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Autophagy. 2011 February; 7(2): 176–187.
Published online 2011 February 1. doi:  10.4161/auto.7.2.14074
PMCID: PMC3039768

Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition


Caffeine is one of the most frequently ingested neuroactive compounds. All known mechanisms of apoptosis induced by caffeine act through cell cycle modulation or p53 induction. It is currently unknown whether caffeine-induced apoptosis is associated with other cell death mechanisms, such as autophagy. Herein we show that caffeine increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, through the use of western blotting, electron microscopy and immunocytochemistry techniques. Phosphorylated p70 ribosomal protein S6 kinase (Thr389), S6 ribosomal protein (Ser235/236), 4E-BP1 (Thr37/46) and Akt (Ser473) were significantly decreased by caffeine. In contrast, ERK1/2 (Thr202/204) was increased by caffeine, suggesting an inhibition of the Akt/mTOR/p70S6K pathway and activation of the ERK1/2 pathway. Although insulin treatment phosphorylated Akt (Ser473) and led to autophagy suppression, the effect of insulin treatment was completely abolished by caffeine addition. Caffeine-induced autophagy was not completely blocked by inhibition of ERK1/2 by U0126. Caffeine induced reduction of mitochondrial membrane potentials and apoptosis in a dose-dependent manner, which was further attenuated by the inhibition of autophagy with 3-methyladenine or Atg7 siRNA knockdown. Furthermore, there was a reduced number of early apoptotic cells (annexin V positive, propidium iodide negative) among autophagy-deficient mouse embryonic fibroblasts treated with caffeine than in their wild-type counterparts. These results support previous studies on the use of caffeine in the treatment of human tumors and indicate a potential new target in the regulation of apoptosis.

Key words: apoptosis, autophagy, PI3K/Akt/mTOR/p70S6K, ERK1/2, caffeine


Caffeine has a diverse range of pharmacological effects.1 In addition to its various effects on the cell cycle and growth arrest, higher (4–10 mM) concentrations of caffeine can induce apoptosis in several cell lines, such as 10 mM caffeine in human neuroblastoma cells,2 4 mM caffeine in human pancreatic adenocarcinoma cells3 and 5 mM caffeine in human A549 lung adenocarcinoma cells.4 Although caffeine has been reported to modulate cell cycle checkpoints and perturb molecular targets of the cell cycle, the exact mechanism of caffeine-induced apoptosis remains unclear.1

Autophagy is a key mechanism in various physiopathological processes, including tumorigenesis, development, cell death and survival.5,6 It has also been shown to have a complex relationship with apoptosis, especially in tumor cell lines.7 Several reports have shown that autophagy not only enhances caspase-dependent cell death, but is also required for it.8 In contrast, it has also been shown that autophagy plays an important role in promoting cell survival against apoptosis.7 Caffeine has been reported to inhibit some kinase activities, including various forms of phosphoinositol-3 kinase and mammalian target of rapamycin (mTOR).9,10 Recently, in food spoilage studies involving yeast, caffeine has been shown to induce a starvation response,11 which is a key regulator of autophagy causing its induction. However, the exact mechanism by which caffeine induces autophagy is still unknown.

Here we report that higher concentrations of caffeine enhance autophagic flux in a dose-dependent manner in various cell lines. Furthermore, we show that caffeine-induced autophagy is mainly dependent on PI3K/Akt/mTOR/p70S6 signaling and eventually results in apoptosis.

