title: Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.
abstract: Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.
authors.affiliation: Tzu Chi University School of Medicine and Department of Dermatology, Buddhist Tzu Chi General Hospital, No.701, Zhongyang Rd., Sec .3, Hualien, 97004 Taiwan, R.O.C. Tel: +88 638565301 jh.med.edu@hotmail.com.
authors.forename: Tsung-Ho
authors.initials: TH
authors.lastname: Ying
authors.forename: Chia-Wei
authors.initials: CW
authors.lastname: Chen
authors.forename: Yu-Ping
authors.initials: YP
authors.lastname: Hsiao
authors.forename: Sung-Jen
authors.initials: SJ
authors.lastname: Hung
authors.forename: Jing-Gung
authors.initials: JG
authors.lastname: Chung
authors.forename: Jen-Hung
authors.initials: JH
authors.lastname: Yang
mesh_terms: D021581:Active Transport, Cell Nucleus; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D019343:Citric Acid / Q000494:pharmacology*; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000201:enzymology; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D017382:Reactive Oxygen Species; D019343:Citric Acid; D020169:Caspases
keywords: Citric acid; HaCaT; apoptosis; cell cycle; cytotoxic effect