肝脏移植瘤缺氧的实效性及与血管生成的相关性做分析。
结果发现,HAL短期(2天)内增加肿瘤组织HIF-1α蛋白表达及缺氧细胞数量(P<0.05),但对远期(4周)无明显影响(P>0.05)。术后2天,HAL组血清VEGF水平(922.5±59.3 pg／mL vs.349.6±46.5 pg／mL,P<0.01)或移植瘤组织VEGF表达较假手术组升高(P<0.05),而4周时上述指标无显著差异(P>0.05)。HAL组移植瘤内微血管密度(Microvessel density,MVD)在术后2天较对照组有增加趋势,但与4周时类似,两组差异均不具有统计学意义(P>0.05)
结果证实,HAL增加肝癌组织短期而并非远期的缺氧,可能与缺氧促进肿瘤血管新生及侧枝循环形成,重塑肿瘤血供有关。新生肿瘤血管形成是一个持久过程,上述短暂缺氧更可能激活某些早期促转移机制(如粘附性降低)提高残癌侵袭、转移潜能。
上皮-间质转化(Epithelial-mesenchymal transition, EMT)是肿瘤转移的起始环节,被视为癌细胞入血前的特征性改变。发生EMT的肿瘤细胞获得间质表型,具有更强的侵袭能力,从而利于其脱离原发病灶,侵入周围血管或淋巴管系统,形成远处转移。缺氧促进肿瘤细胞发生EMT,增加侵袭潜能的证据已有报道,本实验研究“去血供”与EMT的关联性是基于：(1)上述实验结果(中华实验外科杂志,2009,26：988-990)——“去血供”通过诱导肿瘤早期而并非长期缺氧,最终增加残癌转移,提示短暂缺氧可能激活某些转移早期机制如EMT,促进转移。(2)文献报道TAE/TACE能引起肝癌组织间质化,出现肉瘤样变,其分子特征符合EMT变化,如上皮分子E-cadherin表达减少和间质分子Vimentin上调等。
结果显示,(1)在100μmol/L CoCl2诱导的体外缺氧环境中,PLC/PRF/5、HepG2、Hep3B和MHCC97 4种肝癌细胞均出现类似EMT的形态学变化——由紧密联结、铺路石样的生长模式向分散、纺锤样多极化的形态转化。EMT相关分子表达也发生改变：①上皮分子E-cadherin下调和间质分子N-cadherin、Vimentin表达增加；②EMT相关转录因子Snail、Slug和Twist表达增加。并且上述改变与缺氧肝癌细胞增强的运动和侵袭能力有关。(2)在体内模型上,HAL不但导致肿瘤组织E-cadherin表达减少,而且促其细胞内移位；同时N-cadherin、Snail、Slug和Twist等EMT相关分子上调。该变化与HAL后残余肝癌增加的局部侵袭和远处转移相关。另外,HAL加重瘤内缺氧的同时还诱导癌旁组织缺氧,癌旁正常肝细胞上类似的EMT分子变化也被观察到,并通过正常肝细胞L02的体外缺氧实验得到证实。
结果证实,“去血供治疗”及其诱导的缺氧通过激活肝癌细胞EMT而促进转移。
众所周知,肿瘤细胞过度增殖是肝癌最重要的恶性表型。但我们研究发现,肝动脉断流抑制残癌细胞增殖(Cancer Science,2010,101：120-128),似乎与“去血供”后增强的肿瘤侵袭、转移潜能相矛盾。因为Wnt/β-catenin通路是调控肝癌细胞增殖的关键信号,β-catenin为其核心分子,本实验拟研究β-catenin在缺氧环境中的表达与功能的变化。另外,我们前期工作显示,肝癌细胞内β-catenin下游靶分子c-Myc和cyclinD1在缺氧环境中表达减少；而Slug, Twist以及肝癌干细胞标记分子EpCAM等上调(Cancer Science,2010,101：120-128；中华普通外科杂志,2009,24：790-793),提示β-catenin在缺氧促癌机制中的复杂性。
结果显示,(1)β-Catenin异常表达：缺氧不改变肝癌细胞β-catenin mRNA水平,但影响其蛋白表达和／或细胞内定位：缺氧PLC/PRF/5和HepG2细胞内β-catenin蛋白总量增加(＞2.3倍),而缺氧MHCC97及Hep3B细胞则出现明显的β-catenin胞内积聚(＞1.7倍)。上述β-catenin的稳定表达与缺氧下调肝癌细胞内GSK-3β,抑制β-catenin蛋白降解有关。(2)β-Catenin功能转化：缺氧环境中异常表达的β-catenin不上调主管增殖的c-Myc和cyclinD1基因,而增加侵袭相关的靶分子Slug及Twist表达,诱导缺氧相关EMT的发生。敲除β-catenin显著抑制缺氧促癌效应并逆转缺氧细胞EMT。提示β-catenin促进缺氧恶性潜能并发生功能转化——从促增殖向促侵袭转化。(3)β-catenin与HIF-1α的相关性：免疫共沉淀技术发现缺氧肝癌细胞内β-catenin与HIF-1α形成分子复合物,但β-catenin的异常表达与HIF-1α无关。而敲除β-catenin却能减少HIF-1α蛋白表达,提示缺氧细胞内HIF-1α增加受p-catenin调控。在临床肝癌样本上,β-catenin与HIF-1α显著相关(P=0.034 &Pearson'sχ2 test, P= 0.013),共表达β-catenin和HIF-1α的肝癌病人更易出现术后转移,具有较短的总生存时间和肿瘤复发时间。
结果证实,缺氧诱导肝癌β-catenin激活及功能转化,调控细胞EMT,促进肿瘤侵袭、转移。
根据EMT发生和β-catenin活化与缺氧恶性表型的相关性,本实验分别将EMT和β-catenin作为干预靶点,协同“去血供”治疗裸鼠肝癌。
结果显示：(1)将PI3K特异性抑制剂LY294002作为阻断细胞EMT的药物[39],发现100μg/g LY294002虽然不能进一步缩小HAL治疗后的移植瘤体积,但显著减少了肿瘤转移(P<0.05),并延长荷瘤裸鼠生存时间(70.7±5.5天vs.59.2±5.9天,P=0.006)。机制分析显示LY294002逆转缺氧诱导的肝癌细胞EMT相关分子表达,并通过阻止GSK-3β降解而抑制β-catenin表达。(2)选定干扰素α(interferon-α, IFN-α)为细胞内β-catenin的有效抑制剂,发现IFN-α不但减少肝癌β-catenin表达,还有效逆转缺氧细胞内上皮分子E-cahderin的下调及间质分子N-cadherin增加。体内实验显示IFN-α剂量依赖性地增加HAL疗效：中等剂量(7.5×106U/kg)的IFN-α即显著抑制HAL增加的肿瘤肺转移(20%[1/5]vs.100%[5/5],P=0.048),延长荷瘤动物生存时间(68.7±5.5天57.2±3.7天,P=0.001)。
结果证实,LY294002和IFN-α均能抑制β-catenin表达,阻断肝癌细胞EMT,增强HAL疗效。
Hepatocellular carcinoma (HCC) is one of the most common cancer worldwide with nearly 600,000 deaths each year[2,3,41]. Its mortality is almost equal to its morbidity. Although short-term survival of patients with HCC has been improved due to advances in surgical techniques and perioperative management, long-term survival after surgical resection remains unsatisfactory because of the high rate of recurrence and metastasis[34]. The mechanisms underlying the development of HCC remain unclear.
