臺灣博碩士論文加值系統

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。

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.

第一章 中文摘要..............................................4

第二章 英文摘要..............................................6

第三章 縮寫表 (Abbreviation).................................8

第四章 緒論.................................................9
一 肺癌..................................................9
二 細胞自體吞噬..........................................10
三 Vesicular H+-ATPases與ATP6V0A1......................14
四 端粒酶與腫瘤的關係.....................................15
五 靈芝之醫藥研究.........................................18
六 靈芝免疫調節蛋白的抗癌研究..............................19

第五章 研究動機.............................................21

第六章 實驗設計.............................................22
ㄧ 以MTT Assay分析靈芝免疫調節蛋白GMI對非小細胞肺癌細胞之影響...22
二 使用西方點墨法分析靈芝免疫調節蛋白GMI對細胞自噬標的基因LC3蛋白質
變化的影響...........................................22
三 使用p53 siRNA與p53過量表現證實p53對GMI誘發細胞自噬之影響...22
四 使用Acridine orange染色分析靈芝免疫調節蛋白GMI對細胞中酸性囊
狀胞器(Acidic vesicular organelles)表現量的影響........23
五 以GFP-LC3轉染分析靈芝免疫調節蛋白GMI對細胞中細胞自噬小體的影響23
六 透過內質網壓力指標基因的變化分析靈芝免疫調節蛋白GMI是否誘發細胞產
生內質網壓力..........................................24
七 以細胞中鈣離子螯合劑BAPTA-AM分析靈芝免疫調節蛋白GMI是否透過鈣離
子誘發細胞自噬........................................24
八 以持續活化態之Akt (constitutively active Akt)觀察Akt於GMI
所誘發之細胞自噬現象中所扮演隻角色........................25
九 透過GFP-LC3蛋白變化以及溶酶體抑制劑bafilomycin-A1與
chloroquine作用分析靈芝免疫調節蛋白GMI誘發的細胞自噬為穩定態細
胞自噬(steady-state autophagy)或是細胞自噬流(autophgic
flux)..............................................25
十 使用溶酶體抑制劑bafilomycin-A1與chloroquine分析累積自噬小體對
GMI細胞毒殺能力之影響..................................26
十一 以VSV-G pseudotyped lentivirus-shRNA系統降低ATP6V0A1表現
量分析ATP6V0A1對溶酶體功能與自噬小體累積之影響..............26
十二 以VSV-G pseudotyped lentivirus-shRNA系統降低LC3表現量分析
靈芝免疫調節蛋白GMI誘發之細胞自噬對細胞的影響...............27
十三 以細胞群落生成試驗(Clonogenic assay)分析靈芝免疫調節蛋白GMI誘
發之細胞自噬對癌細胞致腫瘤能力的影響.......................27
十四 利用染色體端粒酶重覆增幅步驟（Telomeric Repeat Amplification
Protocol, TRAP assay）分析靈芝免疫調節蛋白GMI對A549肺癌細胞端
粒酶活性的影響及與細胞自噬間的關係.........................28
十五 使用流式細胞儀與西方點墨法分析GMI與cisplatin促進肺癌細胞發生凋亡
現象.................................................28

