Abstract:

Abstract:Objective
To construct the pshuttle-Egr-1-hSmac plasmid and transfect human breast cancer MDA-MB-435 cells, and to observe its radiotherapy enhancing effect on tumor cells.Methods The empty vector pshuttle and pshuttle-Egr-1-hSmac plasmid were transfected into MDA-MB-435 cells by liposomal.At different time（4,8,12,24 and 48 h） after irradiation with 2.0 Gy X-ray and 24 h after irradiation with 0.5―5.0 Gy,the total RNA and protein were collected and extracted from these cells to analyze the Smac mRNA and protein expression levels with RT-PCR and Western blotting methods.The cells were divided into control,pshuttle,pshuttle-Egr-1-hSmac,2.0 Gy irradiation group,pshuttle + 2.0 Gy irra diation and pshuttle-Egr-1-hSmac+2.0 Gy irradiation groups.MTT method was used to evalua te cell proliferation,and the cell survival ability  was measured with clone formation assay;Annexin Ⅴ/PI double staining and PI single staining were used to examine the  apoptosis and cell cycle of MDA-MB-435 cells.Results There was no Smac mRNA expression in MDA-MB-435 cells in control and pshuttle groups,but the Smac mRNA expression levels in  MDA-MB-435 cells in  pshuttle-Egr-1-hSmac plasmid group were gradually increased with the time prolongation,and reached the maximum at 24 and 48 h;the Smac mRNA expression levels in MDA-MB-435 cells  were increased gradually 24 h after irradiation of 0.5―5.0 Gy X-ray with the increasing of irradiation doses,and reached the maximum after 2.0 and 5.0 Gy irradiation.The Smac protein expr ession levels in  pshuttle-Egr-1-hSmac plasmid group were increased gradually with the time prolongation,and reached the maximum at 24 h.The Smac protein expression lervels were increased 24 h afer irradiation of 0,0.5,1.0,2.0 and 5.0 Gy X-ray,especially in 5.0 Gy group.The MTT results showed that the  A490 values  in 2.0 Gy,pshuttle+2.0 Gy and    pshuttle-Egr-1-hSmac groups 24,48,and 72 h after  irradiation were lower than th ose in control group(P<0.01);the  A490 values  of MDA-MB-435 cells in  pshuttle-Egr-1-hSmac group after 1.0-5.0 Gy X-ray irradiation were lower than those in 0 Gy group (P<0.05 or P<0.01);the survival fraction(SF) in pshuttle-Egr-1-hSmac group was lower than those in control group(P<0.01). The percentages of the cells at G0/G1 and S phase in pshuttle-Egr-1-hSmac group were lower than those in 2.0 Gy group(P<0.01)，the percentage of the cells at G2/M phase was higher than that in 2.0 Gy group (P<0.01);the apoptotic rate of the cells  in pshuttle-Egr-1-hSmac group was higher than that in 2.0 Gy group (P<0.01).Conclusion X-ray irradiation can significantly increase the Smac mRNA and protein expression levels in MDA-MB-435 cells transfected with pshuttle-Egr-1-hSmac plasmid,inhibit the  cell survival rate,and  induce G2/M arrest and apoptotic increasing;Smac gene combined with radiotherapy could  significantly increase the  radiosensitivity of breast cancercells.

Key words: Smac gene, Egr-1 promoter, X-ray, gene-radiotherapy, apoptosis