Rooted in the belief that blocking vessel supply starves tumors to death, multiple strategies for obstruction of hepatic arterial blood flow have achieved a pronounced therapeutic effect for HCC, especially unresectable HCC. These measures include hepatic artery ligation (HAL), transarterial embolization (TAE) and transarterial chemoembolization (TACE)[1,2].To some extent, recent rising antiangiogenic therapies such as sorafenib also develops conceptually from this theory. Although overall survival of most patients was prolonged after treatment, this success was transient, without offering enduring cure. Even in some cases, a higher incidence of pulmonary metastasis has been reported following hepatic artery occlusion[5-8]. However, the precise mechanisms responsible for treatment failure are not yet clear, and also is the key point of this study.
Most of the in vivo studies analyzing the effects of blood blockade on HCC metastatic potential have been performed in animals. Unfortunately, they focused mainly on non-human tumors[13-15] and even several studies were based on "experimental metastasis" induced by intravenous injection of cancer cells, so circumventing the steps of the invasion-metastasis cascade[16]. We therefore used a "patient-like" metastatic human HCC orthotopic nude mouse model to investigate whether HAL enhanced the metastatic potential of the residual HCC and its molecular background.
Results:All nude mice with xenograft tumors exhibited 100%(56/56) orthotopic transplantability. The spontaneously metastatic rate of MHCC97 HCC cells in lung is 40%, and both abdominal disseminated rate of HepG2 or Hep3B cells are> 50%. Two weeks after orthotopic implantation were considered as the optimal appoint for second operation by observing tumor growth and angiogenesis. HAL operating successful rate was 93.3%(28/30). Immunostaining and western blot of tumor tissues showed that the expression level of Pimonidazole and HIF-1αwere significantly higher in HAL group than that of in sham-operation group (P< 0.05), suggesting HAL was effective. In vitro, CoCl2 was considered as a inductor for cellular hypoxia and its effective concentration was 100μmol/L. The high expression of HIF-la in HCC cells induced by 100μmol/L CoCl2 was clearly seen after 12 hours, and its maximum effect was achieved at 48-72 hours.
Conclusion:After orthotopic tumor implantation, HAL could reduce the arterial blood provision and consequently induce intratumoral hypoxia as well as HIF-1αoverexpression. This is therefore an appropriate model to illuminate the biological effects of arterial blood shortage of human HCC. In addition, 100μmol/L CoCl2 can lead to cellular hypoxia in vitro and is used in hypoxic model in vitro.
The number of potentially confounding variables means that clinical studies are unlikely to provide sufficient evidence to clarify the effect of hepatic artery occlusion on the metastatic potential of the residual HCC. We therefore used the above in vivo or in vitro models to investigate this significance.
