Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells

Biochemical and Biophysical Research Communications 382 (2009) 451–456

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells Ya Chun Wu a,b,1, William Ka Kei Wu b,c,d,1, Youming Li a, Le Yu b, Zhi Jie Li b, Clover Ching Man Wong b, Hai Tao Li b, Joseph Jao Yiu Sung c,d, Chi Hin Cho b,c,* a

Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, PR China Department of Pharmacology, Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, PR China c Institute of Digestive Diseases, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, PR China d Department of Medicine & Therapeutics, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, PR China b

a r t i c l e

i n f o

Article history: Received 26 February 2009 Available online 14 March 2009

Keywords: Bafilomycin A1 Macroautophagy Proliferation Colon cancer

a b s t r a c t Macroautophagy is a process by which cytoplasmic content and organelles are sequestered by doublemembrane bound vesicles and subsequently delivered to lysosomes for degradation. Macroautophagy serves as a major intracellular pathway for protein degradation and as a pro-survival mechanism in time of stress by generating nutrients. In the present study, bafilomycin A1, a vacuolar type H+-ATPase inhibitor, suppresses macroautophagy by preventing acidification of lysosomes in colon cancer cells. Diminished macroautophagy was evidenced by the accumulation of undegraded LC3 protein. Suppression of macroautophagy by bafilomycin A1 induced G0/G1 cell cycle arrest and apoptosis which were accompanied by the down-regulation of cyclin D1 and cyclin E, the up-regulation of p21Cip1 as well as cleavages of caspases-3, -7, -8, and -9 and PARP. Further investigation revealed that bafilomycin A1 increased the phosphorylation of ERK, JNK, and p38. In this regard, p38 inhibitor partially reversed the anti-proliferative effect of bafilomycin A1. To conclude, inhibition of macroautophagy by bafilomycin A1 lowers G1–S transition and induces apoptosis in colon cancer cells. Our results not only indicate that inhibitors of macroautophagy may be used therapeutically to inhibit cancer growth, but also delineate the relationship between macroautophagy and apoptosis. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Macroautophagy is an evolutionarily conserved catabolic process by which the cell degrades its cellular content through the lysosomal system. Macroautophagy is initiated by the formation of a double-membrane bound vacuole, the autophagosome, which nonselectively sequesters cytosolic proteins and organelles such as mitochondria, endoplasmic reticulum, and ribosomes [1]. In the late stage of macroautophagy, autophagosomes fuse with acidic lysosomes to produce autolysosomes where the engulfed content is digested by lysosomal hydrolases. The resulting degraded components are then returned to the cytoplasm by permeases in the lysosomal membrane for reuse [1]. While overactivation of this cellular process can lead to cell death [2], macroautophagy serves as a crucial pro-survival mechanism in time of stress by generating nutrients [3]. For instances, protein degradation and macroauto* Corresponding author. Address: Department of Pharmacology, Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, PR China. Fax: +86 852 2603 5139. E-mail addresses: [email protected] (W.K.K. Wu), [email protected] (C.H. Cho). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.051

phagy are enhanced in livers of mice undergoing starvation [4]. Dysregulation of macroautophagy has been implicated in many human diseases, such as degenerative neuronal diseases and cancers [5–7]. In carcinogenesis, the role of macroautophagy is oxymoronic. As a tumor suppressing mechanism, macroautophagy serves as an alternative to apoptosis to eliminate transformed cells [1,8]. Moreover, genes that are involved in the execution of macroautophagy are important tumor suppressors [1,8]. Nevertheless, it has also been reported that macroautophagy may facilitate tumor growth and survival during nutrient starvation and may contribute to tumor dormancy [9]. In colon cancer, increased expression of beclin-1 and LC3, two biochemical markers of macroautophagy, have been documented [10,11]. The exact role of macroautophagy in colon carcinogenesis, however, is unknown. Bafilomycin A1, a macrolide antibiotic isolated from Streptomyces griseus, is a potent inhibitor of vacuolar type H+-ATPase which functions to acidify intracellular compartments including lysosomes and to transport protons across the plasma membrane [12,13]. In relation to macroautophagy, vacuolar type H+-ATPase is involved in the maturation of autolysosomes and its inhibitors including bafilomycin A1 have been used as inhibitors of macroautophagy [14]. Macroautophagy plays a crucial part in the protein

