J Breast Cancer.  2018 Jun;21(2):103-111. 10.4048/jbc.2018.21.2.103.

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

• Zhou FM
• Huang YY
• Tian T
• Li XY
• Tang YB

• KMID: 2413928
• DOI: http://doi.org/10.4048/jbc.2018.21.2.103

Abstract

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.

Keyword

Breast neoplasms; Cell proliferation; Chloride channel-3; Insulin-like growth factor 1

MeSH Terms

Animals
Blotting, Western
Breast Neoplasms*
Breast*
Bromodeoxyuridine
Cell Cycle
Cell Line
Cell Proliferation
Chloride Channels
Cyclin D1
Cyclin E
Cyclins
Flow Cytometry
Heterografts
Humans
Immunoprecipitation
In Vitro Techniques*
Insulin-Like Growth Factor I
Methods
Mice
Proliferating Cell Nuclear Antigen
Protein Kinases
Real-Time Polymerase Chain Reaction
RNA, Messenger
Tumor Burden
Up-Regulation
Bromodeoxyuridine
Chloride Channels
Cyclin D1
Cyclin E
Cyclins
Insulin-Like Growth Factor I
Proliferating Cell Nuclear Antigen
Protein Kinases
RNA, Messenger

Figure

• 

  Figure 1 Chloride channel-3 (CIC-3) expression in breast tumor tissues. (A) Chloride channels (ClC-1–7, CFTR) are expressed in human breast cancer tissues from clinical postoperative patients, as indicated by qRT-PCR mRNA analysis. The data are presented as relative fold changes in breast cancer tissues versus normal tissues. (B) Expression of ClC-3 was examined by western blotting, (C) ClC-3 protein is expressed in breast cancer tissues. The result shows a significant increase in ClC-3 expression in breast cancer tissues vs. normal tissues. CFTR=cystic fbrosis transmembrane conductance regulator; qRT-PCR=quantitative real - time polymerase chain reaction; mRNA=messenger RNA; GAPDH=glyceraldehyde 3-phosphate dehydrogenase. *p<0.01 vs. control.

• 

  Figure 2 Insulin-like growth factor 1 (IGF-1) and chloride channel-3 (CIC-3) expression in human breast cancer cells. (A) Effect of 30–300 ng/mL IGF-1 on the expression of ClC-3 in MDA-MB-231 cells. (B) Effect of 30–300 ng/mL IGF-1 on the expression of ClC-3 in MCF-7 cells (n=5). GAPDH=glyceraldehyde 3-phosphate dehydrogenase. *p<0.05 vs. control; †p<0.01 vs. control.

• 

  Figure 3 Effects of chloride channel-3 (ClC-3) knockdown on insulin-like growth factor 1 (IGF-1)-dependent cell proliferation. (A) Increase in cell growth induced by 100 ng/mL IGF-1 was determined by counting the number of cells, and was significantly inhibited by incubation with ClC-3 small interfering RNA (siRNA) for 24 hours, but not by transfection with negative siRNA. (B) The effects of ClC-3 siRNA on cell proliferation induced by IGF-1 were further determined by assaying bromodeoxyuridine (BrdU) incorporation. Incubation with ClC-3 siRNA for 24 hours also significantly decreased BrdU incorporation. (C) Quiescent MDA-MB-231cells incubated in 100 ng/mL IGF-1 were transfected with ClC-3 siRNA or negative siRNA control (IGF-1+negative siRNA) plus transfecting agent lipofectamine. The cells were trypsinized, rinsed with phosphate-buffered saline, and treated with 20 µg/mL RNase. DNA was stained with propidium iodide, and 1×106 cells were analyzed by fluorescence-activated cell sorter analysis. Bars represent mean±SD (n=5). Analysis of variance was used for comparison. *p<0.01 vs. control; †p<0.01 vs. IGF-1.

• 

  Figure 4 Effects of chloride channel-3 (ClC-3) knockdown on the expression of cell cycle-regulatory proteins. (A) Representative western blot images of Ki-67, proliferating cell nuclear antigen (PCNA), cyclins, and p21. (B) Densitometric analysis of the effects of ClC-3 knockdown on expression of Ki-67, PCNA, cyclin D1, cyclin E, and p21 (n=5). GAPDH=glyceraldehyde 3-phosphate dehydrogenase; IGF-1=insulin-like growth factor 1; siRNA=small interfering RNA. *p<0.01 vs. control; †p<0.01 vs. IGF-1.

• 

  Figure 5 Effect of insulin-like growth factor 1 (IGF-1) induced extracellular regulated protein kinases 1/2 (ERK1/2) activation and the signal-responsive interaction of chloride channel-3 (CIC-3) with insulin-like growth factor receptor (IGF-R). (A) Effects of ClC-3 knockdown on the expression of ERK1/2 and phosphorylated ERK1/2 (pERK1/2). Bars represent mean±SD (n=5). Analysis of variance was employed for comparison. (B) Co-immunoprecipitation assay of ClC-3/IGF-R interaction in MDA-MB-231 cells (n=5). Cells were treated with or without IGF-1 (100 ng/mL). Protein extracts were immunoprecipitated (IP) with anti-IGF-R or anti-ClC-3 antibody, followed by immunoblotting (IB) with anti-IGF-R or anti-ClC-3. siRNA=small interfering RNA. *p<0.01 vs. control; †p<0.01 vs. IGF-1+Neg ClC-3.

• 

  Figure 6 Effects of chloride channel-3 (ClC-3) knockdown on the growth of human breast tumors in vivo. (A) Representative images of tumors on day 20 from the control and ClC-3 short hairpin RNA (shRNA) groups. (B) Changes in tumor volume were calculated postinoculation every 4 days. Dots represent mean±SD. Seven mice were included in each group. Analysis of variance was employed for comparison. (C) Immunohistochemistry was performed to assess the expression of Ki-67 in tumor tissues isolated from control and ClC-3 shRNA mice, tumor tissues were cut and stained with hematoxylin-eosin, and observed with phase contrast microscope (×200). (D) Western blot analysis was performed to measure the expression of Ki-67, proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin E, and phosphorylated extracellular regulated protein kinases1/2 (pERK1/2); glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as loading control (n=7). *p<0.01 vs. control.

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