Results:HAL inhibited tumor growth:MHCC97-R:2.0±0.2 cm3 in HAL group vs.4.1±0.6 cm3 in sham-operation group (P< 0.01); HepG2-R:2.8±0.5 cm3 in HAL group vs.4.6±0.9 cm3 in sham-operation group (P< 0.05). However, both the intrahepatic and extrahepatic metastasis of MHCC97-R or HepG2-R xenografts were increased by bioluminescence analysis. Histological analyses revealed that the primary tumor in HAL-treated mice had a much thinner capsule, with areas of breakage, or the capsule was completely absent, whereas the majority of control tumors were predominantly encapsulated or had thicker capsules. Venous invasion and tumor thrombi were also significantly more common in the xenografts in HAL-treated mice. Analysis of serial lung sections in animal with MHCC97-R xenografts indicated a significantly higher incidence of pulmonary metastasis in HAL group compared with that in sham-operated group (10/12 vs. 4/12, P= 0.036) and the numbers of pulmonary metastatic foci in each mouse were 66.1±15.6 vs.49.3±6.8 (P= 0.016). In contrast to that of the MHCC97, the induction of pulmonary metastasis by HAL was not evident in nude mice bearing HepG2-R xenografts. These animals showed enhanced intrahepatic dissemination and increased peritoneal seeding after HAL with respect to controls. The mean survival times of the MHCC97-R-bearing mice were similar between the two groups (56.0±4.6 days in HAL group vs.60.7±5.8 days in sham-operated group, P= 0.153). Also no significant difference was observed in animals with HepG2-R xenografts, regardless of whether receiving HAL or not. Further analysis in vitro revealed that hypoxia due to 100μmol/L CoCl2 cause arrest of cell proliferation, rather than induction of apoptosis, but concomitantly, enhanced migration and invasiveness.
Conclusion:HAL inhibits tumor growth but promotes invasiveness and distant metastasis, and fails to prolong survival. Its significance may be associated with biological effects of hypoxia.
It is well-established that hypoxia, as a selective mechanism, induces cell death by apoptosis[45]. This is one of the most important antitumoal mechanism of the above strategies such as HAL. However, hypoxia has yet been demonstrated to promote tumor invasion or metastasis as evidenced by recent studies[23,24].Cumulative evidences revealed that tumor angiogenesis due to hypoxia played a vital role in this process, which could reversely offset the significance of hypoxia[22,25]. To explore the significance of blocking vessel supply on the metastatic potential of HCC, the relationship between intratumoral hypoxia and angiogenesis after HAL is needed to examine.
Results:HAL increased dramaticly the expression level of pimonidazole (9.33±2.34 vs.5±2.10, P< 0.05) and HIF-1α(+++-++++vs.+-++,P< 0.05) at 2 days after hepatic artery occlusion, comparing with sham-operation group. However, it failed to augment long-term (4 weeks) hypoxia in xenografts. Consistent with these, VEGF level in tumor tissues or serum (922.5±59.3 pg/mL vs.349.6±46.5 pg/mL, P< 0.01) was significant higher at early-stage in HAL group than that of in control, but no significant difference at later stage. Notablely, the intratumoral microvessel density (MVD) in HAL group was similar to that of in sham-operation group, regardless of 2 days or 4 weeks after second operation (P> 0.05).
Conclusion:HAL induces dramaticly short-term hypoxia in HCC xenograft and overexpression of VEGF, but has no effect on long-term hypoxia or angiogenesis in tumor. However, angiogenesis is usually regarded as a long-term adaptation to tumor hypoxia [22,46] and other cell signalling activated by short-term hypoxia after HAL might contribute to the enhanced metastatic potential of HCC.
Epithelial-mesenchymal transition (EMT) is an initial step of metastatic cascades and plays a critical role in tumor progression[26-28]. Reduction of adhesion ability and enhanced invasiveness during EMT may facilitate cancer cells falling off from primary tumor and invading into vascular or lymphatic vessels. We here investigated whether hepatic artery occlusion induced the changes consistent with EMT in the residual HCC cells. Also the relevance between EMT and hypoxia was examined in this study.
Results:Western blot and immunostaining of xenograft tissues showed that HAL induced EMT-related molecular changes, characterized by the loss of epithelial molecule E-cadherin as well as upregulation of N-cadherin, as the mesenchymal marker. Also the EMT-related transcription factors such as Snail, Slug and Twist were increased in HAL-treated mice, compared with those of sham-operated mice. Similar results were also appeared in peritumoral tissues and associated with hypoxia due to HAL. In vitro, four days after initiation of hypoxia, the morphologies of MHCC97 and HepG2 cells were altered from a typical epithelial cobblestone appearance to an elongated/irregular fibroblastoid shape. Accompanying these phenotypic changes, western blot demonstrated a reduction of E-cadherin expression in these hypoxic cells, relative to the normoxic controls. At the same time, the levels of the mesenchymal cell markers vimentin and N-cadherin were increased. Moreover, qRT-PCR showed a trend toward increase of the transcription factors Snail, Slug and Twist mRNA in hypoxic cells with respect to their controls.
Conclusion:Hepatic arterial occlusion induces intratumoral hypoxia and subsequently contributes to enhanced metastatic potential of residual HCC by eliciting EMT.
Consistent with the above findings, growth of HCC cells and xenografts were unexpectedly suppressed by hypoxia in vivo and in vitro models. Because cell proliferation is the most important mechanism of HCC progression, it is hard to explain that HAL inhibited cell proliferation, but concomitantly, enhanced metastatic potential. Wnt/β-catenin pathway plays a dominated role in HCC proliferation andβ-catenin is the chief downstream effector of this pathway. It has been reported that one third of HCCs are associated with aberrant expression ofβ-catenin. Given the significance ofβ-catenin in HCC biology and the well-characterized association betweenβ-catenin and proliferation, it is essential to investigate the role ofβ-catenin in hypoxia.
