Inhibition of AMPK-Associated Autophagy Enhances Caffeic Acid Phenethyl Ester-Induced Cell Death in C6 Glioma Cells

• Abstract
• Introduction
• Materials and Methods
• Chemicals and reagents
• Cell culture
• Cell viability assay
• Western blot analysis
• Measurement of the mitochondrial membrane potential (ΔΨ _m)
• Double staining with annexin-V and propidium iodide (PI) using flow cytometry
• siRNA transfection
• Electron microscopic analyses
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

An increasing number of studies show that AMP-activated protein kinase (AMPK) activation can inhibit apoptosis. To clarify the antitumor mechanism of caffeic acid phenethyl ester (CAPE) and achieve increased therapeutic efficiency, we investigated the potential roles of AMPK and autophagy in CAPE treatment against C6 glioma cells. The roles of AMPK and autophagy inhibition in CAPE's cytotoxic action were investigated. Phosphorylation of AMPK and mitogen-activated protein kinases (MAPKs) were observed in tumor cells following CAPE treatment. A combination of CAPE and the AMPK inhibitor, compound C, resulted in augmented cell death. Similar effects of compound C were observed in response to changes in the mitochondrial membrane potential (ΔΨ _m). Small interfering RNA-mediated AMPK downregulation increased CAPE-induced cell death. The results suggest that AMPK activation plays a role in diminishing apoptosis. CAPE treatment induced an increase in LC3 conversion as represented by the LC3-II/LC3-I ratio. Enlarged lysosomes and autophagosomes were present according to electron microscopy. The autophagy inhibitor, 3-MA, caused increased CAPE cytotoxicity, which suggests that autophagy induction protected glioma cells from CAPE. The combination of CAPE with autophagy and AMPK inhibitors markedly enhanced the cytotoxicity toward C6 glioma cells. Accordingly, CAPE-triggered activation of AMPK and the autophagic response protected tumor cells from apoptotic death. This provides new insights for combined therapy to enhance the therapeutic potential of cancer treatments.

Introduction

Autophagy is characterized by the appearance of double-membraned cytoplasmic vesicles which engulf portions of the cytosol or organelles and form autophagosomes which are then destroyed by the lysosomal system. Increasing evidence supports the link between apoptosis and autophagy, including both positive and negative interactions [1]. Autophagy, described as a form of cell death distinct from apoptosis and necrosis, is a process in which cells generate energy and metabolites by digesting their own organelles and macromolecules in response to nutrient deprivation. However, the role of autophagy in anticancer therapy is controversial. In patients undergoing chemotherapy, autophagy can promote resistance to cell death, whereas blocking this cellular adaptive response results in increased tumor killing [2]. Preventing autophagy could become a potential strategy to enhance anticancer therapeutic efficacy.

Malignant gliomas are among the most lethal cancers, and they are notoriously difficult to treat. Accumulating evidence indicates that invasive glioma cells show a decreased proliferation rate and a relative resistance to apoptosis, which may contribute to their resistance to chemotherapy and radiation [3]. Activation of AMP-activated protein kinase (AMPK) was recently found to induce apoptosis in experimental brain tumors by suppressing the mammalian target of the rapamycin (mTOR) pathway [4]. Glioma cell death induced by pharmacological activation of AMPK was shown to be mediated by c-Jun N-terminal kinase (JNK) activation, mitochondrial depolarization, and oxidative stress. Caffeic acid phenethyl ester (CAPE), the active ingredient in honeybee propolis, was found to have beneficial effects due to its biological and pharmacological activities [5]. Recently, several reports indicated that CAPE is cytotoxic to tumor cells, and it was also shown to inhibit cell invasion and cell migration through matrix metalloproteinase downregulation. The cytotoxic potential of CAPE and the molecular mechanism of its action in C6 glioma cells were investigated, and the results indicated that CAPE-induced apoptosis was associated with mitochondrial dysfunction, serine phosphorylation of p53, and the expressions of Bax, Bak, and Bcl-2 [6]. To clarify the antitumor mechanism of CAPE and achieve increased therapeutic efficiency, we investigated the potential roles of AMPK and autophagy in CAPE treatment against C6 glioma cells, and the results provide insights for more efficacious therapeutic applications.