Results and Discussion

Caffeine (Fig. 1A) is a widely used psychoactive drug that has been used for centuries to increase alertness and energy. It has been reported that caffeine induces autophagy in Zygosaccharomyces bailii in association with a starvation response, caused by a unknown mechanism.11 However, it remains unknown whether caffeine affects autophagy in mammalian cells. To determine if caffeine regulates autophagy at a steady state, we first examined levels of the microtubule-associated protein 1 light chain 3 (LC3)-II, which is an LC3-phosphatidyl-ethanolamine conjugate and a promising autophagosomal marker.12 LC3-II levels (compared to actin loading controls) increased with 525 mM caffeine treatment over 48 hours in SH-SY5Y (Fig. 1B and C), PC12D and HeLa cells (Suppl. Fig. S1A and B). The LC3-II/actin ratio also increased in a time-dependent manner in SH-SY5Y (Fig. 1D and E) and HeLa cells (data not shown). Using an electron microscopy technique, the numbers of autophagic vacuoles (AVs) were markedly increased in SH-SY5Y cells treated with 10 or 25 mM caffeine, but not in the control (Fig. 1F and G). Morphometric analysis revealed that the number of AVs per 100 µm2 of SH-SY5Y cytoplasm in control (Mean ± standard deviation: 1.3 ± 0.50), whereas that in caffeine-treated cells (10 mM: 8.0 ± 0.82; 25 mM: 15 ± 1.9) for 24 hours. Expression levels of p62, a well-known autophagic substrate, were also decreased by caffeine treatment in SH-SY5Y (Fig. 1H and I) and HeLa cells (Suppl. Fig. S1C and D). Furthermore, 10 mM caffeine treatment markedly increased the number of EGFP-LC3-positive vesicles in SH-SY5Y cells transiently transfected with EGFP-LC3 (data not shown) and HeLa cells stably expressing EGFP-LC3 (Figs. 1J and K).12,13 This effect was confirmed by the observation that caffeine administration also increased the number of vesicles positive to endogenous LC3 (Suppl. Fig. S1E).

Figure 1A G
Caffeine increases autophagic flux in various cell lines. (A) structural formula of caffeine. (B and C) SH-SY5Y cells treated with various concentrations of caffeine for 24 or 48 hours were analyzed by immunoblotting (B) with antibodies against LC3 and ...
Figure 1H K
Caffeine increases autophagic flux in various cell lines. (H and I) SH-SY5Y cells treated with various concentrations of caffeine for 24 or 48 hours were analyzed by immunoblotting with antibodies against p62 and actin. Densitometry analysis of p62 levels ...

Endogenous LC3 is post-transcriptionally processed into LC3-I, which is found in the cytosol. LC3-I is in turn lipidated to LC3-II, which then associates with autophagosome membranes.14 LC3-II can accumulate due to increased upstream autophagosome formation or impaired downstream autophagosome-lysosome fusion. To distinguish between these two possibilities, we assayed LC3-II in the presence of E64D plus pepstatin A or bafilomycin A1, which inhibits lysosomal proteases or blocks downstream autophagosome-lysosome fusion and lysosomal pro-teases, respectively.15,16 Caffeine significantly increased LC3-II levels in the presence of E64d plus pepstatin A or bafilomycin compared to E64d plus pepstatin A or bafilomycin alone in (Fig. 2A and B; Suppl. Fig. S1F and G) and HeLa cells (Fig. 2C and D; Suppl. Fig. S1H and I). A saturating dosage of bafilomycin A1 was used in this assay and no further increases in LC3-II levels were observed when cells were treated with higher concentrations. Similar results were observed in PC12D cell lines (data not shown). To confirm the caffeine effect on autophagic flux, we assessed the numbers of autolysosomes and autophagosomes in HeLa cells. The ratio of the numbers of autolysosomes (positive to both LC3 and LAMP2) to autophagosomes (positive to LC3) was increased by 10 mM caffeine treatment for 48 hours (Fig. 2E). Quantification data using ImageJ also showed significant increase of the ratio (Fig. 2F). These results strongly indicate that high concentration of caffeine treatment enhances autophagic flux.

Figure 2
Caffeine does not block autophagosome-lysosome fusion. (A–D) SH-SY5Y (A) or heLa (C) cells treated with 10 mM caffeine with or without E64d (10 µg/ml) and pepstatin A (10 µg/ml) were analyzed by immunoblotting with antibodies against ...

