Blocking transforming growth factor-beta up-regulates E-cadherin and reduces migration and invasion of hepatocellular carcinoma cells
Blocking transforming growth factor-beta up-regulates E-cadherin and reduces migration and invasion of hepatocellular carcinoma cells
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DOI：
10.1002/hep.22201
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发表时间：
2008-05-01
期刊：
影响因子：
13.5
通讯作者：
Giannelli, Gianluigi
中科院分区：
文献类型：
--
作者：
Fransvea, Emilia;Angelotti, Umberto;Giannelli, Gianluigi
肝细胞癌（HCC）的治疗具有挑战性，因为肿瘤进展的潜在机制在很大程度上仍然未知。转化生长因子（TGF）-β1被认为是肝细胞癌发生过程中的一个关键分子，因为患者血清和尿液中该物质水平升高与疾病进展相关。本研究的目的是探讨TGF -β信号通路的抑制及其对肝细胞癌进展的影响。用人TGF -β受体激酶抑制剂（LY2109761）处理人肝细胞癌细胞系，该抑制剂的选择性通过激酶测定来确定。外源性TGF -β1使TGF -β受体磷酸化，从而激活Smad - 2，而该药物在0.001至0.1 μM的浓度范围内选择性地阻断这种作用，并使自分泌的p - Smad - 2去磷酸化。通过3 - [4,5 - 二甲基噻唑 - 2 - 基] - 2,5 - 二苯基四氮唑溴盐（MTT）、台盼蓝和碘化丙啶染色试验证实，在10 μM时观察到细胞毒性作用，而在浓度高达0.1 μM时，该药物抑制（P < 0.001）肝细胞癌细胞在纤连蛋白、层粘连蛋白 - 5和玻连蛋白上的迁移以及通过基质胶的侵袭（P < 0.001）。LY2109761上调（P < 0.001）E - 钙粘蛋白的mRNA和蛋白质水平。这种增加定位于细胞膜，在细胞膜上E - 钙粘蛋白介导依赖细胞 - 细胞的锚定。一致地，一种抑制E - 钙粘蛋白依赖性细胞 - 细胞接触的功能性单克隆抗体恢复了迁移和侵袭活性。最后，将7名患者的非转移性肝细胞癌组织在有或无LY2109761的情况下用TGF -β1培养。通过对显微切割组织进行实时定量PCR和对连续切片进行免疫组织化学检测，发现TGF -β1降低了E - 钙粘蛋白的表达，而LY2109761处理显著（P < 0.0001）增加了E - 钙粘蛋白的表达。在72名患者中，转移性肝细胞癌中E - 钙粘蛋白的组织表达比非转移性肝细胞癌中更弱（P < 0.0001）。结论：LY2109761通过上调E - 钙粘蛋白来阻断肝细胞癌细胞的迁移和侵袭，这表明该分子在临床试验中可能具有机制上的用途。
Hepatocellular carcinoma (HCC) treatment is challenging because the mechanisms underlying tumor progression are still largely unknown. Transforming growth factor (TGF)-beta 1 is considered a crucial molecule in HCC tumorigenesis because increased levels of patients' serum and urine are associated with disease progression. The aim of the present study was to investigate the inhibition of TGF-beta signaling and its impact on HCC progression. Human HCC cell lines were treated with a TGF-beta receptor kinase inhibitor (LY2109761) whose selectivity was determined in a kinase assay. Exogenous TGF-beta 1 phosphorylates the TGF-beta receptor, consequently activating Smad-2, whereas the drug selectively blocks this effect and dephosphorylates autocrine p-Smad-2 at concentrations ranging from 0.001 to 0.1 mu M. A cytotoxic effect documented by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), trypan blue, and propidium iodide staining assays was observed at 10 mu M, whereas the drug inhibits (P < 0.001) the migration of HCC cells on fibronectin, laminin-5, and vitronectin and invasion through Matrigel (P < 0.001) at concentrations up to 0.1 mu M. LY2109761 up-regulates (P < 0.001) E-cadherin mRNA and protein levels. This increase was localized at the cellular membrane where E-cadherin mediates anchorage that is cell-cell dependent. Consistently, a functional monoclonal antibody that inhibits E-cadherin-dependent cell-cell contact restores the migratory and invasive activity. Finally, nonmetastatic HCC tissues from 7 patients were cultured with TGF-beta 1 in the presence or absence of LY2109761. E-cadherin expression was reduced by TGF-beta 1 and was significantly (P < 0.0001) increased by LY2109761 treatment, measured by quantitative real-time PCR on microdissected tissues and by immunohistochemistry on serial sections. In 72 patients, E-cadherin tissue expression was more weakly expressed in metastatic than in nonmetastatic HCC (P < 0.0001). Conclusion: LY2109761 blocks migration and invasion of HCC cells by upregulating E-cadherin, suggesting that there could be a mechanistic use for this molecule in clinical trials.