第七章 研究材料與研究方法.....................................29
ㄧ 細胞株 (Cell lines)..................................29
二 藥品.................................................30
三 GMI表現與純化.........................................31
四 細胞存活試驗..........................................31
五 細胞週期分析..........................................32
六 西方點墨法............................................34
七 酸性囊狀胞器分析.......................................36
八 GFP-LC3表現載體轉染與細胞自噬小體觀察.....................37
九 反轉錄酶鏈聚合酶連鎖反應(RT-PCR)........................38
十 VSV-G pseudotyped lentivirus–shRNA system...........40
十一 細胞群落生成能力分析(Clonogenic assay)..................43
十二 染色體端粒酶重覆增幅步驟（Telomeric Repeat Amplification
Protocol, TRAP assay）...............................44
十三 報導基因分析...........................................45
十四 動物腫瘤實驗...........................................46
十五 細胞死亡試驗...........................................48
十六 Annexin V- Propidium iodide細胞凋亡試驗................48
十七 細胞螢光染色...........................................49
十八 藥物合併作用分析.......................................50
第八章 結果................................................51
ㄧ 靈芝免疫調節蛋白GMI抑制非小細胞肺癌細胞存活...............51
二 GMI促使肺癌細胞產生細胞自體吞噬現象......................51
三 GMI誘發肺癌細胞產生酸性囊狀胞器.........................53
四 GMI誘發肺癌細胞產生細胞自噬小體.........................53
五 GMI透過鈣離子訊息傳遞路徑誘發細胞自體吞噬.................53
六 GMI抑制Akt/mTOR訊息傳遞路徑而誘發細胞自體吞噬............55
七 細胞自噬抑制劑3-methyladenine增加GMI的細胞毒殺能力非透過細胞
凋亡...............................................55
八 GMI誘發肺癌細胞產生細胞自噬流(Autophgic flux)...........56
九 溶酶體抑制劑增加GMI對肺癌細胞之毒殺能力...................57
十 以VSV-G pseudotyped lentivirus-shRNA系統降低LC3表現量阻止
細胞自體吞噬發生......................................58
十一 GMI透過誘發細胞自體吞噬抑制肺癌細胞存活與細胞群落生成能力....59
十二 抑制細胞自體吞噬不影響GMI造成之細胞週期停滯................60
十三 GMI透過誘發細胞自體吞噬抑制端粒酶活性.....................61
十四 以VSV-G pseudotyped lentivirus-shRNA系統降低ATP6V0A1表現
量大幅降低肺癌細胞生長能力..............................61
十五 以VSV-G pseudotyped lentivirus-shRNA系統降低ATP6V0A1表現
量並不影響溶酶體酸性化..................................63
十六 降低ATP6V0A1表現量增加GMI之細胞毒殺能力..................63
十七 降低ATP6V0A1表現量增加細胞中自噬小體累積.................64
十八 ATP6V0A1媒介自噬小體與溶酶體之融合反應...................65
十九 GMI對裸鼠腫瘤形成的影響................................66
二十 GMI增加非小細胞肺癌細胞對cisplatin的感受性...............68
二十一 合併使用Cisplatin與GMI有效抑制肺癌細胞群落生成...........68
二十二 GMI與cisplatin獨立與共同作用下對細胞週期之影響...........69
二十三 GMI與cisplatin共同作用促進細胞凋亡發生..................70
二十四 GMI不影響DNA修復基因ERCC1的表現.......................71
二十五 GMI抑制肺癌細胞中NF-κB活性............................71
二十六 Cisplatin與GMI透過細胞自噬增加非小細胞肺癌細胞走向細胞凋亡.73

第九章 討論...............................................74

第十章 圖表...............................................87

第十一章 附圖..............................................144

第十二章 參考文獻...........................................145

Andriani F, Perego P, Carenini N, Sozzi G, Roz L (2006) Increased sensitivity to cisplatin in non-small cell lung cancer cell lines after FHIT gene transfer. Neoplasia 8: 9-17

Aoki H, Iwado E, Eller MS, Kondo Y, Fujiwara K, Li GZ, Hess KR, Siwak DR, Sawaya R, Mills GB, Gilchrest BA, Kondo S (2007a) Telomere 3'' overhang-specific DNA oligonucleotides induce autophagy in malignant glioma cells. FASEB J 21: 2918-2930

Aoki H, Takada Y, Kondo S, Sawaya R, Aggarwal BB, Kondo Y (2007b) Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways. Mol Pharmacol 72: 29-39

Baehrecke EH (2005) Autophagy: dual roles in life and death? Nature reviews 6: 505-510