The class I phosphatidylinositol 3-phosphate kinase (PI3K)/ Akt/mTOR/p70ribosomal protein S6 kinase (p70S6K) signaling pathway and the Ras/Raf-1/mitogen-activated protein kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway are two well-known pathways involved in the regulation of autophagy. Both are associated with tumorigenesis and often activated in numerous types of tumors.17 Therefore, we examined the effect of caffeine on both of these pathways, using western blotting, according to the protocol by Inoki and colleagues.18 After a 24 hour treatment with caffeine, there was a significant decrease in the levels of phosphorylated p70 S6 kinase, S6 ribosomal protein and 4E-BP1, compared with total normal levels in SH-SY5Y (Fig. 3A), HeLa and PC12D cells (data not shown). Consistent with these results, nonphosphorylated 4E-BP1 proteins were increased by caffeine treatment (Fig. 3A). To further investigate the upstream inhibition of mTOR by caffeine, we examined Ser473 phosphorylation of Akt, which measures both Akt/mTOR and mTORC2 activity. As shown in Figure 3B, treatment with caffeine also decreased the level of phosphorylated Akt in SH-SY5Y cells, which was consistent with a previous report.19 Similar findings were obtained in HeLa (Suppl. Fig. S2A) and PC12D cells (data not shown). Subsequently, we examined whether caffeine increases the phosphorylation of ERK1/2, a key regulator of autophagy downstream of Akt. As shown in Figure 3C, treatment with caffeine increased phosphorylated ERK1/2. The effects of caffeine on mTOR inhibition were initially detected 3 hours after the addition of caffeine and reached a maximal level after 6 hours in SH-SY5Y (Fig. 3D) and 9 hours in HeLa cells (Suppl. Fig. S2B and C).

Figure 3
Caffeine inhibits the Akt/mTOR/p70S6 signaling pathway and activates ERK1/2 signaling. (A and B) SH-SY5Y cells treated with or without 10 mM caffeine for 24 hours were analyzed for mTOR activity by immunoblotting for levels of phosphor- and total p70 ...

Caffeine has been shown to inhibit PI3K and components of the PI3K/Akt pathway.9,20 Next, we performed experiments to confirm whether caffeine-induced autophagy is activated through the PI3K/Akt pathway. Insulin or insulin-like growth factor upregulates PI3K and its downstream targets including Akt and mTOR, resulting in the inactivation of autophagy.2123 As shown in Figure 4A and B, insulin treatment for 30 minutes significantly phosphorylated Akt at Ser473, whereas the phosphorylation was completely abolished by additional treatment with caffeine. No significant differences of the LC3-II/actin ratio between caffeine treatment and caffeine treatment with insulin were observed. Also, caffeine and Akt1/2 inhibitors did not have additive effects on the levels of LC3-II/actin ratio compared to the single treatment of caffeine or Akt inhibitors (Fig. 4C and D). To further confirm the caffeine effects on this pathway, cells were transiently transfected with myristoylated Akt (myr-Akt), a constitutively active form of Akt.24 Caffeine treatment of both cells transfected with control vector and myr-Akt markedly decreased the levels of the phosphorylated Akt (Fig. 3E), indicating that caffeine directly inhibits the Akt phosphorylation. If caffeine facilitates autophagy through PI3K/Akt and ERK1/2 signalings, the autophagy should be partially blocked by ERK1/2 inhibition using the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, U0126. U0126 significantly but mildly reversed the levels of LC3-II/actin ratio (Fig. 4F and G). The failure of U1026 to reverse completely the caffeine effect can be explained by the autophagy induction through Akt/mTOR signaling. In addition, only Akt knockdown with inducible short hairpin RNAs (shRNAs) to specifically and stably knock down all three Akt isoforms sufficiently increases autophagic flux.25 Therefore, we concluded that the caffeine-induced autophagy is mainly dependent on the PI3K/Akt/mTOR pathway.

Figure 4
Caffeine-induced autophagy is dependent on PI3K/Akt/mTOR pathway. (A) SH-SY5Y cells treated with 25 mM caffeine for 3 hours followed by treatment with or without 200 nM insulin for 30 minutes were analyzed by immunoblotting. (B) Densitometry analysis ...

Because caffeine induces autophagy dependently of mTOR inhibition, we hypothesized that combination treatment of caffeine with rapamycin would not have additive effects on autophagy. However, caffeine and rapamycin showed an additive effect on the enhancement of LC3-II/actin ratio compared to the single treatment of caffeine or rapamycin (Fig. 5A and B). Several lines of evidences support the hypothesis that resistance to rapamycin results from a positive feedback loop from mTOR/S6K1 to Akt, resulting in enhancement of Akt phosphorylation at Ser 473.2628 Recently, mutual suppression of the PI3K/Akt/ mTOR pathway by combination of rapamycin with perifosine, an Akt inhibitor, induces synergistic effects on autophagy-induced apoptosis as well as enhancement of autophagy, suggesting that dual inhibition of the PI3K/Akt/mTOR by rapamycin with caffeine would be also a rational treatment for cancer.29

Figure 5
Rapamycin treatment with caffeine has an additive effect on enhancement of autophagy. (A) SH-SY5Y cells treated with various concentrations of rapamycin with or without 10 mM caffeine for 48 hours were analyzed by immunoblotting. (B) Densitometry analysis ...

