The tyrosine kinase inhibitor imatinib (Gleevec, Novartis Pharmaceuticals Corporation; Basel, Switzerland) is a powerful drug for treatment of chronic myelogenous leukemia (CML) and other malignancies. It selectively targets various tyrosine kinases, thereby leading to growth arrest of respective cancer cells. Given its wide application, it is of high importance to know all related underlying molecular mechanisms. We had previously found that imatinib increases the cellular clearance of intracellular protein aggregates by targeting the abl pathway and thereby upregulating lysosomal activity. Here, we describe that imatinib dose dependently activates the cellular autophagy machinery in mammalian cells, independently of tissue type, species origin or immortalization status of cells. Autophagy is an archetypical cellular degradation mechanism implicated in many physiological and pathophysiological conditions. Our data link for the first time the process of autophagy with the mode of action of imatinib. Induction of autophagy might represent an additional mechanism of imatinib to induce growth arrest, promote apoptosis in cancer cells and eventually even promote tumour regression.
Imatinib (also known as Gleevec; Novartis Pharmaceuticals Corporation, Basel, Switzerland) is a very selective tyrosine kinase inhibitor highly efficient in the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours (GIST). The phenylaminopyrimidine derivative imatinib, originally designed to inhibit the CML-specific tyrosine kinase bcr-abl, has been shown to target also c-abl and Arg (abl-related gene) kinases,1, 2 the platelet-derived growth factor receptor a and b (PDGF-Ra and b), the stem cell factor receptor (c-kit)3 and the macrophage colony-stimulating factor receptor (c-fms).4 As these kinases are involved in very important pathways influencing cell growth and survival, their irregular or constitutive activation causes an activation of a number of signal transduction pathways leading finally to uncontrolled cell growth and cancer. Downstream targets for bcr-abl, for example, may include Ras and Raf, Stat, Jun amino terminal kinase, the myc transcription factor and phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt).5, 6, 7 Imatinib binds to the ATP-binding site of the kinase, thereby inhibiting the phosphorylation of its targets and the activation of growth-promoting signal transduction pathways. By targeting the kinases bcr-abl in CML and c-kit in GIST, imatinib very effectively impairs the proliferation of tumour cells. Additionally, it has been shown that imatinib inhibits the growth of PDGF-R-mediated glioblastoma cells8 and sensitizes glioma cells to radiation-induced apoptosis.9 The observation that imatinib inhibits the β-amyloid production in a cell culture model, which could make the inhibitor a useful basis for the development of a novel therapy for Alzheimer's disease,10 points to a possible implication of imatinib also in neurodegenerative diseases. In line with this, we previously published that imatinib can induce the cellular clearance of prion-infected cells from PrPSc, the pathological isoform of the cellular prion protein (PrPc), by activating its lysosomal degradation.11 Here, we show that treatment with imatinib leads to a dose-dependent activation of cellular autophagy in immortalized as well as primary cells. These results possibly identify an additional mechanism by which imatinib induces growth arrest and promotes apoptosis in tumour cells.
Imatinib (Novartis Pharmaceuticals Corporation; Basel, Switzerland) was dissolved in dimethylsulphoxide at a stock solution of 10 mM and stored at −20°C. Pefabloc proteinase inhibitor was from Roche Diagnostics GmbH (Mannheim, Germany). The inhibitor CT52923 was kindly provided by Millenium Pharmaceuticals (Cambridge, MA, USA). Rapamycin, vinblastine, 3-methyladenine, wortmannin, AG957, anti-β-actin-antibody and all other chemicals were from Sigma (Munich, Germany). Immunoblotting was carried out using the enhanced chemiluminescence blotting technique (ECL plus) from Amersham Corporation (Buckinghamshire, UK). The mammalian expression vector pGFP-LC3 and the antiserum against mammalian LC3 have been described previously.12 Rat anti-lamp-1 antibody was purchased from Santa Cruz (Santa Cruz, CA, USA). Cell culture media and solutions were obtained from Gibco BRL (Karlsruhe, Germany). G418 was purchased from PAA (Marburg, Germany).
