Effects Of Eupatolide On The Proliferation Of HeLa Cells And Related Mechanisms

Cancer are the second deadly disease after cardio-cerebral vascular diseases and seriously threatens human health. With the development of medicine, the mechanism of occurrence and development of tumors is under deep investigation, and numerous anticancer drugs have emerged in clinical practice. However, the toxic and side effects of chemotherapeutic drugs limit their clinical application. To develop cheap chemotherapeutic drugs with little toxic and side effects become a big challenge for medical workers.Study have shown that Inula helenium (compositae) is effective in strengthening stomach, diuresis and expelling phlegm. Inula japonica is indicated for the treatment of cold with cough, ascending stomach-qi of years, urination difficulty and water retention. Currently, various sesquiterpene lactone components such as alantolactone, isoalantolactone and dihydro-alant- olactone have been extracted from Inula helenium and Inula japonica. These compounds have been validated to possess a variety of biological activities such as antitumor, anti-inflammatory, worms-expelling, antimicrobial, loweri- ng blood-glucose and pain-killing. The present experiment investigated the effects of six sesquiterpene lactone compounds extracted from Inula helenium and Inula japonica—Eupatolide(1),3,9-Diacetoxy-1,4(5)-germacra-dien-12, 6α-olide(2), 2-Hydroxy-11,13-dihydroxyisoalantolactone(3), 11α,13-Dihydro- xyisoalantolactone(4), Britannilactone(5) and 1-O-acetylbritannilactone(6)—on the proliferation of HeLa cells. The compounds with obvious inhibitory activity were screened out. And the mechanism of anti-proliferation of HeLa cells was investigated in order to provide experimental basis and theoretical foundation for developing new antitumor drugs and clinical application.1 The inhibitory effects of six sesquiterpene lactone compounds on proliferation of HeLa cells Objective:To observe the inhibitory effects of six sesquiterpene lactone compounds—Eupatolide(1), 3,9-Diacetoxy-1,4(5)-germacra-dien-12,6α- olid -e(2),2-Hydroxy-11,13-dihydroxyisoalantolactone(3), 11α,13-Dihydroxyisoala -ntolactone(4), Britannilactone(5) and 1-O-acetylbritannilactone(6)—on the proliferative activity of HeLa cells; To explore the structure-activity relationsh -ip so as to screen out the compounds with obvious inhibitory activity.Methods: The proliferation of HeLa cells was measured by monotetrazolium (MTT) colorimetric assay. HeLa cells treated with solvent control (final concentration: 0.1%DMSO), positive control (1μmol/L, 10μmol/L and 100μmol/L Cisplatinum and Taxol) and Six sesquiterpene lactone compounds(final concentrations: 1μmol/L, 10μmol/L and 100μmol/L) were inoculated in 96-pore plate(1×104 cells in each pore). There were three multiple pores in each group, and they were cultured in incubators of 37℃, 5%CO2 and saturated humidity for 48 hours. 5 mg/mL MTT was added at the time of four hours before ending. At the end of the experiment, the OD values of each pore was tested and Origin 7.0 was adopted to make the statistical analysis. The result was presented with mean±standard deviation ( x±s). The concentration effect curve of experimental compounds on the tumor cells was fit by Hill mathematical model to calculate the IC50 of medicine.Results: (1) 100μmol/L cisplatinum inhibited the proliferation of HeLa cells strongly. The inhibition rate of cell proliferation was 66.16%; HeLa cells were treated by six sesquiterpene lactone compounds 1~6 at respective final concentrations of 1μmol/L, 10μmol/L and 100μmol/L. We found that only compound 1 (Eupatolide) inhibited HeLa cells proliferation significantly. The inhibition rate was 65.38%. There was significant difference compared with the control group (p<0.01), while compounds(2-6) demonstrated weak anti-proliferative activity(p>0.05 vs control group); (2) 1μmol/L, 10μmol/L and 100μmol/L cisplatinum and taxol inhibited the proliferation of HeLa cells. The inhibition rates of cell proliferation were 19.13%, 43.15%, 66.1% and 51.66%, 77.18%, 84.77% respectively. Dose-dependence was presented. There was significant difference compared with control group(p<0.01,p<0.05); 1 μmol/L, 10μmol/L and 100μmol/L Eupatolide inhibited the proliferation of HeLa cells. The inhibition rates of cell proliferation were 10.17%,54.96% and 70.39%, demonstrating dose-dependence. The group at the concentration of 100μmol/L had significant difference compared with control group(p<0.01). Through logarithm regression equation of anti-proliferative rate of tumor cells on the concentration of Eupatolide (y=10.215ln(x)+19.297, y=7.1897ln(x) +54.648 and y=13.076ln(x)+15.063), IC50 values of cisplatinum, taxol and Eupatolide on HeLa cell proliferation were 20.19μmol/L,0.52μmol/L and 14.47μmol/L respectively.Conclusions: Eupatolide significantly inhibited the proliferation of HeLa cells in a dose-dependence manner. The IC50 value was 14.47μmol/L. Its anti-proliferative activity on tumor cells were speculated to be related to the structure of the unsaturated five-membered lactone rings. 