Results:Hypoxia induced a pronounced increase in totalβ-catenin levels in PLC/PRF/5 and HepG2 cells (> 2.3-fold), as well as intracellular translocation ofβ-catenin (> 1.7-fold) in Hep3B and MHCC97 cells. The elevatedβ-catenin level in HCC cells was attributed to downregulation of GSK-3βexpression and proteasome inhibition. In hypoxic HCC cells,β-catenin interacted HIF-1α. Knockdownβ-catenin by shRNA reduced HIF-1αprotein level, whereas silencing HIF-1αfailed to affect the expression ofβ-catenin. Loss ofβ-catenin in HCC cells suppressed hypoxia-induced metastatic potential, which was associated with the inhibition of hypoxia-triggered EMT. Positive expression ofβ-catenin in HCC TMA coincided with expression of HIF-1α(P= 0.034; Pearson'sχ2 test, P= 0.013), and co-expression ofβ-catenin and HIF-1αin HCC was correlated with shorter overall survival and time to recurrence.
Conclusion:P-Catenin in HCC is activated by hypoxia and regulates EMT in hypoxic cells, contributing to enhanced metastatic potential.
Consistent with the above findings, hypoxia-induced EMT orβ-catenin aberrant activation in HCC contribute to enhanced metastatic potential. It provides the promising targets for sensitization of HAL. PI3K selective inhibitor, LY294002, has been demonstrated to suppress hypoxia-induced EMT and interferon-a (IFN-a) was reported to reduceβ-catenin expression in cells. We thus investigated the effects of LY294002 or IFN-a on enhanced metastastic potential of HCC due to hepatic arterial occlusion, respectively.
Results:HAL inhibited tumor growth (2.0±0.3 cm3 vs.3.9±0.8 cm3, P< 0.05), but promoted pulmonary metastatsis (83% vs.33%, P< 0.05). HAL combined with LY294002 (100μg/g body weight) repressed significantly enhanced distant metastasis by HAL alone (P< 0.05) and prolonged survival. IFN-αincreased synergistically the therapeutic effects of HAL in dose-dependent manner. The maximum effect on tumor growth, lung metastases and life-span was achieved by 1.5×107 U/kg of IFN-a. Moderate-dose IFN-α(7.5×106 U/kg) suppressed pulmonary metastasis in HAL-treated mice as well as prolonged survival, but it failed to inhibit tumor growth. Western blot of tumor tissues showed that both LY294002 or IFN-a reversed hypoxia-induced EMT. These significances were further demonstrated by the in vitro response of hypoxic cells to the both agents.
Conclusion:Inhibition of EMT orβ-catenin aberrant expression in hypoxic HCC cells could repress enhanced metastastic potential due to hepatic arterial occlusion.
基于上述发现,本实验拟研究“去血供治疗”对残余肝癌侵袭、转移潜能的影响,分析其分子机制及可干预靶点,为进一步提高“去血供”疗效提供理论依据。
由于获取“去血供治疗”后临床肝癌样本较为困难,开展“去血供”后残余肝癌生物学特性的研究多通过动物实验来完成,主要选用动物源性肿瘤,不具有人源性背景；且一般采用经血管直接注入癌细胞的“实验性转移模式(Experimental metastatis)”,不能完整反映肝癌侵袭、转移的全过程。本实验用较高转移潜能的人肝癌细胞(MHCC97)及较低转移潜能的HepG2或Hep3B细胞行裸鼠原位种植,继以HAL模拟临床“去血供”,为开展相关研究提供了合适的体内实验平台。
