The Growth Inhibition Effect Of Emodin On Leukemia Cell Lines Of K562 And U937 And Its Mechanism

Objective: Leukemia is a group of malignant clonal diseases derived from the hematopoietic stem cells (HSC), or from leukemic stem cells (LSC). These cells lose their normal ability of differentiation and maturation and accumulated in bone marrow and other hematopoietic tissues, such as liver and spleen. Leukemia cells can also infiltrate in other organs such as central nervous system (CNS) and testis. Growing of normal hematopoietic cells is suppressed by leukemia cells. The incidence of leukemia in our country is 2.76 per 100 thousands people. The mortality of leukemia occupies the top 6th for male and 8^th for female among the overall mortalities of adult malignant tumors. However, it rises to highest among children malignant tumors. Although several new effective drugs have been developed and the skills for hematopoietic stem cell transplantation improved during last decade, there are still many leukemia patients who can not achieve complete remission or relapse sooner or later after the complete remission. The treatment of leukemia becomes more difficult with the appearance of the primary or secondary drug resistance, especially multidrug resistance (MDR). Therefore, it is a long-term and pressing task to find new effective drugs without or with slight side-effect for the treatment of leukemia.Emodin is a kind of hydroxyanthraquinone chemical compound. It has many functions, such as anti-inflammation, bacteriostasis, immune regulation, inhibiting the secretion of trypsin, protecting the hepatic function and renal function, as well as anti-tumor effect. The anti-tumor effect of Emodin, including anti-proliferation and inducing apoptosis of tumor cells, reversing multidrug resistance, becomes the focus of medical research recent years.The present study is thus designed to explore the effects of Emodin, at different concentrations, on the proliferation inhibition of leukemia cell lines K562 and U937, by observing the morphological changes, apoptosis bodies, measuring the change of cell cycle distributions and the apoptosis rates, and detecting the expression levels of bcl-2 and NF-κB mRNA by reverse transcriptase polymerase chain reaction (RT-PCR).Methods:1 Cell culture: K562 cells and U937 cells were cultured in RPMI-1640 medium which supplemented with 10% newborn bovine serum, at 37℃, 5% CO₂, and fully humidified atmosphere. Cells at logarithmic growth phase were treated with Emodin (at different final concentrations for 48 hours) as different treatment groups or without Emodin treatment as control group for 48 hours, then, they used for the following experiments. 2 Cells proliferation assay: A cell counting kit-8 (CCK-8) was used to measure the growing inhibition effect of Emodin on K562 cells or U937 cells. After the cells were treated with or without Emodin for 48 hours, 10μl of CCK8 solution were added to each group of cells, incubated at 37℃for another 2 hours. The absorbance of each sample was measured at 450 nm by using an enzyme-labeling measuring instrument. The inhibition rates were calculated based on the following formula: the inhibition rate (%) = (the absorbance value of control group - the absorbance value of experiment group)/the absorbance value of control group×100 % . The 50% inhibiting concentration (IC50) were also calculated from the result of this experiment.3 Hoechst 33342 staining was performed to measure the index of cell apoptosis: K562 cells and U937 cells which had been treated with Emodin at IC50 concentration for 48h were collected, washed with PBS, then stained with Hoechst 33342. At least 400 cells were examined under a fluorescence microscope to observe morphological changes of apoptosis.4 Flow cytometry (FCM) was used to measure the changes of cell cycle distribution and apoptosis rate: For FCM analysis, K562 cells and U937 cells were treated with Emodin at different concentrations for 48h. The cells were collected, washed with PBS and fixed with 70% ethanol. After the cells were washed with PBS again, RNAase were added to destroy RNA, then stained with propidium iodide (PI) for 30 minutes. 1×10⁶ fixed cells were examined by FCM to measure the change of cell cycle distribution and apoptosis rate.5 Measurement of enzyme activity of caspase3/7: K562 cells and U937 cells were treated with Emodin at different concentrations for 48h. Then 100μl of caspase3/7 enzyme activity measurement reagent were added to each cell culture and the cells were cultured for another 2 hours. The absorbance value was measured at 405 nm by using enzyme-labeling measuring instrument.6 RT-PCR was performed to measure the mRNA expression levels of Bcl-2 and NF-κB: Total RNA was extracted from each group of cells by Trizol. The reverse transcription reaction was performed to synthesize cDNA by using M-MLV enzyme and random primer. The cDNA and the special primers were then used to amplify the target genes by PCR. PCR products were examined by agarose gel electrophoresis with Goldview staining, and the relative expression levels of Bcl-2 and NF-κB mRNA were calculated according to the expression level ofβ-action.Results:1 Emodin inhibited the growth of K562cells and U937 cells in a dose dependent manner and the IC50 of Emodin for K562 cells was 51.58μmol/L and 48.00μmol/L for U937 cells.2 After cells had been treated with Emodin for 48 hours, morphological changes of cell apoptosis were observed clearly. The percentage of cells with bright blue fluorescence increased in the Emodin treated groups compared with that of control group, observed under a fluorescence microscope.3 FCM examination results showed that the percentage of cells in S phase increased and the percentage of cells in G0/G1 phase and G2/M phase decreased in Emodin treated K562 cells compared with that of control cells (all P values are <0.05) in a dose-dependence manner. In U937 cell line cells, the percentage of cells in G0/G1 phase increased and the percentages of cells of S phase and G2/M phase decreased in Emodin treated cells compared with that of control cells (all P values are <0.05). Moreover, Emodin increased significantly the apoptosis rate of both cell lines in a dose-dependent manner (P values are <0.05).4 The enzyme activity of caspase3/7 increased significance in both cell lines, after the treatment of Emodin (P values are <0.05).5 RT-PCR results showed that the mRNA expression levels of bcl-2 and NF-κB decreased significantly in both K562 and U937 cells with the treatment of Emodin compared with that of control cells.Conclusion: Emodin inhibited in vitro the growth of K562 cells and U937 cells efficiently. It probably played its role through down-regulating the expression of bcl-2 and NF-κB mRNA, blocking cells cycle progression and inducing the apoptosis of cells.