Materials and Methods

Chemicals and reagents

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5′-monophosphate (AICAR, > 98 %), CAPE (> 97 %), small interfering (si)RNAs, and N‐TER transfection reagents were purchased from Sigma. 3-Methyladenine (3-MA, > 98 %), 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine (compound C, 98 %), and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT, 98.3 %) were from EMD Chemicals. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1) was purchased from BD Biosciences. All of the solvents used in this study were from Merck. Dulbecco's modified Eagle's medium (DMEM) and penicillin-streptomycin-amphotericin were obtained from Gibco BRL. Antibodies for the Western blot analysis were from Cell Signaling Technology. The annexin-V-FITC apoptosis detection kit was purchased from BioVision. Fetal bovine serum (FBS) was from Hyclone.

Cell culture

Human non-small cell lung carcinoma H1299 cells, human colon carcinoma HT-29 cells, human breast carcinoma MCF-7 cells, human hepatocellular carcinoma (HCC) HepG2 cells, and rat glioma C6 cells were all obtained from American Tissue Culture Collection (ATCC) and grown in DMEM supplemented with 10 % FBS, 1.5 g/L sodium bicarbonate, 1 % penicillin-streptomycin-amphotericin, 1 % sodium pyrophosphate, and 1 % nonessential amino acids at 37 °C in a humidified atmosphere with 5 % CO₂.

Cell viability assay

The MTT assay to test the cytotoxicity of reagents and cell viability was performed as described previously, based on the conversion of the yellow tetrazolium salt to the purple formazan product [7]. Cells (1 × 10⁴ cells/well) were grown on a 96-well plate supplemented with culture medium. Cells were treated with CAPE (0 ∼ 50 µM) for 24 h, and an MTT stock solution (5 mg of MTT/mL of phosphate-buffered saline; PBS) was added to the growing cultures (at a final concentration of 0.5 mg/mL) for 2 h. The absorbance was measured with a spectrophotometer (Thermo Varioskan Flash) at 560 nm. A blank with DMSO alone was measured and subtracted from all values.

Western blot analysis

Cells were lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 137 mM NaCl, 1 mM sodium orthovanadate, 1 mM EGTA, 10 mM NaF, 1 mM sodium pyrophosphate, 10 % glycerol, and a protease inhibitor cocktail) on ice for 30 min and briefly sonicated. The mixtures were then centrifuged (12 000 rpm) for 20 min, and the supernatants were collected as whole-cell extracts. Samples of each lysate (30 µg proteins/lane) were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with the primary antibody at 4 °C overnight, and then incubated with a horseradish peroxidase-conjugated secondary immunoglobulin G (IgG) antibody [8]. The immunoreactive bands were visualized with enhanced chemiluminescence reagents (PerkinElmer), and photographed using the UVP Biospectrum AC imaging system.

Measurement of the mitochondrial membrane potential (ΔΨ _m)

The JC-1 dye undergoes a reversible change in fluorescence emission from red to green as the mitochondrial membrane potential decreases. Cells with a high membrane potential promote the formation of dye aggregates, due to the physiological ΔΨ _m, which fluoresce red; cells with a low potential contain monomeric JC-1 and fluoresce green. Therefore, the green/red ratio can help identify a cell's status. Cells were grown until the desired confluence and treated with the test compounds for 12 h, exposed to a 1× JC-1 dye solution (BD Biosciences) for 20 min, then examined by confocal microscopy (Leica TCS SP5). JC-1 fluorescence was viewed with excitation at 488 nm and emission at 585 ∼ 590 nm for the aggregate form (red) and 510 ∼ 527 nm for the monomeric form (green) [9]. Cell samples were also analyzed on a FACSCanto II flow cytometer (BD Biosciences). Green and red fluorescences were measured in the logarithmic mode.