The mouse neuroblastoma cell line N2a (ATCC CCL-131) and the murine neuronal septum cell line SN56 were maintained in OptiMEM medium containing 10% fetal calf serum, antibiotics and glutamine. GFP-LC3 N2a stably expressing GFP-LC3 were generated by transfecting N2a-cells with the plasmid pGFP-LC3 using Fugene 6 (Roche Diagnostics GmbH, Mannheim, Germany). Stably transfected cells were selected by adding 1000 μg/ml G418 to the culture medium. The mouse hypothalamic cell line GT1 was cultured in MEM medium containing 10% fetal calf serum, antibiotics and glutamine. The monkey kidney cell lines COS-7 (ATCC CRL-1651) and Vero (ATCC, CCL-81), the Chinese hamster ovary cells CHO (ATCC CCL-61), the mouse muscle cells C2C12 (ATCC, CRL-1772), the murine fibroblast cell line NIH3T3 (ATCC, CRL-6361), the human lung carcinoma cell line A549 (ATCC CCL-185) and semi-permanent human foreskin fibroblasts (HFF) (ATCC CRL-2522) were maintained in DMEM containing 10% fetal calf serum, antibiotics and glutamine. Human peripheral blood mononuclear cells were freshly prepared using Ficoll Paque plus (Amersham Corporation, Buckinghamshire, UK) according to the manufacturer's protocol. Cells were kept for 24 h in RPMI medium containing 10% fetal calf serum, antibiotics and glutamine and either mock-treated or treated with 10 μ M imatinib. Then cells were lysed as described below.
Drug treatment of cells and immunoblotting
If not otherwise stated, drug treatment of cells was performed by adding 10 μ M imatinib, 10 mM 3-methyladenine, 100 nM wortmannin, 5 μ M AG957, 20 μ M CT52923 or 2 μg/ml rapamycin to the culture medium for 24 h. The substance vinblastine was added 90 min before cell lysis. For detection of LC3 or lamp-1, cells were lysed with 1% Triton X-100 in Tris-buffered saline (pH 7.5) for 20 min on ice. Cell debris was removed by centrifugation and protein concentration was measured using Coomassie Protein Assay Reagent (Pierce, Bonn, Germany). Equal amounts of lysates were analyzed by 12.5% SDS-PAGE followed by immunoblot analysis using anti-LC3-antiserum. In all experiments mock-treated control cells were used, which were treated with solvent in an identical fashion to drug-treated cells.
Confocal microscopy and indirect immunofluorescence
Cells were grown on glass coverslips (Marienfeld, Bad Mergentheim, Germany) and transfected with pGFP-LC3 using Fugene 6 reagent (Roche Diagnostics GmbH, Mannheim, Germany) for N2a, SN56, Cos-7 and HFF cells. The transfection of GT1 cells was performed with Nanofectin (PAA, Cölbe, Germany). A549 cells were transfected with jetPei (Morgan Irvine, CA, USA). Twenty-four hours after transfection cells were treated as indicated and analysed after additional 24 h. Cells were fixed with 3% paraformaldehyde in PBS for 30 min at room temperature. After sequential treatment with NH4Cl (50 mM in 20 mM glycine), Triton X-100 (0.3% in PBS), and gelatine (0.2% in PBS) for 10 min each at room temperature, anti-lamp-1 antibody (1:100 in PBS) was added and incubated for 30 min at room temperature. After an additional incubation for 30 min at room temperature with Cy3-conjugated secondary antibody (Dianova, Hamburg, Germany) (1:100 in PBS), the slides were mounted in anti-fading solution (Permafluor, Beckman Coulter, Krefeld, Germany) and stored at 4°C. Confocal laser scanning microscopy was carried out using a Zeiss LSM 510 Confocal system (Carl Zeiss, Göttingen, Germany).
For electron microscopy cells were fixed by immersion in a mixture of 2.5% glutaraldehyde, 2.5% paraformaldehyde and 0.05% picric acid in 0.067 M cacodylate buffer (pH 7.4) according to Ito and Karnovsky.13 Postfixation was performed in 1% osmium tetroxide followed by an overnight immersion with 0.3% uranylacetate dissolved in 50 mM maleate buffer (pH 5.0). Standard procedures for dehydration and embedding in Epon were employed. Thin sections were further stained with lead citrate, and were examined in a Zeiss EM 9S electron microscope.
