Anti-tumour effects of a dual cancer-specific oncolytic adenovirus on Breast Cancer Stem cells

Abstract

Apoptin can specifically kill cancer cells but has no toxicity to normal cells. Human telomerase reverse transcriptase (hTERT) can act as a tumour-specific promoter by triggering the expression of certain genes in tumour cells. This study aims to investigate the inhibitory effects and to explore the inhibitory pathway of a dual cancer-specific recombinant adenovirus (Ad-apoptin-hTERTp-E1a, Ad-VT) on breast cancer stem cells. Breast cancer cell spheres were obtained from MCF-7 cells through serum-free suspension culture. The cell spheres were detected by flow cytometry for CD44⁺ CD24^- cell subsets. The stemness of MCF-7-CSC cells was confirmed by in vivo tumorigenesis experiments. The inhibitory effect of the recombinant adenoviruses on MCF-7-CSC cells was evaluated by CCK-8 assay. In addition, the stemness of adenovirus-infected MCF-7-CSC cells was analysed by testing the presence of CD44⁺ CD24^- cell subsets. The ability of the recombinant adenovirus to induce MCF-7-CSC cell apoptosis was detected by staining JC-1, TMRM and Annexin V. Our results showed that a significantly higher proportion of the CD44⁺ CD24^- cell subsets was present in MCF-7-CSC cells with a significantly increased expression of stem cell marker proteins. The MCF-7-CSC cells, whlist exhibited a strong tumorigenic ability with a certain degree of stemness in mice, were shown to be strongly inhibited by recombinant adenovirus Ad-VT through cell apoptosis. In addition, Ad-VT was shown to exert a killing effect on BCSCs. These results provide a new theoretical basis for the future treatment of breast cancer.

Keywords: apoptin; apoptosis; breast cancer stem cells; recombinant adenovirus.

FIGURE 1

Isolation and identification of MCF‐7‐CSC. (A) A cell cluster in suspension growth formed (200×) after a 10‐day culture of MCF‐7 in serum‐free suspension. (B) Detection of CD44⁺ CD24⁻ cell subsets using flow cytometry. After serum‐free suspension culture, the proportion of CD44⁺ CD24⁻ cell subsets in MCF‐7‐CSC was significantly higher than that of MCF‐7. (C) The expression of ALDH1A1, C‐Myc, OCT4, NANOG, KLF 4 and SOX2 in MCF‐7 and MCF‐7‐CSC cells by Western blot analysis. The protein levels inMCF‐7‐CSC cells were higher than those in MCF‐7 cells. Data are presented as means ± SD (*P < 0.05 and ***P < 0.001)

FIGURE 2

Analysis of MCF‐7‐CSC tumour formation in vivo. (A) Tumour formation in mice. As low as 100 MCF‐7‐CSC cells could induce tumour formation, whilst 1 × 10⁴ MCF‐7 cells could not induce tumour formation. (B‐D) The tumour formation rate of MCF‐7‐CSC was significantly higher than that of MCF‐7. (E) After inoculation of cells, mice in the MCF‐7‐CSC group formed tumours and the tumour volume increased rapidly, whilst mice in the MCF‐7 group formed fewer tumours, and the tumour volume growth trend of mice was slower than that of mice in the MCF‐7‐CSC group. Data are presented as mean ± SD

FIGURE 3

The killing and stemness effects of Ad‐VT on MCF‐7‐CSC cells. (A) After infection, the killing effects of Ad‐VT, Ad‐T, Ad‐VP3 and Ad‐Mock on MCF‐7‐CSC cells were analysed at 24, 48, 72 and 96 h. The killing effects of adenovirus on MCF‐7‐CSC cells were time‐ and dose‐dependent. The killing effects of Ad‐VT, Ad‐T and Ad‐VP3 on MCF‐7‐CSC showed the highest rates at 96 h (82%, 69% and 30%, respectively). (B) After the MCF‐7‐CSC cells were infected with Ad‐VT, the changes of CD44⁺CD24⁻ cell subsets were detected by flow cytometry. Ad‐VT could reduce the CD44⁺CD24⁻ cell subpopulation in MCF‐7‐CSC. (C) Western blot analysis of ALDH1A1, C‐Myc, OCT4, NANOG, KLF 4 and SOX2 protein expression in MCF‐7‐CSC cells infected with Ad‐VT, Ad‐T, Ad‐VP3 and Ad‐Mock. The protein levels in MCF‐7‐CSC cells were higher than those in MCF‐7 cells infected with recombinant adenoviruses. Data are means ± SD (**P < 0.01) when compared with controls

FIGURE 4

Characterization of cell death pathway induced by Ad‐VT on MCF‐7‐CSC cells. (A) Cell morphological changes were visualized by fluorescence microscopy after Annexin V FITC/PI staining. MCF‐7‐CSC cells infected with Ad‐VT, Ad‐T, Ad‐VP3 and Ad‐Mock at 24, 48 and 72 h were stained with Annexin V FITC/PI stain. Cell membrane phospholipid eversion and nuclear fragmentation of Ad‐apoptin group increased significantly over time. (B) MCF‐7‐CSC cell apoptosis was analysed by flow cytometry after Annexin V FITC/PI staining. The apoptosis level of MCF‐7‐CSC cells infected with Ad‐VT, Ad‐T and Ad‐VP3 was significantly higher than that of Ad‐Mock and control groups. Amongst them, the effect of Ad‐VT was the most significant. (C) Western blot analysis of apoptin, E1A and caspase‐3 in MCF‐7‐CSC cell extracts. Both Ad‐VT and Ad‐T could effectively express E1A protein, and Ad‐VT and Ad‐VP3 could effectively express apoptin protein. After infection with Ad‐VT, Ad‐T and Ad‐VP3, the level of cleaved‐caspase‐3 increased overtime. Amongst them, the effect of Ad‐VT was the most significant. The scale bar equals 50 μm. Data are shown as mean ± SD (*P < 0.05, **P < 0.01 and ***P < 0.001) when compared with controls

FIGURE 5

Effect of Ad‐VT on the mitochondrial membrane potential of MCF‐7‐CSC cells. (A‐B) Changes in red and green fluorescence signals measured by fluorescence microscopy after JC‐1 staining. Increased apoptosis resulted in a decrease in the ratio of red to green fluorescence. Quantitative measurement of changes in the ratio of red to green fluorescence after JC‐1 staining. Ad‐VT clearly altered the mitochondrial membrane potential (MMP). Ad‐VT had the strongest ability to induce apoptosis by affecting the MMP. (C‐D) The MMP of MCF‐7‐CSC cells was analysed by fluorescence microscopy and flow cytometry after TMRM staining. The MMP of MCF‐7‐CSC cells was significantly decreased after infection with Ad‐VT. The scale bar equals 50 μm. Data are shown as mean ± SD (*P < 0.05, **P < 0.01 and ***P < 0.001) when compared with control