The Mechanisms Of Inhibitory Effect And Inducing Apoptosis Of D-limonene On Human Leukemia Cells

Background: D-limonene( D-L ) is a naturally occurring monocyclic monoterpene found in the essential oils of citrus fruits, spices, and herbs and is widely used in the perfume industry. In medical aspect , the effect of D-L include antioxidation,anti-inflammatory and so on. In the recent years, it is reported that D-L has the apparently chemopreventive and chemotherapeutic activity against spontaneous and chemically induced rodent mammary,skin,liver,lung and forestomach tumors. The research of the mechanism of the chemopreventive and chemotherapeutic properties of D-L is mostly focused on the solid tumors, while, there is few reports of that kind of properties of D-L on human leukemia. Objective: To evaluate the inhibitory and apoptotic effect induced by D-L at different concentrations on human leukemia cells and to investigate the possible mechanism of inducing apoptosis of D-L. Method: k562 and HL-60 cells were cultured in DMEM supplemented with 10% fetal bovine serum, streptomycin(100ug/ml) and penicillin(100u/ml) under an atmosphere of 95% air and 5%co2 in vitro. When the cells were growing exponentially, subculture of K562 and HL-60 were performed on the 24-hole culture plate and D-L at the final concentration of 0.5,0.6,0.7,0.8,0.9mmol/L respectively was added. The blank control was added with 1%DMSO. All experiments were performed on triplicate wells and repeated at least three times. After 24,48,72 hours of treatment, PI(1mg/ml) was added and the inhibitory rate on the cells was assayed by detected the absorption value at 530nm wavelength. Sub-G1 cells were determined by flow cytometry, the number of apoptosis cell were assessed by Hoechst 33258 dying , and the expression of mutant type p⁵³, bcl-2,bax in K562 and HL-60 cells were detected by immunocytochemistry technique. Results:â‘ the inhibitory effect of D-L on K562 and HL-60 cell increased with the increase of treatment concentration. The difference of inhibitory rates at the final concentration of 0.5,0.6,0.7,0.8,0.9mmol/L were significant(p<0.01). Its effect also increased with the increase of the time, the difference of inhibitory rates 24,48,72 hours after treatment were significant(p<0.01). However, the inhibitory effect of D-L on K562did not differ from HL-60(p>0.05). The IC50 value of d-limonene for K562 and HL-60 cells was 0.75mmol/L. â‘¡The apoptosis apex would appear at concentration of 0.5mmol/L .With the increase of the concentration of D-L the apoptosis apex would increase. While, the apoptosis apex of K562 and HL-60 cell at the concentration of 1.0mmol/L will be decreased compared with the concentration of 0.75mmol/L when the concentration of D-L is over 0.75mmol/L. Apoptosis of K562 and HL-60 cells could be efficiently induced by d-limonene. This kind of effect of D-L on K562 is not different from that on HL-60, the difference of inducing apoptosis of D-L between K562 and HL-60 is not significant(p>0.05).â‘¢the number of apoptotic cells of K562 and HL-60 were observed by fluorescent microscope after dying the Hoechst33258. The nucleus of K562 and HL-60 cells condensed treated with D-L at the concentration of 0.75mmol/L compared with the control cells. â‘£The expression of bcl-2 and mutant type p53 protein in K562 cells decreased with the increase of the concentration of D-L compared with the control cells, at the same time ,bax protein increased in K562 cells treated with D-L(p<0.01) , compared with the control cells. While the expression of bcl-2 protein decreased in HL-60 cell with theincrease of the concentration of D-L , compared with the control cells, the expression of mutant type p53 and bax protein has no discrimination between the control cells and the treated cells in HL-60 cell(P>0.05). Conclusion: D-limonene has the inhibitory effect on K562and HL-60 cells in vitro in a dose and time-dependent manner . D-L can obviously induce apoptosis on K562 and HL-60 cells, and this kind of effect is related to the concentration of D-L. The mechanism of inducing apoptosis of D-L on K562 is different from on HL-60.