探討 Semaphorin 6A 與 FADD 之交互作用及其應用於肺癌治療上之潛力

Abstract

在台灣，肺癌是所有癌症中致死率最高的。目前治療肺癌的方式主要是透過化學療法和放射療法，但是這些治療方式皆會產生嚴重的副作用。從本實驗室先前研究中發現肺癌細胞的Semaphorin 6A (SEMA6A) 蛋白表現量比正常肺細胞來的低，並且將SEMA6A大量表現在肺癌細胞株中， SEMA6A 會誘發細胞凋亡。因此本研究在探討SEMA6A是否會透過與死亡受體 (Death receptor) 交互作用，啟動細胞凋亡訊號傳遞，造成肺癌細胞死亡。實驗結果顯示在 H1299 肺癌細胞株中，當FAS，死亡受體5或腫瘤壞死因數受體1等死亡受體在被抑制的情況下，表現 SEMA6A 細胞質片段仍會誘發細胞凋亡，同時在實驗室先前研究中發現，FADD在被抑制的情況下，SEMA6A 細胞質片段誘發的細胞凋亡會減少。顯示SEMA6A 並不是與死亡受體交互作用，而主要是透過與 FADD 交互作用誘導細胞凋亡。本篇研究深入探討SEMA6A與 FADD 結合區域。在 HEK293T 細胞株中大量表現不同的 SEMA6A 刪減系列蛋白，進而觀察SEMA6A與 FADD 結合能力，誘發的細胞凋亡以及細胞凋亡標記蛋白 caspase-8的活化情形。實驗結果顯示在 SEMA6A蛋白中的第897~906 胺基酸處的α-螺旋是調控細胞凋亡的重要區域，此外927~1031 胺基酸區域中亦含有調控細胞凋亡的重要區域。瞭解 SEMA6A和FADD 的交互作用區域後，進一步分析 SEMA6A 其他domain之作用後發現，SEMA6A膜外的 SEMA domain會減少SEMA6A細胞質片段與 FADD 的交互作用，進而降低細胞凋亡。因此，我們接著探討SEMA domain是否可以透過影響SEMA6A 與FADD的交互作用，抑制正常細胞中放射線或化療藥物治療所活化的 FADD 凋亡訊號進而導致的細胞凋亡，而達到減低副作用的功能。因此利用管柱層析純化出SEMA domain蛋白，並利用有內生性SEMA6A 蛋白表現的神經膠瘤細胞株M059K 進行實驗。細胞先處理SEMA domain蛋白後，再處理化療藥物 Cisplatin或放射線，觀察外加的SEMA domain蛋白是否可以降低Cisplatin或放射線誘發的細胞毒性。初步的實驗結果顯示，在處理 Cisplatin或照射放射線後，SEMA domain對於細胞的生長、凋亡以及存活情形在統計上沒有呈現顯著的差異。因此 SEMA domain 對於降低化療藥物或是放射線誘發的細胞毒性上的應用仍須更多研究。

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.