Analysis of the synergistic effect of combination of Ad-VT and cyclophosphamide
The synergistic inhibiton of combination of Ad-VT and cyclophosphamide was detected by the CCK-8 assay (Fig. 1A-D). The purpose of this experiment was to use a combination of Ad-VT and cyclophosphamide to reduce the cytotoxicity of cyclophosphamide, while improving the therapeutic effect. To screen for an optimal concentration, different concentrations of Ad-VT and cyclophosphamide were used for the treatment of MCF-7 cells. Based on the calcusyn software calculation, we found that the combinations groups, 600 µM + 60 MOI, 600 µM + 40 MOI, 400 µM + 40 MOI, 800 µM + 20 MOI, and 600 µM + 20 MOI, were less than 1, indicating these combinations have synergistic inhibitory effect (Fig. 1A-B). Based on the above results, we further narrowed the concentration range of cyclophosphamide to 200 µM, 400 µM, and 600 µM and the Ad-VT to 20 MOI, 40 MOI, and 60MOI.
We also performed a combined experiment in MDA-MB-231 cells to select the optimal combined concentration of Ad-VT and cyclophosphamide. The analysis of the treated MDA-MB-231 cells showed that, the CI < 1 included the combinations, 400 µM + 40 MOI, 600 µM + 40 MOI, and 400 µM + 60 MOI. The combination group 400 µM + 40 MOI had the lowest CI value and a good synergistic effect, and therefore, was selected for subsequent in vitro assays (Fig. 1C-D).
The combination of Ad-VT and cyclophosphamide inhibits the proliferation of BC cells
Breast cancer cells and normal breast epithelial cells were treated with Ad-VT and cyclophosphamide. After 48 hours, the cells were stained with crystal violet. The results showed that Ad-VT inhibits MCF-7 and MDA-MB-231 cells but had no inhibitory effect on MCF-10A cells; however, cyclophosphamide showed some toxic effects on MCF-10A cells. When cyclophosphamide was combined with Ad-VT, the cytotoxicity of cyclophosphamide on MCF-10A cells was significantly lower than that on MCF-7 and MDA-MB-231 cells (Fig. 2A).
In the CCK-8 assay, we found that the use of Ad-VT alone could inhibit two kinds of breast cancer cells by about 40%, but had no toxic effect on MCF-10A cells (Fig. 2B). On the contrary, cyclophosphamide has obvious cytotoxicity on MCF-10A cells (p < 0.01). The combination of Ad-VT and cyclophosphamide significantly reduced the cytotoxicity that was caused by cyclophosphamide alone (p < 0.05). The inhibition rate of breast cancer cells following the combined treatment was higher than 60%, which was also significantly higher than that of the Ad-VT or Cyclophosphamide group (p < 0.05). Ad-MOCK had no significant killing effect on breast cancer cells (p > 0.05). The above results indicate that the combined use of Ad-VT and cyclophosphamide has a synergistic effect and significantly reduces cytotoxicity in normal breast epithelial cells.
The combination of Ad VT and cyclophosphamide increases apoptosis in BC cells
Hoechst is a type of dye that can enter cells at will and bind to nucleic acids to display blue fluorescence. The results showed the presence of a significant nuclear fragmentation and nuclear overstaining in the cells that were treated with a combination of Ad-VT and cyclophosphamide compared to that in the control and Ad-MOCK groups (Fig. 3A).
The quantitative experiment results of apoptosis showed that the combined treatment, Ad-VT, and cyclophosphamide induce cell apoptosis, but with different degrees. In MDA-MB-231 cells, the apoptosis rates of cyclophosphamide, Ad-VT, and the combined treatment groups were 15.62%, 37.59%, and 51.24%, respectively. In MCF-7 cells, the apoptotic rates of cyclophosphamide, Ad-VT, and combination therapy groups were 19.28%, 45.68%, and 62.76%, respectively. Among these, the apoptotic rate of BC cells in the combined treatment group was significantly higher than that of the other treatment groups (p < 0.05) (Fig. 3B-C).
Induction of the endogenous apoptosis pathway in BC Cells by the combination of Ad-VT and cyclophosphamide
The results of the JC-1 staining showed that the combination of Ad-VT and cyclophosphamide, Ad-VT and cyclophosphamide can cause mitochondrial damage and decrease MMP, but there is no obvious change in MMP of control group cells (Fig. 4A). After calculating the ratio of red fluorescence to green fluorescence, it was found that the red/green ratio in cells of combined treatment group was significantly lower than that of other groups (p < 0.01) (Fig. 4B).
From the results of the caspase and cytochrome c activity assays, it was found that the activities of caspase-3, -7, -9 and cytochrome c in the cells in the combination treatment and the Ad-VT groups are significantly increased, while in the cyclophosphamide group these levels did not significantly change (Fig. 4C). The caspase and cytochrome c activities of the combined group were higher than that of the single drug group (Fig. 4C). These results suggest that the combination of Ad-VT and cyclophosphamide induces apoptosis in BC cells by activating endogenous apoptosis pathway.
The combination of Ad-VT and cyclophosphamide increases the inhibition of BC Cell migration and invasion
The BioCat results showed that the ability of cell invasion in the combination of Ad-VT and cyclophosphamide, Ad-VT and cyclophosphamide groups was significantly lower than that in the control group (p < 0.001), indicating that all treatment groups can inhibit the invasion ability of BC cells. Among them, combination of Ad-VT and cyclophosphamide had the most significant effect (p < 0.05) (Fig. 5A-B).
The results of the scratch test showed that the combination of Ad-VT and cyclophosphamide, Ad-VT and cyclophosphamide significantly inhibits the healing ability of BC cells (p < 0.01). Among them, combination of Ad-VT and cyclophosphamide had the most significant effect (p < 0.01) (Fig. 5C-D). Above results indicated that the inhibiting tumor metastasis effect of the combination group was stronger than that of the single drug groups.
The anti-tumor effect of Ad-VT and cyclophosphamide treatment combination in vivo
During the 6-week monitoring of tumor bioluminescence intensity, it was found that the tumor bioluminescence intensity of control group increased rapidly all the time in the whole experimental period, while the growth rate of tumor bioluminescence intensity of other groups (20 mg/kg and 50 mg/kg cyclophosphamide, Ad-VT (1×109 PFU), 20 mg/kg cyclophosphamide + Ad-VT (1×109 PFU), 50 mg/kg Cyclophosphamide + Ad-VT (1×109 PFU)) were slower. The difference in tumor volume between the treatment group and the control group was statistically significant (p < 0.001). The inhibition rates of each treatment group were 11.2%, 19.7%, 38.5%, 55.4%, and 64.2%, respectively (Fig. 6A-D).
After successful tumor bearing, the survival of nude mice in each group was recorded continuously. The survival rates in the 20 mg / kg cyclophosphamide + Ad-VT (1 × 109 PFU), and the 50 mg/kg cyclophosphamide + Ad-VT (1 × 109 PFU) groups were 70% and 80%, respectively, which were significantly higher than those of the control group (p < 0.001) (Fig. 6E). These results indicate that Ad-VT combined with cyclophosphamide could not only significantly inhibit the growth of tumor in vivo, but also significantly improve the survival rate of mice.