Imatinib affects lysosomal morphology in neuronal and non-neuronal cells
We have found that the tyrosine kinase inhibitor imatinib activates the lysosomal degradation of PrPSc in prion-infected cells.11 To examine a direct effect of imatinib on cellular lysosomes, we analysed the lysosomal morphology under imatinib treatment. Lysosomes of treated and mock-treated cells were labelled with an antibody against the lysosomal membrane protein lamp-1 and analysed by indirect immunofluorescence and confocal microscopy. We initially used the mouse neuroblastoma cell line N2a for this analysis. In imatinib-treated cells, a clear change in the pattern and the intensity of the lysosomal staining compared to mock-treated controls could be observed (Figure 1a, left). This effect could be detected in all subsequently tested cell lines (see also Figure 3, lamp-1). In imatinib-treated cells the size and the amount of the lysosomes were apparently increased. This observation was confirmed by immunoblotting analysis (Figure 1a, right). Cell lysates of N2a cells treated with imatinib showed a stronger signal for lamp-1 than mock-treated cells. These results again indicate an activation of the lysosomal degradation machinery by imatinib.
Imatinib induces the formation of autophagosomes
Given the obvious enlargement of lysosomal compartments induced by treatment with imatinib, we investigated whether the observed effect is connected with an activation of cellular autophagy, a lysosome-dependent degradative process. It has been previously described that the mammalian homologue of the yeast protein Apg8p, the microtubule-associated protein 1 light chain 3 (LC3), is a novel marker for autophagy.12 Newly synthesized LC3 is immediately processed and localizes to the cytosol as LC3-I. Upon induction of autophagy some LC3-I is converted into LC3-II, which is most likely conjugated to phosphatidylethanolamine (PE) and tightly bound to the autophagosomal membranes forming ring-shaped structures in the cytosol.12 In our study N2a cells were transfected with an expression construct for rat-LC3 fused to green fluorescent protein (GFP-LC3). In mock-treated control cells GFP-LC3 was evenly distributed in the entire cytoplasm. After imatinib treatment ring-shaped structures were detectable in the cytosol, indicating the association of GFP-LC3 with autophagosomal membranes following an induction of autophagy (Figure 1b). In N2a cells stably expressing GFP-LC3 (GFP-LC3 N2a cells) we investigated the nature of those vesicles further. The induction of spherical structures by imatinib treatment was reversed when the cells were in parallel treated with 3-methyladenine, an inhibitor of PI3K,14 commonly used for specific inhibition of autophagosome formation15 (Figure 2). In contrast to 3-methyladenine, treatment with the microtubule-depolymerizing reagent vinblastine, which inhibits the fusion between autophagosomes and lysosomes,16 led to an accumulation of autophagosomes under imatinib treatment (Figure 2). A strong formation of autophagosomes was also observed when cells were treated with rapamycin, a well-characterized inhibitor of the mammalian target of rapamycin (mTOR)-kinase that has been described to induce autophagy.17 Interestingly, the rapamycin-induced vesicles were much smaller than those detected after imatinib treatment. This observation was confirmed by electron microscopy studies (data not shown). We investigated a variety of cell lines derived from different tissues like CNS, kidney and lung, and species as well as the semi-permanent human cell line HFF. In all of these cell lines, treatment with imatinib resulted in the appearance of ring-shaped structures in the cytosol, leading to the conclusion that imatinib generally induces the formation of autophagosomes (Figure 3, LC3). Additional staining of the lysosomal membrane protein lamp-1 revealed no or only rare colocalization with LC3-II (Figure 3, lamp-1 and merge). This is consistent with previous publications in which degradation and/or recycling of LC3-II after the fusion of autophagosomes with lysosomes has been reported.12 Ultrastructural analysis of imatinib-treated N2a cells by electron microscopy showed large autophagic vacuoles in contrast to control cells where no such vacuoles could be detected (Figure 4).
The PE-conjugated form of LC3, LC3-II, migrates faster than LC3-I on SDS-PAGE, leading to the appearance of two bands after immunoblotting – LC3-I with an apparent mobility of about 18 kDa and LC3-II with an apparent mobility of 16 kDa. The amount of LC3-II or the ratio between LC3-II and LC3-I correlates well with the number of autophagosomes.12 When we analysed lysates of N2a cells treated for 24 h with imatinib by immunoblotting, we observed a clear increase in the signal for LC3-II in comparison to mock-treated cells (Figure 5a, lane 1+2). When cells were treated in parallel with wortmannin, which is, like 3-methyladenine, also an inhibitor of the PI3K, or 3-methyladenine, the signal for LC3-II was decreased, as expected (Figure 5a, lane 6+7), indicating an inhibition of the LC3-II induction caused by imatinib. In contrast, vinblastine led to a strong increase in the signal intensity of LC3-II in imatinib-treated N2a cells (Figure 5a, lane 5) owing to the accumulation of autophagosomes. Interestingly, the induction of LC3-II by imatinib was even more effective than treatment with rapamycin (Figure 5a, lane 3). Additional treatment with imatinib increased the effect of rapamycin on the formation of LC3-II (Figure 5a, lane 4).