3,9-Diacetoxy1,4(5) -germacra-dien-12,6α-olide(2), 2-Hydroxy-11,13-dihydr-oxyisoalantolactone (3), 11α,13-Dihydroxyisoalantolactone(4), Britannilactone(5) and 1-O-acetyl- britannilactone(6) demonstrated weak anti-proliferative activity (IC50>100μmol/L).2 Effect of Eupatolide on apoptosis of HeLa cells and related mechanismsObjective: Vitro and in vivo experiments have shown that many anticancer drugs can induce different types of sensitive tumor cells apoptosis. Numerous monomeric chemicals extracted from herbs played an important role in anti-tumor by inducing cell apoptosis. The first part of our experiment had demonstrated that Eupatolide significantly inhibited the proliferation of HeLa cells. In this part, to identify the antitumor mechanism of Eupatolide, we explored whether Eupatolide induce HeLa cells apoptosis by FCM and observed expressions of p53, Bax, Bcl-2 protein by Western blot.Methods: We examined cell apoptosis by FCM. Cells were incubated by Eupatolide at final concentrations of 0μmol/L, 5μmol/L, 10μmol/L, 20μmol/L and collected. They were washed by PBS three times respectively, digested by trypsin, fixed by 70% ethanol, added with 10% chicken erythrocytes as internal reference standard and stained simultaneously with the samples. While added with propidium iodide DNA fluorescent staining(Propidium Iodide, PI:50 mg.L-1,triton-x100 1.0%). The samples were stained away from light for 30 minutes in 4℃refrigerator, filtrated by 500 copper mesh, and made into qualified single cell suspension. Epics-XLⅡFCM produced by American Beckman Coulter was used to detect DNA. (2) We examined the p53, Bcl-2 and Bax protein by Western blot. Cells were incubated by Eupatolide at different final concentrations(0μmol/L, 5μmol/L, 10μmol/L, 20μmol/L)for different time (0 hour, 1 hour, 4 hours, 8 hours), followed by collecting cells, extracting total protein, electrophoretic separation of protein. Then, they were transferred to nitrocellulose membrane and blocked with 2% skim milk. TBS was added with anti-53 polyclonal antibody(1:1000), anti-Bax polyclonal antibody (1:1000) and anti-Bcl-2 polyclonal antibody(1:400)and incubated overnight at 4℃. Afterwards, membrane was washed and secondary antibody(1:20000)was added to incubation for 20 minutes. The protein was detected by chemiluminescence. Image analysis software was used to make semi-quantitative analysis. Data were presented as mean±standard deviation ( x±s) and analyzed by one way ANOVA and least difference test with SPSS 13.0 statistical program. A level of p<0.05 was considered statistically significant.Results: (1) Eupatolide increased apoptosis rate of HeLa cells in a dose-dependent manner. The apoptosis rates of 5μmol/L, 10μmol/L and 20μmol/L groups were 4.45%, 6.62%, 12.26% respectively, and an obvious hypodiploid peak appeared before the G1 peak of DNA. (2) p53, Bax, and Bcl-2 protein were detected in HeLa cells and Eupatolide could increase the expressions of p53, Bax, and Bcl-2 in a time-dependent manner. The expressions of p53, Bax, and Bcl-2 reached the peak at the time of 4 h, 4 h, 8 h after Eupatolide incubation. The expressions of p53, Bax, and Bcl-2 at different time group had significant difference compared with the 0 hour group (p<0.05), The expressions at the peak time group had significant difference compared with the other time points group. P53 protein were detected in HeLa cells. 5μmol/L, 10μmol/L and 20μmol/L Eupatolide could significantly increase the expression of p53 protein. There were significant difference compared with the control group (p<0.05); Bax and Bcl-2 protein were detected in HeLa cells. 5μmol/L, 10μmol/L and 20μmol/L Eupatolide could significantly increase the expression of Bax and inhibit the expression of Bcl-2; The ratio of Bcl-2/Bax tended to decline, and the comparison between each dose group had significant difference compared with the control group.Conclusions: Eupatolide could significantly induce the apotosis of HeLa cells; Eupatolide could increase the expressions of p53 and Bax, down-regulate the expression of Bcl-2, and lower the ratio of Bcl-2/Bax, which was probably one of the mechanisms of HeLa cell apotosis induced by Eupatolide.