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

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 1.02 μM, respectively, which were much lower than that of normal fibroblast cells (SF with LC50 = 5.6 μM) with more than 90% of normal fibroblasts surviving as compared to control at a concentration of AZ-1 as high as 2 μM. It was interesting to note that the LC50 of monotype diaziridinylbenzoquinone compound, diaziquone (AZQ), was 50 μM 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 0.5 μM and then decreased from 1 to 2 μM of AZ-1 treatment as compared with control for 24 h. Both proteins were expressed most abundantly at 0.5 μM 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 2 μM AZ-1 for 48 h 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 2.0 μM in a dose-dependently increasing manner on OEC-M1 as compared with control for 24 and48 h 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 2 μM AZ-1 for a 24 h treatment, yet apoptotic body formation was observed at as low as 0.5 μM AZ-1 and in a dose-dependently increasing manner for a 48 h treatment. The caspase-3 activity was increased 20.6%, 26.8%, and 84.2%, respectively, at 0.5, 1, and 2 μM concentrations of AZ-1 for a 48 h 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 48 h 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.

Bis-type bioreductive compound AZ-1 was synthesized to study the mechanism of death effect in oral cancer cell (OEC-M1). The AZ-1 compound induced two of oral cancer cells, with LC50 = 0.72 μM in OEC-M1 cell and with LC50 = 1.02 μM in KB cell, and with less cytotoxicity to normal fibroblast cell (SF with LC50 = 5.6 μM) which was still with over 90% of survival rate as high as 2 μM of AZ-1. There is significant difference between oral cancer cells (OEC-M1 and KB cells) and normal fibroblasts. The AZ-1 compound induced the cell death of OEC-M1 that was mediated through the G2/M phase arrest of cell cycle first at 24 h and into apoptosis pathway at 48 h which might be associated with p53 protein and bcl-2 protein expression.Figure optionsDownload as PowerPoint slide