The induction of autophagosomes by imatinib is dose dependent
To analyse whether the effect of imatinib on the induction of LC3-II is dose dependent, we treated N2a cells with concentrations of imatinib varying from 0.25 to 20 μ M. After 24 h of treatment, immunoblot analysis revealed an induction of LC3-II formation already at concentrations of imatinib below 1 μ M. With increasing concentrations of imatinib an increasing amount of LC3-II was detected indicating a clear dose dependence of LC3-II production (Figure 5b).
Comparable to the experiments carried out with GFP-LC3, the LC3-II band was also induced by imatinib in all other investigated cell lines (Figure 6). To obtain experimental conditions close to an in vivo situation, freshly prepared human peripheral blood mononuclear cells (PBMC) were treated with imatinib for 24 h (Figure 6, PBMC I+PBMC II). As anticipated, imatinib increased the formation of LC3-II in PBMCs of all tested individuals (PBMC I, PBMC II, representative examples).
In summary, we show that imatinib dose-dependently induces the formation of autophagosomes and activates the process of cellular autophagy, independently of tissue type, and species origin or immortalization status of cells. Induction of autophagy was observed in immortalized cells as well as in the semi-permanent cell line HFF and in freshly prepared PBMC.
Inhibition of c-abl but not of the PDGF-R or the c-kit kinase induces autophagy
In our previous report we described that the clearance of PrPSc in prion-infected cells by imatinib was most probably caused by the inhibition of the tyrosine kinase c-abl. Neither the inhibition of the PDGF-R nor that of the c-kit kinase was able to clear prion-infected cells from PrPSc.11 To investigate the role of those kinases in the induction of autophagosome formation induced by imatinib, we treated N2a cells with CT52923, which is an inhibitor of the PDGF-R and the c-kit kinase,18 and analysed the effect on LC3-II formation in immunoblotting. Whereas the treatment with CT52923 was not able to amplify the formation of LC3-II compared to the mock-treated control (Figure 7a, lane 4 vs lane 1), imatinib and AG957, an additional inhibitor of the c-abl kinase,19 led to an increase in the LC3-II signal (Figure 7a, lane 2+3 vs lane 1). Furthermore, we analysed the formation of LC3-II-positive autophagosomes under the treatment with the different substances by confocal microscopy using GFP-LC3 N2a cells (Figure 7b). In contrast to imatinib, which induces large spherical structures within the cytoplasm, CT92523-treated cells showed the same cytosolic LC3-staining as mock-treated cells (Figure 5b). After treatment with AG957 we observed a punctuated staining pattern for LC3 in the cytosol, caused by small spherical structures (Figure 7b, AG957). Interestingly, the autophagosomes of imatinib-treated cells were larger in size than those in cells treated with AG957. Additionally, cellular autophagy was also induced by two further novel selective c-abl inhibitors (data not shown).
Taken together, we provide experimental evidence that imatinib is a strong inducer of autophagy in mammalian cells and that c-abl is likely to be the responsible tyrosine kinase.
We describe here for the first time that the tyrosine kinase inhibitor imatinib induces pronounced cellular autophagy in mammalian cells. Interestingly, the induction of autophagy upon imatinib treatment occurred in all cell lines tested, strongly suggesting that this is not a cell type or species specific but a general effect. The fact that we observed a strong dose dependence, and that autophagy was already detected at nanomolar concentrations of imatinib, clearly indicates that this is not an unspecific side effect caused by cytotoxicity but a specific process owing to the inhibition of tyrosine kinase activity. In line with this, even after 3 days of treatment with 10 μ M imatinib no cytotoxicity was measured by MTT assay and Trypan blue staining (data not shown). Additionally, further selective c-abl inhibitors tested also showed an induction of autophagy, again excluding unspecific reactions (data not shown).