Double staining with annexin-V and propidium iodide (PI) using flow cytometry

Cells were treated with the test compounds for 24 h, then trypsinized, pelleted, washed in PBS, and resuspended in 1× binding buffer containing the conjugated annexin-V (annexin-V-FITC) antibody and PI (BioVision) according to the manufacturer's protocol. These cell samples were analyzed using a FACS Canto-II flow cytometer (BD Biosciences). The fraction which was both annexin-V- and PI-positive indicated cytotoxicity [10].

siRNA transfection

Cells were seeded in 6-well tissue culture plates at a density of 5 × 10⁴ cells/well and transfected with the N‐TER Nanoparticle siRNA Transfection System (Sigma) at a final siRNA concentration of 20 nM. siRNA targeting AMPK (5′-CUUAUUGGAUUUCCGAAGUTT‐3′ equally mixed with 5′-ACUUCGGAAAUCCAAUAAGTT‐3′) and a nontargeting control (5′-GCAAGCUGACCCUGAAGUUCAU‐3′ equally mixed with 5′-GAACUUCAGGGUCAGCUUGCCG‐3′) were used. The N‐TER peptide (3 µL/mL) was added to the targeted siRNA and incubated at room temperature for 20 min to allow the N‐TER-siRNA nanoparticles to form. N‐TER-siRNA complexes were then diluted in DMEM with serum and incubated at 37 °C in a humidified atmosphere with 5 % CO₂ for 24 h [11].

Electron microscopic analyses

Cells were fixed with 3 % glutaraldehyde in 0.1 M Mops buffer (pH 7.0) for 8 h at room temperature, then in 3 % glutaraldehyde/1 % paraformaldehyde in 0.1 M Mops buffer (pH 7.0) for 16 h at 41 °C, postfixed in 1 % osmium tetroxide for 1 h, embedded, sectioned, and double stained with 2 % uranyl acetate and 0.4 % lead citrate. Electron micrographs were taken with a transmission electron microscope (TEM; Hitachi H-600) at 75 kV [12].

Statistical analysis

Unless otherwise indicated, results are presented as the mean ± SD of four replicates, and at least three separate experiments were performed. One-way analysis of variance (ANOVA) followed by Bonferroni's test was used to determine statistical significance for multiple comparisons, and Student's t-test was used for two groups. * P < 0.05 was accepted as statistically significant.

Results

We examined the cytotoxic effects of CAPE against 5 different cancer cell lines including rat glioma C6, human non-small cell lung carcinoma H1299, human breast carcinoma MCF-7, human HCC HepG2 cells, and human colon carcinoma HT-29 cells. Cells treated with various concentrations (0 ∼ 50 µM) of CAPE were incubated for 24 h, and the number of surviving cells was quantitatively determined using the MTT assay. CAPE treatment decreased cell viability in a dose-dependent manner in all tested cancer cells ([Fig. 1]). The half inhibitory concentration (IC₅₀) values for CAPE were 24.6, 37.1, 43.0, 47.4, and 47.8 µM for the C6 glioma, H1299, MCF-7, HepG2, and HT29 cell lines, respectively. Among the five tested cancer cell lines, CAPE displayed the most sensitive response toward C6 glioma cells; we thus focused on the cytotoxic mechanism induced by CAPE in C6 glioma cells.

CAPE treatment induced an increase in annexin V-FITC and PI fluorescence from 3.3 % in control cells to 14.6 % ([Fig. 2 A]). We further assessed the effect of CAPE on intracellular ROS levels. Using the redox-sensitive fluorescent dye, DHR123, the fluorescence intensity significantly increased in C6 glioma cells treated with CAPE ([Fig. 2 B]). CAPE treatment produced increased ROS levels in C6 glioma cells. The proapoptotic protein, Bax, and antiapoptotic protein, Bcl-2, were analyzed after 24 h of treatment with 10 µM CAPE. The Bax level was fairly constant, whereas Bcl-2 was downregulated in a time-dependent manner. Phosphorylation of MAPKs including ERK, JNK, and p38 was further examined using antibodies against the phosphorylated forms of those kinases. Exposure of cells to 10 µM CAPE induced the phosphorylation of ERK1/2 (Thr²⁰²/Tyr²⁰⁴), p38 (Thr¹⁸⁰/Tyr¹⁸²), and JNK (Thr¹⁸³/Tyr¹⁸⁵) with different timing. ERK phosphorylation began at 1 h of treatment and persistently increased to 24 h; p38 phosphorylation began at 0.5 h of treatment and persisted through the next 1 ∼ 6 h, and then declined to an undetectable level; whereas JNK phosphorylation began at 3 h of treatment, reached a peak level at 6 h, and then declined to an undetectable level. Unphosphorylated AMPK expression remained at a fairly constant level, while its phosphorylation (Thr¹⁷²) began at 0.5 h, persisted through the next 1 ∼ 6 h, and then gradually declined to an undetectable level. AMPK activation was calculated as the ratio of phosphorylated AMPK to total AMPK, and there was a maximum 5-fold change in AMPK activation at 3 h of CAPE treatment. P38 and AMPK displayed similar activation timing in response to CAPE exposure ([Fig. 3]).

