| Metastasis involves several discrete steps, each serving to overcome a physiologic barrier against the spread of tumor cells. One such barrier, which is intrinsic to epithelial cells particularly, is anoikis, or apoptosis induced by unfamiliar (or loss of) cell adhesion signals. Anoikis provides protection against the metastatic spread of tumor cells. TrkB, a neurotrophic tyrosine kinase receptor,serves as a potent and specific suppressor of caspase-associated anoikis of non-malignant epithelial cells by activating the phosphatidylinositol-3-OH kinase/protein kinase B pathway. TrkB overexpression has been found in neuroblastoma, pancreatic and prostate carcinomas, and some other solid and lymphoid cancers. The PI3K pathway acts as an integrator of multiple inputs during tumorigenesis, by regulating mitochondria-dependent pathway of apoptosis which control the release of cytochrome c. High activation of PI3K pathway that increased expression of pro-apoptotic proteins and decreased expression of anti-apoptotic proteins was found in tumor cells. The inhibition of cell death is a crucial characteristic of metastatic cancer cells, metastatic cancer cells could be expected to display increased chemotherapy resistance. Thus, chemotherapy-resistance and metastasis may be the results of suppression of apoptosis in cancer cells, which controlled by the same mechanism.Unlike normal epithelial cells, carcinomas grow, invade and metastasize as disorganized three-dimensional cellular spheroids in which cells are deprived of adhesion to the basement membrane but remain anoikis-resistance. Resistance to anoikis may allow survival of cancer cells during systemic circulation, thereby facilitating secondary formation in distant, and now has been thought to be critical for the progression of the disease and could therefore serve as a carcinoma-specific novel therapeutic target.In this study we revealed that ovarian cancer cells (in monolayer adhesive culture) imitated the status of cancer cells in primary lesions. Multicellular spheroids (in anchorage-independent culture) imitated the status of cancer cells deprived of ECM and surviving in ascites, and ovarian cancer cells (multicellular spheroids were trypsinized and replaced in monolayer adhesive dishes) imitated the the status of cancer cells in matastatic lesions, both of them were survived from anoikis.In the first part of our research, we detected the expression of TrkB and its ligand BDNF in ovarian cancer especially in anoikis-survived ovarian cancer cells, and the results showed that there might be a TrkB and BDNF autocrine-loop in ovarian epithelial cancer. In condition of anoikis- suppression, basing on overexpression of TrkB protein precursor, the expression of full-length TrkB protein increased and TrkB could auto-activate, which was independent on BDNF stimulation.In the second part we investigated the relationship between TrkB overexpression and activation of PI3K/AKT pathway, and the results showed that the overexpressed TrkB could activate PI3K/AKT pathway and mediate anoikis suppression in ovarian cancer.In the third part we studied the mechanism of anoikis-suppression mediated by TrkB in human ovarian cancer, and the results showed that high activated PI3K/AKT pathway by overexpressed TrkB, through increasing the expression of Bcl-2 family, induced anoikis/apoptosis suppression in human ovarian cancer, promoting metastasis and chemotherapy-resistanceOn the summary, we could conclude that TrkB could act as a anoikis-suppressor in human ovarian cancer. Under anoikis-suppression condition, the full-length TrkB expression increased and auto- phorsphorylation took place. With the PI3K/AKT activation mediated by TrkB overexpression, the ovarian cells showed high ability of metastasis and chemotherapy-resistance. Part I: Expression and significance of anoikis-suppressor TrkB in human epithelial ovarian cancerObjective: To detect the expression and significance of anoikis- suppressor TrkB in human epithelial ovarian cancer.Methods: The expression of TrkB and its ligand BDNF was evaluated in epithelial ovarian cancer specimens and OVCAR-3 ovarian cancer cells cultured under different conditions by RT-PCR and real-time PCR, Immunohist-chemistry and Western blot.Results: TrkB and BDNF mRNA were overexpressed in epithelial ovarian cancer specimens especially in greater omentum metastatic lesions and multicellular spheroids in ascites. Theses ratios of TrkB or BNDF toβ-actin were 31.43±1.41% and 97.56±1.50%, 28.20±0.72% and 73.51±0.51% respectively, compared with corresponding lesions that were 18.06±1.09% and 39.66±0.59% (P<0.001). In addition, and there was a significant difference between these ratios of epithelial ovarian cancer specimens and borderline epithelial ovarian tumors, 11.71±2.12% and 21.17±0.76% versus 4.59±0.40% and 12.60±0.88% respectively (P<0.001) TrkB mRNA was more overexpressed in multicellular spheroids than that in OVCAR-3 cells