452

Y.C. Wu et al. / Biochemical and Biophysical Research Communications 382 (2009) 451–456

degradation system [15,16]. In this connection, inhibition of protein degradation, for example, by targeting the proteasome has been shown to lower cell proliferation or induce apoptosis in various types of epithelial and hematological malignancies [17–19]. Whether inhibition of macroautophagy can achieve similar effects on cancer growth, however, is unknown. In the present study, the effects of macroautophagy inhibitor bafilomycin A1 on proliferation and apoptosis of colon cancer cells were investigated.

Methods Reagents. All primary antibodies were purchased from Cell Signaling Technology (Beverley, MA, USA) unless otherwise specified. Acridine orange and 40 ,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Invitrogen, Carlsbad, CA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO, USA) unless otherwise specified. Cell culture and proliferation assay. The human colon adenocarinoma cell lines HT-29, HCT-116, SW1116, and normal colon fibroblasts CCD-18Co were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640, supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cell proliferation was measured by MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. Cells were plated at a density of 5000 cells per well in 96-well plates. After treatment, MTT solution dissolved in the culture medium at the final concentration of 0.5 mmol/L was added to each well and the plates were incubated for another 4 h. Dimethyl sulfoxide was then added to solubilize MTT tetrazolium crystal. Finally, the optical density was determined at 570 nm using a Benchmark Plus microplate reader (Bio-Rad, Hercules, USA). Colony formation assay. HT-29 cells were seeded at the concentration of 2  104 cells per well in 6-well plates. After attachment, cells were treated with various concentrations of bafilomycin A1 for 24 h and incubated in fresh medium for another 6 days, after which the cells were fixed with absolute methanol and stained in haematoxylin for 30 min and the cultures were photographed under white-transillumination using ChemiDoc XRS system (BioRad). Colonies were then counted using Quantity One software (Bio-Rad). Cell cycle analysis. HT-29 cells were fixed with ice-cold 70% ethanol in phosphate buffered saline followed by incubation with 50 lg/ml propidium iodide, 3.8 mmol/L sodium citrate, and 0.5 lg/ml RNase A at 4 °C for 3 h and analyzed by flow cytometry (Beckman Coulter, Fullerton, CA, USA). The resultant DNA histograms were generated using WinMDI 2.8 software. ELISA for cytoplasmic nucleosomes. Apoptotic cell death was measured as the amount of histone-associated DNA-fragments of mono- and oligo-nucleosomes released into cytoplasm. In brief, HT-29 cells were plated at a density of 5000 cells per well in 96well plates. After treatment, cytoplasmic nucleosomes were quantified using the spectrophotometric Cell Death Detection ELISA plus system (Roche Diagnostics Corp.) in accordance with the manufacturer’s protocol. DNA fragmentation. HT-29 cells were plated in six-well plates at the density of 2  105 cells per well. After treatment, DNA was purified using the Wizard SV Genomic DNA kit (Promega) in accordance with the manufacturer’s recommendation. Equal amount (1 lg) of the extracted DNA was resolved over 1.7% agarose gel containing 1 GelRed reagent (Biotium, USA). Images were visualized under UV light using ChemiDoc XRS system (Bio-Rad). Acridine orange staining for acidic vesicular organelles. Acridine orange was added at a final concentration of 1 lg/ml for a period of 15 min. Pictures were obtained with a fluorescence microscope

