Yu Xiaohui, Liu Ling, Cai Bin, He Yinyan, Wan Xiaoping
Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated First People's Hospital, No. 85 Wujin Road, Shanghai 200080, China.
Cancer Sci. 2008 Mar;99(3):543-52. doi: 10.1111/j.1349-7006.2007.00722.x. Epub 2008 Jan 14.
TrkB is a neurotrophic tyrosine kinase receptor (Trk). To investigate its role in anoikis suppression in human ovarian cancer, we used reverse transcription-polymerase chain reaction and real-time polymerase chain reaction, immunohistochemistry, and western blotting to compare the expression levels of TrkB and its ligand brain-derived neurotrophic factor between (i) 20 epithelial ovarian cancers, their multicellular spheroids in ascites or great omentum metastatic lesions, and eight borderline or benign ovarian tumors, as well as four normal ovarian tissues; and (ii) three ovarian cancer cell lines cultured under different conditions: monolayer adhesive culture (adhesive cells), anchorage-independent culture (cell spheroids), and trypsinized cell spheroids placed in monolayer adhesive dishes (cell spheroids replaced). TrkB and brain-derived neurotrophic factor were overexpressed in epithelial ovarian cancers, and full-length TrkB was more often overexpressed in high-grade carcinomas and multicellular spheroids in ascites. Expression of TrkB mRNA was higher in OVCAR-3 cell spheroids than in adhesive cells. The expression of full-length TrkB protein was highest in OVCAR-3 cell spheroids, but its precursor was expressed highly in OVCAR-3 cells under all three culture conditions. The relationship between TrkB overexpression and phosphatidylinositol 3'-kinase (PI3K)-AKT pathway activation in OVCAR-3 cells was studied by western blotting and RNA interference. The PI3K-AKT pathway was highly activated in anoikis-survived cells and was inhibited when TrkB was silenced by small interfering RNA. Finally, the chemosensitivity and invasiveness of OVCAR-3 cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, fluorescence-activated cell sorting, Matrigel invasion assay, and in vivo studies. Adhesive cells showed higher chemosensitivity and lower invasion ability than anoikis-survived cells. Our study suggests that TrkB might mediate anoikis suppression by activating the PI3K-AKT pathway in ovarian cancer cells.
TrkB是一种神经营养性酪氨酸激酶受体（Trk）。为了研究其在人卵巢癌失巢凋亡抑制中的作用，我们采用逆转录-聚合酶链反应和实时聚合酶链反应、免疫组织化学及蛋白质印迹法，比较了（i）20例上皮性卵巢癌、其腹水或大网膜转移灶中的多细胞球体、8例交界性或良性卵巢肿瘤以及4例正常卵巢组织之间TrkB及其配体脑源性神经营养因子的表达水平；以及（ii）在不同条件下培养的三种卵巢癌细胞系：单层贴壁培养（贴壁细胞）、非锚定依赖性培养（细胞球体）以及置于单层贴壁培养皿中的胰酶消化细胞球体（细胞球体替代）。TrkB和脑源性神经营养因子在上皮性卵巢癌中过表达，全长TrkB在高级别癌及腹水中的多细胞球体中更常过表达。TrkB mRNA在OVCAR-3细胞球体中的表达高于贴壁细胞。全长TrkB蛋白在OVCAR-3细胞球体中的表达最高，但其前体在所有三种培养条件下的OVCAR-3细胞中均高表达。通过蛋白质印迹法和RNA干扰研究了OVCAR-3细胞中TrkB过表达与磷脂酰肌醇3'-激酶（PI3K）-AKT途径激活之间的关系。PI3K-AKT途径在失巢凋亡存活细胞中高度激活，当用小干扰RNA使TrkB沉默时受到抑制。最后，通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑、荧光激活细胞分选、基质胶侵袭试验及体内研究检测了OVCAR-3细胞的化学敏感性和侵袭性。贴壁细胞比失巢凋亡存活细胞表现出更高的化学敏感性和更低的侵袭能力。我们的研究表明，TrkB可能通过激活卵巢癌细胞中的PI3K-AKT途径介导失巢凋亡抑制。