under monolayer adhesive culture, 35.29±0.67% versus 23.51±0.51%; but BDNF mRNA was the opposite by RT-PCR, 41.42±0.58% versus 32.23±0.70% (P<0.001). The results of RT-PCR above were confirmed by real-time PCR analysis. The Full-length TrkB protein (glycosylated receptor, 145 000) was more often overexpressed in multicellular spheroids in ascites and high grade carcinomas than that in corresponding primary carcinoma and low grade carcinomas, but full-length TrkB protein precursor (non-glycosylated receptor) was overexpressed extensively in epithelial ovarian cancer specimens by IHC. Full-length TrkB more often was overexpressed in OVCAR-3 multi-cellular spheroids (in anchorage-independent culture) compared with OVCAR-3 cells (in monolayer adhesive culture) (P<0.001), and full-length TrkB protein precursor (about 115 000) was highly overexpressed in OVCAR-3 ovarian cancer cells under different culture conditions without statistical difference by western blot.Conclusion: There may be a TrkB and BDNF autocrine-loop in ovarian epithelial cancer. Under anoikis-suppression condition, basing on overexpression of TrkB protein precursor, expression of full-length TrkB protein increased and consequently can auto-activated. TrkB may acts as a anoikis-suppressor in human ovarian cancer.Part II: Tyrosine kinase receptor B promoted activation of PI3K/AKT pathway and mediate anoikis suppression in ovarian cancer cellsObjective: To investigate the relationship between TrkB overexpress ion and activation of PI3K/AKT pathway, and if TrkB mediate anoikis suppression in ovarian cancer.Methods: Western blot and RNA interference were applied to investigate the difference of full-length TrkB expression and activation of PI3K/AKT pathway in OVCAR-3 ovarian cancer cells cultured in different conditions, and the difference of anoikis in OVCAR-3 cells when TrkB was silenced by siRNA.Results: OVCAR-3 multicellular spheroids (in anchorage-indepen dent culture) displayed the highest full-length TrkB expression and activation of PI3K/AKT pathway, while the OVCAR-3 cells (multicellular spheroids were trypsinized and replaced in monolayer adhesive dishes) and the OVCAR-3 cells (in monolayer adhesive culture) were lower than OVCAR-3 multicellular spheroids (P<0.001). Activation of PI3K/AKT pathway was inhibited, and anoikis was increased in OVCAR-3 cells when TrkB was silenced (P<0.001).Conclusion: TrkB overexpression could activate PI3K/AKT signal pathway and mediate anoikis suppression in human ovarian cancer cells. .PartⅢ:Mechanism of anoikis-suppression induced by TrkB in human ovarian cancerObjective: To study the mechanism of anoikis-suppression induced by TrkB in human ovarian cancer.Methods: Western blot, RNA interference, MTT and FACS were applied to investigate the expression of Bcl-Xl and Bad in OVCAR-3 ovarian cancer cells cultured under different conditions and treated with paclitaxel and cisplatin with or without TrkB silenced were studied. Additionally, we detected the difference of chemosensitivity of OVCAR-3 cells among different culture conditions, and with or without LY294002 combined. The invasion ability of OVCAR-3 cultured in different conditions and with or without TrkB silenced by RNAi was also investigatd by Matrigel invasion assay and In vivo studies.Results: Multicellular spheroids presented higher expression of Bcl-Xl and Bad than OVCAR-3 cells in monolayer adhesive culture (P<0.001). The expression of Bcl-Xl and Bad was reduced when TrkB silenced combined with chemotherapeutic agents (paclitaxel or cisplatin) treatment in OVCAR-3 cells in monolayer adhesive culture (P<0.001). When OVCAR-3 cells treated with paclitaxel, the expression of Bcl-Xl and Bad in OVCAR-3 multicellular spheroids was higher than the expression in OVCAR-3 cells (in monolayer adhesive culture), while treated with cisplatin the result was opposite (P<0.001). Compared with OVCAR-3 cells in monolayer adhesive culture, OVCAR-3 cells of multicellular spheroids trypsinized and replaced in monolayer adhesive dishes again showed the higher chemotherapy-resistance to paclitaxel and cisplatin (P<0.001). Survival rate was decreased (P<0.01), but apoptotic rate increased significantly (P<0.001) in OVCAR-3 cells when treated with LY294002 and paclitaxel than treated with paclitaxel only (P<0.001). OVCAR-3 multicellular spheroids showed the most strong invasion ability among OVCAR-3 cells cultured in three conditions, and the metastasis of OVCAR-3 cells was attenuated whatever TrkB was silenced In vitro and in vivo (P<0.001 and P<0.03).Conclusion: TrkB could act as an anoikis-suppressor in human ovarian cancer. In condition of anoikis-suppression, the full-length TrkB expression increased and auto-phorsphorylation took in ovarian cancer cells, with the PI3K/AKT activation promoted by TrkB overexpression, the ovarian cells showed strong ability of chemotherapy-resistance and metastasis...
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