(Nikon TS100-F) equipped with a 50-W mercury lamp, a 450–490nm band-pass blue excitation filters, a 505-nm dichroic mirror, a 520-nm long pass-barrier filter, and a digital camera (Nikon DS5Mc). Western blot for proteins related to autophagy, cell cycle, and apoptosis. HT-29 cells were harvested in radioimmunoprecipitation buffer [50 mmol/L Tris–HCl (pH 7.5), 150 mmol/L sodium chloride, 0.5% a-cholate acid, 0.1% SDS, 2 mmol/L EDTA, 1% Triton X-100, and 10% glycerol] containing proteinase and phosphatase inhibitors [1 mmol/L phenylmethylsulfonyl fluoride, 1 lg/ml aprotinin, 1 lg/ml leupeptin, 1 lg/ml pepstatin, 1 mmol/L Na3VO4, and 1 mmol/L NaF]. Protein was quantified using protein assay kit (Bio-Rad Laboratories, Hercules, USA). Equal amounts of protein (50 lg/lane) were resolved by SDS–PAGE, and transferred to Hybond C nitrocellulose membranes (Amersham Corporation, Arlington Heights, IL, USA). The membranes were probed with primary antibodies overnight at 4 °C and incubated for 1 h with secondary peroxidase-conjugated antibodies (Invitrogen). Chemiluminescent signals were then developed with Lumiglo reagent (Cell Signaling Technology) and detected by the ChemiDoc XRS gel documentation system (Bio-rad). Statistical analysis. Results were expressed as means ± SEM. Statistical analysis was performed with an analysis of variance

Fig. 1. Suppression of basal macroautophagy by bafilomycin A1 in HT-29 cells. (A) The formation of acidic vesicular organelles, which emitted bright red fluorescence, was visualized by acridine orange staining. Treating the cells with 1 nmol L1 bafilomycin A1 for 24 h remarkably reduced the number and size of acidic vesicular organelles. (B) Bafilomycin A1 at the concentration of 1 nmol L1 time-dependently increased the accumulation of LC3-I and -II protein. (C) Class III PI3 K inhibitor 3methyadenine (10 mmol L1) failed to reverse the up-regulation of LC3 induced by bafilomycin A1 (1 nmol L1). (For interpretation of color mentioned in this figure legend, the reader is referred to the web version of the article.)

Y.C. Wu et al. / Biochemical and Biophysical Research Communications 382 (2009) 451–456

(ANOVA) followed by the Turkey’s t-test. P values less than 0.05 were considered statistically significant.

453

suggesting that the degradation but not the formation of LC3-II was interfered by bafilomycin A1. Bafilomycin A1 inhibited proliferation of colon cancer cells

Results Bafilomycin A1 inhibited the acidification of vesicular organelles and induced accumulation of LC3-II To determine the effect of bafilomycin A1 on macroautophagy of colon cancer cells, the formation of acidic vesicular organelles was determined by acridine orange staining. This lysosomotropic agent emitted bright red fluorescence in acidic vesicles including lysosomes but fluoresced green in cytoplasm and nucleus [20]. Vital staining of HT-29 cells with acridine orange revealed that red fluorescence was reduced in bafilomycin A1-treated cells (Fig. 1A), indicating the acidification of vesicular organelles was prevented. Conversely, the majority of control cells exhibited a moderate level of red fluorescence. To further confirm the inhibitory effect of bafilomycin A1 on macroautophagy, the accumulation of LC3-II protein was determined. LC3-II is a surface protein marker of autophagosomes, which is ultimately degraded by acidic hydrolases after the formation of autolysosomes [21]. Western blot showed that bafilomycin A1 dramatically increased the protein levels of LC3-II in a time-dependent manner (Fig. 1B). In the next step, 3methyadenine was used to inhibit the activity of class III PI3K which is involved in the conversion of LC3-I to LC3-II [22,23]. Results indicated that bafilomycin A1 increased the expression of LC3-II independent of the presence of 3-methyadenine (Fig. 1C),