该动物模型特征如下：人肝癌裸鼠原位种植成瘤率几近100%(56／56)；较高转移潜能的MHCC97(或带红色荧光标记的MHCC97-R)移植瘤自发肺转移率达40%以上[17]；而低转移潜能的HepG2(或带红色荧光标记的HepG2-R)或Hep3B(或带红色荧光标记的Hep3B-R)移植瘤腹腔转移率也超过50%。通过分析肝脏移植瘤生长曲线、瘤内血供(彩色多普勒超声)及侧枝循环建立时间,选定原位接种肝癌术后2周为HAL手术时机。HAL手术成功率达到93.3%(28／30)HAL后,移植瘤内缺氧细胞(Pimonidazole阳性染色)及组织缺氧诱导因子-1α(Hypoxia-induced factor-1α, HIF-1α)蛋白水平显著增加(P<0.05),证实“去血供”有效。
考虑到“去血供治疗”主要的生物学效应是诱发或加重瘤内缺氧,为进一步分析缺氧对肿瘤细胞生物学特性的影响,本研究用氯化钴(CoCl2)诱导肝癌细胞缺氧建立了体外模型。通过分析其量效性,选定100μmol/L作为CoCl2诱导肝癌细胞缺氧的有效浓度；同时对比其时效性,发现处理肝癌细胞12h后,细胞内HIF-1α蛋白表达即增加,48h-72h达到高峰。因此以48h-72h作为体外实验的有效时间段。
利用上述HAL模型,本研究观察“去血供治疗”对肝癌原发瘤生长和残癌细胞侵袭、转移潜能的影响。
结果发现,(1)HAL后肿瘤组织坏死增加,残癌细胞增殖抑制,原发瘤体积缩小——MHCC97-R移植瘤体积：HAL组2.0±0.2 cm3 vs.假手术组4.0±0.6cm3(P<0.01)；HepG2-R移植瘤体积：HAL组2.8±0.2 cm3 vs.假手术组4.6±0.9mm3(P<0.05)。(2)但残癌细胞的局部侵袭和远处转移明显增加,表现为：①肝脏原发瘤组织学显示,HAL导致肿瘤包膜破坏、残癌细胞局部侵袭,微血管侵犯和癌栓形成。②体视荧光显微镜显示,HAL增加肝癌移植瘤肝内、外转移(MHCC97-R:肝内播散+腹膜种植+肺转移；HepG2-R:肝内播散+腹膜种植)。③荷瘤裸鼠肺组织连续切片发现,MHCC97-R肝癌细胞肺转移率和转移灶数量也在HAL后显著增加(HAL组83.3%[10／12]与66.1±15.6个／只vs.假手术组33.3%[4／12]与49.3±6.8个／只,P<0.05)。(3)生存分析发现,HAL不能延长动物生存时间——MHCC97-R:HAL组56.0±4.6天vs.假手术组60.7±5.8天(P>0.05)：HepG2-R:HAL组54.3±4.5天vs.假手术组60.7±5.8天(P>0.05)。类似结果也见于体外实验,进一步证实“去血供”促癌效应与缺氧的相关性：100μmol/L CoCl2诱导缺氧显著抑制肝癌细胞增殖,但提高了细胞运动和侵袭潜能：(1)缺氧培养48h后,肝癌细胞迁移距离较常氧组增加：MHCC97细胞：缺氧组472.5±87.2μm vs.常氧组346.8±65.6μm(P=0.022)；HepG2细胞：缺氧组474.9±65.4μm vs.常氧组388.3±67.7μm(P=0.041)。(2)缺氧培养72h后,肝癌细胞侵袭数目也较常氧组增加(个／200x视野)：MHCC97:缺氧组75.8±12.7 vs.常氧组43.3±4.9(P=0.003)；HepG2:缺氧组52.0±4.6 vs.常氧组26.7±5.5(P<0.0001)。
结果证实,“去血供治疗”虽然抑制肝癌原发瘤生长,但促进了残癌细胞的侵袭、转移,与缺氧的生物学效应有关。
缺氧是“去血供治疗”的主要抗癌机制,但也有上述促癌效应。近年来缺氧增加肿瘤细胞侵袭、转移潜能及诱导放、化疗抵抗等均被广泛证实,并以促进肿瘤血管新生最为重要。然而瘤内缺氧具有波动性,新生肿瘤血管及侧枝循环反过来缓解缺氧,也减弱了其促癌效应。本研究对HAL后肝脏移植瘤缺氧的实效性及与血管生成的相关性做分析。
结果发现,HAL短期(2天)内增加肿瘤组织HIF-1α蛋白表达及缺氧细胞数量(P<0.05),但对远期(4周)无明显影响(P>0.05)。术后2天,HAL组血清VEGF水平(922.5±59.3 pg／mL vs.349.6±46.5 pg／mL,P<0.01)或移植瘤组织VEGF表达较假手术组升高(P<0.05),而4周时上述指标无显著差异(P>0.05)。HAL组移植瘤内微血管密度(Microvessel density,MVD)在术后2天较对照组有增加趋势,但与4周时类似,两组差异均不具有统计学意义(P>0.05)
结果证实,HAL增加肝癌组织短期而并非远期的缺氧,可能与缺氧促进肿瘤血管新生及侧枝循环形成,重塑肿瘤血供有关。新生肿瘤血管形成是一个持久过程,上述短暂缺氧更可能激活某些早期促转移机制(如粘附性降低)提高残癌侵袭、转移潜能。
上皮-间质转化(Epithelial-mesenchymal transition, EMT)是肿瘤转移的起始环节,被视为癌细胞入血前的特征性改变。发生EMT的肿瘤细胞获得间质表型,具有更强的侵袭能力,从而利于其脱离原发病灶,侵入周围血管或淋巴管系统,形成远处转移。缺氧促进肿瘤细胞发生EMT,增加侵袭潜能的证据已有报道,本实验研究“去血供”与EMT的关联性是基于：(1)上述实验结果(中华实验外科杂志,2009,26：988-990)——“去血供”通过诱导肿瘤早期而并非长期缺氧,最终增加残癌转移,提示短暂缺氧可能激活某些转移早期机制如EMT,促进转移。(2)文献报道TAE/TACE能引起肝癌组织间质化,出现肉瘤样变,其分子特征符合EMT变化,如上皮分子E-cadherin表达减少和间质分子Vimentin上调等。
结果显示,(1)在100μmol/L CoCl2诱导的体外缺氧环境中,PLC/PRF/5、HepG2、Hep3B和MHCC97 4种肝癌细胞均出现类似EMT的形态学变化——由紧密联结、铺路石样的生长模式向分散、纺锤样多极化的形态转化。EMT相关分子表达也发生改变：①上皮分子E-cadherin下调和间质分子N-cadherin、Vimentin表达增加；②EMT相关转录因子Snail、Slug和Twist表达增加。并且上述改变与缺氧肝癌细胞增强的运动和侵袭能力有关。(2)在体内模型上,HAL不但导致肿瘤组织E-cadherin表达减少,而且促其细胞内移位；同时N-cadherin、Snail、Slug和Twist等EMT相关分子上调。该变化与HAL后残余肝癌增加的局部侵袭和远处转移相关。另外,HAL加重瘤内缺氧的同时还诱导癌旁组织缺氧,癌旁正常肝细胞上类似的EMT分子变化也被观察到,并通过正常肝细胞L02的体外缺氧实验得到证实。
结果证实,“去血供治疗”及其诱导的缺氧通过激活肝癌细胞EMT而促进转移。