Autophagy is a degradation mechanism mainly involved in the recycling and the turnover of cytoplasmic constituents by delivering cytoplasmic cargo to the lysosomes. During autophagy, portions of the cytosol are engulfed by a membrane sac resulting in a double-membrane vesicle, called autophagosome. After fusion with lysosomes, the protein and organelle contents of the autophagosome are degraded and recycled.20, 21 Beyond its classical role in the turnover of organelles and proteins, autophagy has various physiological and pathophysiological functions. Upon environmental stress, nutrient starvation or pathogen infection, cellular autophagy is activated resulting in either adaption and survival or death.
Increasing evidence points at the importance of autophagy in cancer. However, the role of autophagy in cancer is still not completely understood. Therefore, it is not clear whether autophagy kills cancer cells or protects them from adverse conditions. It is widely believed that there is a link between carcinogenesis and decreased levels of basal autophagy.22, 23 The tumour-suppressor beclin 1 (BECN1), a mammalian homologue of yeast Atg6/Vps30, which is essential for the induction of autophagy in response to nitrogen deprivation, is mono-allelically deleted in 40–75% of human sporadic breast, prostate and ovarian cancers.24 Introduction of BECN1 into MCF-7 cells induced autophagy and inhibited cell proliferation, in vitro clonogenicity and tumourigenicity.25 During later stages of tumour progression, however, autophagy seems to be upregulated. This upregulation may ensure the nutrient supply of tumour cells localized in poorly vascularized areas of the tumour.26
Induction of autophagy can promote cell adaption and survival, but under some circumstances it can also lead to cell death. Besides type-1 cell death, known as apoptosis27 and type-3, known as necrosis,28 autophagy represents the type-2 cell death also called autophagic cell death. This type is characterized by the accumulation of autophagic vacuoles in the cytosol, mitochondria dilation and enlargement of the endoplasmic reticulum and the Golgi apparatus. Various cancer therapies have been shown to induce autophagy or autophagic cell death in cancer cells derived from tissues such as breast, colon, prostate and brain. Such therapies include for example tamoxifen,29 temozolomide,30 γ-irradiation,31, 32, 33 sodium butyrate and suberoylanilide hydroxamic acid,34 hyperthermia,35 arsenic trioxide,36, 37 resveratrol,38 soybean B-group triterpenoid saponins39 and rapamycin.40 We provide evidence that, like the above-mentioned cancer therapy substances, also the anticancer drug imatinib can induce autophagy in cells. Unfortunately, the induction of autophagy in cancer cells following cancer therapy is not strictly resulting in cell death. Apparently, it can also represent a protective mechanism of the cancer cell to the treatment, by recycling proteins and cellular components41, 42 and by removing organelles damaged by the anticancer treatment. Therefore, it is important to determine in which cancer cells autophagy is induced following cancer therapy, and whether autophagy protects the cells by blocking the apoptotic pathway or kills them by leading to autophagic cell death.
Recent publications report that resistance of cancer cells against imatinib treatment can be overcome by a combined treatment with imatinib and rapamycin. In these studies, cell cycle arrest in cancer cells induced by imatinib was blocked by the activation of the PI3K/Akt/mTor pathway. Treatment with the mTor-inhibitor rapamycin abrogated this activation and hence the resistance to imatinib.43, 44 Interestingly, in one study imatinib increased the rapamycin-induced cell death of serum-deprived small cell lung cancer cells.44 We also observed an increase in autophagosome formation when cells were treated with imatinib in combination with rapamycin. Considering our results and that the PI3K/Akt/mTor pathway is also the main pathway-regulating autophagy, it would be very interesting whether the apoptosis observed in the starved small cell lung cancer cells treated with rapamycin and imatinib is also accompanied by autophagic cell death.
We demonstrate that neither c-kit nor the PDGF-R is targeted by imatinib in the induction of cellular autophagy. In line with this, the inhibition of c-Abl by the inhibitor AG957 and the two novel selective inhibitors dasatinib and nilotinib (data not shown) showed similar effects on autophagosome formation as observed with imatinib. This strongly indicates that c-Abl is the molecular target and that its inhibition by imatinib leads to the induction of autophagy. At the moment we do not know which downstream signalling pathway is involved in the activation of autophagy induced by imatinib. Given the fact that oncogenic Abl kinases interact with the PI3K class I thereby activating the Akt/mTor pathway, it might be possible that also the cellular c-abl kinase might be involved in the regulation of that pathway. Another very important pathway regulating-autophagy is exerted by the PI3K class II. An interference of imatinib with one of these two main pathways by the inhibition of c-Abl or another yet unknown target kinase resulting in the induction of autophagy is quite possible.
