Knockdown of Chloride Channel-3 Inhibits Breast Cancer Growth In Vitro and In Vivo / 한국유방암학회지

PURPOSE: Chloride channel-3 (ClC-3) is a member of the chloride channel family and plays a critical role in a variety of cellular activities. The aim of the present study is to explore the molecular mechanisms underlying the antitumor effect of silencing ClC-3 in breast cancer. METHODS: Human breast cancer cell lines MDA-MB-231 and MCF-7 were used in the experiments. Messenger RNA and protein expression were examined by quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was measured by the bromodeoxyuridine method, and the cell cycle was evaluated using fluorescence-activated cell sorting. Protein interaction in cells was analyzed by co-immunoprecipitation. Tumor tissues were stained with hematoxylin-eosin and tumor burden was measured using the Metamorph software. RESULTS: Breast cancer tissues collected from patients showed an increase in ClC-3 expression. Knockdown of ClC-3 inhibited the secretion of insulin-like growth factor (IGF)-1, cell proliferation, and G1/S transition in breast cancer cells. In the mouse xenograft model of human breast carcinoma, tumor growth was significantly slower in animals injected with ClC-3-deficient cells compared with the growth of normal human breast cancer cells. In addition, silencing of ClC-3 attenuated the expression of proliferating cell nuclear antigen, Ki-67, cyclin D1, and cyclin E, as well as the activation of extracellular signal-regulated protein kinases (ERK) 1/2, both in vitro and in vivo. CONCLUSION: Together, our data suggest that upregulation of ClC-3 by IGF-1 contributes to cell proliferation and tumor growth in breast cancer, and ClC-3 deficiency suppresses cell proliferation and tumor growth via the IGF/IGF receptor/ERK pathway.

Chloride channel involved in the regulation of curcumin-induced apoptosis of human breast cancer cells

Objective: To investigate the role of ClC-3 chloride channel in the proliferation of breast cancer cell line Mcf-7 treated with curcumin and its specific mechanism. Methods: MTT assay was used to detect the effect of chloride channel blocker (DIDS) and curcumin on Mcf-7 and human normal cell viability. Patch-clamp technique was used to determine the current density before and after drug treatment. Apoptosis assay by flow cytometry was performed for further examination of cell apoptosis. Results: Curcumin had toxicity on Mcf-7 and HUVEC cells and DIDS reduced the survival rate of Mcf-7 cells by inhibiting proliferation. Curcumin could activate the chloride ion current on MCF-7 cell membrane, which would be inhibited by DIDS. Finally, curcumin in low concentration combined with DIDS could significantly promote the MCF-7 cells apoptosis. Conclusions: Our results suggest that ClC-3 protein is involved in the regulation of curcumin induced proliferation inhibiting in breast cancer cells through inducing cell apoptosis. ClC-3 may be a potential target of tumor therapy.

Chloride channel involved in the regulation of curcumin-induced apoptosis of human breast cancer cells

Objective:To investigate the role of ClC-3 chloride channel in the proliferation of breast cancer cell line Mcf-7 treated with curcumin and its specific mechanism.Methods:MTT assay was used to detect the effect of chloride channel blocker (DIDS) and curcumin on Mcf-7 and human normal cell viability. Patch-clamp technique was used to determine the current density before and after drug treatment. Apoptosis assay by flow cytometry was performed for further examination of cell apoptosis.Results:Curcumin had toxicity on Mcf-7 and HUVEC cells and DIDS reduced the survival rate of Mcf-7 cells by inhibiting proliferation. Curcumin could activate the chloride ion current on MCF-7 cell membrane, which would be inhibited by DIDS. Finally, curcumin in low concentration combined with DIDS could significantly promote the MCF-7 cells apoptosis.Conclusions:Our results suggest that ClC-3 protein is involved in the regulation of curcumin induced proliferation inhibiting in breast cancer cells through inducing cell apoptosis. ClC-3 may be a potential target of tumor therapy.

Establishment of a hepatic carcinoma cell line with stable ClC-3 silencing by lentivirus-mediated RNA interference and its influence on invasion and migration / 中国药理学通报

Aim To establish a hepatic carcinoma cell line with stable voltage-gated chloride channel 3 ( ClC-3 ) gene silencing through the lentivirus-mediated short-hairpin RNA ( shRNA ) method and investigate the effects of gene silencing on invasion and migration. Methods Three lentiviral vectors coding shRNA tar-geting ClC-3 gene were constructed, the recombinant plasmids were packaged into mature lentivirus by 293FT cells, and then the lentiviruses were harvested, concentrated and titrated. MHCC97H cells were infec-ted with the recombinant lentiviruses and then were se-lected to obtain cell lines stably expressing ClC-3 shR-NA. The efficiency of ClC-3 mRNA and protein ex-pression interference were determined by real-time flu-orescence quantitative PCR and Western blot, respec-tively. The effects of ClC-3 gene interference on inva-sion and migration of MHCC97 H cells were performed by Transwell chamber assays with or without Matrigel and cell scratch assay. Results The recombinant lentiviral vectors were successfully constructed and four lentiviruses were acquired after packaged by 293 FT cells. One negative control cell line and three cell lines with ClC-3 gene interference ( MHCC97 H/shClC-3-1 , shClC-3-2 and shClC-3-3 ) were successfully construc-ted after MHCC97 H cells were infected with lentivirus-es. The expression level of ClC-3 mRNA and protein in three ClC-3-silenced cells were obviously lower than the negative control cells ( P <0. 01 ) , MHCC97 H/shClC-3-2 cells showed the greatest inhibition of ClC-3 mRNA and protein expressions. As compared with the negative control cells, the ClC-3 gene interference sig-nificantly decreased invasion and migration of MH-CC97 H cells in vitro ( P <0. 01 ) . Conclusion The hepatic carcinoma cell lines with stable ClC-3 gene si-lencing were successfully established and the ClC-3 gene interference could significantly inhibit invasion and migration of MHCC97H cells.