Several anti-cancer agents are known to inhibit the PI3K/Akt/ mTOR/p70S6K pathway and simultaneously activate ERK1/2, resulting in induction of autophagy in tumor cell lines.30,31 The upregulation of this process has beneficial effects in neurodegenerative diseases, such as Parkinson and Huntington diseases, whereas an excess of autophagy can lead to cell death.32,33 Therefore, we decided to investigate whether caffeine-induced autophagy rescues or induces cell death. Using PC12D cells treated with 1-methyl-4-phenylpyridinium (MPP+), a well-established Parkinson disease model,34 we determined that 1 mM caffeine treatment was not sufficient for the induction of autophagy (Suppl. Fig. S4 and B) and promoted increased cell viability, whereas >2.5 mM caffeine decreased cell viability (Fig. 6A). In addition, a significant decrease in cell viability was noted in cells treated with >2.5 mM caffeine without MPP+. Also, mitochondrial membrane potentials assessed by JC-1 were significantly preserved by 1 mM caffeine treatment compared to the control with MPP+, while those were lost by >5 mM caffeine treatment (Fig. 6B and Suppl. Fig. S5A). These data suggest that caffeine-induced autophagy is not protective in these cell lines and leads to cell death.

Figure 6
Caffeine induces apoptosis by enhancement of autophagy. (A) After PC12D cells were treated with 0, 1, 2.5, 5 or 10 mM caffeine with DMSO or MPP+ for 72 hours, cell viability was measured using trypan blue dye exclusion assay. Data are the means of triplicate ...

Autophagy and apoptosis may act independently in parallel pathways or may influence one another.7 To confirm the relationship between these pathways in cells treated with caffeine, we examined caffeine effects on the cell cycle with a propidium iodide (PI) staining assay. Treatment with 2.5–10 mM caffeine increased the percentage of cells in the sub-G1 peak, which is indicative of apoptosis (Fig. 6C). To confirm whether caffeine-induced cell death is apoptotic, we examined the activity of caspase-3, a well-known inducer of apoptosis. Treatment with 10 mM caffeine markedly increased levels of cleaved caspase-3 and decreased full-length caspase-3 in PC12D cells (Fig. 6D), consistent with previous reports on the induction of apoptosis by caffeine.3537

To test whether caffeine-induced apoptosis is dependent on autophagy, we determined whether the inhibition of autophagy by 3-methyladenine (3-MA) or Atg7 siRNA knockdown affects caffeine-induced cytotoxicity in PC12D cells. Treatment with 1 or 5 mM 3MA or Atg7 knockdown significantly decreased the percentage of cell death or cells with reduced mitochondrial membrane potentials caused by caffeine treatment (5 or 10 mM) (Fig. 6E and F and Suppl. Fig. S6B). As can be seen from the increased caffeine-induced apoptosis shown in Figure 6A and C, our data suggests that caffeine-induced autophagy is necessary for apoptotic cell death. To further confirm this, we compared autophagy-deficient mouse embryonic fibroblasts (MEFs), lacking the Atg7 gene (Atg7−/−), without LC3-II expression (Suppl. Fig. S4E), and matched wild-type (Atg7+/+) MEFs, in which autophagy is induced by caffeine in a dose-dependent manner (Suppl. Fig. S4C and D). As expected, the level of caffeine-induced cell death (positive to trypan blue staining) in Atg7−/− MEFs was less than that in Atg7+/+ MEFs (Fig. 7A). The numbers of early apoptotic cells (annexin V positive, PI negative) were significantly increased in both a time-dependent and dose-dependent manner by caffeine treatment of Atg7+/+ MEFs compared to Atg7−/− MEFs (Fig. 7B–D). Also, apoptotic or necrotic cells (annexin V positive) were significantly increased by caffeine treatment of Atg7+/+ MEFs compared to Atg7−/− MEFs (Suppl. Fig. S6). Together, these results indicate that caffeine-induced autophagy partly occurs upstream of apoptosis and is not a protective response to caffeine.

