The lethal effect of bis-type azridinylnaphthoquinone derivative on oral cancer cells (OEC-M1) associated with anti-apoptotic protein bcl-2

Abstract

Several drugs of aziridinylbenzoquinone analogs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill tumor cells preferentially within the hypoxic microenvironment. From our previous reported data, it was found that the synthesized 2-aziridin-1-yl-3-[(2-{2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy}ethyl)thio]naphthoquinone (AZ-1) is a bioreductive compound with potent lethal effect on oral cancer cell, OEC-M1. It was found in this study that the lethal effect of the oral cancer cell lines OEC-M1 induced by AZ-1 was mediated through the cell cycle arrest and apoptosis pathway.The LC50 values of OEC-M1 and KB cells induced by AZ-1 compound were 0.72 and 1.02μM, respectively, which were much lower than that of normal fibroblast cells (SF with LC50=5.6μM) with more than 90% of normal fibroblasts surviving as compared to control at a concentration of AZ-1 as high as 2μM. It was interesting to note that the LC50 of monotype diaziridinylbenzoquinone compound, diaziquone (AZQ), was 50μM on OEC-M1 cells. Comparing the cytotoxicity of AZ-1 and AZQ on OEC-M1 cells, AZ-1 is approximately 70 times more potent than AZQ.By using Western blot, both G2/M phase cell cycle arresting protein, cyclin B, and anti-apoptotic protein, bcl-2, were expressed in OEC-M1 cell when the concentrations of AZ-1 were increased from 0.125 to 0.5μM and then decreased from 1 to 2μM of AZ-1 treatment as compared with control for 24h. Both proteins were expressed most abundantly at 0.5μM AZ-1. However, the expression of bcl-2 protein in OEC-M1 was significantly decreasing in a dose-dependent manner and was only about 50% protein level at 2μM AZ-1 for 48h as compared with control. The cell survival check protein p53 increased from 1.72- to 2.8-fold and 1.36- to 2.16-fold at concentrations of AZ-1 from 0.125 to 2.0μM in a dose-dependently increasing manner on OEC-M1 as compared with control for 24 and48h treatments, respectively. The apoptotic-related phenomena were observed, which included apoptotic body formation and the enzyme activity change of caspase-3. The apoptotic bodies and caspase-3 activity of OEC-M1 were induced only at 2μM AZ-1 for a 24h treatment, yet apoptotic body formation was observed at as low as 0.5μM AZ-1 and in a dose-dependently increasing manner for a 48h treatment. The caspase-3 activity was increased 20.6%, 26.8%, and 84.2%, respectively, at 0.5, 1, and 2μM concentrations of AZ-1 for a 48h treatment as compared with control. These results indicate that AZ-1 induced the cell death of OEC-M1 through the G2/M phase arrest of cell cycle and anti-apoptosis first and then apoptosis following a 48h treatment. All of the pathway might be associated with bcl-2 and p53 protein expression. We propose that the AZ-1 could be used as anti-oral cancer drug for future studies with animal models.