We examined the cytotoxicity of C6 cells transiently transfected with siRNA against AMPK (si-AMPK). C6 glioma cells were transfected with si-AMPK for 24 h, and protein levels of AMPK showed an 80 % reduction in knockdown si-AMPK cells ([Fig. 4 A]). The viability of cells exposed to CAPE treatment with knockdown of si-AMPK was determined. si-Scrambled and si-AMPK cells were exposed to CAPE (10 µM) for another 24 h, and the cell viability was 85 % in si-AMPK cells without CAPE exposure, while it was 51 % for si-scrambled cells exposed to CAPE for 24 h, and 33 % for si-AMPK cells ([Fig. 4 B]). The combination of CAPE and si-AMPK knockdown treatment showed synergistic activity toward C6 glioma cell cytotoxicity. The AMPK inhibitor, compound C, was used to verify the role of AMPK in the cytoprotective effect. [Fig. 5 A] shows that CAPE induced an increase in both annexin-V and PI fluorescence from 4.5 % in control cells ([Fig. 5 A], left) to 30.8 % (middle). Inhibition of AMPK activity with compound C co-treatment resulted in an increase to 58 % (right) from 30.8 % with CAPE treatment. The Δψ was detected under laser scanning confocal microscopy with JC-1 staining to confirm the roles of compound C and AICAR in CAPE treatment. Control cells showed heterogeneous staining with both red and green fluorescence coexisting in the same cell which produced a nearly orange color in the merged picture ([Fig. 5 B]). Exposure of C6 cells to CAPE and compound C co-treatment induced marked changes in ΔΨ _m as evidenced by the increase in green fluorescence in most cells. Co-treatment with the AMPK activator, AICAR, showed a similar green/red ratio as the control. The green/red ratio was quantitated via flow cytometry, and those cells showed an increase in the ratio in CAPE treatment from 0.05 (untreated control) to 0.2. CAPE-treated cells co-incubated with compound C had a ratio of 0.67, and of 0.1 with co-treatment with AICAR ([Fig. 5 C]). These data support the role of AMPK activation elicited by CAPE treatment in protecting C6 glioma cells from apoptosis. Inhibition of the elicited AMPK activation contributed to the enhanced cytotoxicity of CAPE.

The conversion of microtubule-associated protein light chain (LC) 3 is widely used to monitor autophagy. Along with the downregulation of Bcl-2 in CAPE-treated C6 cells for 24 h, the LC3-II/LC3-I ratio increased in a time-dependent manner from 1.0 at time 0 to 3.2 at 24 h ([Fig. 6 A]). To confirm elicitation of autophagy, organelle structures were inspected using TEM. Compared to untreated control cells ([Fig. 6 B–a]), enlarged lysosomes ([Fig. 6 B–b]) and autophagosomes (organelles enclosed in a vacuole) ([Fig. 6 B–c]) were observed in CAPE-treated C6 cells. The elicited autophagic response represented by beclin-1 expression was diminished in cells co-treated with compound C ([Fig. 6 C]). The results suggest that the autophagic response elicited by CAPE is associated with AMPK activation.

Single-agent treatment with CAPE (10 µM) reduced cell viability of C6 glioma cells to 49 %. The combination of CAPE and the AMPK inhibitor, compound C (10 µM), reduced viability to 22 %, and the combination of CAPE with the autophagy inhibitor, 3-MA (2 mM), reduced the viability to 31 %. The combined treatment of CAPE, compound C, and 3-MA led to a statistically significant decrease in cell viability (15 %) compared to the effects of each drug alone (p < 0.01 for all groups) ([Fig. 7]). This result indicates the synergistic effects of CAPE with the AMPK inhibitor and/or autophagy inhibitor on the cytotoxicity toward glioma cells.