Figure 7
Cells without Atg7 expression are more resistant to caffeine-induced apoptosis. (A) After Atg7+/+ or −/− mouse embryonic fibroblasts (MEFs) were treated with 0, 1, 2.5, 5, 10, 25 mM caffeine for 24 hours, the cell viability was measured ...

In various tumor cell lines, higher concentrations of caffeine alone induce p53-dependent G1 phase arrest and under certain conditions apoptosis can also occur in a p53-independent manner.1 Furthermore, disruption at the G2/M checkpoint by caffeine allows cells time to repair DNA damage by driving them through mitosis, eventually resulting in apoptosis.36,38,39 Consistent with these reports, the results of our study indicate that increased concentrations of caffeine treatment cause a dose-dependent increase in apoptosis. More recently, autophagy, a process long known to provide a survival advantage to cells undergoing nutrient deprivation and other stresses, has also been linked to the cell death process.7 The cross-talk between apoptosis and autophagy is complex and sometimes contradictory; however, it is critical to the overall fate of the cell. In this study, we have shown that autophagy is induced by higher concentrations of caffeine without starvation, mainly via the inhibition of PI3K/Akt/mTOR/p70S6K signaling. Likewise, when caffeine-induced autophagy is blocked by 3-MA treatment or Atg7 knockout, apoptosis is partially attenuated, suggesting that caffeine-induced autophagy occurs upstream of caffeine-induced apoptosis. It also indicates the involvement of other pathways in caffeine-induced apoptosis. These results provide new insight into the effects of caffeine on cell death and survival and its use as a possible intervention strategy for the upregulation of apoptosis by a harnessing of its autophagic activity in tumor treatment.

Materials and Methods

Cell line.

HeLa cells were maintained in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 100 U/ml penicillin/streptomycin (Sigma) at 37°C and 5% CO2. PC12D and SH-SY5Y cells were maintained in DMEM (Sigma) supplemented with 10% FBS (Sigma), 5% horse serum and 100 U/ml penicillin/streptomycin at 37°C and 5% CO2. All experiments with PC12D were performed after differentiation with NGF treatment for 48 hours. Atg7+/+ and −/− MEFs were maintained in DMEM (Sigma) supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 1% sodium pyruvate (Gibco, 11360), 1% non-essential amino acid (NEAA) and 4.2 µl 2% beta-mercaptoethanol at 37°C.

To establish a HeLa GFP-LC3 stable cell line, proliferating HeLa cells were transfected with a GFP-LC3 plasmid.14 Forty-eight hours post-transfection with Lipofectamine 2000 (Invitrogen), positive stable clones were selected by growing cells with G418 (400 µg/ml) for 2 weeks and maintained in DMEM (Sigma) supplemented with 10% FBS (Sigma), 100 U/ml penicillin/streptomycin and 200 µg/ml G418 at 37°C and 5% CO2. All cellular experiments were performed with cells cultured in complete medium with FBS as explained above.

Cell viability assays.

A trypan blue dye (Invitrogen, 15250-061) exclusion assay was used to examine cell viability and performed according to previously reported protocols.40,41 Changes of mitochondrial membrane potentials were assessed also with the lipophilic cationic membrane potential-sensitive dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraehylbenzimidazolylcarbocyanineiodide) (Wako, 106-00131) according to the manufacturer's protocol. Detection of early apoptotic cells was determined using an annexin V/propidium iodide (PI) detection kit (Invitrogen), according to the manufacturer's protocol. Briefly, 0.5 × 106 Atg7+/+ or −/− MEFs were exposed to caffeine (0–25 mM) for 24 hours and washed twice. Then, they were incubated at room temperature with annexin V/Alexa488 and PI for 15 minutes. Annexin V+PI cells, considered as early apoptotic cells, were enumerated using FACScan (BD Biosciences). Data were analyzed with CellQuest (BD Biosciences) and FlowJo softwares (Tree Star Inc.). Cells positive or negative for annexin V were regarded as apoptotic or non-apoptotic cells, respectively.

Cell cycle analysis.

To examine apoptosis, 1.0 × 104 cells/well PC12D cells were seeded onto 96-well culture plate and incubated for 48 h in DMEM with NGF and treated with caffeine for 72 h. The cells were harvested and washed with PBS and fixed with ice-cold 70% ethanol at 4C for 2 h. The cells were then stained with PI solution according to previously reported protocol.41 DNA content was analyzed by flow cytometry using FACScan and CellQuest software (BD Biosciences).


