Part A. 第三號誘餌受體經由PI3K/Akt/NF-kappa B的訊號路徑調控在AsPC-1人類胰臟癌細胞株的表現之研究 Part B. Denbinobin 藉由Src 去活性化抑制乳癌細胞移行及轉移作用之研究

Abstract

Part A Many cancers develop different means of escaping destruction by the immune system, such as resistance to FasL-Fas interaction-mediated apoptotic signals. Decoy receptor 3 (DcR3), a soluble receptor for FasL, is highly expressed in cancer cells and plays a significant role in immune suppression and tumor progression. However, how DcR3 expression is modulated is unclear. In this study, immunoprecipitation and ELISA assays using human pancreatic cancer cells showed the presence of high levels of DcR3 protein in AsPC-1 cells, but not in PANC-1 cells. Treatment with herbimycin A (a tyrosine kinase inhibitor), LY294002 or wortmannin (PI3K inhibitors), PDTC (an NF-kappa B inhibitor), or AG1024 (an IGF-1 inhibitor) significantly reduced endogenous DcR3 levels in AsPC-1 cells. Furthermore, transfection of AsPC-1 cells with Akt or I-kappa-B dominant negative plasmids also markedly reduced DcR3 levels. In contrast, 48 h transfection of PANC-1 cells with a constitutively active Akt induced DcR3 expression. Flow cytometry assays indicated that apoptosis was not seen in AsPC-1 cells incubated with sFasL or membrane-bound FasL, but was seen when DcR3 siRNA-transfected AsPC-1 cells underwent the same treatment. In addition, PANC-1 cells incubation with conditioned medium from AsPC-1 cells transfected with dominant negative Akt or I-kappa-B plasmids or DcR3 siRNA showed increased sFasL-mediated apoptosis compared to the control group. Our results show that IGF-1-induced activation of the PI3K/Akt/NF-kappa B signaling pathway is involved in the modulation of endogenous DcR3 expression in AsPC-1 cells and that reducing endogenous DcR3 levels increases FasL-induced apoptosis of human pancreatic cancer cells. Part B Breast cancer is the most common malignancy of women, and the highly metastasis ability of breast cancer is now a major problem of therapy. Src is a non receptor tyrosine kinase protein, plays an important role in tumor progression such as cell survival, adhesion, motility, invasion and metastasis, and researchers found that Src is overexpressed and activated in breast cancer. Recently, Src becomes a target of anti-breast cancer therapy. Denbinobin, a bioactive compound isolated from E. lonchophylla, has been reported to induce cell death in many cancer cells, and the xenograft model shows that denbinobin has anticancer efficacy in vivo. Src kinase activity is elevated in many cancers, including breast cancer, and is often associated with progressive disease. In this study, we examined the anti-metastatic effect of denbinobin. Using EGF to induce Src and downstream protein activation in breast cancer cells, denbinobin reduced the phosphorylation of Src and downstream signals such as FAK, CAS130 and paxillin, whereas this effect could be prevented by constant active of Src kinase. Furthermore, denbinobin inhibits Src kinase activity and breast cancer cell migration in a dose-dependent manner. Then we labeled cells with FITC-antibodies against the phosphorylation Src, FAK and paxillin, and found the immunofluorescence intensity of phosphorylated Src decreases significantly. In addition, the 4T1/luc metastatic mouse model revealed antimetastatic activity effect of denbinobin, and we further proved that Src/FAK pathway mediated the effect of denbinobin in vivo. In summary, our study demonstrates that denbinobin inhibits breast cancer cell migration, reduces the Src/FAK signaling pathway with no changes in cell proliferation, and shows in vivo efficacy in the mouse metastatic model to prevent tumor migration. Our results show that, acting as a potent Src inhibitor, denbinobin inhibits signaling pathways involved in controlling breast cancer migration and metastasis, suggesting that it has therapeutic potential in breast cancer treatment.