| Objective:Endometrial cancer is one of the most common gynecologic malignant tumor.In recent years with the population aging,population growth,population obesity,the incidence of endometrial cancer increases year by year.There are approximately 320000 new cases of endometrial cancer and 76000 related deaths are reported in the world every year.Although stage I of endometrial cancer was the most common,and the 5-year survival rate could reach 85-90% after surgery.But the patients with stage III-IV of endometrial cancer even received standardized surgery,radiotherapy and chemotherapy treatment,the 5-year survival rate was only about 30-40%.Combination chemotherapy can improve the disease-free survival of patients with advanced or recurrent period,but it did not improve the overall survival time.America's latest survey shows that the endometrial cancer 5-year survival rate was about 81.8%.There are nearly 4489 stage II-IV of endometrial cancer patients died every year.The 5-year survival of developing countries is only 67%.Thus the new anti-tumor drugs remains to be explored.NK-1 receptor is a neurokinin-1 receptor and a G protein–coupled receptor.It is widespread in central nervous system and peripheral organs,including the striatum and brain stem,spinal cord,gastrointestinal tract,bladder,thyroid,a variety of immune cells,lung airway epithelium and submucosal gland,and plays an important role in many physiological and pathological process,such as pain,migraine,inflammation,emotion regulation,anxiety,drug abuse,nausea,vomiting.In recent decades,the scholars' study discovered that NK-1 receptor played an important role in the development process of tumor.NK-1 receptors are involved in a wide variety of tumor proliferation,metastasis and angiogenic.After the binding to the NK-1 receptor,NK-1 receptor antagonists have anti-tumor effect in a variety of tumor cells.L-733060 is a efficent,selective and long-acting NK-1 receptor antagonist.It has anti-tumor effect in a wide variety of tumor cells.Whether NK-1 receptor antagonists play a role on endometrial carcinoma is no related reports.We used MTT assay,flow cytometry and Transwell to investigate the effect of NK-1 specific receptor antagonist(L-733060)on proliferation,apoptosis and invasion of human endometrial cancer cells in vitro culture system,seeking new targeted therapeutic drugs for endometrial cancer.Methods:1 Ishikawa cells culture: The Ishikawa cells provided by Animal Centre of the No.4 affiliated hospital of Hebei Medical University.The Ishikawa cell line was routinely grown in RPMI-1640,supplemented with 10% fetal bovine serum(FBS),100U/ml penicillin and 100U/ml streptomycin at 37 ?in a 5% CO2 incubator until the cell confluence rate reach at 50%-60%.2 Ishikawa cells proliferation change was detected by MTT assay after incubated with L-733060: Ishikawa cells were divided into blank control group and different concentrations of L-733060(5,10,20,40 ?M)group.The experiment was repeated three times each group.According to the group,put 5×104 exponential growth of Ishikawa cells in 96-well format plates.After 48 h,MTT assay was used to determine inhibition rate by absorbance of Multimode Reader.It is judged the effect of the L-733060 on Ishikawa cells and the optimal concentration.3 Ishikawa cells cycle and apoptosis was tested by flow cytometry after incubated with L-733060: Ishikawa cells were divided into blank control group and different concentrations of L-733060(5,10,20,40 ?M)group.The experiment was repeated three times each group.According to the group,Ishikawa cells in vitro incubated 48 h.(1)apoptosis: putting 5?L Annexin V-FITC and 10?L PI in Ishikawa cells for dyeing.Flow cytometry detected cell apoptosis rate.(2)cells cycle: after 70% ethanol precooling 4? for 12 h,the Binding Buffer suspend processed cells.After PI dyeing Flow cytometry detected the effect of L-733060 on Ishikawa cell cycle.4 The invasion ability of Ishikawa cells after incubated with L-733060 was detect by Transwell: Ishikawa cells were divided into blank control group and different concentrations of L-733060(5,10,20,40 ?M)group.According to the group,put 5×105 exponential growth of Ishikawa cells in 6-well format plates with 48 h.Through calculating the cell membrane in Ishikawa cells invade evaluation L-733060.The number of invaded cells reflected the invasion of Ishikawa cells.5 The SPSS 13.0 software was employed to analyze the data.And it was analyzed by one-way ANOVA,using LSD pairwise comparison difference between the groups.P<0.05 was considered as statistical significance.The statistical figure portrayed using GraphPad Prism 5 and SPSS 13.0 software.Results:1 Various concentration of L-733060(5,10,20,40?M)were applied in Ishikawa cells during 48 h.MTT assay were used to detect the inhibition rates,the results were as follows 10.260±2.746%,22.633±3.050%,61.180±5.346%,91.873±8.905%.There existed significantly statistical difference(P<0.01).At the concentration of 5 to 40 ?M L-733060 inhibited cell growth in a dose dependent manner.2 Flow cytometry detected that apoptosis rates for Ishikawa cells incubated in culture medium containing different concentrations L-733060(0,5,10,20,40 ?M)during 48 h,the results were as follows 4.437%±0.952%,7.767%±1.725%,11.82%±1.350%,19.723%±1.957%,26.39%±2.168%.There existed significantly statistical difference(P<0.01).The higher concentration of L-733060,the higher the apoptosis rates was.3 Various concentration of L-733060(0,5,10,20,40?M)were applied in Ishikawa cells during 48 h.Flow cytometry were used to detect the Ishikawa cell cycle percentage,the results were as follows: G0-G1 phase 45.390±0.948%,47.253±1.546%,54.287±1.826%,63.730±1.721%,69.293± 1.895%.There existed significantly statistical difference(P<0.01).G2-M phase 16.323±0.441%,15.593±0.402%,13.160±0.881%,10.637±0.497%,8.183±1.204%.There existed significantly statistical difference(P<0.01).S phase 38.967±1.670%,37.203±1.491%,32.400±1.213%,25.633±1.245%,22.523±0.725%.There existed significantly statistical difference(P<0.01).L-733060 blocked the cycle in G0-G1 period and increased the cycle in S period and G2-M period.4 Transwell detected that number of invaded cells for Ishikawa cells incubated in culture medium containing different concentrations L-733060(0,5,10,20,40 ?M)during 48 h,the results were as follows177.333±30.989,152.000±27.875%,94.333%±19.604%,51.000±12.000,10.333±4.163.There existed significantly statistical difference(P<0.01).The higher concentration of L-733060,the lower the number of invaded cells was.Conclusions:1 Combination with NK-1 receptor,L-733060 can blunt cell growth.Along with the increasing concentration of L-733060,the inhibitory role increased gradually in the experimental concentration range.NK-1 receptor antagonists would become the new targeted therapies for endometrial cancer.2 L-733060 can cause the Ishikawa cell apoptosis.L-733060 increase the Ishikawa apoptosis in a dose dependent manner.L-733060 can blunt the cycle in G0-G1 period and increase the rates of cell apoptosis,NK-1 receptor can play a role of anti-tumor by inducing tumor cell apoptosis.Its anti-tumor effect may be related to the endometrial cancer cell cycle.3 L-733060 can decrease the number of invaded cells.With the concentration increasing the Ishikawa cells has the lower cell invasive ability.NK-1 receptor antagonist may be inhibiting tumor cell metastasis drug for endometrial cancer. |
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