Biessmann H, Mason JM (1992) Genetics and molecular biology of telomeres. Adv Genet 30: 185-249

Biroccio A, Benassi B, Amodei S, Gabellini C, Del Bufalo D, Zupi G (2001) c-Myc down-regulation increases susceptibility to cisplatin through reactive oxygen species-mediated apoptosis in M14 human melanoma cells. Mol Pharmacol 60: 174-182

Bredesen DE, Rao RV, Mehlen P (2006) Cell death in the nervous system. Nature 443: 796-802

Carew JS, Medina EC, Esquivel JA, 2nd, Mahalingam D, Swords R, Kelly K, Zhang H, Huang P, Mita AC, Mita MM, Giles FJ, Nawrocki ST (2010) Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation. J Cell Mol Med 14: 2448-2459

Caro LH, Plomp PJ, Wolvetang EJ, Kerkhof C, Meijer AJ (1988) 3-Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism. Eur J Biochem 175: 325-329

Chang CP, Yang MC, Liu HS, Lin YS, Lei HY (2007) Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model. Hepatology 45: 286-296

Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB (2008) Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ 15: 171-182

Chiu HW, Lin JH, Chen YA, Ho SY, Wang YJ (2010) Combination treatment with arsenic trioxide and irradiation enhances cell-killing effects in human fibrosarcoma cells in vitro and in vivo through induction of both autophagy and apoptosis. Autophagy 6: 353-365

Chu TT, Benzie IF, Lam CW, Fok BS, Lee KK, Tomlinson B (2012) Study of potential cardioprotective effects of Ganoderma lucidum (Lingzhi): results of a controlled human intervention trial. Br J Nutr 107: 1017-1027

Coates JM, Galante JM, Bold RJ (2009) Cancer Therapy Beyond Apoptosis: Autophagy and Anoikis as Mechanisms of Cell Death. J Surg Res

Crighton D, Wilkinson S, O''Prey J, Syed N, Smith P, Harrison PR, Gasco M, Garrone O, Crook T, Ryan KM (2006) DRAM, a p53-induced modulator of autophagy, is critical for apoptosis. Cell 126: 121-134

De Mejia EG, Prisecaru VI (2005) Lectins as bioactive plant proteins: a potential in cancer treatment. Crit Rev Food Sci Nutr 45: 425-445

Dean EJ, Ward T, Pinilla C, Houghten R, Welsh K, Makin G, Ranson M, Dive C (2010) A small molecule inhibitor of XIAP induces apoptosis and synergises with vinorelbine and cisplatin in NSCLC. Br J Cancer 102: 97-103

Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, Mukherjee C, Shi Y, Gelinas C, Fan Y, Nelson DA, Jin S, White E (2006) Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 10: 51-64

Falchetti ML, Levi A, Molinari P, Verna R, D''Ambrosio E (1998) Increased sensitivity and reproducibility of TRAP assay by avoiding direct primers interaction. Nucleic Acids Res 26: 862-863

Fan QW, Cheng C, Hackett C, Feldman M, Houseman BT, Nicolaides T, Haas-Kogan D, James CD, Oakes SA, Debnath J, Shokat KM, Weiss WA (2010) Akt and autophagy cooperate to promote survival of drug-resistant glioma. Sci Signal 3: ra81

Feng Z, Zhang H, Levine AJ, Jin S (2005) The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci U S A 102: 8204-8209

Forgac M (1999) Structure and properties of the vacuolar (H+)-ATPases. J Biol Chem 274: 12951-12954

Fu L, Kim YA, Wang X, Wu X, Yue P, Lonial S, Khuri FR, Sun SY (2009) Perifosine inhibits mammalian target of rapamycin signaling through facilitating degradation of major components in the mTOR axis and induces autophagy. Cancer Res 69: 8967-8976

Fujita N, Hayashi-Nishino M, Fukumoto H, Omori H, Yamamoto A, Noda T, Yoshimori T (2008) An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol Biol Cell 19: 4651-4659