Compounds used included caffeine (Wako, 031-06792), E64d (Sigma, E8640), pepstatin A (Sigma, P5318), rapamycin (LC Laboratories, R5000), CCI-779 (Selleck Chemicals, S1044), MPP+ (Sigma, M0896), bafilomycin A1 (Sigma, B1793), 3-methyladenine (Sigma, M9281), insulin (Sigma, I0516), U0126 (Sigma, U120), Akt1/2 inhibitors (Sigma, A6730), staurosporine (Cell Signaling Technology, 9953) and DMSO (Sigma, D2650).

Plasmid DNAs.

Myrystoylated Akt (21–151), a constitutively active form of Akt, was purchased from Millipore.

siRNA knockdown experiments. PC12D cells were transfected with rat Atg7 siRNAs (Invitrogen, 10620318-9) using Lipofectamine RNAiMAX (Invitrogen, 13778-075) according to the manufacturer's protocol.

Western blotting.

Cell pellets were lysed on ice in RIPA buffer for 20 minutes in the presence of protease inhibitor (Roche). Western blotting was performed according to a previously published report.42 The antibodies used were as follows: anti-p70 ribosomal protein (Cell Signaling Technology, 2708), anti-ribosomal protein (Cell Signaling Technology, 2217), anti-4E-BP1 (Cell Signaling Technology, 9452), anti-Akt (Cell Signaling Technology, 9272), anti-p44/42 MAP kinase (Cell Signaling Technology, 9102), anti-phospho-p70 ribosomal protein (Thr389) (Cell Signaling Technology, 9205), anti-phospho-S6 ribosomal protein (Ser235/236) (Cell Signaling Technology, 2211), anti-phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology, 9459), anti-phospho-p44/p42 MAPK (Thy202/Tyr204) (Cell Signaling Technology, 9101), anti-Atg7 (Cell Signaling Technology, 2631), anti-phospho-Akt (Cell Signaling Technology, 4060), anti-actin (Millipore, clone C4), anti-LC3 (MBL, clone 4E12), anti-p62 (Progen Biotechnik, GP62-C) antibodies. Antibody signals were enhanced with chemifluorescent methods from GE HealthCare.

Immunofluorescent microscopy.

Cells were embedded with 4% paraformaldehyde for 20 minutes. Following this, they were permeabilized with 0.1% Triton-X in 1x PBS. After incubation with 10% FBS and 1% bovine serum albumin in 1x PBS for 30 minutes, cells were immunostained with anti-LC3B (x500) (Sigma, L7543), anti-LAMP2 (x50) (Development Studies Hybridoma Bank, clone H4B4) overnight and incubated with anti-rabbit IgG tagged with AlexaFluor 488 or anti-mouse IgG tagged with AlexaFluor 546 for 1 hour. The cover slips were embedded with VectaShield, stained with DAPI and images were acquired on a Zeiss LSM510 META confocal microscope (63 × 1.4 NA) or a Leica TCS SP5 confocal microscope at room temperature using Zeiss LSM510 v.3.2 software or Leica LAS AF software. Adobe Photoshop 7.0 (Adobe Systems Inc.) was used for subsequent image processing. For colocalization assay in HeLa cells, an appropriate confocal image was taken with Leica LAS AF software. Then, these images were analyzed automatically with the ImageJ “Colocalization” Plugin (Settings: Each threshold: 25, Ratio: 75%) followed by “Analyze particles” (Settings: threshold 25; Pixel: 1) between endogenous LC3 positive and LAMP2 vesicles. Experiments were done in triplicate at least twice.

Quantification of cells with GFP-LC3 vesicles.

HeLa cells stable expressing GFP-LC3 were treated with various concentrations of caffeine for 24 or 48 hours and then fixed as described above. Analyses in triplicate were done for counting the proportion of GFP-positive cells with GFP-LC3 vesicles as previously described in reference 43.

Electron microscopy.

SH-SY5Y cells treated with various concentrations of caffeine were prefixed in 2% glutaraldehyde in PBS at 4°C, treated with 1% OsO4 for 3 hours at 4°C, dehydrated in a graded series of ethanol and flat embedded in epon. Ultra-thin sections were doubly stained with uranyl acetate and observed using a JEOL JEM-2000EX electron microscopy at 80 kV.

