ClC-5 Downregulation Induces Osteosarcoma Cell Apoptosis by Promoting Bax and tBid Complex Formation

Abstract

Osteosarcoma is the most common malignant bone tumor. Chloride (Cl−) channels-mediated Cl− movement plays an important role in regulating the functions of various cancer cells, but its role in osteosarcoma remains unclear. In this study, we found that ClC-5 was increased in osteosarcoma tissues compared with normal bone tissues. Patients with high ClC-5 expression showed poor overall survival relative to those patients with low ClC-5 expression. Higher ClC-5 expression and lower intracellular Cl− concentration ([Cl−]i) were observed in osteosarcoma cells compared with normal osteoblasts. Lowering [Cl−]i increased the viability of osteosarcoma cells, which was markedly blocked by ClC-5 downregulation. Knockdown of ClC-5 significantly induced osteosarcoma cell apoptosis and increased the release of cytochrome c from mitochondria to cytosol, concomitantly with cleavage of caspase-9, caspase-3, and PARP. The effect of ClC-5 downregulation on osteosarcoma cell apoptosis and viability was abolished by caspase-3 and caspase-9 inhibitors, but not caspase-8 inhibitor. Furthermore, ClC-5 inhibition promoted Bax translocation from cytosol to mitochondria. Immunoprecipitation showed that ClC-5 interacted with Bax and ClC-5 downregulation enhanced Bax and tBid complex formation. Collectively, we demonstrate that ClC-5 downregulation induces osteosarcoma cell apoptosis via mitochondria-dependent apoptotic pathway activation by promoting Bax and tBid association and subsequent Bax translocation.

Inhibition of ClC-5 suppresses proliferation and induces apoptosis in cholangiocarcinoma cells through the Wnt/β-catenin signaling pathway

“…The decrease of mitochondrial membrane potential can cause mitochondrial hyperpolarization, and release of Cyt-c, leading to caspase-3 activation in cells, and finally to the occurrence of apoptosis (16,17). As reported, ClC-5 knockdown can promote apoptosis in osteosarcoma cells through mitochondrial apoptotic pathway activation (11). Similarly, in the present study, we also found that ClC-5 depression could inhibit Bcl-2 expression, promote Bax expression and Cyt-c release, and decrease mitochondrial membrane potential in CCA cells.…”

“…3A and B , the percentages of apoptotic cells in both shClC-5#1 and shClC-5#2 groups were increased (all P < 0.01) compared with the control group. The mitochondrial apoptotic pathway is the most important pathway of apoptosis ( 11 , 17 ). Here, we found that ClC-5 knockdown inhibited Bcl-2 expression, promoted Bax expression, and cytochrome-c (Cyt-c) release (the expression of Cyt-c was decreased in mitochondria while increased in the cytoplasm), decreased mitochondrial membrane potential (MMP; evaluation was performed with JC-1 staining), and ulti-mately led to Caspase 3 activation (the ratio of Cleaved caspase-3/Caspase-3 was increased) in both QBC939 and SNU-869 cells ( Fig.…”

“…Increasing studies have shown that ClC family proteins are involved in the pathology of many diseases, including cancer (8)(9)(10). As an idominant member of the ClC family, ClC-5 played a crucial role in the proliferation, fate, and chemotherapy resistance of various tumor cells (11)(12)(13)(14)(15). Peng et al (11) pointed out that ClC-5 was up-regulated in patients' osteosarcoma tissues compared with para-cancer tissues, and ClC-5 knockdown inhibited proliferation and induced cell death in osteosarcoma cells through promoting Bax translocation.…”

“…As an idominant member of the ClC family, ClC-5 played a crucial role in the proliferation, fate, and chemotherapy resistance of various tumor cells (11)(12)(13)(14)(15). Peng et al (11) pointed out that ClC-5 was up-regulated in patients' osteosarcoma tissues compared with para-cancer tissues, and ClC-5 knockdown inhibited proliferation and induced cell death in osteosarcoma cells through promoting Bax translocation. Similarly, ClC-5 has been reported to participate in the regulation of proliferation in glioma and leukemic cell models (12,13).…”

“…Increasing studies have shown that ClC family proteins are involved in the pathology of many diseases, including cancer ( 8 - 10 ). As an idominant member of the ClC family, ClC-5 played a crucial role in the proliferation, fate, and chemotherapy resistance of various tumor cells ( 11 - 15 ). Peng et al .…”