Gao M, Yeh PY, Lu YS, Hsu CH, Chen KF, Lee WC, Feng WC, Chen CS, Kuo ML, Cheng AL (2008) OSU-03012, a novel celecoxib derivative, induces reactive oxygen species-related autophagy in hepatocellular carcinoma. Cancer Res 68: 9348-9357

Gao Y, Zhang R, Zhang J, Gao S, Gao W, Zhang H, Wang H, Han B (2011) Study of the extraction process and in vivo inhibitory effect of ganoderma triterpenes in oral mucosa cancer. Molecules 16: 5315-5332

Geng Y, Kohli L, Klocke BJ, Roth KA (2010) Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent. Neuro Oncol 12: 473-481

Green DR, Kroemer G (2009) Cytoplasmic functions of the tumour suppressor p53. Nature 458: 1127-1130

Greider CW (1996) Telomere length regulation. Annu Rev Biochem 65: 337-365

Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100: 57-70

Harley CB (2008) Telomerase and cancer therapeutics. Nature reviews 8: 167-179

Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345: 458-460

Harley CB, Villeponteau B (1995) Telomeres and telomerase in aging and cancer. Curr Opin Genet Dev 5: 249-255

Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, Yamamoto A (2009) A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 11: 1433-1437

Herbst RS, Heymach JV, Lippman SM (2008) Lung cancer. N Engl J Med 359: 1367-1380

Hikino H, Konno C, Mirin Y, Hayashi T (1985) Isolation and hypoglycemic activity of ganoderans A and B, glycans of Ganoderma lucidum fruit bodies. Planta Med: 339-340

Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek MA, Shay JW, Ishioka S, Yamakido M (1995) Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 155: 3711-3715

Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, Bianchi K, Fehrenbacher N, Elling F, Rizzuto R, Mathiasen IS, Jaattela M (2007) Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell 25: 193-205

Hoyer-Hansen M, Jaattela M (2007) Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium. Cell death and differentiation 14: 1576-1582

Hung JY, Hsu YL, Li CT, Ko YC, Ni WC, Huang MS, Kuo PL (2009) 6-Shogaol, an active constituent of dietary ginger, induces autophagy by inhibiting the AKT/mTOR pathway in human non-small cell lung cancer A549 cells. J Agric Food Chem 57: 9809-9816

Jiang J, Jedinak A, Sliva D (2011) Ganodermanontriol (GDNT) exerts its effect on growth and invasiveness of breast cancer cells through the down-regulation of CDC20 and uPA. Biochem Biophys Res Commun 415: 325-329

Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19: 5720-5728

Kartalou M, Essigmann JM (2001) Mechanisms of resistance to cisplatin. Mutat Res 478: 23-43

Klionsky DJ et al. (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4: 151-175

Lee I, Ahn B, Choi J, Hattori M, Min B, Bae K (2011) Selective cholinesterase inhibition by lanostane triterpenes from fruiting bodies of Ganoderma lucidum. Bioorg Med Chem Lett 21: 6603-6607

Letai AG (2008) Diagnosing and exploiting cancer''s addiction to blocks in apoptosis. Nat Rev Cancer 8: 121-132

Levine B (2007) Cell biology: autophagy and cancer. Nature 446: 745-747

Levine B, Abrams J (2008) p53: The Janus of autophagy? Nat Cell Biol 10: 637-639

Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6: 463-477

Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132: 27-42

Levine B, Yuan J (2005) Autophagy in cell death: an innocent convict? J Clin Invest 115: 2679-2688

Li F, Zhang Y, Zhong Z (2011) Antihyperglycemic effect of ganoderma lucidum polysaccharides on streptozotocin-induced diabetic mice. Int J Mol Sci 12: 6135-6145

