| 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.
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