The expression and induction of metallothionein has been associated with protection against oxidative stress and apoptosis. This study examines the effect of tumour suppressor protein p53 on metallothionein expression following CdCl 2 treatment in eight human epithelial breast cancer cell lines differing in p53 and oestrogen-receptor status. Cells were treated with 10 mM CdCl 2 for 24 h and metallothionein protein levels were measured by cadmium binding assay. MCF7 cells which are p53-positive (p53+) and oestrogen-receptor-positive showed a large induction in metallothionein synthesis by 10.79+1.36-fold. Other breast cancer cell lines which are p53-negative (p537) and oestrogen-receptor-negative or weakly oestrogenreceptor-positive showed a small induction ranging from 1.40+0.10 to 3.65+0.30-fold. RT -PCR analysis showed an induction of metallothionein mRNA in MCF7 cells by about 1.61+0.08-fold, while in HCC1806 cells (p537, oestrogenreceptor-negative) by 1.11+0.13-fold, and in MDA-MB-231 (p537, oestrogen-receptor-negative) by 1.25+0.06-fold. Metallothionein localisation was determined by immunohistochemical staining. Prior to metal treatment, metallothionein was localised mainly in the cytoplasm of MCF7 and MDA-MB-231 cells. After treatment with 10 mM CdCl 2 for 24 h, MCF7 cells showed intense nuclear and cytoplasmic staining for metallothionein, while MDA-MB-231 cells showed staining in the cytoplasm with weak nuclear staining. Apoptosis induced by 10 -40 mM CdCl 2 at time points between 4 and 48 h was examined with TUNEL assay. In MCF7 cells, apoptosis increased with higher concentrations of CdCl 2 , it peaked at 6 -8 h and appeared again at 48 h for all concentrations of CdCl 2 tested. In MDA-MB-231 cells, apoptosis remained at low levels for 10 -40 mM CdCl 2 at all time points. Studies on cadmium uptake showed similar uptake and accumulation of cadmium at 8 and 24 h in all the cell lines. The data demonstrate that treatment of epithelial breast cancer cells with 10 mM CdCl 2 for 24 h caused a greater induction of metallothionein protein and mRNA expression in p53+ and oestrogen-receptor-positive cells as compared to p537 and oestrogen-receptor-negative or weakly oestrogen-receptor-positive cells. This effect may be associated with the occurrence of apoptosis and suggests a role for p53 and oestrogen-receptor on the expression and induction of metallothionein in epithelial cells.

miR-381 suppressed CX43 expression by directly targeting the 3′-UTR of C/EBPα, a novel transcription factor of Cx43 in human breast cancer cells. The miR-381–Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.

SynopsisBoth miRNAs (miRs) and connexin 43 (Cx43) were important regulators of the metastasis of breast cancer, whereas the miRs regulating Cx43 expression in breast cancer cells were still obscure. In the present study, we scanned and found miR-1, miR-206, miR-200a, miR-381, miR-23a/b and miR-186 were functional suppressors of human Cx43 mRNA and protein expression. Specially, we demonstrated that only miR-200a could directly target the 3 -untranslated region (3 -UTR) of human Cx43 gene. Functionally, overexpression of Cx43 in MCF cells potentiated the migration activity, whereas additional miR-200a treatment notably prevented this effect. Finally, we demonstrated that decreased levels of miR-200a and elevated expression of Cx43 in the metastatic breast cancer tissues compared with the primary ones. Thus, we are the first to identify miR-200a as a novel and direct suppressor of human Cx43, indicating that miR200a/Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.Key words: breast cancer, connexin 43 (Cx43), metastasis, micro ribonucleic acid (miR)-200a, micro ribonucleic acid (miR)-1, 3 -untranslated region (3 -UTR).