Liao CH, Hsiao YM, Hsu CP, Lin MY, Wang JC, Huang YL, Ko JL (2006) Transcriptionally mediated inhibition of telomerase of fungal immunomodulatory protein from Ganoderma tsugae in A549 human lung adenocarcinoma cell line. Mol Carcinog 45: 220-229

Liao CH, Hsiao YM, Lin CH, Yeh CS, Wang JC, Ni CH, Hsu CP, Ko JL (2008) Induction of premature senescence in human lung cancer by fungal immunomodulatory protein from Ganoderma tsugae. Food Chem Toxicol 46: 1851-1859

Liao CH, Hsiao YM, Sheu GT, Chang JT, Wang PH, Wu MF, Shieh GJ, Hsu CP, Ko JL (2007) Nuclear translocation of telomerase reverse transcriptase and calcium signaling in repression of telomerase activity in human lung cancer cells by fungal immunomodulatory protein from Ganoderma tsugae. Biochem Pharmacol 74: 1541-1554

Lin CC, Yu YL, Shih CC, Liu KJ, Ou KL, Hong LZ, Chen JD, Chu CL (2011) A novel adjuvant Ling Zhi-8 enhances the efficacy of DNA cancer vaccine by activating dendritic cells. Cancer Immunol Immunother 60: 1019-1027

Lin CH, Sheu GT, Lin YW, Yeh CS, Huang YH, Lai YC, Chang JG, Ko JL (2010) A new immunomodulatory protein from Ganoderma microsporum inhibits epidermal growth factor mediated migration and invasion in A549 lung cancer cells. Process Biochemistry 45: 1537–1542

Lin WH, Hung CH, Hsu CI, Lin JY (1997) Dimerization of the N-terminal amphipathic alpha-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis. J Biol Chem 272: 20044-20048

Liu LZ, Zhou XD, Qian G, Shi X, Fang J, Jiang BH (2007) AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/p70S6K1 pathway. Cancer Res 67: 6325-6332

Ma JQ, Liu CM, Qin ZH, Jiang JH, Sun YZ (2011) Ganoderma applanatum terpenes protect mouse liver against benzo(alpha)pyren-induced oxidative stress and inflammation. Environ Toxicol Pharmacol 31: 460-468

Mabuchi S, Ohmichi M, Nishio Y, Hayasaka T, Kimura A, Ohta T, Saito M, Kawagoe J, Takahashi K, Yada-Hashimoto N, Sakata M, Motoyama T, Kurachi H, Tasaka K, Murata Y (2004) Inhibition of NFkappaB increases the efficacy of cisplatin in in vitro and in vivo ovarian cancer models. J Biol Chem 279: 23477-23485

Maclean KH, Dorsey FC, Cleveland JL, Kastan MB (2008) Targeting lysosomal degradation induces p53-dependent cell death and prevents cancer in mouse models of lymphomagenesis. J Clin Invest 118: 79-88

Maiuri MC, Criollo A, Kroemer G Crosstalk between apoptosis and autophagy within the Beclin 1 interactome. EMBO J 29: 515-516

Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, Burden SJ, Di Lisi R, Sandri C, Zhao J, Goldberg AL, Schiaffino S, Sandri M (2007) FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab 6: 458-471

Martinez-Montemayor MM, Acevedo RR, Otero-Franqui E, Cubano LA, Dharmawardhane SF (2011) Ganoderma lucidum (Reishi) inhibits cancer cell growth and expression of key molecules in inflammatory breast cancer. Nutr Cancer 63: 1085-1094

Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, Bray K, Reddy A, Bhanot G, Gelinas C, Dipaola RS, Karantza-Wadsworth V, White E (2009) Autophagy suppresses tumorigenesis through elimination of p62. Cell 137: 1062-1075

Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140: 313-326

Nakanishi C, Toi M (2005) Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer 5: 297-309

Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K, Shiosaka S, Hammarback JA, Urano F, Imaizumi K (2006) Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 26: 9220-9231

