title: Knockdown of CLIC4 enhances ATP-induced HN4 cell apoptosis through mitochondrial and endoplasmic reticulum pathways.
abstract: BACKGROUND
  Human head and neck squamous carcinoma is the 6th most prevalent carcinoma worldwide. Although many novel therapies have been developed, the clinical treatment for patients remains non-ideal. Chloride intracellular channel 4 (CLIC4), one of the seven members of the CLIC family, is a newly found Cl(-) channel that participates in various biological processes, including cellular apoptosis and differentiation. Accumulating evidence has revealed the significant role of CLIC4 in regulating the apoptosis of different cancer cells. Here, we investigated the functional role of CLIC4 in the apoptosis of HN4 cells, a human head and neck squamous carcinoma cell line.
  
  
  RESULTS
  In the present study, we used immunohistochemical staining to demonstrate that the expression level of CLIC4 is elevated in the tissue of human oral squamous carcinoma compared with healthy human gingival tissue. Specific CLIC4 small interfering RNA was used to knockdown the expression of CLIC4. The results showed that knockdown of CLIC4 with or without 100 μM adenosine triphosphate (ATP) treatment significantly increased the expression of Bax, active caspase 3, active caspase 4 and CHOP but suppressed Bcl-2 expression in HN4 cells. Moreover, the results from the TdT-mediated dUTP nick end labeling assay indicated that CLIC4 knockdown induced a higher apoptotic rate in HN4 cells under the induction of ATP. In addition, knockdown of CLIC4 dramatically enhanced ATP-induced mitochondrial membrane depolarization in HN4 cells. Moreover, intracellular Ca(2+) measurement revealed that Ca(2+) release induced by ATP and thapsigargin, a Ca(2+)-ATPase inhibitor of the endoplasmic reticulum, was significantly enhanced by the suppression of CLIC4 in HN4 cells.
  
  
  CONCLUSIONS
  Knockdown of CLIC4 enhanced ATP-induced apoptosis in HN4 cells. Both the pathways of mitochondria and endoplasmic reticulum stress were involved in CLIC4-mediated cell apoptosis. Based on our finding, CLIC4 may be a potential and valuable target for the clinical treatment of head and neck squamous carcinoma.
authors.affiliation: Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022 Anhui China.
authors.forename: Haowei
authors.initials: H
authors.lastname: Xue
authors.affiliation: Department of Physiology, Anhui Medical University, 81 Meishan Road, Hefei, 230032 Anhui China.
authors.forename: Jinsen
authors.initials: J
authors.lastname: Lu
authors.affiliation: Department of Physiology, Anhui Medical University, 81 Meishan Road, Hefei, 230032 Anhui China.
authors.forename: Renxiang
authors.initials: R
authors.lastname: Yuan
authors.affiliation: Department of Dermatology, Anhui Provincial Hospital, Hefei, 230001 Anhui China.
authors.forename: Jinli
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022 Anhui China.
authors.forename: Yehai
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022 Anhui China.
authors.forename: Kaile
authors.initials: K
authors.lastname: Wu
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022 Anhui China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Physiology, Anhui Medical University, 81 Meishan Road, Hefei, 230032 Anhui China.
authors.forename: Juan
authors.initials: J
authors.lastname: Du
authors.affiliation: Department of Physiology, Anhui Medical University, 81 Meishan Road, Hefei, 230032 Anhui China.
authors.forename: Bing
authors.initials: B
authors.lastname: Shen
keywords: Adenosine triphosphate; Apoptosis; Chloride intracellular channel 4; Endoplasmic reticulum stress; Head and neck squamous carcinoma; Mitochondrial membrane potential
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