众所周知,肿瘤细胞过度增殖是肝癌最重要的恶性表型。但我们研究发现,肝动脉断流抑制残癌细胞增殖(Cancer Science,2010,101：120-128),似乎与“去血供”后增强的肿瘤侵袭、转移潜能相矛盾。因为Wnt/β-catenin通路是调控肝癌细胞增殖的关键信号,β-catenin为其核心分子,本实验拟研究β-catenin在缺氧环境中的表达与功能的变化。另外,我们前期工作显示,肝癌细胞内β-catenin下游靶分子c-Myc和cyclinD1在缺氧环境中表达减少；而Slug, Twist以及肝癌干细胞标记分子EpCAM等上调(Cancer Science,2010,101：120-128；中华普通外科杂志,2009,24：790-793),提示β-catenin在缺氧促癌机制中的复杂性。
结果显示,(1)β-Catenin异常表达：缺氧不改变肝癌细胞β-catenin mRNA水平,但影响其蛋白表达和／或细胞内定位：缺氧PLC/PRF/5和HepG2细胞内β-catenin蛋白总量增加(＞2.3倍),而缺氧MHCC97及Hep3B细胞则出现明显的β-catenin胞内积聚(＞1.7倍)。上述β-catenin的稳定表达与缺氧下调肝癌细胞内GSK-3β,抑制β-catenin蛋白降解有关。(2)β-Catenin功能转化：缺氧环境中异常表达的β-catenin不上调主管增殖的c-Myc和cyclinD1基因,而增加侵袭相关的靶分子Slug及Twist表达,诱导缺氧相关EMT的发生。敲除β-catenin显著抑制缺氧促癌效应并逆转缺氧细胞EMT。提示β-catenin促进缺氧恶性潜能并发生功能转化——从促增殖向促侵袭转化。(3)β-catenin与HIF-1α的相关性：免疫共沉淀技术发现缺氧肝癌细胞内β-catenin与HIF-1α形成分子复合物,但β-catenin的异常表达与HIF-1α无关。而敲除β-catenin却能减少HIF-1α蛋白表达,提示缺氧细胞内HIF-1α增加受p-catenin调控。在临床肝癌样本上,β-catenin与HIF-1α显著相关(P=0.034 &Pearson'sχ2 test, P= 0.013),共表达β-catenin和HIF-1α的肝癌病人更易出现术后转移,具有较短的总生存时间和肿瘤复发时间。
结果证实,缺氧诱导肝癌β-catenin激活及功能转化,调控细胞EMT,促进肿瘤侵袭、转移。
根据EMT发生和β-catenin活化与缺氧恶性表型的相关性,本实验分别将EMT和β-catenin作为干预靶点,协同“去血供”治疗裸鼠肝癌。
结果显示：(1)将PI3K特异性抑制剂LY294002作为阻断细胞EMT的药物[39],发现100μg/g LY294002虽然不能进一步缩小HAL治疗后的移植瘤体积,但显著减少了肿瘤转移(P<0.05),并延长荷瘤裸鼠生存时间(70.7±5.5天vs.59.2±5.9天,P=0.006)。机制分析显示LY294002逆转缺氧诱导的肝癌细胞EMT相关分子表达,并通过阻止GSK-3β降解而抑制β-catenin表达。(2)选定干扰素α(interferon-α, IFN-α)为细胞内β-catenin的有效抑制剂,发现IFN-α不但减少肝癌β-catenin表达,还有效逆转缺氧细胞内上皮分子E-cahderin的下调及间质分子N-cadherin增加。体内实验显示IFN-α剂量依赖性地增加HAL疗效：中等剂量(7.5×106U/kg)的IFN-α即显著抑制HAL增加的肿瘤肺转移(20%[1/5]vs.100%[5/5],P=0.048),延长荷瘤动物生存时间(68.7±5.5天57.2±3.7天,P=0.001)。
结果证实,LY294002和IFN-α均能抑制β-catenin表达,阻断肝癌细胞EMT,增强HAL疗效。
Hepatocellular carcinoma (HCC) is one of the most common cancer worldwide with nearly 600,000 deaths each year[2,3,41]. Its mortality is almost equal to its morbidity. Although short-term survival of patients with HCC has been improved due to advances in surgical techniques and perioperative management, long-term survival after surgical resection remains unsatisfactory because of the high rate of recurrence and metastasis[34]. The mechanisms underlying the development of HCC remain unclear.
Rooted in the belief that blocking vessel supply starves tumors to death, multiple strategies for obstruction of hepatic arterial blood flow have achieved a pronounced therapeutic effect for HCC, especially unresectable HCC. These measures include hepatic artery ligation (HAL), transarterial embolization (TAE) and transarterial chemoembolization (TACE)[1,2].To some extent, recent rising antiangiogenic therapies such as sorafenib also develops conceptually from this theory. Although overall survival of most patients was prolonged after treatment, this success was transient, without offering enduring cure. Even in some cases, a higher incidence of pulmonary metastasis has been reported following hepatic artery occlusion[5-8]. However, the precise mechanisms responsible for treatment failure are not yet clear, and also is the key point of this study.