In summary, with the ability to induce cellular autophagy, we identified a further very important effect of imatinib. As the induction of autophagy may promote cell death in several cancer cells, this feature is in accordance with the anticancer potential of imatinib and may strengthen its pro-apoptotic function. The potential of imatinib to induce cellular autophagy should be further investigated in various cancer cells and in vivo experiments. In cancer cells where autophagy is not a protective mechanism but leads to cell death, the application of imatinib in combination with other autophagy-inducing drugs might not only inhibit tumour progression but even promote tumour regression.
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996; 2: 561–566.
Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Druker BJ et al. Inhibition of the Abl protein-tyrosine kinase in vitro by a 2-phenylaminopyrimidine derivative. Cancer Res 1996; 56: 1000–1004.
Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ . Inhibition of c-kit receptor tyrosine kinase activity by STI571, a selective tyrosine kinase inhibitor. Blood 2000; 96: 925–932.
Dewar AL, Zannettino AC, Hughes TP, Lyons AB . Inhibition of c-fms by imatinib: expanding the spectrum of treatment. Cell Cycle 2005; 4: 851–853.
Sawyers CL . Chronic myeloid leukaemia. N Engl J Med 1999; 340: 1330–1340.
Kurzrock R, Kantarjian HM, Drucker BJ, Talpaz M . Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics. Ann Int Med 2003; 138: 819–830.
Pendergast AM . The Abl family kinases: mechanisms of regulation and signalling. Adv Cancer Res 2002; 85: 51–100.
Kilic T, Alberta J, Zdunek PR, Acar M, Iannarelli P, O’Reilly T et al. Intracranial inhibition of platelet-derived growth factor-mediated glioblastoma cell growth by an orally active kinase inhibitor of the 2-phenylaminopyrimidine class. Cancer Res 2000; 60: 5143–5150.
Russell J, Brady K, Burgan W, Cerra MA, Oswald KA, Camphausen K et al. STI571-mediated inhibition of Rad51 expression and enhancement of tumor cell radiosensitivity. Cancer Res 2003; 63: 7377–7383.
Netzer WJ, Dou F, Cai D, Veach D, Jean S, Li Y et al. Gleevec inhibits beta-amyloid production but not Notch cleavage. Proc Natl Acad Sci USA 2003; 100: 12444–12449.
Ertmer A, Gilch S, Yun SW, Flechsig E, Klebl B, Stein-Gerlach M et al. The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells. J Biol Chem 2004; 279: 41918–41927.
Kabeya Y, Mizushima N, Ueno T, Yamamotot A, Kirisako T, Noda T et al. LC3, a mammalian homologue of yeast Apg8p is localized in autophagosome membranes after processing. EMBO J 2000; 19: 5720–5728.
Ito S, Karnovsky MJ . Formaledhyde-glutaraldehyde fixatives containing trinitro compounds. J Cell Biol 1968; 39: 168a–169a.
Blommaart EF, Luiken JJ, Meijer AJ . Autophagic proteolysis: control and specificity. Histochem J 1997; 29: 365–385.
Seglen PO, Bohley P . Autophagy and other vacuolar protein degradation mechanisms. Experientia 1992; 48: 158–172.
Hoyvik H, Gordon PB, Seglen PO . Use of a hydrolysable probe, [14C]lactose, to distinguish between pre-lysosomal and lysosomal steps in the autophagic pathway. Exp Cell Res 1986; 166: 1–14.
Raught B, Gingras AC, Sonenberg N . The target of rapamycin (TOR) proteins. Proc Natl Acad Sci USA 2001; 98: 7037–7044.
Yu JC, Lokker NA, Hollenbach S, Apatira M, Li J, Betz A et al. Efficacy of the novel selective platelet derived growth factor receptor antagonist CT52923 on cellular proliferation, migration, and suppression of neointima following vascular injury. J Pharmacol Exp Ther 2001; 298: 1172–1178.
Sun X, Layton JF, Elefanty A, Lieschke GJ . Comparison of effects of the tyrosine kinase inhibitors AG957, AG490 and STI571 on BCR-ABL-expressing cells, demonstrating synergy between AG490 and STI571. Blood 2001; 97: 2008–2015.