Discussion

A number of studies demonstrated a protective role of autophagy against ROS-mediated cell death. It was shown that DNA damage induced by oxidative stress is involved in activating autophagy which initiates a cell-protective pathway [13], [14]. Stimulation of ROS production in mitochondria induced apoptosis following changes in the membrane potential. However, autophagy also takes part in removing damaged mitochondria and prevents apoptosis resulting from blockage of the release of proapoptotic substances, such as cytochrome (Cyt) c and Apaf-1, from mitochondria [15]. Recent studies demonstrated that upregulation of Bax and/or downregulation of Bcl-2, followed by Bax incorporation into mitochondrial membranes and release of Cyt c, are crucial events in tumor cell apoptosis induced by CAPE in other cell lines [16]. In this study, CAPE induced an increase in the Bax/Bcl-2 ratio, suggesting that the apoptotic signal is transmitted through a mitochondrion-dependent pathway.

MAPK pathways of ERK1/2, JNK, and p38 are involved in various biological responses including differentiation, proliferation, and cell death. Descriptions of the functional roles of the activation of these kinases are often controversial. CAPE-mediated p38 activation in C6 glioma cells was coordinated with AMPK phosphorylation ([Fig. 3]). Since AMPK activation is associated with autophagy, these results suggest that p38 is also involved in regulating autophagy. The ERK signaling cascade plays a role in many distinct, and even opposing, cellular processes, including proliferation, survival, and also apoptosis [17]. JNKs are characterized as stress-activated protein kinases on the basis of their activation in response to stress, and they contribute to multiple forms of cell death including apoptosis, necrosis, and autophagy [18]. The role of JNK activation in apoptosis shows opposite effects in response to different stimuli resulting in either cell survival or cell apoptosis. Lee et al. reported that transient activation of JNK is antiapoptotic, whereas sustained activation of JNK is proapoptotic [19]. Herein, activation of JNK likely contributed to the cell survival effect because JNK was transiently activated at about 6 h of CAPE treatment of C6 glioma cells. However, JNK activation occurred later than p38 and AMPK phosphorylation, so the exact role of JNK activation in CAPE treatment remains to be further identified.

Vucicevic et al. described the antiglioma effects of compound C through mechanisms involving both AMPK-dependent and ‐independent pathways [20]. Harhaji-Trajkovic et al. recently demonstrated that AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated C6 glioma cells [21]. They showed that inhibition of autophagy markedly augmented cisplatin-triggered oxidative stress and caspase activation, leading to increases in DNA fragmentation and cell apoptosis. This suggests that cisplatin-triggered activation of AMPK can induce an autophagic response that protects tumor cells from cisplatin-mediated apoptotic death. Clinical studies on cholesterol-lowering agents (statins) showed their apoptotic induction activities in HCC and colorectal carcinoma therapies. Atorvastatin-induced AMPK activation caused an endoplasmic reticular (ER) stress response leading to the induction of autophagy, which promotes cancer cell survival. The combination of atorvastatin with an autophagic inhibitor enhanced atorvastatin-induced cytotoxicity and apoptosis [22]. ER stress is associated with the BCL-2 protein and mitochondrial membrane functions to regulate diverse cellular processes including autophagy [23]. In accordance with these findings, compound C treatment or siRNA-mediated AMPK inhibition significantly reduced the cell viability with CAPE treatment, supporting the role of AMPK inhibition in augmenting glioma cell apoptosis. In conclusion, CAPE-treated C6 glioma cells were demonstrated to be associated with AMPK activation. AMPK and autophagy inhibition showed significantly increased cytotoxicity. Therefore, the intracellular energy sensor, AMPK, and autophagy were demonstrated to have protective effects in CAPE-treated glioma cells. This provides new insights for combined therapy during cancer treatment to enhance the therapeutic potential by targeting AMPK and autophagy.

Acknowledgements

This study was supported by grants from the National Science Council, Taiwan (NSC99-2113-M-038-001) and Taipei Medical University Hospital (97TMU-TMUH-09).

