在肺癌及許多癌症中，激活的磷脂醯肌醇3-激酶 (PI3K)/蛋白激酶B (Akt)訊息傳遞路徑會調控許多細胞功能。LY294002 (LY, 2-嗎啉-4-基-8-苯基-4H-色烯-4-酮)是一種能有效抑制PI3K活性的小分子抑制劑，並且已證實LY294002在活體內與活體外實驗中皆具有抑制腫瘤的良好效果。但其抑制專一性、不佳水溶性和未知的副作用等特性侷限LY294002在生物體上的應用。然而在癌症治療上，LY294002在開發良好有效的藥物時提供了關鍵的角色。聚乳酸聚乙醇酸共聚物 (poly lactic-co-glycolic acid; PLGA)是一種可被生物降解的奈米材料，常被用於藥物傳遞的載體。本研究使用ethanol titration method 和two emulsion method兩種不同的合成方式，將LY294002包裹於無表面活性劑的PLGA中，進而發展出獨特的奈米藥物 (LY294002@PLGA) 以改善LY294002的療效。在先前的研究中，意外地發現LY294002@PLGA能增進細胞死亡的現象與其誘發顯著的內質網壓力(endoplasmic reticulum stress)有關，但目前對於LY294002@PLGA如何誘發內質網壓力和其相關機轉仍待研究。因此本實驗使用四種內質網壓力誘導劑 (tunicamycin、thapsigargin、brefeldin A和DBeQ)處理不同的肺癌細胞 (H460、H157和H1650) 藉此探索其相關作用機制；比較上述的內質網壓力誘導劑、LY294002和LY294002@PLGA對細胞的反應，進而了解內質網壓力所誘導的相關機轉。使用動態光散射儀和高效液相層析儀分析LY294002@PLGA的特性，分析結果顯示，使用ethanol titration method合成的LY294002@PLGA，平均直徑和藥物包裹率為94.02±5.05 nm和6.35±0.03%；而使用two emulsion method合成的LY294002@PLGA，平均直徑和藥物包裹率為174.5±3.29 nm和54.66%±3.81%。經由MTT試驗證實在三株肺癌細胞中，LY294002@PLGA能增進對細胞的毒殺效果，並且PLGA無明顯的細胞毒性。此外，經過LY294002@PLGA處理後的細胞， Akt和ERK的活性能同時且持續地被抑制。Unfolded protein response (UPR)包括ATF6、PERK以及IRE1三條訊息傳遞路徑，誘發不同程度的內質網壓力會活化不同的UPR傳遞路徑趨使細胞存活或走向死亡。三株肺癌細胞分別經過tunicamycin和thapsigargin處理後，所有UPR路徑皆被活化使細胞存活，然而部分的PERK-ATF4-CHOP路徑活化卻趨使細胞走向死亡。Brefeldin A活化IRE1及PERK路徑，並且由於CHOP和磷酸化JNK表現量上升趨使細胞傾向凋亡。而DBeQ只活化PERK路徑增進細胞凋亡。細胞處理LY294002後只會誘導輕微的內質網壓力，但經過LY294002@PLGA處理後，細胞不僅誘發嚴重的內質網壓力產生，更藉由PERK-ATF4-CHOP路徑的活化促使細胞趨向凋亡。與上述的內質網壓力誘導劑比較，只有含缬酪肽蛋白(VCP)的抑制劑DBeQ與LY294002@PLGA具有相似的內質網壓力機制而增進細胞死亡。因此，VCP在LY294002@PLGA所誘發的細胞死亡機制中可能扮演重要的角色。我們的研究證實，LY294002@PLGA由於誘導嚴重的內質網壓力產生及持續地抑制Akt和ERK的活性，達到加強細胞的毒殺效果，並且藉由活化PERK-ATF4-CHOP路徑增進細胞凋亡。在未來癌症研究上，LY294002@PLGA奈米藥物具有藥物開發的潛力。

The phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) signaling pathway has been activated and regulated a variety of cellular processes in many cancers and lung cancer as well. LY294002 is a potent inhibitor of PI3K activity and also has been demonstrated having antitumor activity in vitro and in vivo. While its restrictive bio-application such as poor specificity, solubility, potential side effects and cytotoxicity offer us the key to an understanding of therapeutic efficacy of LY294002. PLGA is a biodegradable nanomaterial that can be used as a favorable carrier for drug delivery. We have developed LY294002-loaded surfactant-free poly lactic-co-glycolic acid nanoparticles (LY294002@PLGA) synthesized by two methods, ethanol titration and two emulsion, to improve therapeutic effects. In our research, LY294002@PLGA unexpectedly increases cancer cell death with its profound endoplasmic reticulum (ER) stress. However, insufficient information is available about ER stress induced and its underlying mechanisms activated by LY294002@PLGA. In this study, lung cancer cell lines (H460, H157 and H1650) treated with various ER stress-related inducers (tunicamycin, thapsigargin, brefeldin A, and DBeQ) for exploring the underlying mechanisms. The cellular responses between these agents, free form LY294002 and LY294002@PLGA will be compared and reserved for further study. Characterizations of nanoparticles synthesized by two methods were analyzed using dynamic light scattering (DLS) and high-performance liquid chromatography (HPLC). The mean diameter and encapsulation efficiency of LY294002@PLGA synthesized by ethanol titration method was 94.02±5.05 nm and 6.35±0.03%, by two emulsion method was 174.5±3.29 nm and 54.66%±3.81%. Determined by MTT assay, the cell cytotoxicity of LY294002@PLGA was enhanced, and no obvious toxicity of PLGA nanoparticles in three lung cancer cell lines. In addition, LY294002@PLGA treatment sustained suppressed Akt and ERK activations simultaneously. Unfolded protein response (UPR) including activating transcription factor 6 (ATF6), PKR-like ER kinase (PERK) and inositol requiring enzyme 1 (IRE1), are activated for cell survival or death in different ER stress conditions. After tunicamycin and thapsigargin treatments, all of UPR pathways were induced for survival, but PERK-ATF4-CHOP pathway was activated for cell death in three lung cancer cell lines. Cells treated with brefeldin A were induced to express PERK and IRE1 pathways but not ATF6 pathway, and were more susceptible to apoptosis because of CHOP and phosphorylated JNK elevation. Only PERK pathway was activated by DBeQ to increase apoptotic cell death. Cells treated with LY294002 caused slight ER stress for cell survival. By LY294002@PLGA, cells were induced profound ER stress and caused apoptotic cell death through PERK-ATF4-CHOP pathway. Compared with the tested ER stress-related inducers, only DBeQ, valosin-containing protein (VCP) inhibitor, had similar ER stress-derived mechanism of action with LY294002@PLGA. Therefore, VCP may play an important role in LY294002@PLGA-derived cell death. These findings confirmed that both ER-stress induction and sustained suppression of Akt and ERK by LY294002@PLGA resulted in profound cytotoxic activity and cell apoptosis improved through PERK-ATF4-CHOP pathway. LY294002@PLGA may be a potential developed drug for cancer therapy in the future.

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