Statistical analysis.

Densitometry analysis was performed using ImageJ 1.43 on immunoblots from three independent experiments. A t-test was performed with SYSTAT software (Hulinks).


We thank Dr. Takashi Ueno (Department of Biochemistry, Juntendo University) for critical comments and Drs. Masaaki Komatsu and Yu-Shin Sou for providing Atg7+/+ and −/− MEFs. We are very grateful for a grant from Hayashi Memorial Foundation for Female Natural Scientists (Y.S.), the Grant-in-Aid for Young Scientists (B) (S. Saiki and F. Sato), grants from the All Japan Coffee Association (S. Saiki), the Takeda Scientific Foundation (S. Saiki) and the Nagao Memorial Fund (S. Saiki).


phosphoinositide-3 kinase
eukaryotic initiation factor 4-binding protein 1
extracellular signal-regulated kinase
mammalian target of rapamycin
mouse embryonic fibroblasts
70-kDa ribosomal protein S6 kinase
propidium iodide


Supplementary Material

Supplementary Material:


1. Bode AM, Dong Z. The enigmatic effects of caffeine in cell cycle and cancer. Cancer Lett. 2007;247:26–39. [PMC free article] [PubMed]
2. Jang MH, Shin MC, Kang IS, Baik HH, Cho YH, Chu JP, et al. Caffeine induces apoptosis in human neuroblastoma cell line SK-N-MC. J Korean Med Sci. 2002;17:674–678. [PMC free article] [PubMed]
3. Gururajanna B, Al-Katib AA, Li YW, Aranha O, Vaitkevicius VK, Sarkar FH. Molecular effects of taxol and caffeine on pancreatic cancer cells. Int J Mol Med. 1999;4:501–507. [PubMed]
4. Qi W, Qiao D, Martinez JD. Caffeine induces TP53-independent G(1)-phase arrest and apoptosis in human lung tumor cells in a dose-dependent manner. Radiat Res. 2002;157:166–174. [PubMed]
5. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature. 2008;451:1069–1075. [PMC free article] [PubMed]
6. Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature. 2006;443:780–786. [PubMed]
7. Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A. Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 2009;16:966–975. [PubMed]
8. Espert L, Denizot M, Grimaldi M, Robert-Hebmann V, Gay B, Varbanov M, et al. Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4. J Clin Invest. 2006;116:2161–2172. [PMC free article] [PubMed]
9. Foukas LC, Daniele N, Ktori C, Anderson KE, Jensen J, Shepherd PR. Direct effects of caffeine and theophylline on p110delta and other phosphoinositide 3-kinases. Differential effects on lipid kinase and protein kinase activities. J Biol Chem. 2002;277:37124–37130. [PubMed]
10. Kudchodkar SB, Yu Y, Maguire TG, Alwine JC. Human cytomegalovirus infection alters the substrate specificities and rapamycin sensitivities of raptor- and rictor-containing complexes. Proc Natl Acad Sci USA. 2006;103:14182–14187. [PubMed]
11. Winter G, Hazan R, Bakalinsky AT, Abeliovich H. Caffeine induces macroautophagy and confers a cytocidal effect on food spoilage yeast in combination with benzoic acid. Autophagy. 2008;4:28–36. [PubMed]
12. Rubinsztein DC, Cuervo AM, Ravikumar B, Sarkar S, Korolchuk V, Kaushik S, Klionsky DJ. In search of an “autophagomometer” Autophagy. 2009;5:585–589. [PubMed]
13. Tanida I, Ueno T. Kominami Em LC3 and Autophagy. Methods Mol Biol. 2008;445:77–88. [PubMed]
14. Kabeya Y, Mizushima N, Ueno T, Yamamoto 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. [PubMed]
15. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 1998;23:33–42. [PubMed]
16. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 140:313–326. [PMC free article] [PubMed]
17. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. [PubMed]
18. Ikenoue T, Hong S, Inoki K. Monitoring mammalian target of rapamycin (mTOR) activity. Methods Enzymol. 2009;452:165–180. [PubMed]
19. Sinn B, Tallen G, Schroeder G, Grassl B, Schulze J, Budach V, Tinhofer I. Caffeine confers radiosensitization of PTEN-deficient malignant glioma cells by enhancing ionizing radiation-induced G1 arrest and negatively regulating Akt phosphorylation. Mol Cancer Ther. 9:480–488. [PubMed]
20. Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM, Abraham RT. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. 1999;59:4375–4382. [PubMed]
21. Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 2003;17:1829–1834. [PubMed]
22. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002;4:648–657. [PubMed]
23. Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M, Roccio M, Stocker H, et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell. 2003;11:1457–1466. [PubMed]
24. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N. Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol-3-kinase/Akt-dependent pathway. J Biol Chem. 1998;273:29864–29872. [PubMed]
25. Degtyarev M, De Maziere A, Orr C, Lin J, Lee BB, Tien JY, et al. Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents. J Cell Biol. 2008;183:101–116. [PMC free article] [PubMed]
26. Wan X, Harkavy B, Shen N, Grohar P, Helman LJ. Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism. Oncogene. 2007;26:1932–1940. [PubMed]
27. Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H, Khuri FR. Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res. 2005;65:7052–7058. [PubMed]
28. O'Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006;66:1500–1508. [PubMed]
29. Cirstea D, Hideshima T, Rodig S, Santo L, Pozzi S, Vallet S, et al. Dual inhibition of akt/mammalian target of rapamycin pathway by nanoparticle albumin-bound-rapamycin and perifosine induces antitumor activity in multiple myeloma. Mol Cancer Ther. 2010;9:963–975. [PMC free article] [PubMed]
30. Aoki H, Takada Y, Kondo S, Sawaya R, Aggarwal BB, Kondo Y. 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. 2007;72:29–39. [PubMed]
31. Ellington AA, Berhow MA, Singletary KW. Inhibition of Akt signaling and enhanced ERK1/2 activity are involved in induction of macroautophagy by triterpenoid B-group soyasaponins in colon cancer cells. Carcinogenesis. 2006;27:298–306. [PubMed]
32. Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ. Potential therapeutic applications of autophagy. Nat Rev Drug Discov. 2007;6:304–312. [PubMed]
33. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet. 2004;36:585–595. [PubMed]
34. Kotake Y, Ohta S. MPP+ analogs acting on mitochondria and inducing neuro-degeneration. Curr Med Chem. 2003;10:2507–2516. [PubMed]
35. Hagan MP, Hopcia KL, Sylvester FC, Held KD. Caffeine-induced apoptosis reveals a persistent lesion after treatment with bromodeoxyuridine and ultraviolet-B light. Radiat Res. 1997;147:674–679. [PubMed]
36. Efferth T, Fabry U, Glatte P, Osieka R. Expression of apoptosis-related oncoproteins and modulation of apoptosis by caffeine in human leukemic cells. J Cancer Res Clin Oncol. 1995;121:648–656. [PubMed]
37. Shinomiya N, Takemura T, Iwamoto K, Rokutanda M. Caffeine induces S-phase apoptosis in cis-diamminedichloroplatinum-treated cells, whereas cis-diamminedichloroplatinum induces a block in G2/M. Cytometry. 1997;27:365–373. [PubMed]
38. Lau CC, Pardee AB. Mechanism by which caffeine potentiates lethality of nitrogen mustard. Proc Natl Acad Sci USA. 1982;79:2942–2946. [PubMed]
39. Takagi M, Shigeta T, Asada M, Iwata S, Nakazawa S, Kanke Y, et al. DNA damage-associated cell cycle and cell death control is differentially modulated by caffeine in clones with p53 mutations. Leukemia. 1999;13:70–77. [PubMed]
40. Ormerod MG, Collins MK, Rodriguez-Tarduchy G, Robertson D. Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods. J Immunol Methods. 1992;153:57–65. [PubMed]
41. Kawatani M, Uchi M, Simizu S, Osada H, Imoto M. Transmembrane domain of Bcl-2 is required for inhibition of ceramide synthesis, but not cytochrome c release in the pathway of inostamycin-induced apoptosis. Exp Cell Res. 2003;286:57–66. [PubMed]
42. Kawajiri S, Saiki S, Sato S, Sato F, Hatano T, Eguchi H, Hattori N. PINK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy. FEBS Lett. 2010;584:1073–1079. [PubMed]
43. Sarkar S, Davies JE, Huang Z, Tunnacliffe A, Rubinsztein DC. Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein. J Biol Chem. 2007;282:5641–5652. [PubMed]

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