References

• 1 Maiuri M C, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis.  Nat Rev Mol Cell Biol. 2007;  8 741-752
  CrossrefPubMedSearch in Google Scholar
• 2 Hotchkiss R S, Strasser A, McDunn J E, Swanson P E. Cell death.  N Engl J Med. 2009;  361 1570-1583
  CrossrefPubMedSearch in Google Scholar
• 3 Sathornsumetee S, Reardon D A, Desjardins A, Quinn J A, Vredenburgh J J, Rich J N. Molecularly targeted therapy for malignant glioma.  Cancer. 2007;  110 13-24
  CrossrefPubMedSearch in Google Scholar
• 4 Mukherjee P, Mulrooney T J, Marsh J, Blair D, Chiles T C, Seyfried T N. Differential effects of energy stress on AMPK phosphorylation and apoptosis in experimental brain tumor and normal brain.  Mol Cancer. 2008;  7 37
  CrossrefPubMedSearch in Google Scholar
• 5 Ilhan A, Akyol O, Gurel A, Armutcu F, Iraz M, Oztas E. Protective effects of caffeic acid phenethyl ester against experimental allergic encephalomyelitis-induced oxidative stress in rats.  Free Radic Biol Med. 2004;  37 386-394
  CrossrefPubMedSearch in Google Scholar
• 6 Lee Y J, Kuo H C, Chu C Y, Wang C J, Lin W C, Tseng T H. Involvement of tumor suppressor protein p 53 and p 38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells.  Biochem Pharmacol. 2003;  66 2281-2289
  CrossrefPubMedSearch in Google Scholar
• 7 Hansen M B, Nielsen S E, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.  J Immunol Methods. 1989;  119 203-210
  CrossrefPubMedSearch in Google Scholar
• 8 Chan A L, Chang W S, Chen L M, Lee C M, Chen C E, Lin C M, Hwang J L. Evodiamine stabilizes topoisomerase I-DNA cleavable complex to inhibit topoisomerase I activity.  Molecules. 2009;  14 1342-1352
  CrossrefPubMedSearch in Google Scholar
• 9 Dedov V N, Cox G C, Roufogalis B D. Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy.  Micron. 2001;  32 653-660
  CrossrefPubMedSearch in Google Scholar
• 10 Rasola A, Geuna M. A flow cytometry assay simultaneously detects independent apoptotic parameters.  Cytometry. 2001;  45 151-157
  CrossrefPubMedSearch in Google Scholar
• 11 Deshayes S, Morris M, Heitz F, Divita G. Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy.  Adv Drug Deliv Rev. 2008;  60 537-547
  CrossrefPubMedSearch in Google Scholar
• 12 Huang S H, Lin C M, Chiang B H. Protective effects of Angelica sinensis extract on amyloid beta-peptide-induced neurotoxicity.  Phytomedicine. 2008;  15 710-721
  CrossrefPubMedSearch in Google Scholar
• 13 Munoz-Gamez J A, Rodriguez-Vargas J M, Quiles-Perez R, Aguilar-Quesada R, Martin-Oliva D, de Murcia G, Menissier de Murcia J, Almendros A, Ruiz de Almodovar M, Oliver F J. PARP-1 is involved in autophagy induced by DNA damage.  Autophagy. 2009;  5 61-74
  CrossrefPubMedSearch in Google Scholar
• 14 Moreau K, Luo S, Rubinsztein D C. Cytoprotective roles for autophagy.  Curr Opin Cell Biol. 2010;  22 206-211
  CrossrefPubMedSearch in Google Scholar
• 15 Sasnauskiene A, Kadziauskas J, Vezelyte N, Jonusiene V, Kirveliene V. Damage targeted to the mitochondrial interior induces autophagy, cell cycle arrest and, only at high doses, apoptosis.  Autophagy. 2009;  5 743-744
  CrossrefPubMedSearch in Google Scholar
• 16 Chen Y J, Shiao M S, Hsu M L, Tsai T H, Wang S Y. Effect of caffeic acid phenethyl ester, an antioxidant from propolis, on inducing apoptosis in human leukemic HL-60 cells.  J Agric Food Chem. 2001;  49 5615-5619
  CrossrefPubMedSearch in Google Scholar
• 17 Shaul Y D, Seger R. The MEK/ERK cascade: from signaling specificity to diverse functions.  Biochim Biophys Acta. 2007;  1773 1213-1226
  CrossrefPubMedSearch in Google Scholar
• 18 Weston C R, Davis R J. The JNK signal transduction pathway.  Curr Opin Cell Biol. 2007;  19 142-149
  CrossrefPubMedSearch in Google Scholar
• 19 Lee Y J, Shukla S D. Pro- and anti-apoptotic roles of c-Jun N-terminal kinase (JNK) in ethanol and acetaldehyde exposed rat hepatocytes.  Eur J Pharmacol. 2005;  508 31-45
  CrossrefPubMedSearch in Google Scholar
• 20 Vucicevic L, Misirkic M, Janjetovic K, Harhaji-Trajkovic L, Prica M, Stevanovic D, Isenovic E, Sudar E, Sumarac-Dumanovic M, Micic D, Trajkovic V. AMP-activated protein kinase-dependent and -independent mechanisms underlying in vitro antiglioma action of compound C.  Biochem Pharmacol. 2009;  77 1684-1693
  CrossrefPubMedSearch in Google Scholar
• 21 Harhaji-Trajkovic L, Vilimanovich U, Kravic-Stevovic T, Bumbasirevic V, Trajkovic V. AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumor cells.  J Cell Mol Med. 2009;  13 3644-3654
  CrossrefPubMedSearch in Google Scholar
• 22 Yang P M, Liu Y L, Lin Y C, Shun C T, Wu M S, Chen C C. Inhibition of autophagy enhances anticancer effects of atorvastatin in digestive malignancies.  Cancer Res. 2010;  70 7699-7709
  CrossrefPubMedSearch in Google Scholar
• 23 Hetz C A. ER stress signaling and the BCL-2 family of proteins: from adaptation to irreversible cellular damage.  Antioxid Redox Signal. 2007;  9 2345-2355
  CrossrefPubMedSearch in Google Scholar