Olaussen KA, Dunant A, Fouret P, Brambilla E, Andre F, Haddad V, Taranchon E, Filipits M, Pirker R, Popper HH, Stahel R, Sabatier L, Pignon JP, Tursz T, Le Chevalier T, Soria JC (2006) DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 355: 983-991

Paroutis P, Touret N, Grinstein S (2004) The pH of the secretory pathway: measurement, determinants, and regulation. Physiology (Bethesda) 19: 207-215

Pena-Llopis S, Vega-Rubin-de-Celis S, Schwartz JC, Wolff NC, Tran TA, Zou L, Xie XJ, Corey DR, Brugarolas J (2011) Regulation of TFEB and V-ATPases by mTORC1. EMBO J 30: 3242-3258

Peri F, Nusslein-Volhard C (2008) Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell 133: 916-927

Ramser B, Kokot A, Metze D, Weiss N, Luger TA, Bohm M (2009) Hydroxychloroquine modulates metabolic activity and proliferation and induces autophagic cell death of human dermal fibroblasts. J Invest Dermatol 129: 2419-2426

Ravikumar B, Moreau K, Jahreiss L, Puri C, Rubinsztein DC (2010) Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat Cell Biol 12: 747-757

Rong Y, Distelhorst CW (2008) Bcl-2 protein family members: versatile regulators of calcium signaling in cell survival and apoptosis. Annu Rev Physiol 70: 73-91

Rosell R, Lord RV, Taron M, Reguart N (2002) DNA repair and cisplatin resistance in non-small-cell lung cancer. Lung Cancer 38: 217-227

Roudier E, Mistafa O, Stenius U (2006) Statins induce mammalian target of rapamycin (mTOR)-mediated inhibition of Akt signaling and sensitize p53-deficient cells to cytostatic drugs. Mol Cancer Ther 5: 2706-2715

Sakaki K, Wu J, Kaufman RJ (2008) Protein kinase Ctheta is required for autophagy in response to stress in the endoplasmic reticulum. J Biol Chem 283: 15370-15380

Scarlatti F, Granata R, Meijer AJ, Codogno P (2009) Does autophagy have a license to kill mammalian cells? Cell Death Differ 16: 12-20

Scarlatti F, Maffei R, Beau I, Codogno P, Ghidoni R (2008) Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells. Cell Death Differ 15: 1318-1329

Scott CC, Bissig C, Gruenberg J (2011) Duelling functions of the V-ATPase. EMBO J 30: 4113-4115

Shay JW, Wright WE (2002) Telomerase: a target for cancer therapeutics. Cancer cell 2: 257-265

Shen HM, Codogno P (2011) Autophagic cell death: Loch Ness monster or endangered species? Autophagy 7: 457-465

Shvets E, Fass E, Elazar Z (2008) Utilizing flow cytometry to monitor autophagy in living mammalian cells. Autophagy 4: 621-628

Smith DM, Patel S, Raffoul F, Haller E, Mills GB, Nanjundan M (2010) Arsenic trioxide induces a beclin-1-independent autophagic pathway via modulation of SnoN/SkiL expression in ovarian carcinoma cells. Cell Death Differ 17: 1867-1881

Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1: 84-91

Tasdemir E, Maiuri MC, Galluzzi L, Vitale I, Djavaheri-Mergny M, D''Amelio M, Criollo A, Morselli E, Zhu C, Harper F, Nannmark U, Samara C, Pinton P, Vicencio JM, Carnuccio R, Moll UM, Madeo F, Paterlini-Brechot P, Rizzuto R, Szabadkai G, Pierron G, Blomgren K, Tavernarakis N, Codogno P, Cecconi F, Kroemer G (2008) Regulation of autophagy by cytoplasmic p53. Nat Cell Biol 10: 676-687

