Recombinant adenovirus of human p66Shc inhibits MCF-7 cell proliferation

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.

Figure 1. Construction and expression of human recombinant p66Shc adenovirus.

(A) Schematic presentation of p66Shc homologous recombination with pAdenoX-CMV. CH1: collagen-homology region; PTB: phosphotyrosine-binding domain; SH2: Src-homology2 domain; P_{CMV IE}: cytomegalovirus immediate early promoter; SV40 polyA: simian virus 40 polyA signals; ITR: inverted terminal repeat; AMP: ampicillin. (B) HEK293A, HUVECs, HeLa and MCF-7 cells were infected with Ad-p66Shc or negative control (NC) for 48 h. The expression of p66Shc protein was detected by Western blot.

Figure 2. p66Shc inhibited MCF-7 cell viability.

(A) MCF-7 cells were infected with various concentrations of Ad-p66Shc or negative control (20, 40, 80, 160, or 320 MOI) for 48 h. (B) MCF-7 cells were infected with 100 MOI Ad-p66Shc for different amounts of time (12, 24, 36, 48, or 60 h), and cell viability was detected using the MTT method. The data represent the means ± SEM, n=6 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs NC (NC: negative control).

Figure 3. p66Shc increased intracellular ROS, accompanied by an enhanced level of 8-OHdG.

(A) MCF-7 cells were treated with NC, 80 MOI Ad-p66Shc and 160 MOI Ad-p66Shc for 48 h. DCFH-DA staining showed that p66Shc significantly induced intracellular ROS. *p < 0.05, ***p < 0.001 vs Negative control. (B) MCF-7 cells were infected with 100 MOI Ad-p66Shc or negative control for 48 h. The upregulation of p66Shc also increased the level of 8-OHdG. The data represent the means ± SEM, n = 3 independent experiments. **p < 0.01 vs NC (NC: negative control).

Figure 4. Upregulation of p66Shc induced the expression and phosphorylation of p53.

MCF-7 cells were infected with Ad-p66Shc or negative control for 48 h. (A) The cell extracts were prepared and analyzed by Western blot with the corresponding antibodies. The quantification of the digital images was performed using ImageJ software. (B) MCF-7 cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. The data represent the means ± SEM, n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs NC (NC: negative control).

Figure 5. p66Shc induced MCF-7 cell cycle arrest at the G2/M phase.

MCF-7 cells were infected with Ad-p66Shc or negative control for 48 h. (A) After treatment, the cells were examined by flow cytometer for cell cycle analysis. (B) The cell extracts were prepared and analyzed by Western blot with the corresponding antibodies. The quantification of the digital images was performed using ImageJ software. The data represent the means ± SEM, n = 3 independent experiments. **p < 0.01, ***p < 0.001 vs NC (NC: negative control).