泌蛋白質。當內質網受到破壞後，會有大量折疊錯誤的蛋白質聚集或鈣 離子通透失衡，會產生所謂的內質網壓力 (ER stress)25。當 ER stress 時，
GRP78 (glucose-regulated protein 78) 和 GADD153 (growth arrest and DNA damage inducible gene 153) 這兩種基因會大量表現68。有文獻提及，
GADD153 能抑制 Bcl-2 及促進 Bax 的表現以調控細胞凋亡66，因此鈣 (Intrinsic pathway) 以及內質網壓力 (Endoplasmic reticulum stress, ER stress )17。內在路徑細分為凋亡蛋白酶依賴 (caspase dependent) 路徑以即 凋亡蛋白非依賴 (caspase independent) 路徑。凋亡蛋白酶依賴路徑主要 受到 Bcl-2 家族的調控，因其結合至粒線體膜上影響膜電位，使粒線體 功能喪失進而釋放 cytochrome c，並與 caspase-9 結合，活化 caspase-3 而走向細胞凋亡；凋亡蛋白非依賴路徑主要是因 Bcl-2 蛋白家族結合至 粒線體膜上形成孔洞，導致粒線體間質釋出 apoptosis-inducing factor
(AIF) 和 endonuclease G (Endo G) 至細胞質中，進而轉位進入細胞核 內，造成 DNA 受損及斷裂，最後走向細胞凋亡69。這些蛋白的釋放被認 為與粒線體膜上稱為 mitochondrial permeability transition pore (MPTP) 的巨大通道有關，有研究顯示， MPTP 的開啟被認為是使粒線體膜電位 下降的主要因素之一。在本研究中，NCI-H460 細胞經魚藤素加藥處理 後，可活化促凋亡相關蛋白 (Bid, Bax, Bak) 和抑制抗凋亡蛋白 (Bcl-2, Bcl-X) 的表現 (圖十八)，至使粒腺體膜電位下降 (圖十七) ，釋出 AIF 和 cytochrome c (圖十八)，cytochrome c, Apaf-1 和 caspase-9 結合形成凋 亡體，活化caspase-3 (圖十八)，且利用 CaspaLux kit 偵測 caspase-3 的 活性，亦也證實 caspase-3 之活性明顯上升 (圖二十)；另一方面， AIF 轉 位至細胞核中造成 DNA 受損引發細胞凋亡 (圖十九)。以上結果證實魚 藤素能經由粒線體內在路徑誘導 NCI-H460 細胞凋亡。
第五章 結論
魚藤素能有效抑制人類非小細胞肺癌 NCI-H460 細胞株的增生，造成 細胞死亡而降低細胞存活率；並且誘發細胞凋亡和 DNA 受損，致使磷 脂絲胺酸蛋白外翻 , DNA 斷裂和染色質濃縮形成凋亡小體。
經由西方墨點法和免疫螢光染色法證實，魚藤素會抑制 p-AKT 蛋白 表現，促進 BAD 的活化，並且使之從細胞質轉位至粒腺體外膜上，與 Bcl-2 或 Bcl-X 結合，進而影響促細胞凋亡相關蛋白 (Bid, Bax, Bak) 和 抑制抗凋亡蛋白 (Bcl-2, Bcl-X) 的表現，至使粒腺體膜電位下降，釋出 AIF 和 cytochrome c。cytochrome c, Apaf-1 和 caspase-9 結合形成凋亡 體，活化caspase-3，最後誘發細胞凋亡；然而，AIF 轉移至細胞核中造 成 DNA 受損。
總觀以上結果，我們認為魚藤素會引起人類非小細胞肺癌 NCI-H460 細胞株之粒腺體功能喪失並誘導細胞經由 Akt 和 Bad 相關粒腺體內在 路徑走向細胞凋亡。
圖二十七 魚藤素誘導人類非小細胞肺癌 (NCI-H460) 細胞凋亡路徑圖
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