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.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Autophagy/physiology , Caffeic Acids/pharmacology , Phenylethyl Alcohol/analogs & derivatives , Pyrazoles/pharmacology , Pyrimidines/pharmacology , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/drug effects , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Apoptosis , Autophagy/drug effects , Cell Death/drug effects , Cell Line, Tumor , Down-Regulation , Drug Synergism , Glioma/pathology , Humans , Membrane Potential, Mitochondrial/drug effects , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Oxidative Stress , Phenylethyl Alcohol/pharmacology , Phosphorylation/drug effects , RNA, Small Interfering/metabolism , RatsABSTRACT
Caffeic acid is a xanthine oxidase (XO) inhibitor that binds to the molybdopterin region of its active site. Caffeic acid phenethyl ester (CAPE) has higher hydrophobicity and exhibits stronger inhibition potency toward XO. Chlorogenic acid is a quinyl ester of caffeic acid that has increased hydrophilicity and also shows stronger XO inhibitory activity compared with caffeic acid. Caffeic acid and CAPE showed competitive inhibition against XO, whereas chlorogenic acid displayed mixed-type inhibition, implying that it binds to sites other than the active site. Structure-based molecular modeling was performed to account for the different binding characteristics of the hydrophobic and hydrophilic esters of caffeic acid. Chlorogenic acid showed weak binding to the molybdopterin region of XO, while it more strongly bound the flavin adenine dinucleotide region than it did the molybdopterin region. These results provide the basis for interactions of caffeic acid analogues with XO via various binding domains.