To study the effect of blockade of macroautophagy by bafilomycin A1 on proliferation of colon cancer cells, we examined changes in MTT tetrazolium salt formation in HT-29, HCT-116, and SW1116 cells. As shown in Fig. 2A, bafilomycin A1 significantly reduced MTT tetrazolium salt formation in all three colon cancer cell lines in a concentration-dependent manner. At the dose of 5 nmol L1, 24h treatment of bafilomycin A1 inhibited HT-29 cell proliferation by about 76%. The anti-mitogenic effect of bafilomycin A1 could be detected at the concentration as lowest as 1 nmol L1. In contrast, bafilomycin A1 slightly increased the proliferation of normal colon fibroblasts CCD-18Co. To further confirm the anti-mitogenic effect of bafilomycin A1 on colon cancer cells, colony-formation assay and cell cycle analysis were performed. Results showed that 24-h treatment of bafilomycin A1 significantly reduced the colony-forming ability of HT-29 (Fig. 2B). Moreover, flow cytometry revealed that treating HT-29 cells with bafilomycin A1 for 12 or 24 h reduced the number of cells in S- and G2/M-phases, suggesting that bafilomycin A1 lowered G1–S transition in colon cancer cells. Prolonging bafilomycin A1 treatment to 36 or 48 h increased the number of cells in the sub-G1-phase which was indicative of apoptotic cell death (Fig. 2C). Consistent with these observations, bafilomycin A1 altered the expression of a number of cell cycle regulators related to G1–S transition. In this regard, bafilomycin A1 down-regulated the expression of cyclin D1 and cyclin E but en-

Fig. 2. Inhibition of colon cancer cell proliferation and cell cycle progression by bafilomycin A1. (A) Incubation of bafilomycin A1 for 24 h dose-dependently lowered cell proliferation as determined by MTT assay in colon cancer cell lines HT-29, HCT-116, and SW1116. The anti-proliferative effect was not observed in normal colon fibroblasts CCD18-Co. (B) Bafilomycin A1 reduced the colony-forming ability of HT-29. Cells were treated with various concentrations of bafilomycin A1 for 24 h and then incubated in fresh medium with bafilomycin A1 for another 6 days. (C) DNA histogram shows that 12-h and 24-h treatment with bafilomycin A1 (1 nmol L1) induced he accumulation of G0/G1-phase cells in HT-29. Increased sub-G1-phase was also observed for prolonged treatment (36 h and 48 h). DNA contents were determined by propidium iodide staining followed by flow cytometry analysis. (D) Bafilomycin A1 (1 nmol L1) time-dependently decreased cyclin D1 and cyclin E and increased p21Cip1 protein expression. **P < 0.01, significantly different from respective control group.

454

Y.C. Wu et al. / Biochemical and Biophysical Research Communications 382 (2009) 451–456

hanced the expression of p21Cip1. The expression of p27Kip1, however, was not significantly affected (Fig. 2D). Bafilomycin A1-induced apoptosis in colon cancer cells To further confirm the pro-apoptotic effects of bafilomycin A1 on colon cancer cells, we determined the changes in apoptotic cell death by detection of DNA fragmentation, PARP cleavage and cytoplasmic nucleosomes. As shown in Fig. 3A, 24-h treatment of bafilomycin A1 substantially enhanced DNA ladder formation in HT-29 and HCT-116 cells. Bafilomycin A1 also remarkably increased the cleavage of PARP in a time-dependent manner (Fig. 3B). Quantitatively, apoptosis of HT-29 cells was increased by about 19-fold in response to 24-h treatment of bafilomycin A1 (1 nmol L1) as determined by the enrichment of cytoplasmic nucleosomes (Fig. 3C). Activation of caspases plays a central role in the execution of apoptosis. To elucidate which caspase(s) mediated bafilomycin A1-induced apoptosis, cleavage of caspases-3, -7, -8 and -9 were determined by Western blot. As shown in Fig. 3D, the cleavage of procaspase-3, -7, -8, -9 and the formation of corresponding active forms were observed. Bafilomycin A1-induced the phosphorylation of MAPKs MAPKs (ERK, p38, and JNK) were reported to be activated during apoptosis of colon cancer cells [24,25]. We therefore studied

