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

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.

Key words

AMPK - autophagy - CAPE - glioma - oxidative stress

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
• 2 Hotchkiss R S, Strasser A, McDunn J E, Swanson P E. Cell death.  N Engl J Med. 2009;  361 1570-1583
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 10 Rasola A, Geuna M. A flow cytometry assay simultaneously detects independent apoptotic parameters.  Cytometry. 2001;  45 151-157
• 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
• 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
• 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
• 14 Moreau K, Luo S, Rubinsztein D C. Cytoprotective roles for autophagy.  Curr Opin Cell Biol. 2010;  22 206-211
• 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
• 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
• 17 Shaul Y D, Seger R. The MEK/ERK cascade: from signaling specificity to diverse functions.  Biochim Biophys Acta. 2007;  1773 1213-1226
• 18 Weston C R, Davis R J. The JNK signal transduction pathway.  Curr Opin Cell Biol. 2007;  19 142-149
• 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
• 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
• 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
• 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
• 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

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