Induction of cell cycle arrest and apoptosis in human b-lymphoma cells by citral

Abstract

This study intended to evaluate anti-tumor activity of citral on human B-lymphoma cells, Ramos cells. The effects of citral on apoptotic induction, on cell cycle as well as its mechanism of action on apoptotic induction were studied. The apoptotic induction of citral on Ramos cells was examined by annexin V-FITC and propidium iodide (PI) staining monitored by fluorescence flow cytometer. The results demonstrated that 37.5, 75 and 150 µM citral induced Ramos cell death in a concentration- and time-dependent manner. It induced mainly apoptosis on Ramos cells. Other types of cell death were detected only when the cells were treated with 150 µM citral. It induced Ramos cell apoptosis after 3 h of exposure. The apoptotic activity of citral was confirmed by assessing apoptotic cells with hypodiploid DNA when compare to diploid DNA in viable cells by PI staining. The content of DNA in the cells was detected by flow cytometer. Citral, at 37.5, and 75 µM, induced normal PBMC death much less than Ramos cell death. The effect of citral on the cell cycle was investigated by washing citral-treated Ramos cells and allowed them to proliferate in fresh medium. Distribution of the treated cells in the cell cycle was detected by PI staining. There was no change in the cell cycle pattern in the citral treated cells, at all concentrations of citral. The mechanism of apoptotic induction of citral was also investigated. Apoptotic induction of citral via death receptor pathway was determined by using anti-FAS ligand antibody to inhibit Fas-Fas ligand binding. The results showed that anti-Fas ligand didn’t inhibit citral activity. The effect of citral on the mitochondrial pathway was evaluated by analyzing the mRNA expression of the BCL-2 family proteins (BCL-2, BCL-XL, BAX, BAK) and p53, which control the mitochondrial pathway of apoptosis. It was revealed that citral only inhibited BCL-2 mRNA expression. It didn’t have any effect on the mRNA expression of the other proteins in the study. To study the degree of dependency on caspase activation of citral activity, a pan caspase inhibitor, Z-VAD-FMK, was used. This inhibitor significantly decreased apoptotic induction activity of citral. The results in this study indicate that citral can induced human B-lymphoma cells apoptosis via the mitochondrial pathway by down regulating BCL-2 expression and depending mainly on caspases activation.