the involvement of MAPKs in bafilomycin A1-induced apoptosis by Western blot. Results revealed that the phosphorylation of ERK, p38, and JNK were up-regulated by bafilomycin A1 (Fig. 4A). To examine whether activation of MAPKs was required for the pro-apoptotic effect of bafilomycin A1, several specific MAPK inhibitors (U0126, SB203580, and SP600125) were used. In this regard, p38 inhibitor SB203580 but not ERK (U0126) or JNK (SP600125) inhibitors significantly reversed bafilomycin A1-induced reduction of MTT tetrazolium salt formation (Fig. 4B), suggesting that p38 was involved in the anti-mitogenic action of bafilomycin A1. Discussion Here we show that inhibition of the macroautophagy by vacuolar type H+-ATPase inhibitor bafilomycin A1 lowers G1–S transition and induces apoptosis in colon cancer cells. The inhibition of macroautophagy is evidenced by the reduced formation of acidic vesicular organelles and the accumulation of undigested LC3-II protein. These data suggest that bafilomycin A1 may inhibit the function of lysosomal hydrolases by preventing acidification of lysosomes. In addition, bafilomycin A1 when used at high concentration (100 nmol L1) has been reported to block the fusion of autophagosomes with lysosomes in the rat hepatoma H-4-II-E cell line [26]. This finding together with our data suggest that the vacuolar type H+-ATPase play an essential role in the regulation of macroautophagy, especially in the late stage.

Fig. 3. Induction of apoptosis by bafilomycin A1 in colon cancer cells. (A) Incubation of bafilomycin A1 for 48 h induced the formation of DNA ladder in HT-29 and HCT-116. (B) Forty eight-hour treatment of bafilomycin A1 enhanced PARP cleavage in HT-29. (C) Treating HT-29 with bafilomycin A1 for 48 h increased the cytosolic content of monoand oligo-nucleosomes as determined by ELISA. (D) Treating HT-29 with bafilomycin A1 for 48 h enhanced the cleavages of caspases-3, -7, -8, and -9. **P < 0.01, significantly different from the control group.

Y.C. Wu et al. / Biochemical and Biophysical Research Communications 382 (2009) 451–456

455

phagy by leupeptin enhances neuronal apoptosis in a murine model of Alzheimer’s disease and the mechanism is possibly mediated by the accumulation of activated caspase-3 which is normally digested by the macroautophagy pathway [31]. Moreover, inhibition of macroautophagy by beclin-1 and atg7 siRNAs aggravates TRAILinduced apoptosis in MCF10A breast epithelial cells [32]. Intriguingly, inhibition of apoptosis has been shown to induce macroautophagy in a reciprocal manner. In this respect, caspase-3 inhibitor ZDEVD up-regulated macroautophagy in the mouse lung cancer cells [33]. These findings suggest that apoptosis and macroautophagy are two complementary, reciprocally regulated pathways for determination of cell fate between death and survival. To conclude, our study not only demonstrates for the first time that suppression of macroautophagy by bafilomycin A1 inhibits cell proliferation and induces apoptosis in colon cancer cells, but also possibly opens up novel therapeutic avenues for the treatment of colon cancer with the inhibitors of macroautophagy. References

Fig. 4. Involvement of MAPKs in the anti-proliferative effect of bafilomycin A1 in HT-29. (A) Bafilomycin A1 (1 nmol L1) time-dependently increased the phosphorylation of ERK, p38 and JNK. (B) p38 inhibitor SB203580 (3 lmol L1) but not ERK (U0126; 3 lmol L1) or JNK (SP600125; 5 lmol L1) inhibitors reversed the antimitogenic effect of bafilomycin A1. Cells with pre-treated with respective inhibitors for 12 h followed by 24-h treatment of bafilomycin A1 (1 nmol L1). **P < 0.01, significantly different from respective control group.  P < 0.05, significantly different from bafilomycin A1-treated group.

