Newcastle disease virus suppress glycolysis pathway and induce breast cancer cells death

Abstract

Newcastle disease virus (NDV) can modulate cancer cell signaling pathway and induce apoptosis in cancer cells. Cancer cells increase their glycolysis rates to meet the energy demands for their survival and generate ATP as the primary energy source for cell growth and proliferation. Interfering the glycolysis pathway may be a valuable antitumor strategy. This study aimed to assess the effect of NDV on the glycolysis pathway in infected breast cancer cells. Oncolytic NDV attenuated AMHA1 strain was used in this study. AMJ13 and MCF7 breast cancer cell lines and a normal embryonic REF cell line were infected with NDV with different multiplicity of infections (moi) to determine the IC50 of NDV through MTT assay. Crystal violet staining was done to study the morphological changes. NDV apoptosis induction was assessed using AO/PI assay. NDV interference with the glycolysis pathway was examined through measuring hexokinase (HK) activity, pyruvate, and ATP concentrations, and pH levels in NDV infected and non-infected breast cancer cells and in normal embryonic cells. The results showed that NDV replicates efficiently in cancer cells and spare normal cells and induce morphological changes and apoptosis in breast cancer cells but not in normal cells. NDV infected cancer cells showed decreased in the HK activity, pyruvate and ATP concentrations, and acidity, which reflect a significant decrease in the glycolysis activity of the NDV infected tumor cells. No effects on the normal cells were observed. In conclusion, oncolytic NDV ability to reduce glycolysis pathway activity in cancer cells can be an exciting module to improve antitumor therapeutics.

Keywords: Cancer metabolism; Oncolytics; Pyruvate; Virotherapy; Warburg effect.

Fig. 1

NDV induced significant cytotoxicity in breast cancer cells but not in normal cells. a NDV cytotoxicity (CT %) in REF normal cells; b NDV CT % in AMJ13 cell line; c NDV CT % in MCF-7 cell-line

Fig. 2

Morphological changes in REF, AMJ13, and MCF7 cell lines treated with NDV for 72 h and stained with crystal violet, photographed under ×40 magnification (a–f). Morphological changes in REF, AMJ13, and MCF7 cell lines after treatment with NDV: untreated cells (control), treated cells with NDV. The quantitative image analysis revealed a significant decrease in treated cell density, which reflects the destructive effect induced by NDV infection, in which there was no decrease in the infected normal cells REF

Fig. 3

All cells were stained with AO/PI and viewed under fluorescence microscopy at ×40 magnification to determine the NDV induction of apoptosis. Yellow-orange cells are representing apoptosis, and green cells are viable cells. Quantitative image analysis for fluorescence intensity revealed significant apoptosis induction in the NDV infected cells while there was no induction in normal REF cells

Fig. 4

The effect of NDV on glycolysis inhibition. The activity of HK (U/ml) in REF, AMJ13, and MCF7 cell lines after NDV treatment with MOI 2 was compared with that of the control. a REF, b AMJ13, and c MCF7, at values represent the (mean ± SD) at *P < 0.05, **P < 0.01

Fig. 5

Effect of NDV on pyruvate. REF, AMJ13 and MCF7 cell lines under NDV treatment (MOI 2) in comparison with that under the control at *P < 0.05, **P < 0.01 and ***P < 0.001. a REF, b AMJ13, and c MCF7. Pyruvate μmol/ml

Fig. 6

Effect of NDV on ATP production in REF, AMJ13, and MCF7 cell lines under treatment with NDV (MOI 2) compared with that in cell lines under the control treatment at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. a REF, b AMJ13, and c MCF7. (ATP mmol/L)

Fig. 7

Levels of acidity of NDV infected and non-infected cells, normal cells have neutral pH value in both infected and non-infected cells. Non-infected cancer cells showing a high level of acidity, but this situation reversed by NDV infection to show a rise to alkaloid levels. a pH values of the REF cell line, b pH values of the AMJ13 cell line and c pH values of the MCF7 cell line). Values represent the (mean ± SD) at t *P < 0.05, **P < 0.01 and ***P < 0.001