Klionsky DJ, Ohsumi Y . Vacuolar import of proteins and organelles from the cytoplasm. Annu Rev Cell Dev Biol 1999; 15: 1–32.
Yoshimori T . Autophagy : a regulated bulk degradation process inside cells. Biochem Biophys Res Commun 2004; 313: 453–458.
Otsuka H, Moskowitz M . Differences in the rates of protein degradation in untransformed and transformed cell lines. Exp Cell Res 1978; 112: 127–135.
Schwarze PE, Seglen PO . Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats. Exp Cell Res 1985; 157: 15–28.
Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 2003; 112: 1809–1820.
Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999; 402: 672–676.
Cuervo AM . Autophagy: in sickness and in health. Trends Cell biol 2004; 14: 70–77.
Kerr JF, Wyllie AH, Currie AR . Apoptosis: a baxic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239–257.
Syntichaki P, Tavernakis N . Death by necrosis. Uncontrollable catastrophe, or is there order behind the chaos? EMBO Rep 2002; 3: 604–609.
Bursch W, Ellinger A, Kienzl H, Torok L, Pandey S, Sikorska M et al. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis 1996; 17: 1595–1607.
Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S . Role of autophagy in temozolomide-inducedcytotoxicity for malignant glioma cells. Cell Death Differ 2004; 11: 448–457.
Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res 2001; 61: 439–444.
Ito H, Daido S, Kanzawa T, Kondo S, Kondo Y . Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells. Int J Oncol 2005; 26: 1401–1410.
Yao KC, Komata T, Kondo Y, Kanzawa T, Kondo S, Germano IM . Molecular response of human glioblastoma multiforme cells to ionizing radiation: cell cycle arrest modulation of the expression of cyclin-dependent kinase inhibitors, and autophagy. J Neurosurg 2003; 98: 378–384.
Shao Y, Gao Z, Marks PA, Jiang X . Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci USA 2004; 101: 18030–18035.
Komata T, Kanzawa T, Nashimoto T, Aoki H, Endo S, Nameta M et al. Mild heat shock induces autophagic growth arrest, but not apoptosis in U251-MG and U87-MG human malignant glioma cells. J Neurooncol 2004; 68: 101–111.
Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I . Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res 2003; 63: 2103–2108.
Kanzawa T, Zhang L, Xiao L, Germano IM, Kondo Y, Kondo S . Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3. Oncogene 2005; 24: 980–991.
Opipari Jr AW, Tan L, Boitani AE, Sorenson DR, Aurora A, Liu JR . Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer Res 2004; 64: 696–703.
Ellington AA, Berhow M, Singletary KW . Induction of macroautophagy in human colon cancer cells by soybean B-group triterpenoid saponins. Carcinogenesis 2005; 26: 159–167.
Takeuchi H, Kondo Y, Fujiwara K, Kanzawa T, Aoki H, Mills GB et al. Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-phosphate kinase/protein kinase B inhibitors. Cancer Res 2005; 65: 3336–3346.
Ogier-Denis E, Codogno P . Autophagy: a barrier or an adaptive response to cancer. Biochim Biophys Acta 2003; 1603: 113–128.
Gozuacik D, Kimchi A . Autophagy as a cell death and tumor suppressor mechanism. Oncogene 2004; 23: 2891–2906.
Burchert A, Wang Y, Cai D, von Bubnoff N, Paschka P, Muller-Brusselbach S et al. Compensatory PI3-kinase/Akt/mTor activation regulates imatinib resistance development. Leukemia 2005; 19: 1774–1782.
Tsurutani J, West KA, Sayyah J, Gills JJ, Dennis PA . Inhibition of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway but not the MEK/ERK pathway attenuates laminin-mediated small cell lung cancer cellular survival and resistance to imatinib mesylate or chemotherapy. Cancer Res 2005; 65: 8423–8432.
We thank Dr Max Nunziante for helpful discussions and critically reading of the manuscript. This work was supported by the SFB-596 (project A8), FORPRION grant number LMU 5* and the DFG (SCHA 594/3-4), and was performed within the framework of the EU FP6 Network of Excellence Neuroprion.
About this article
Multiple molecular pathways stimulating macroautophagy protect from alpha-synuclein-induced toxicity in human neurons
Pediatric Surgery International (2019)
Current Opinion in Pharmacology (2019)
European Journal of Pharmaceutical Sciences (2019)