In mammalian cells, macroautophagy serves complementarily with the ubiquitin-proteasome system as two important intracellular pathways for protein degradation [15,16]. In time of nutrient deprivation, macroautophagy also functions as a pro-survival mechanism by liberating free amino acids into the cytosol as a result of self-digestion [27]. Macroautophagy is also considered to be cytoprotective based on the findings that it protects the cells against the accumulation of damaged organelles or protein aggregates [2,7], the loss of interaction with the extracellular matrix [28], and the toxicity of cancer therapies [29]. In the present study, suppression of macroautophagy by bafilomycin A1 inhibits the proliferation of colon cancer cells at doses in the low nanomolar range. The prominent anti-mitogenic effect is paralleled by the down-regulation of cyclin D1 and cyclin E and up-regulation of the cyclindependent kinase inhibitor p21Cip1. Prolonged treatment of bafilomycin A1 also induces apoptotic cell death as evidenced by the appearance of DNA fragmentation, the accumulation of cells in the sub-G1 phase, increased levels of cytoplasmic nucleosomes, and the cleavages of caspases and PARP. The anti-proliferative effect of bafilomycin A1 is mediated in part by the p38 MAPK. These data indicate that the growth and survival of colon cancer cells are incompatible with the abstinence from macroautophagy. Indeed, macroautophagy is remarkably up-regulated in colon cancer tissues when compared with the surrounding non-cancerous counterparts [10,11]. In this study, we demonstrate that inhibition of macroautophagy elicits apoptosis in colon cancer cells. The relationship between macroautophagy and apoptosis has also been reported in other cell types [30]. For instances, impairment of macroauto-

[1] A.J. Meijer, P. Codogno, Regulation and role of autophagy in mammalian cells, Int. J. Biochem. Cell Biol. 36 (2004) 2445–2462. [2] N. Mizushima, B. Levine, A.M. Cuervo, D.J. Klionsky, Autophagy fights disease through cellular self-digestion, Nature 451 (2008) 1069–1075. [3] F. Scarlatti, R. Granata, A.J. Meijer, P. Codogno, Does autophagy have a license to kill mammalian cells?, Cell Death Differ 16 (2009) 12–20. [4] G.E. Mortimore, N.J. Hutson, C.A. Surmacz, Quantitative correlation between proteolysis and macro- and microautophagy in mouse hepatocytes during starvation and refeeding, Proc. Natl. Acad. Sci. USA 80 (1983) 2179–2183. [5] J.J. Shacka, K.A. Roth, J. Zhang, The autophagy-lysosomal degradation pathway: role in neurodegenerative disease and therapy, Front. Biosci. 13 (2008) 718– 736. [6] M.C. Maiuri, E. Tasdemir, A. Criollo, E. Morselli, J.M. Vicencio, R. Carnuccio, G. Kroemer, Control of autophagy by oncogenes and tumor suppressor genes, Cell Death Differ. 16 (2009) 87–93. [7] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (2008) 27–42. [8] S. Bialik, A. Kimchi, Autophagy and tumor suppression: recent advances in understanding the link between autophagic cell death pathways and tumor development, Adv. Exp. Med. Biol. 615 (2008) 177–200. [9] R.K. Amaravadi, Autophagy-induced tumor dormancy in ovarian cancer, J. Clin. Invest. 118 (2008) 3837–3840. [10] C.H. Ahn, E.G. Jeong, J.W. Lee, M.S. Kim, S.H. Kim, S.S. Kim, N.J. Yoo, S.H. Lee, Expression of beclin-1, an autophagy-related protein, in gastric and colorectal cancers, APMIS 115 (2007) 1344–1349. [11] A. Yoshioka, H. Miyata, Y. Doki, M. Yamasaki, I. Sohma, K. Gotoh, S. Takiguchi, Y. Fujiwara, Y. Uchiyama, M. Monden, LC3, an autophagosome marker, is highly expressed in gastrointestinal cancers, Int. J. Oncol. 33 (2008) 461–468. [12] V. Marshansky, M. Futai, The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function, Curr. Opin. Cell Biol. 20 (2008) 415–426. [13] S. Gagliardi, M. Rees, C. Farina, Chemistry and structure activity relationships of bafilomycin A1, a potent and selective inhibitor of the vacuolar H+-ATPase, Curr. Med. Chem. 6 (1999) 1197–1212. [14] J.J. Shacka, B.J. Klocke, K.A. Roth, Autophagy, bafilomycin, cell death: the ‘‘a-Bcs” of plecomacrolide-induced neuroprotection, Autophagy 2 (2006) 228–230. [15] D.C. Rubinsztein, The roles of intracellular protein-degradation pathways in neurodegeneration, Nature 443 (2006) 780–786. [16] A.L. Goldberg, Protein degradation and protection against misfolded or damaged proteins, Nature 426 (2003) 895–899. [17] W.K. Wu, J.J. Sung, Y.C. Wu, Z.J. Li, L. Yu, C.H. Cho, Bone morphogenetic protein signalling is required for the anti-mitogenic effect of the proteasome inhibitor MG-132 on colon cancer cells, Br. J. Pharmacol. 154 (2008) 632–638. [18] W.K. Wu, J.J. Sung, L. Yu, C.H. Cho, Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling, Biochem. Biophys. Res. Commun. 371 (2008) 209–214. [19] K. Tobinai, Proteasome inhibitor, bortezomib, for myeloma and lymphoma, Int. J. Clin. Oncol. 12 (2007) 318–326. [20] S. Paglin, T. Hollister, T. Delohery, N. Hackett, M. McMahill, E. Sphicas, D. Domingo, J. Yahalom, A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles, Cancer Res. 61 (2001) 439–444. [21] Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (2000) 5720–5728. [22] J.M. Backer, The regulation and function of Class III PI3Ks: novel roles for Vps34, Biochem. J. 410 (2008) 1–17. [23] A. Petiot, E. Ogier-Denis, E.F. Blommaart, A.J. Meijer, P. Codogno, Distinct classes of phosphatidylinositol 30 -kinases are involved in signaling pathways that control macroautophagy in HT-29 cells, J. Biol. Chem. 275 (2000) 992– 998.

