Abstract
The aim of this work was to construct a human recombinant p66Shc adenovirus and to investigate the inhibition of recombinant p66Shc adenovirus on MCF-7 cells. The recombinant adenovirus expression vector was constructed using the Adeno-X Adenoviral System 3. Inhibition of MCF-7 cell proliferation was determined by MTT. Intracellular ROS was measured by DCFH-DA fluorescent probes, and 8-OHdG was detected by ELISA. Cell apoptosis and the cell cycle were assayed by flow cytometry. Western blot were used to observe protein expression. p66Shc expression was upregulated in 4 cell lines after infection. The inhibitory effect of p66Shc recombinant adenovirus on MCF-7 cells was accompanied by enhanced ROS and 8-OHdG. However, no significant differences were observed in the cell apoptosis rate. The ratio of the cell cycle G2/M phase showed a significant increase. Follow-up experiments demonstrated that the expressions of p53, p-p53, cyclin B1 and CDK1 were upregulated with the overexpression of p66Shc. The Adeno-X Adenoviral System 3 can be used to efficiently construct recombinant adenovirus containing p66Shc gene, and the Adeno-X can inhibit the proliferation of MCF-7 cells by inducing cell cycle arrest at the G2/M phase. These results suggested that p66Shc may be a key target for clinical cancer therapy.
Highlights
P66Shc, a 66 kDa proto-oncogene Src homologous-collagen homologue (Shc) adaptor protein, is an important protein that regulates the levels of reactive oxygen species (ROS) and lifespan in mammals[1,2]
To further validate the efficiency of the recombinant p66Shc adenovirus, HEK293A, Human umbilical vein endothelial cells (HUVECs), HeLa and MCF-7 cells were infected by AdenoX-p66Shc (Ad-p66Shc) or a negative control for 48 h
Western blot analysis revealed that the expression of p66Shc in all of the cells infected by AdenoX-p66Shc was dramatically increased compared with the negative control (Fig. 1B)
Summary
P66Shc, a 66 kDa proto-oncogene Src homologous-collagen homologue (Shc) adaptor protein, is an important protein that regulates the levels of reactive oxygen species (ROS) and lifespan in mammals[1,2]. Its effect on tumor cells has attracted the attention of researchers. To increase the level of p66Shc expression, only plasmid transfection methods can be utilized to import the exogenous gene into cell lines. Due to the limited gene transfection efficiency, further research of the function of p66Shc, especially in primary cells and in vivo studies, is restricted. We observed a significantly higher expression of p66Shc in AdenoX-p66Shc infected cells, including primary cells, indicating that this tool could potentially be used to research the function of p66Shc in vitro and in vivo in the future. Correspondence and requests for materials should be addressed to Y.L
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