Knockdown of CLIC4 enhances ATP-induced HN4 cell apoptosis through mitochondrial and endoplasmic reticulum pathways

Abstract

BackgroundHuman 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.ResultsIn 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 Ca2+ measurement revealed that Ca2+ release induced by ATP and thapsigargin, a Ca2+-ATPase inhibitor of the endoplasmic reticulum, was significantly enhanced by the suppression of CLIC4 in HN4 cells.ConclusionsKnockdown 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0070-1) contains supplementary material, which is available to authorized users.

Head and neck squamous cancer progression is marked by CLIC4 attenuation in tumor epithelium and reciprocal stromal upregulation of miR-142-3p, a novel post-transcriptional regulator of CLIC4

“…Our finding that CLIC4 is expressed in several HNSCC cell lines in vitro but is downregulated upon shifting from culture to growth as in vivo xenografts suggests that CLIC4 expression is altered by the transition to anchorage-independent growth or exposure to factors from the host microenvironment. Xue et al also reported CLIC4 expression in HN4, an additional HNSCC cell line, but suggested that CLIC4 is elevated in HNSCC and its knockdown sensitizes HN4 cells to apoptosis [109]. However, we contend that the study design and exclusive use of in vitro manipulations, in light of the dynamic regulation of CLIC4 in vivo, does not tell the entire story.…”

Intracellular Chloride Channels: Novel Biomarkers in Diseases

“…Given the association with TGF-β pathway, CLIC4 could be directly involved in ROS mediated mechanisms for tumor growth (Yao et al, 2009). CLIC4 expression is shown to be elevated in human oral squamous carcinoma compared to normal controls (Xue et al, 2016). This study demonstrated that knockdown of CLIC4 expression by siRNA leads to increased apoptosis, mediated by enhanced ATP and thapsigargin induced calcium release from endoplasmic reticulum calcium stores (Xue et al, 2016).…”

“…CLIC4 expression is shown to be elevated in human oral squamous carcinoma compared to normal controls (Xue et al, 2016). This study demonstrated that knockdown of CLIC4 expression by siRNA leads to increased apoptosis, mediated by enhanced ATP and thapsigargin induced calcium release from endoplasmic reticulum calcium stores (Xue et al, 2016). It would be interesting to investigate how endoplasmic reticulum/mitochondrial localization of CLIC4 could possibly be affecting tumor metabolism.…”

TDB protects vascular endothelial cells against oxygen-glucose deprivation/reperfusion-induced injury by targeting miR-34a to increase Bcl-2 expression

“…Previous studies13141516 have revealed that activation of anti-apoptotic Bcl-2 protein or inactivation of pro-apoptotic Bax protein could disturb the process of vascular endothelial cell apoptosis under ischemic conditions. In this study, we found that TDB significantly increased Bcl-2 protein expression in a dose-dependent manner without affecting Bax expression.…”

“…Recent evidences13141516 suggest that Bcl-2 protein family members are potent regulators of the mitochondrial changes during apoptosis, and two general drug intervention strategies of preventing mitochondrial depolarisation are practicable. The first is to enhance expression of mitochondria-related anti-apoptosis proteins, such as Bcl-2 and Bcl XL .…”