Effect Of Thalidomide On Proliferration And VEGF Expression In Human Endometrial Carcinoma Cell Line

Objective: Endometrial cancer is a frequent malignant tumor in genital organs, its incidence rate present an unremitting assurgent tendency in the world. Not with standing, the survival rate has an obviously elevation through operation, radiotherapy and chemo in the pristine Endometrial cancer, the patient of advanced stage are difficult to cure, so we need to research a new therapeutic tool. Along with the investigative deep-going of the pathogenesy of the tumor, people have known that tumorous invasion and aversion are closely correlated with neovascular- ization. The antiangiogenic therapy aim directly at the intervention of some factors and key steps about angiopoietic, accordingly intercepting blood supply of tumor endothelial cells, make them go into resting state moreover induce apoptosis, at last prevent tumor growth and metastasis, and achieve therapy tumor. Now antiangiogenic therapy has already become a hot spot in tumor therapy. Thalidomide is a kind of biosynthesis antiangiogenesis, which possess inhibiting the activity of vascular endothelial growth factor and inducing cell apoptosis. It has entered phaseⅠandⅡclinical trials for diverse solid tumors, but its antiangiogenic and antitumor effects disagree. There would be present specificity of genus and organ. The purpose of this study is to ascertain if it is feasibility in the therapy of endometrial cancer by thalidomide from foundational angle, and provide a new available drug of targeted therapy for the therapy of endometrial cancer.Materials and Methods: The human endometrial carcinoma cell line ishikawa were cultivated with RPMI-1640 including 10%FCS on the condition of the 37℃, saturated humidity and 5%CO2 , the cell in the experiment retained logarithmic growth. Different concentrations of thalidomide were used to treat ishikawa cells. the cell proliferation or inhibition was measured by MTT;the expression of VEGF was measured by ELISA;the ratio of apoptosis and cell cycle was quantitated by flow cytometry.Cell apoptosis was observed by inverted phase contrast microscope after dyeing by Giemsa.The statistical significance of the change in different models were analysed by SPSS 13.0 statistics software. A value of P<0.05 was accepted as statistically significant, and P<0.01 was accepted as significant differences.Results: 1 MTT: The ishikawa cell were effected by different concentrations of thalidomide for 24h,48h and 72h, the proliferation inhibition was observed, and it presented concentrations dependent and time dependent. The maximum concentration dimethyl sulfoxide (DMSO) of dissolving the thalidomide didn't effect cell proliferation (P>0.05). The rate of cell inhibition by treated with thalidomide at various concentrations (25μg/ml,50μg/ml,100μg/ml,200μg/ml,400μg/ml) after 24h: 11.90%,17.14%,28.90%,39.24%,51.00%;after 48h: 16.64%,27.1%,39.69%,49.64%,58.18%;after 72h: 26.48%, 35.92%, 45.63%, 56.76%, 62.82%. 2 Effect of thalidomide on VEGF produced by ishikawa cell line : according to the illustration of human VEGF-ELISA kiting prepare standard preparation and drawing standard curve , the expression of VEGF (pg/ml) in different concentrations and different effect-times was obtained, the result show: at 24h, 48h and 72h,there were significant difference among the various concentrations (25μg/ml,50μg/ml,100μg/ml,200μg/ml,400μg/ml) and control (P<0.01): 356.7±2.5, 299.3±2.4, 242.8±1.6, 180.3±2.0, 135.7±2.2, 411.5; 320.4±1.6, 271.7±3.1, 207.7±2.5, 153.4±2.9, 107.4±2.1, 418.5±2.7; 307.5±1.4, 250.7±1.0, 192.3±2.5, 129.1±1.6, 92.1±2.0, 416.1±2.4. The untreated controls in three different points didn't show statistical differences (P>0.05), and the various concentrations showed significant differences in three different points (P<0.01). 3 The cell morphology changes after the cells treated with thalidomide: In the control group , the cell shape is well-stacked, cell karyotin uniformity and round or ellipse, plasmosome clear; at 48h treated with thalidomide , the cell-substance concentrated and meta-round, and the mark morphology changes of cell apoptosis such as cell nucleus concentrated , nuclear fragmentation and so on, but didn't see a great quantity apoptotic body . Along with the concentration increasing, the living cell decrease, and the phenomenon above-mentioned became obviously. 4 Effect of thalidomide on the ratio of apoptosis and cell cycle:After 48h of various concentrations (50μg/ml,100μg/ml,200μg/ml) thalidomide treatment, the rate of Ishikawa cell apoptosis were (7.96±0.29)%, (12.14±0.77)% and (19.06±0.39)% , and the rate of apoptosis in untreated controls were (2.59±0.47)%, therewere significant differences among the four groups (P<0.01); the percentage of cell in G0-G1 stage : (44.75±1.54)%, (46.45±0.81)%, (52.45±1.84)% and (40.38±1.54)%, significant differences were found in the various concentrations compared with untreated controls(P<0.01), but not found between 50μg/ml group and 100μg/ml group(P>0.05); the percentage of cell in S stage : (41.40±1.34)%, (26.55±1.21)%, (19.75±1.05)% and (51.80±1.81)%, there were significant differences among the four groups (P<0.01); the percentage of cell in G2-M stage: (13.85±0.68)%, (27.00±1.53)%, (27.85±1.99)% and (7.81±0.52) %, significant differences were found in the various concentrations compared with untreated controls(P<0.01), but not found between 100μg/ml group and 200μg/ml group (P>0.05); the cell proliferation index:(55.25±0.71)%, (53.55±0.81)%, (47.55±1.84)% and (59.61±1.55)% , significant differences were found in the various concentrations compared with untreated controls (P<0.01), but not found between 50μg/ml group and 100μg/ml group(P>0.05).Conclusion : 1 The human endometrial carcinoma cell line ishikawa can secrete VEGF in vitro. Moreover, thalidomide can visibly inhibit ishikawa cell proliferation and expression of VEGF in vitro, and the inhibitory effect is correlated with concentration and time. These will provide a theoretical proof for the experiment in vivo and clinical application with thalidomide. 2 Thalidomide educe the effect of intimating anti-tumor through inducing apoptosis of the human endometrial carcinoma cell line ishikawa and G1 stage growth retardation. 3 Thalidomide can obviously decrease the percentage of cell in S stage, and showing concentration dependent. This also provided an experimental proof for clinical therapy endometrial cancer with thalidomide.