Fransvea Emilia, Angelotti Umberto, Antonaci Salvatore, Giannelli Gianluigi
Department of Internal Medicine, Immunology and Infectious Diseases, Section of Internal Medicine, University of Bari Medical School, Bari, Italy.
Hepatology. 2008 May;47(5):1557-66. doi: 10.1002/hep.22201.
Hepatocellular carcinoma (HCC) treatment is challenging because the mechanisms underlying tumor progression are still largely unknown. Transforming growth factor (TGF)-beta1 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-beta1 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 microM. 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 10microM, 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 microM. 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-beta1 in the presence or absence of LY2109761. E-cadherin expression was reduced by TGF-beta1 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).
LY2109761 blocks migration and invasion of HCC cells by up-regulating E-cadherin, suggesting that there could be a mechanistic use for this molecule in clinical trials.
肝细胞癌（HCC）的治疗具有挑战性，因为肿瘤进展的潜在机制仍大多未知。转化生长因子（TGF）-β1被认为是HCC肿瘤发生中的关键分子，因为患者血清和尿液中其水平升高与疾病进展相关。本研究的目的是研究TGF-β信号传导的抑制及其对HCC进展的影响。用人HCC细胞系用TGF-β受体激酶抑制剂（LY2109761）处理，其选择性在激酶测定中确定。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微摩尔的浓度范围内选择性地阻断这种作用并使自分泌的磷酸化Smad-2去磷酸化。在10微摩尔时观察到由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记录的细胞毒性作用，而该药物在浓度高达0.1微摩尔时抑制（P <0.001）HCC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-钙黏蛋白mRNA和蛋白水平。这种增加定位于细胞膜，其中E-钙黏蛋白介导细胞间依赖的锚定。一致地，一种抑制E-钙黏蛋白依赖性细胞间接触的功能性单克隆抗体恢复了迁移和侵袭活性。最后，将来自7名患者的非转移性HCC组织在有或没有LY2109761的情况下用TGF-β1培养。通过对显微切割组织进行定量实时PCR以及对连续切片进行免疫组织化学测定，TGF-β1降低了E-钙黏蛋白的表达，而LY2109761处理使其显著增加（P <0.0001）。在72名患者中，E-钙黏蛋白组织表达在转移性HCC中比在非转移性HCC中表达更弱（P <0.0001）。
LY2109761通过上调E-钙黏蛋白来阻断HCC细胞的迁移和侵袭，表明该分子在临床试验中可能有机制性用途。