Most of the in vivo studies analyzing the effects of blood blockade on HCC metastatic potential have been performed in animals. Unfortunately, they focused mainly on non-human tumors[13-15] and even several studies were based on "experimental metastasis" induced by intravenous injection of cancer cells, so circumventing the steps of the invasion-metastasis cascade[16]. We therefore used a "patient-like" metastatic human HCC orthotopic nude mouse model to investigate whether HAL enhanced the metastatic potential of the residual HCC and its molecular background.
Results:All nude mice with xenograft tumors exhibited 100%(56/56) orthotopic transplantability. The spontaneously metastatic rate of MHCC97 HCC cells in lung is 40%, and both abdominal disseminated rate of HepG2 or Hep3B cells are> 50%. Two weeks after orthotopic implantation were considered as the optimal appoint for second operation by observing tumor growth and angiogenesis. HAL operating successful rate was 93.3%(28/30). Immunostaining and western blot of tumor tissues showed that the expression level of Pimonidazole and HIF-1αwere significantly higher in HAL group than that of in sham-operation group (P< 0.05), suggesting HAL was effective. In vitro, CoCl2 was considered as a inductor for cellular hypoxia and its effective concentration was 100μmol/L. The high expression of HIF-la in HCC cells induced by 100μmol/L CoCl2 was clearly seen after 12 hours, and its maximum effect was achieved at 48-72 hours.
Conclusion:After orthotopic tumor implantation, HAL could reduce the arterial blood provision and consequently induce intratumoral hypoxia as well as HIF-1αoverexpression. This is therefore an appropriate model to illuminate the biological effects of arterial blood shortage of human HCC. In addition, 100μmol/L CoCl2 can lead to cellular hypoxia in vitro and is used in hypoxic model in vitro.
The number of potentially confounding variables means that clinical studies are unlikely to provide sufficient evidence to clarify the effect of hepatic artery occlusion on the metastatic potential of the residual HCC. We therefore used the above in vivo or in vitro models to investigate this significance.
Results:HAL inhibited tumor growth:MHCC97-R:2.0±0.2 cm3 in HAL group vs.4.1±0.6 cm3 in sham-operation group (P< 0.01); HepG2-R:2.8±0.5 cm3 in HAL group vs.4.6±0.9 cm3 in sham-operation group (P< 0.05). However, both the intrahepatic and extrahepatic metastasis of MHCC97-R or HepG2-R xenografts were increased by bioluminescence analysis. Histological analyses revealed that the primary tumor in HAL-treated mice had a much thinner capsule, with areas of breakage, or the capsule was completely absent, whereas the majority of control tumors were predominantly encapsulated or had thicker capsules. Venous invasion and tumor thrombi were also significantly more common in the xenografts in HAL-treated mice. Analysis of serial lung sections in animal with MHCC97-R xenografts indicated a significantly higher incidence of pulmonary metastasis in HAL group compared with that in sham-operated group (10/12 vs. 4/12, P= 0.036) and the numbers of pulmonary metastatic foci in each mouse were 66.1±15.6 vs.49.3±6.8 (P= 0.016). In contrast to that of the MHCC97, the induction of pulmonary metastasis by HAL was not evident in nude mice bearing HepG2-R xenografts. These animals showed enhanced intrahepatic dissemination and increased peritoneal seeding after HAL with respect to controls. The mean survival times of the MHCC97-R-bearing mice were similar between the two groups (56.0±4.6 days in HAL group vs.60.7±5.8 days in sham-operated group, P= 0.153). Also no significant difference was observed in animals with HepG2-R xenografts, regardless of whether receiving HAL or not. Further analysis in vitro revealed that hypoxia due to 100μmol/L CoCl2 cause arrest of cell proliferation, rather than induction of apoptosis, but concomitantly, enhanced migration and invasiveness.
Conclusion:HAL inhibits tumor growth but promotes invasiveness and distant metastasis, and fails to prolong survival. Its significance may be associated with biological effects of hypoxia.
It is well-established that hypoxia, as a selective mechanism, induces cell death by apoptosis[45]. This is one of the most important antitumoal mechanism of the above strategies such as HAL. However, hypoxia has yet been demonstrated to promote tumor invasion or metastasis as evidenced by recent studies[23,24].Cumulative evidences revealed that tumor angiogenesis due to hypoxia played a vital role in this process, which could reversely offset the significance of hypoxia[22,25]. To explore the significance of blocking vessel supply on the metastatic potential of HCC, the relationship between intratumoral hypoxia and angiogenesis after HAL is needed to examine.
Results:HAL increased dramaticly the expression level of pimonidazole (9.33±2.34 vs.5±2.10, P< 0.05) and HIF-1α(+++-++++vs.+-++,P< 0.05) at 2 days after hepatic artery occlusion, comparing with sham-operation group. However, it failed to augment long-term (4 weeks) hypoxia in xenografts. Consistent with these, VEGF level in tumor tissues or serum (922.5±59.3 pg/mL vs.349.6±46.5 pg/mL, P< 0.01) was significant higher at early-stage in HAL group than that of in control, but no significant difference at later stage. Notablely, the intratumoral microvessel density (MVD) in HAL group was similar to that of in sham-operation group, regardless of 2 days or 4 weeks after second operation (P> 0.05).
