The Inhibition Of Emodin On Smooth Muscle Cell Proliferation Mechanism

Part I. The antiproliferative activity of emodin is through ROS-dependent and p53-dependent pathway in human smooth muscle cells Background: Emodin (1, 3, 8-Trihydroxy-6-methylanthraquinone), a natural anthraquinoid compound, has the remarkably suppressing activity on the various tumor cells proliferation. However, the mechanism of effect of emodin on the smooth muscle cells has remained largely unknown. Emodin can induce apoptotic response in the human hepatocellular carcinoma cell line Mahlavu and human promyeloleukemic HL-60 cells. Preincubation of Mahlavu cells with catalase (CAT) showed partially preventive effect on emodin-induced apoptotic responses. However, preincubation of HL-60 cells with free radical scavenging agents including catalase showed no preventive effect. Whether the antiproliferative activity of emodin is through reactive oxygen species (ROS)-dependent apoptotic pathway is still in argument. Meanwhile, emodin exhibits mutagenicity in the Salmonella typhimurium mutagenicity assay and genotoxicity in the mammalian test systems. DNA damage-inducing protein p53 can inhibit cell growth, by arresting proliferation or inducing apoptosis. So, the activation of p53 may be another pathway participated in the inhibition of proliferation induced by emodin. Emodin can be transformed into at least 10 anthraquinoid metabolites through cytochrome P450 enzyme (CYP). 2-hydroxyemodin, a main metabolite converted from emodin, is a little more cytotoxic than emodin. The characteristic of antiproliferation of emodin might be influenced by CYP activity which can be enhanced by CYP inducer or suppressed by CYP inhibitor. In the present study, we investigated whether emodin has antiproliferative effect on the smooth muscle cells and whether the effect of emodin is through ROS-dependent and p53-dependent pathway and whether metabolic transformation of emodin is an important step for the effect of emodin. Materials and methods: Vascular smooth muscle cells were cultured in M199 medium supplemented with 15% fetal bovine serum (FBS). The antiproliferative activity of emodin was observed by several tests, such as total cell count, MTT assay, lactate dehydrogenase (LDH) release, cell cycle analysis, and cell migration. The types of cell death induced by emodin were determined by annexin V-PE and 7-AAD staining. To investigate ROS pathway,celluar ROS was measured. When ROS could be suppressed by inhibitor, the types of cell death were measured again. To investigate p53 pathway, the level of protein p53 was measured by western blot. Transmission electron microscope and laser confocal microscope had also been used. To investigate emodin metabolic pathway, CYP inducer or inhibitor was used to alter CYP enzyme activity. cDNA MicroArray was used to determined which gene expression is altered after emodin treatment.Results: (1) The loss in viability of VSMCs was an emodin dose-dependent manner. Emodin delayed the number of VSMCs entering DNA synthesis (S) phase in a concentration-dependent manner. The percent of S in 25.0 ug/ml emodin group was significantly lower than that in control group (7.40±1.65% vs. 39.05±5.88%, p<0.05). VSMCs migration was also inhibited by emodin, 89.6% decrease of migrated cell numbers in 25 μg/ml emodin group. (2) ROS generation increased in emodin dose-dependent manner (r=0.97, p<0.01). The cellular ROS level remained unchanged during a 72 h period. Increased ROS could be inhibited by NADPH oxidase inhibitor DPI or PDTC, but not other inhibitors (CsA, ABT, α-NF). The change of cellular antioxidantive potential was not significantly. Two types of cell death, apoptosis and necrosis, had been found in VSMCs treated with emodin for 24 hours. When VSMCs was precubated with DPI before emodin added in, the ratio of apoptotic cells significantly decreased from 14.55±2.29% to 8.77±0.83% (p<0.05) and the ratio of necrotic cells didn't change significantly (1.18±0.14% vs. 1.25±0.28%). DPI could also significantly inhibit the cell death induced by 200μM H₂O₂ (apoptotic cell: 12.6+0.98% vs. 4.0±1.00%, p<0.05; necrotic cell: 32.2±2.99% vs. 4.87±2.22%, p<0.05). LDH release can be enhanced by emodin and can be partially inhibited by DPI pretreatment. (3) Unshedueled DNA synthesis, a symbol of DNA damage, was observed after emodin treatment and can not be eliminated by DPI. The level of protein p53 in 25 ug/ml emodin group was 1.69-fold more than that in control group. Heterochromatin was also increased. (4) The intensity of fluorescence of emodin increased rapidly and reached plateau in 15s. However, the distribution of emodin was selective, mostly in cytoplasma and scarcely in nucleus. (5) Neither CYP inducers (ABT, α-NF) nor CYP inhibitors (3-MC, β-NF) had impact on the effects of emodin, such as ROS generation and proliferation inhibition. (6) Emodin accelerated cellsenescence in a dose and time dependent manner. Cell autophagy had also been observed.Conclusions: (1) Emodin can inhibite human VSMCs proliferation and induce cell death (majority as apoptosis and minority as necrosis ). (2) The effect of emodin is through ROS-dependent and p53-dependent pathway. ROS pathway mainly participated in the apoptotic response and p53 pathway participated both in the aopototic response and in the proliferation inhibition. (3) There is no evidence to support the existence of CYP. Emodin played the role of inhibitor of VSMCs by itself, not its metabolite.Part II. Evaluation of MTT assay for measurement of emodin-inducedcytotoxicity.MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay is widely used for measuring cytotoxicity induced by emodin. Since the specifity and sensitivity of MTT assay may be influenced by many factors including coloring substances, we therefore investigated whether the absorption spectrum of emodin partially overlapped with that of formazan and the reasons for these influences in the present study. We cultured vascular smooth muscle cells (VSMCs) in M199 medium. The optical density (O.D.) of emodin or formazan was measured by spectrophotometer. We observed that emodin has different absorption spectrum in different solvent. The solvents containing water induced shift of the absorption curve of emodin to right, which increased the overlap of absorption curve of emodin and formazan. The increase of the formazan, which was converted from MTT tetrazolium salt by emodin, was paralleled to the concentration of emodin. The intrinsic reductive potential of emodin can be partially suppressed by serum. Emodin in cells was very tiny and its metabolic transformation was quite slow. These data suggest that emodin can alter the accuracy of MTT assay and that modified MTT assay is valuable in emodin-induced cytotoxicity.