title: A prolyl-hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, induces cell autophagy and apoptosis in esophageal squamous cell carcinoma cells via up-regulation of BNIP3 and N-myc downstream-regulated gene-1.
abstract: The protocatechuic acid ethyl ester ethyl-3,4-dihydroxybenzoate is an antioxidant found in the testa of peanut seeds. Previous studies have shown that ethyl-3,4-dihydroxybenzoate can effectively reduce breast cancer cell metastasis by inhibiting prolyl-hydroxylase. In this study, we investigated the cytotoxic effect of ethyl-3,4-dihydroxybenzoate on esophageal squamous cell carcinoma cells in vitro and identified key regulators of ethyl-3,4-dihydroxybenzoate-induced esophageal cancer cell death through transcription expression profiling. Using flow cytometry analysis, we found that ethyl-3,4-dihydroxybenzoate induced S phase accumulation, a loss in mitochondrial membrane permeabilization, and caspase-dependent apoptosis. Moreover, an expression profile analysis identified 46 up- and 9 down-regulated genes in esophageal cancer KYSE 170 cells treated with ethyl-3,4-dihydroxybenzoate. These differentially expressed genes are involved in several signaling pathways associated with cell cycle regulation and cellular metabolism. Consistent with the expression profile results, the transcriptional and protein expression levels of candidate genes NDRG1, BNIP3, AKR1C1, CCNG2 and VEGFA were found to be significantly increased in treated KYSE 170 cells by reverse-transcription PCR and western blot analysis. We also found that protein levels of hypoxia-inducible factor-1α, BNIP3, Beclin and NDRG1 were increased and that enriched expression of BNIP3 and Beclin caused autophagy mediated by microtubule-associated protein 1 light chain 3 in the treated cells. Autophagy and apoptosis were activated together in esophageal cancer cells after exposed to ethyl-3,4-dihydroxybenzoate. Furthermore, knock-down of NDRG1 expression by siRNA significantly attenuated apoptosis in the cancer cells, implying that NDRG1 may be required for ethyl-3,4-dihydroxybenzoate-induced apoptosis. Together, these results suggest that the cytotoxic effects of ethyl-3,4-dihydroxybenzoate were mediated by the up-regulation of NDRG1, BNIP3, Beclin and hypoxia-inducible factor-1α, initiating BNIP3 and Beclin mediated autophagy at an early stage and ultimately resulting in esophageal cancer cell apoptosis.
authors.affiliation: State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Han
authors.affiliation: State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Yulin
authors.initials: Y
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Lanping
authors.initials: L
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Xiaohang
authors.initials: X
authors.lastname: Zhao
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001343:Autophagy / Q000187:drug effects; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy*; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004938:Esophageal Neoplasms / Q000188:drug therapy*; D000077277:Esophageal Squamous Cell Carcinoma; D006801:Humans; D062385:Hydroxybenzoates / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis; D051336:Mitochondrial Membranes / Q000502:physiology; D064800:Prolyl-Hydroxylase Inhibitors / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D034622:RNA Interference; D034741:RNA, Small Interfering; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects; D000099259:N-myc Downstream-Regulated Gene 1 Protein
chemical_list: D018797:Cell Cycle Proteins; D062385:Hydroxybenzoates; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D064800:Prolyl-Hydroxylase Inhibitors; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D000099259:N-myc Downstream-Regulated Gene 1 Protein; C090121:BNIP3 protein, human; C018063:ethyl protocatechuate