Conclusion:HAL induces dramaticly short-term hypoxia in HCC xenograft and overexpression of VEGF, but has no effect on long-term hypoxia or angiogenesis in tumor. However, angiogenesis is usually regarded as a long-term adaptation to tumor hypoxia [22,46] and other cell signalling activated by short-term hypoxia after HAL might contribute to the enhanced metastatic potential of HCC.
Epithelial-mesenchymal transition (EMT) is an initial step of metastatic cascades and plays a critical role in tumor progression[26-28]. Reduction of adhesion ability and enhanced invasiveness during EMT may facilitate cancer cells falling off from primary tumor and invading into vascular or lymphatic vessels. We here investigated whether hepatic artery occlusion induced the changes consistent with EMT in the residual HCC cells. Also the relevance between EMT and hypoxia was examined in this study.
Results:Western blot and immunostaining of xenograft tissues showed that HAL induced EMT-related molecular changes, characterized by the loss of epithelial molecule E-cadherin as well as upregulation of N-cadherin, as the mesenchymal marker. Also the EMT-related transcription factors such as Snail, Slug and Twist were increased in HAL-treated mice, compared with those of sham-operated mice. Similar results were also appeared in peritumoral tissues and associated with hypoxia due to HAL. In vitro, four days after initiation of hypoxia, the morphologies of MHCC97 and HepG2 cells were altered from a typical epithelial cobblestone appearance to an elongated/irregular fibroblastoid shape. Accompanying these phenotypic changes, western blot demonstrated a reduction of E-cadherin expression in these hypoxic cells, relative to the normoxic controls. At the same time, the levels of the mesenchymal cell markers vimentin and N-cadherin were increased. Moreover, qRT-PCR showed a trend toward increase of the transcription factors Snail, Slug and Twist mRNA in hypoxic cells with respect to their controls.
Conclusion:Hepatic arterial occlusion induces intratumoral hypoxia and subsequently contributes to enhanced metastatic potential of residual HCC by eliciting EMT.
Consistent with the above findings, growth of HCC cells and xenografts were unexpectedly suppressed by hypoxia in vivo and in vitro models. Because cell proliferation is the most important mechanism of HCC progression, it is hard to explain that HAL inhibited cell proliferation, but concomitantly, enhanced metastatic potential. Wnt/β-catenin pathway plays a dominated role in HCC proliferation andβ-catenin is the chief downstream effector of this pathway. It has been reported that one third of HCCs are associated with aberrant expression ofβ-catenin. Given the significance ofβ-catenin in HCC biology and the well-characterized association betweenβ-catenin and proliferation, it is essential to investigate the role ofβ-catenin in hypoxia.
Results:Hypoxia induced a pronounced increase in totalβ-catenin levels in PLC/PRF/5 and HepG2 cells (> 2.3-fold), as well as intracellular translocation ofβ-catenin (> 1.7-fold) in Hep3B and MHCC97 cells. The elevatedβ-catenin level in HCC cells was attributed to downregulation of GSK-3βexpression and proteasome inhibition. In hypoxic HCC cells,β-catenin interacted HIF-1α. Knockdownβ-catenin by shRNA reduced HIF-1αprotein level, whereas silencing HIF-1αfailed to affect the expression ofβ-catenin. Loss ofβ-catenin in HCC cells suppressed hypoxia-induced metastatic potential, which was associated with the inhibition of hypoxia-triggered EMT. Positive expression ofβ-catenin in HCC TMA coincided with expression of HIF-1α(P= 0.034; Pearson'sχ2 test, P= 0.013), and co-expression ofβ-catenin and HIF-1αin HCC was correlated with shorter overall survival and time to recurrence.
Conclusion:P-Catenin in HCC is activated by hypoxia and regulates EMT in hypoxic cells, contributing to enhanced metastatic potential.
Consistent with the above findings, hypoxia-induced EMT orβ-catenin aberrant activation in HCC contribute to enhanced metastatic potential. It provides the promising targets for sensitization of HAL. PI3K selective inhibitor, LY294002, has been demonstrated to suppress hypoxia-induced EMT and interferon-a (IFN-a) was reported to reduceβ-catenin expression in cells. We thus investigated the effects of LY294002 or IFN-a on enhanced metastastic potential of HCC due to hepatic arterial occlusion, respectively.
Results:HAL inhibited tumor growth (2.0±0.3 cm3 vs.3.9±0.8 cm3, P< 0.05), but promoted pulmonary metastatsis (83% vs.33%, P< 0.05). HAL combined with LY294002 (100μg/g body weight) repressed significantly enhanced distant metastasis by HAL alone (P< 0.05) and prolonged survival. IFN-αincreased synergistically the therapeutic effects of HAL in dose-dependent manner. The maximum effect on tumor growth, lung metastases and life-span was achieved by 1.5×107 U/kg of IFN-a. Moderate-dose IFN-α(7.5×106 U/kg) suppressed pulmonary metastasis in HAL-treated mice as well as prolonged survival, but it failed to inhibit tumor growth. Western blot of tumor tissues showed that both LY294002 or IFN-a reversed hypoxia-induced EMT. These significances were further demonstrated by the in vitro response of hypoxic cells to the both agents.
Conclusion:Inhibition of EMT orβ-catenin aberrant expression in hypoxic HCC cells could repress enhanced metastastic potential due to hepatic arterial occlusion.
引文
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