| Summary: | 碩士 === 輔仁大學 === 基礎醫學研究所碩士班 === 101 === Tumor metastasis is a complicated process with multiple stages. During the initial stage of metastasis upon detachment from primary site, the tumor cells are kept in anchorage-independent condition. In order to accomplish successful metastasis, the tumor cells must be resistant to detachment-induced apoptosis, so-called anoikis resistance. Melanoma is one kind of skin cancers with high metastatic ability, poor prognosis, high mortality, and resistance to chemotherapy. Possibility some key proteins or intracellular molecular mechanisms are changed under dissemination or anchorage-independent stress. The understanding of molecular mechanisms for anchorage-independent melanoma to exhibit anoikis resistance, would be beneficial to treat melanoma.
In this study, we found the detachment of different human melanoma cells conferred the characteristics of anoikis resistance - no significant caspase activation and substrate PARP cleavage. Syndecan-2 is the transmembrane protein and associates with metastatic potential of melanoma cells. By using qPCR and western blot analysis, we found the upregulation of syndecan-2 under suspension condition, which is regulated by PKCδ. In order to investigate the importance of syndecan-2, we manipulated syndecan-2 expression level in melanoma A375 cells by transfection of the syndecan-2 construct to overexpress syndecan-2; and transfection of shRNAs to reduce syndecan-2 expression. Although the PARP cleavage remained unchanged upon syndecan-2 overexpression or downregulation, the increased in syndecan-2 expression did suppress caspase-8 activation, which determined their anoikis resistance.
To identify the potential kinase and signaling pathways involved, western blot and cDNA microarray analysis were used. We found the hyper-phosphorylation of specific kinases, such as ERK, PI3K, Akt, JNK, and GSK-3β, in anchorage-independent melanoma cells, which might correlated with cell survival and anoikis resistance. The increase in syndecan-2 expression did increased ERK phosphorylation and GSK-3β phosphorylation at serine9, which all contributed to anoikis resistance of melanoma cells. However, increase in syndecan-2 expression under anchorage-independency did not result in hyper-phosphorylation of Akt and PI3K.
In addition, we use camptothecin to induced DNA damage and cell apoptosis. Under the camptothecin treatment, anchorage-independent melanoma cells caused higher level of cell apoptosis then adherent melanoma cells. This indicated the anchorage-independent condition promoted the sensitivity toward chemotherapy agents. Interestingly, the increased syndecan-2 expression in melanoma cells under anchorage-independency sensitized the camptothecin-induced apoptosis. Treatments of specific kinase inhibitors demonstrated that ERK phosphorylation and GSK-3β phosphorylation at serine9 protected cells from camptothecin-induced apoptosis; while PI3K phosphorylation sensitized cells to camptothecin-induced apoptosis.
In summary, syndecan-2 was upregulated in human melanoma cells under anchorage-independency, which suppressed caspase-8 activation, increased ERK phosphorylation and GSK-3β phosphorylation at serine9, and achieved anoikis resistance. However, this syndecan-2 upregulation, as well as PI3K activation, sensitized camptothecin-induced apoptosis. The role of syndecan-2 in anoikis of human melanoma cells might act as "double-edged sword" to protect from and sensitize to apoptosis.
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