Effect Of β-elemene On The Proliferation, Apoptosis, Angiogenesis And The Activity Of MMP-2, MMP-9 In Vascular Endothelial Cells

IntroductionThere are two stages in the development of tumor:when the tumor is smaller than 1 mm3,its nutrition mainly depends on infiltration, which is called prevascular phase; when the tumor is bigger than 1 mm3, its nutrition mainly depends on the formation of vessels, which is called vascular phase.From the point of the formation of vessels, progressing tumor can be seen as a developing organ, and in which process, vascularization is very important. If there is not enough blood, oxygen, or other nutrients, the tumor can't grow bigger than 2-3mm3. Therefore, anti-vascularization is very important in the therapy of tumor.In the process of the formation of vessels, promoting the proliferation and inducing the apoptosis of vascular endothelial cells are necessary for the inhibition of vascularization in tumor.Matrix metalloproteinase (MMP) can selectively digest extensive extracellular matrix, and typeⅣcollagen is the most important protein in the extracellular matrix and basement membrane. So typeⅣcollagenase MMP-2 and MMP-9 play an important role in the degradation of basement membrane in the early phase of vascularization. Therefore, the inhibition of MMP-2 and MMP-9 can effectively decrease the vessels formation in tumor.β-elemene is a kind of non-cytotoxic anti-cancer drugs. Lots of researches indicated that P-elemene could inhibit the growth and proliferation of many kinds of tumor cells.The main characters of its acting mechanism are inhibiting the DNA synthesis and inducing apoptosis and differentiation of tumor cells. However, there is few research focusing on the anti-vascularization action ofβ-elemene. Mathods1. Cell cultureECV-304 cells were cultured with DMEM medium supplemented with 100 ml/L calf serum, 1×105 U/L penicillin and streptomycin at 37℃in a humidified atmosphere containing 5% CO2 in air.2. Evaluation of the angiogenic ability of ECV-304 cellsECV-304 cells were seeded into 24-well plates at a density of 1×105/ml, and incubated for 24,48,72h with different concentrations ofβ-elemene(0,20,40,80, 160μg/ml). Collected and planted cells into 24-well plates with Matrigel, and continued to culture for 24 hours. Then observed the formation of the tubes under the microscope (×40) and counted the number of the tubes formed.3. Evaluation of the proliferation of ECV-304 cellsThe ECV-304 cells were seeded in 96-well plates at a density of 1×104/ml and incubated for 24,48,72h with different concentrations ofβ-elemene(0,20,40,80, 160μg/ml), followed by incubation with 5 mg/mL of MTT for 4h. Then the supernatant was removed following centrifugation, and 100μl of DMSO was added. Absorbance at 490 nm was measured with an enzyme-labeling instrument.4. Analysis of cell cycle by flow cytometryThe ECV-304 cells were seeded into 24-well plates at a density of 1×104/ml and incubated for 24,48,72h with different concentrations ofβ-elemene(0,20,40,80, 160μg/ml). After fixation with 75% ethanol, cells were digested with DNAse-free RNAse in PBS containing 50 mg/ml propidium iodide for DNA staining. Cell cycle was detected by using a flow cytometer.5. Analysis of apoptosis by flow cytometryThe apoptosis was detected by FCM using Annexin-V-FITC and PI staining. The test protocol was performed according to the instruction of the detection Kit. The cultured cells were collected through centrifugation, washed twice with PBS, re-suspended with PBS and adjusted to a density of 5×105 cells/ml. Subsequently 195μl cell suspension was incubated with 5μl Annexin-V-FITC for 10 min at room temperature and rinsed with PBS one time. After the rinsed cell was re-suspended with 190μl PBS and 10μl PI, cell fluorescence level was analyzed by FCM. Living cells were Annexin-V-FITC and PI double negative, while late apoptotic cells were double positive. Early apoptotic cells were Annexin-V-FITC single positive, while necrotic cells were PI single positive. Data was got and analyzed by Cell Quest software.6. Evaluation of the activity of MMP-2 and MMP-9 in ECV-304 cells with zymographyThe ECV-304 cells were seeded into 24-well plates at a density of 1×104/ml and incubated for 24,48,72h with different concentrations ofβ-elemene(0,20,40,80, 160μg/ml). Following determination of the protein in the supernatant by the Lowry's method, protein samples were mix with the 5×loading buffer and electrophoresed on SDS-PAGE at 120V for 1.5 hours. And then the gel underwent elution (40min, twice), rinsing (20min, twice), incubation (48hours,37℃), dying (4hours) and decolorization in turn. Finally, semi-quantitative analysis was performed to the gel.7. Statistical analysisAnalysis of the data was performed with a SPSS 13.0 software package. The differences among the groups were compared by one way ANOVA and t test. The level of significance was set at P<0.05.Results1. Evaluation of the angiogenic ability of ECV-304 cells20μg/mlβ-elemene acted on ECV-304 cells for 24 hours, the number of tubes formed was similar with that of the control group. Whileβ-elemene of other concentration acted on ECV-304 cells for 24,48,72 hours, the numbers of tubes formed were apparently decreased compared with the control group in a in a dose and time-dependent manner.2. Evaluation of the proliferation of ECV-304 cells20μg/mlβ-elemene acted on ECV-304 cells for 24 hours, the proliferation of cells was promoted. Whileβ-elemene of other concentration acted on ECV-304 cells for 24,48,72 hours, the proliferation of cells were obviously inhibited in a dose and time-dependent manner.3. Analysis of cell cycle by flow cytometry 20μg/mlβ-elemene acted on ECV-304 cells for 24 hours, cells in G1 phase were decreased compared with the control group. Whileβ-elemene of other concentration acted on ECV-304 cells for 24,48,72 hours, FCM assays showed that cells in G1 phase were increased,while in G2 phase and S phase were decreased, which indicated that G1 phase arrest happened.4. Analysis of apoptosis by flow cytometryTogether with the dose and acting time increasing,the apoptosis of ECV-304 cells was increased notably.5. Evaluation of the activity of MMP-2 and MMP-9 in ECV-304 cells with zymography80μg/ml and 160μg/mlβ-elemene could significantly decrease the activity of MMP-2 and MMP-9 in endothelial cell. But 20μg/ml and 40μg/mlβ-elemene acted on ECV-304 cells for 24 hours, the activity of MMP-2 and MMP-9 was similar with that of the control group.Conclusion1.β-elemene may inhibit the angiogenic ability of vascular endothelial cells cultured in vitro.2.β-elemene may inhibit the proliferation of vascular endothelial cells cultured in vitro, arrest cells in G1 phase and increase apoptosis.3.β-elemene may inhibit the activity of MMP-2 and MMP-9 in vascular endothelial cells cultured in vitro.