To study the interactions between FADD and Semaphorin 6A and its potential applications to lung cancer therapy

• Main Authors: Gao-Ming Chang, 張高銘
• Other Authors: Eric Y. Chuang
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/438cg2

碩士 === 國立臺灣大學 === 生命科學系 === 105 === In Taiwan, the lung cancer has the highest mortality rate in all cancers. Although chemotherapy and radiation therapy are the main treatments of lung cancer, these therapies usually induce severe side effects. Our previous research has shown that the levels of Semaphorin 6A (SEMA6A) were lower in the lung cancer tissues than in the adjacent normal tissues. Furthermore, we overexpressed SEMA6A in the lung cancer cells and demonstrated that the cytoplasmic region of SEMA6A could interact with FADD to induce apoptosis. In the present research, we were aiming to study if SEMA6A could interact with death receptors (DRs) to activate apoptotic signaling. Our data showed that the overexpression of the cytoplasmic domain of SEMA6A could promote apoptosis in H1299 following down-regulation of DRs including FAS, DR5 and TNFR1. In the meantime, we have found that silencing of FADD inhibits cytoplasmic domain of SEMA6A induced apoptosis. It suggests that SEMA6A induces apoptosis through the interaction with FADD rather than with DRs. To map the binding region of FADD on SEMA6A, we overexpressed a series of deletion constructs of SEMA6A in HEK293T cells. Then, we determined the cell proliferation, apoptosis rate, and the level of cleaved caspase-8. Our results reveal that the α-helix locating on amino acids 897-906 of SEMA6A and the amino acids 927-1031 of SEMA6A are critical in the regulation of SEMA6A-induced apoptosis. After understanding the interaction region of FADD and SEMA6A, we further analyzed the functions of different domains of SEMA6A. The results indicated that the extracellular domain could reduce apoptosis by suppressing the interaction between the FADD and the intracellular domain of SEMA6A. We subsequently study if the extracellular domain of SEMA6A could reduce the side effect of chemotherapy or radiation therapy by interacting with endogenous SEMA6A to inhibit irradiation or Cisplatin (chemotherapy drug) induced FADD-dependent death signaling in normal lung cells. Therefore, we purified the extracellular domain of SEMA6A by affinity chromatography, and selected M059K, the cell line with the endogenous expression of SEMA6A, for the further study. The M059K cells were cultured with the extracellular domain of SEMA6A followed by the treatments of irradiation or Cisplatin. The results showed that the proliferation, apoptosis and survival were no significant difference between M059K cells cultured with and without the extracellular domain of SEMA6A. Therefore, the further study is still needed to elucidate the application of SEMA domain in the reduction of chemotherapy drugs or radiation-induced cytotoxicity.