靈芝免疫調節蛋白誘發肺癌細胞自體吞噬與輔助療法應用之機制探討

細胞凋亡、細胞壞死與細胞自體吞噬是三種主要的程序性細胞死亡方式。細胞自體吞噬是一種細胞自我消化細胞質內物質的過程。免疫調節蛋白是靈芝的有效成分之一，已知具有抗癌的功效。重組靈芝免疫調節蛋白GMI是由小孢子靈芝中所選殖並純化而得。於本研究中，我們發現GMI透過誘發細胞自體吞噬抑制肺癌細胞生長，而非造成細胞凋亡。使用西方點墨法分析，GMI能增加LC3的轉變以及減少p53的表現量，並與GMI的處理劑量及處理時間有正相關的關係。p53過度表現可抑制GMI誘發之細胞自噬。以細胞中鈣離子螯合劑BAPTA-AM證實GMI經由鈣離子訊息傳遞路徑促進細胞產生自體吞噬。我們發現GMI透過抑制Akt/mTOR訊息傳遞路徑活化細胞自噬。透過溶酶體抑制劑Bafilomycin-A1與chloroquine可增加GMI誘發之自噬型細胞死亡。我們發現GMI能抑制V-ATPase次單元ATP6V0A1表現。使用VSV-G pseudotyped lentivirus-shRNA系統抑制ATP6V0A1的表現，發現GMI之毒殺細胞與誘發自噬小體堆積能力明顯增加。以shRNA系統破壞細胞自噬能力後，可顯著回復GMI抑制細胞生長、端粒酶活性及細胞群落生成能力的效果。以3-methyladenine或shRNA系統抑制細胞自噬後，GMI依然無法刺激細胞走向凋亡。利用小鼠腫瘤模式，口服GMI能抑制腫瘤生長並誘發腫瘤產生細胞自噬。此外，我們發現GMI能透過誘發細胞自噬促進cisplatin造成的細胞凋亡，增加肺癌細胞對cisplatin的感受性。本研究提供靈芝免疫調節蛋白抗癌的新機制，證實GMI有潛力成為對抗非小細胞肺癌的化學預防藥劑。

關鍵字

靈芝；免疫調節蛋白；細胞自體吞噬；肺癌

並列摘要

Apoptosis, necrosis, and autophagy are three major types of programmed cel death. Autophagy is a self-digestive process that degrades the cytoplasmic constituents. Immunomodulatory protein, one major bioactive component of Ganoderma, has anti-tumor activity. In this study, recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. We demonstrated that GMI inhibits lung cancer cell proliferation by activating autophagy, but does not induce apoptotic cell death. On western blot, GMI increased LC3 conversion and decreased p53 expression in a time- and concentration-dependent manner. p53 overexpression inhibited GMI mediated autophagy. Cytoplasmic calcium chelator BAPTA-AM was used to prove that GMI promotes autophagy via calcium-mediated signaling pathway. GMI induced autophagy through inhibiting Akt/mTOR pathway. Bafilomycin-A1 and chloroquine, the lysosome inhibitors, enhanced GMI mediated autophagic cell death. We found that GMI inhibits the expression of ATP6V0A1, a subunit of V-ATPase. Using VSV-G pseudotyped lentivirus-shRNA system for ATP6V0A1 silencing, cytotoxicity and autophagosome accumulation induced by GMI were enhanced. The capabilities of GMI to suppress cell growth, telomerase activity, and colony formation were abolished in autophagy-defective cells. GMI did not stimulate apoptosis after blocking of autophagy by 3-MA or shRNA knockdown system. In xenograft studies, oral administration of GMI inhibited the tumor growth and induced autophagy significantly in nude mice that had received a subcutaneous injection of A549 cells. Furthermore, GMI increases susceptibility to cisplatin through inducing apoptosis by autophagy. This study reveals the novel anti-cancer function of GMI. GMI may be a potential chemopreventive agent against non-small cell lung cancer.

並列關鍵字

Ganoderma ； Immunomodulatory Protein ； Autophagy ； Lung cancer

參考文獻

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靈芝免疫調節蛋白誘發肺癌細胞自體吞噬與輔助療法應用之機制探討

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