456

Y.C. Wu et al. / Biochemical and Biophysical Research Communications 382 (2009) 451–456

[24] M. Serova, A. Ghoul, K.A. Benhadji, S. Faivre, C. Le Tourneau, E. Cvitkovic, F. Lokiec, J. Lord, S.M. Ogbourne, F. Calvo, E. Raymond, Effects of protein kinase C modulation by PEP005, a novel ingenol angelate, on mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling in cancer cells, Mol. Cancer Ther. 7 (2008) 915–922. [25] H.C. Thoms, M.G. Dunlop, L.A. Stark, P38-mediated inactivation of cyclin D1/ cyclin-dependent kinase 4 stimulates nucleolar translocation of RelA and apoptosis in colorectal cancer cells, Cancer Res. 67 (2007) 1660–1669. [26] A. Yamamoto, Y. Tagawa, T. Yoshimori, Y. Moriyama, R. Masaki, Y. Tashiro, Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4II-E cells, Cell Struct. Funct. 23 (1998) 33–42. [27] J.J. Lum, R.J. DeBerardinis, C.B. Thompson, Autophagy in metazoans: cell survival in the land of plenty, Nat. Rev. Mol. Cell Biol. 6 (2005) 439–448. [28] C. Fung, R. Lock, S. Gao, E. Salas, J. Debnath, Induction of autophagy during extracellular matrix detachment promotes cell survival, Mol. Biol. Cell 19 (2008) 797–806.

[29] M.J. Abedin, D. Wang, M.A. McDonnell, U. Lehmann, A. Kelekar, Autophagy delays apoptotic death in breast cancer cells following DNA damage, Cell Death Differ. 14 (2007) 500–510. [30] A. Thorburn, Apoptosis and autophagy: regulatory connections between two supposedly different processes, Apoptosis 13 (2008) 1–9. [31] D.S. Yang, A. Kumar, P. Stavrides, J. Peterson, C.M. Peterhoff, M. Pawlik, E. Levy, A.M. Cataldo, R.A. Nixon, Neuronal apoptosis and autophagy cross talk in aging PS/APP mice, a model of Alzheimer’s disease, Am. J. Pathol. 173 (2008) 665– 681. [32] K.J. Park, S.H. Lee, T.I. Kim, H.W. Lee, C.H. Lee, E.H. Kim, J.Y. Jang, K.S. Choi, M.H. Kwon, Y.S. Kim, A human scFv antibody against TRAIL receptor 2 induces autophagic cell death in both TRAIL-sensitive and TRAIL-resistant cancer cells, Cancer Res. 67 (2007) 7327–7334. [33] K.W. Kim, M. Hwang, L. Moretti, J.J. Jaboin, Y.I. Cha, B. Lu, Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer, Autophagy 4 (2008) 659–668.