1 Szu-Hsu Yu and Yung-Ta Kao contributed equally to this work.

Dr. Chun-Mao Lin

Department of Biochemistry
School of Medicine, Taipei Medical University

250 Wu-Xing Street

Taipei 110

Taiwan

Phone: +88 62 27 36 16 61 ext. 31 65

Fax: +88 62 27 38 73 48

Email: cmlin@tmu.edu.tw

References

• 1 Maiuri M C, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis.  Nat Rev Mol Cell Biol. 2007;  8 741-752
  CrossrefPubMedSearch in Google Scholar
• 2 Hotchkiss R S, Strasser A, McDunn J E, Swanson P E. Cell death.  N Engl J Med. 2009;  361 1570-1583
  CrossrefPubMedSearch in Google Scholar
• 3 Sathornsumetee S, Reardon D A, Desjardins A, Quinn J A, Vredenburgh J J, Rich J N. Molecularly targeted therapy for malignant glioma.  Cancer. 2007;  110 13-24
  CrossrefPubMedSearch in Google Scholar
• 4 Mukherjee P, Mulrooney T J, Marsh J, Blair D, Chiles T C, Seyfried T N. Differential effects of energy stress on AMPK phosphorylation and apoptosis in experimental brain tumor and normal brain.  Mol Cancer. 2008;  7 37
  CrossrefPubMedSearch in Google Scholar
• 5 Ilhan A, Akyol O, Gurel A, Armutcu F, Iraz M, Oztas E. Protective effects of caffeic acid phenethyl ester against experimental allergic encephalomyelitis-induced oxidative stress in rats.  Free Radic Biol Med. 2004;  37 386-394
  CrossrefPubMedSearch in Google Scholar
• 6 Lee Y J, Kuo H C, Chu C Y, Wang C J, Lin W C, Tseng T H. Involvement of tumor suppressor protein p 53 and p 38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells.  Biochem Pharmacol. 2003;  66 2281-2289
  CrossrefPubMedSearch in Google Scholar
• 7 Hansen M B, Nielsen S E, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.  J Immunol Methods. 1989;  119 203-210
  CrossrefPubMedSearch in Google Scholar
• 8 Chan A L, Chang W S, Chen L M, Lee C M, Chen C E, Lin C M, Hwang J L. Evodiamine stabilizes topoisomerase I-DNA cleavable complex to inhibit topoisomerase I activity.  Molecules. 2009;  14 1342-1352
  CrossrefPubMedSearch in Google Scholar
• 9 Dedov V N, Cox G C, Roufogalis B D. Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy.  Micron. 2001;  32 653-660
  CrossrefPubMedSearch in Google Scholar
• 10 Rasola A, Geuna M. A flow cytometry assay simultaneously detects independent apoptotic parameters.  Cytometry. 2001;  45 151-157
  CrossrefPubMedSearch in Google Scholar
• 11 Deshayes S, Morris M, Heitz F, Divita G. Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy.  Adv Drug Deliv Rev. 2008;  60 537-547
  CrossrefPubMedSearch in Google Scholar
• 12 Huang S H, Lin C M, Chiang B H. Protective effects of Angelica sinensis extract on amyloid beta-peptide-induced neurotoxicity.  Phytomedicine. 2008;  15 710-721
  CrossrefPubMedSearch in Google Scholar