Wang SH, Liang CJ, Weng YW, Chen YH, Hsu HY, Chien HF, Tsai JS, Tseng YC, Li CY, Chen YL (2012) Ganoderma lucidum polysaccharides prevent platelet-derived growth factor-stimulated smooth muscle cell proliferation in vitro and neointimal hyperplasia in the endothelial-denuded artery in vivo. J Cell Physiol 227: 3063-3071

Wang SH, Shih YL, Ko WC, Wei YH, Shih CM (2008) Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway. Cell Mol Life Sci 65: 3640-3652

Wong CH, Iskandar KB, Yadav SK, Hirpara JL, Loh T, Pervaiz S (2010) Simultaneous induction of non-canonical autophagy and apoptosis in cancer cells by ROS-dependent ERK and JNK activation. PLoS One 5: e9996

Wu CT, Lin TY, Hsu HY, Sheu F, Ho CM, Chen EI (2011) Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway. Carcinogenesis 32: 1890-1896

Wu TC, Lin P, Hsu CP, Huang YJ, Chen CY, Chung WC, Lee H, Ko JL (2003) Loss of telomerase activity may be a potential favorable prognostic marker in lung carcinomas. Lung Cancer 41: 163-169

Wu YT, Tan HL, Shui G, Bauvy C, Huang Q, Wenk MR, Ong CN, Codogno P, Shen HM (2010) Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem 285: 10850-10861

Xu ZX, Liang J, Haridas V, Gaikwad A, Connolly FP, Mills GB, Gutterman JU (2007) A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase. Cell Death Differ 14: 1948-1957

Yahata N, Ohyashiki K, Ohyashiki JH, Iwama H, Hayashi S, Ando K, Hirano T, Tsuchida T, Kato H, Shay JW, Toyama K (1998) Telomerase activity in lung cancer cells obtained from bronchial washings. J Natl Cancer Inst 90: 684-690

Yang C, Kaushal V, Shah SV, Kaushal GP (2008) Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells. Am J Physiol Renal Physiol 294: F777-787

Yang PM, Liu YL, Lin YC, Shun CT, Wu MS, Chen CC (2010) Inhibition of autophagy enhances anticancer effects of atorvastatin in digestive malignancies. Cancer Res 70: 7699-7709

Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12: 814-822

Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L, Brunner T, Simon HU (2006) Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 8: 1124-1132

Yue GG, Fung KP, Tse GM, Leung PC, Lau CB (2006) Comparative studies of various ganoderma species and their different parts with regard to their antitumor and immunomodulating activities in vitro. Journal of alternative and complementary medicine (New York, NY 12: 777-789

Zhang Q, Yang W, Man N, Zheng F, Shen Y, Sun K, Li Y, Wen LP (2009) Autophagy-mediated chemosensitization in cancer cells by fullerene C60 nanocrystal. Autophagy 5: 1107-1117

Zhou WJ, Deng R, Zhang XY, Feng GK, Gu LQ, Zhu XF (2009a) G-quadruplex ligand SYUIQ-5 induces autophagy by telomere damage and TRF2 delocalization in cancer cells. Mol Cancer Ther 8: 3203-3213

Zhou X, Zhang Z, Yang X, Chen W, Zhang P (2009b) Inhibition of cyclin D1 expression by cyclin D1 shRNAs in human oral squamous cell carcinoma cells is associated with increased cisplatin chemosensitivity. Int J Cancer 124: 483-489

Zhu CQ, Cutz JC, Liu N, Lau D, Shepherd FA, Squire JA, Tsao MS (2006) Amplification of telomerase (hTERT) gene is a poor prognostic marker in non-small-cell lung cancer. Br J Cancer 94: 1452-1459

Zhuang SR, Chiu HF, Chen SL, Tsai JH, Lee MY, Lee HS, Shen YC, Yan YY, Shane GT, Wang CK (2012) Effects of a Chinese medical herbs complex on cellular immunity and toxicity-related conditions of breast cancer patients. Br J Nutr 107: 712-718

Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12: 21-35