Differentiation Of U937 Cells Induced By Matrine And Its Mechanism

Objectives: Leukemia is one of the most commonly seen malignant tumors, characterized in excessive proliferation, differentiation failure and apoptosis disorder. U937 cell is a common acute monocytic leukemia cells used in basic research. Chemotherapy is prevalent method for leukemia, but the drugs are selectively limited and have toxic side effect which will cause severe damage to normal immune and hemopoietic system. From this aspect, a new drug with high selectivity and minor toxic side effect is critical to improving clinic efficiency. Matrine is a alkaloid separated from leguminous plant, too abundant species in west China. Years of pharmacological and clinical research proved that Matrine is antiviral and anti-inflammatory as well as controlling arrhythmia and cancer spreading. Some research reported that matrine can induce differentiation of tumor cell into normal cell, but no reference on inducing differentiation of U937 by matrine is published.This paper aim at illustrating the influence of Mat. on U937 proliferation by observing drug toxicity and the changes of cell cycle; evaluating cell differentiation from cell function, morphologic changes and the cell surface markers changes; and discussing the mechanism of Mat-induced U937 differentiation by recording the expression of cyclin and its inhibitors.Methods: 1. The effect of Mat on proliferation in U937 cells was observed by trypanblue staining, followed by determining differentiation concentration by investigating the viability rate of U937 cells, and measuring the distribution of cell cycle by flow cytometry.2. The function of U937 cells was detected by NBT reduction test; expression of cell surface markers CD11b and CD14 was measured by flow cytometry, and the morphologic changes was observed by inverted microscope and light microscope. All of the three methods were used to evaluate cell differentiation.3. Immunocytochemical method and western blot were used to detect the protein expression of cyclin E, cyclin A and p21Waf1/Cip1.Results: 1. Mat could inhibit U937 cells proliferation. After being treated with 0.1mg/mL Mat for 48h, the proliferation of U937 cells was inhibited significantly, the effect of inhibition was time and dose-dependent. After the various times of treatment with 0.2mg/mL Mat, the number of cells in G0/G1-phase decreased, whereas the number in S-phase increased significantly.2. After being treated with 0.2mg/mL Mat, the NBT positive cells and the expression of CD11b increased obviously, cells aggregated and adhered, pseudopodia could be found also. Light microscope showed reduction of karyoplasmic ratio, chromatin margination and prominent nucleoli were not seen.3. The expression of cyclin E, cyclin A was down-regulated, whereas p21Waf1/Cip1, was up-regulated by immunocytochemical method and western blot.Conclusions:1. Mat could inhibit U937 cells proliferation. the effect of inhibition was time and dose-dependent, and cells were arrested in S-phase.2. After being treated with Mat, U937 cells changed in function, cell surface markers and morphology, and partial differentiation was induced.3. The mechanism of inducing U937 cells differentiation by Mat might be related to the down-regulation of cyclin E, cyclin A and up-regulation of p21Waf1/Cip1.