Background: Most cancer cells exhibit increased glycolysis and use this metabolic pathway cell growth and proliferation. Targeting cancer cells' metabolism is a promising strategy in inhibiting cancer cell progression. We used D-Mannoheptulose, a specific hexokinase inhibitor, to inhibit glycolysis to enhance the Newcastle disease virus antitumor effect. Methods: Human breast cancer cells were treated by NDV and/or hexokinase inhibitor. The study included cell viability, apoptosis, and study levels of hexokinase enzyme, pyruvate, ATP, and acidity. The combination index was measured to determine the synergism of NDV and hexokinase inhibitor. Results: The results showed synergistic cytotoxicity against breast cancer cells by combination therapy but no cytotoxic effect against normal cells. The effect was accompanied by apoptotic cell death and hexokinase downregulation and inhibition to glycolysis products, pyruvate, ATP, and acidity. Conclusions: The combination treatment showed safe significant tumor cell proliferation inhibition compared to monotherapies suggesting a novel strategy for anti-breast cancer therapy through glycolysis inhibition by hexokinase downregulation.

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Newcastle Disease Virus (NDV) can modulate cancer cell signaling pathways and induce apoptosis in cancer cells. The laboratory-based studies of the oncolytic NDV requires a reliable protocol for the propagation of the oncolytic NDV. A comprehensive protocol is provided for virus propagation in fertile chicken eggs, which consistently yields high titer viral stock. Aim: Propagation of oncolytic NDV AMHA1 attenuated strain in Embryonated Chicken Eggs (ECE) and tissue culture infective dose 50% (TCID50) determination protocol of the virus. Method: Specific pathogen-free fertilized chicken eggs were incubated at 37 °C and 55-60% humidity for 9’ 10 days. Over this period, embryo death was monitored using an egg candle regularly. Virus inoculation is carried out by injection of the diluted virus stock into the allantoic cavity using a needle. embryo death was recorded every two hours and the egg rushed to the refrigerator and fluids collected after four to six hours. Hemagglutination assay (HA) was used to determine the preliminary titer of the virus to collect the high titer egg fluids only which is about 128 to 256HAU. The Vero cell line was exposed to NDV at tenfold serial dilutions to determine TCID50 of the virus. The number of viruses in 1 ml of allantoic fluid was measured of embryonated chicken eggs. Results: NDV Iraqi virulent strain has the ability to kill all the chicken embryos through (24-72) h of inoculation. A high titer of NDV was achieved from the infected eggs. Conclusion: Oncolytic NDV propagated in embryonated chicken eggs in high titers as indicated by TCID50 value.

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