• 13 Munoz-Gamez J A, Rodriguez-Vargas J M, Quiles-Perez R, Aguilar-Quesada R, Martin-Oliva D, de Murcia G, Menissier de Murcia J, Almendros A, Ruiz de Almodovar M, Oliver F J. PARP-1 is involved in autophagy induced by DNA damage.  Autophagy. 2009;  5 61-74
  CrossrefPubMedSearch in Google Scholar
• 14 Moreau K, Luo S, Rubinsztein D C. Cytoprotective roles for autophagy.  Curr Opin Cell Biol. 2010;  22 206-211
  CrossrefPubMedSearch in Google Scholar
• 15 Sasnauskiene A, Kadziauskas J, Vezelyte N, Jonusiene V, Kirveliene V. Damage targeted to the mitochondrial interior induces autophagy, cell cycle arrest and, only at high doses, apoptosis.  Autophagy. 2009;  5 743-744
  CrossrefPubMedSearch in Google Scholar
• 16 Chen Y J, Shiao M S, Hsu M L, Tsai T H, Wang S Y. Effect of caffeic acid phenethyl ester, an antioxidant from propolis, on inducing apoptosis in human leukemic HL-60 cells.  J Agric Food Chem. 2001;  49 5615-5619
  CrossrefPubMedSearch in Google Scholar
• 17 Shaul Y D, Seger R. The MEK/ERK cascade: from signaling specificity to diverse functions.  Biochim Biophys Acta. 2007;  1773 1213-1226
  CrossrefPubMedSearch in Google Scholar
• 18 Weston C R, Davis R J. The JNK signal transduction pathway.  Curr Opin Cell Biol. 2007;  19 142-149
  CrossrefPubMedSearch in Google Scholar
• 19 Lee Y J, Shukla S D. Pro- and anti-apoptotic roles of c-Jun N-terminal kinase (JNK) in ethanol and acetaldehyde exposed rat hepatocytes.  Eur J Pharmacol. 2005;  508 31-45
  CrossrefPubMedSearch in Google Scholar
• 20 Vucicevic L, Misirkic M, Janjetovic K, Harhaji-Trajkovic L, Prica M, Stevanovic D, Isenovic E, Sudar E, Sumarac-Dumanovic M, Micic D, Trajkovic V. AMP-activated protein kinase-dependent and -independent mechanisms underlying in vitro antiglioma action of compound C.  Biochem Pharmacol. 2009;  77 1684-1693
  CrossrefPubMedSearch in Google Scholar
• 21 Harhaji-Trajkovic L, Vilimanovich U, Kravic-Stevovic T, Bumbasirevic V, Trajkovic V. AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumor cells.  J Cell Mol Med. 2009;  13 3644-3654
  CrossrefPubMedSearch in Google Scholar
• 22 Yang P M, Liu Y L, Lin Y C, Shun C T, Wu M S, Chen C C. Inhibition of autophagy enhances anticancer effects of atorvastatin in digestive malignancies.  Cancer Res. 2010;  70 7699-7709
  CrossrefPubMedSearch in Google Scholar
• 23 Hetz C A. ER stress signaling and the BCL-2 family of proteins: from adaptation to irreversible cellular damage.  Antioxid Redox Signal. 2007;  9 2345-2355
  CrossrefPubMedSearch in Google Scholar

1 Szu-Hsu Yu and Yung-Ta Kao contributed equally to this work.

Dr. Chun-Mao Lin

Department of Biochemistry
School of Medicine, Taipei Medical University

250 Wu-Xing Street

Taipei 110

Taiwan

Phone: +88 62 27 36 16 61 ext. 31 65

Fax: +88 62 27 38 73 48

Email: cmlin@tmu.edu.tw