Study Of Genes Related To Radiosensitivity And Angiogeneisis In Endometrial Cancer Using Oligonuleotide Mircroarrys

Partâ… Identification of Differentially Expressed Genes Response to Ionizing Radiation in Human Endometrial Cancer Cell Lines with Distinct Radiosensitivities by Oligonucleotide MicroarraysChapterâ… Experimental Study of Radiosensitivity for Human Endometrial Cancer Cell LinesOBJECTIVE:Endometrial cancer is one of the most common gynecological malignancies worldwide,which morbidity is increasing year by year.High-risk patients are at risk for local-regional relapse and therefore adjuvant radiotherapy is essential for local control.Although there are two-thirds of patients need adjuvant radiotherapy,quite a few patients recurrent within 5 years.It is generally believed that the efficacy of this therapeutic modality depends on the individual inherent radiation sensitivity.The aim of the present study is to investigate the radiosensitivities of 4 human endometrial cancer cell lines ISK,RL95-2,HHUA and KLE and select two cell lines with different radiosensitivity for later analysis.METHODS:Human endometrial cancer cells from the ISK(well differentiated), RL95-2(moderately differentatied),HHUA(well differentiated) and KLE(poorly differentiated) cell lines were irradiated by 6MV X-rays with various doses:0,1,2,3, 4,6,8,10Gy,and plating efficiency was calculated from the postirradiated cells by colony formation assay.Survival curves were determined by 'single hit multi target theory' and radiosensitivity parameters were calculated by GraphPad prism software. Cell cycle distributions and apoptosis after irradiation were analyzed using a FACScan flow cytometer.RESULTS:After irradiation,the cells became round in morphology and appeared anoikis comparing with normal cell.Colonies formed after 10 days of incubation.The SF₂ values were 0.572,0.510,0.446,and 0.328,respectively.Significant difference was found between ISK and KLE cells(P=0.017).The cell cycle distribution showed lower fractions of G₂/M phase cells in radioresistant cell line ISK before radiation compared with radiosensitive cell line KLE(13.6%vs.37.0%,P＜0.05).Irradiation caused cell cycle arrest at G₂/M phases in both cell lines.12 hours after radiation, higher fractions of G₂/M phase cells were found in ISK compared with KLE(50.0% vs.14.1%,P＜0.05).Apoptosis assessment also showed significant elevation in the percentage of early apoptosis cells in KLE cells(P＜0.05).CONCLUSION:From detection of clonogenic survival curves for the four cell lines exposed by X-ray radiation,we select two cell lines with different radiosensitivity.Cell line KLE was classified as radiation-sensitive,and cell lines ISK were classified as relatively radiation resistant. Chapterâ…¡Differential Expression Profiling of Genes Response to Ionizing Radiation in Endometrial Cancer Cell LinesOBJECTIVE:The efficacy of radiotherapy depends on the individual inherent radiosensitivity which related to gene expression induced by irradiation.Identification of the genes differentially expressed between radiosensitive and radioresistant cancers may provide new insights into the mechanisms underlying clinical radioresistance and improve the efficacy of radiotherapy.The aim of the present study is to examine global expression patterns induced by X-ray irradiation of two cell lines with different radiosensitivities and elucidate the further molecular events involved in radiosensitivity of endometrial cancer.METHODS:Microarray experiments were performed using 22K Human genome oligonucleotide microarrays containing 21,329 well-characterized Homo sapiens genes to screen gene expression changes after X-ray exposure in ISK and KLE cells.Differential expression was determined using the combined basis of t-test with P＜0.05 and fold changes(either up or down) of≥2-fold.All differentially expressed genes were analyzed using Molecular Annotation System 5.0 which integrates three pathway resources—KEGG,BioCarta and GenMAPP.To show the reproducibility of the microarray analysis,10 genes were selected at random,and these changes were validated by real-time RT-PCR and Western blot.RESULTS:227 and 354 genes that exhibited≥2-fold difference were identified in ISK and KLE,respectively.However,only 53 genes showing differences more than double the median expression value between the two groups were defined as radiosensitivity(or radioresistance) related genes.Among these,genes associated with DNA-repair,apoptosis,growth factor,signal transduction,cell cycle and cell adhesion were obviously predominant.The validity of the expression level of 10 randomly selected genes was confirmed by real-time PCR and/or Western blotting.CONCLUSION:The molecular mechanisms of radiosensitivity in endometrial cancer are very complex and involve expression changes of multiple genes associated with DNA-repair,cell cycle,apoptosis,signal transduction,cell adhesion and stress reaction.The differential gene expression changes that occur after radiation in the two cell lines will not only provide insight into molecular mechanisms of radioresistance in endometrial carcinoma,but also as a means to find potential targets to achieve further gains in therapeutic benefit. Partâ…¡Isolation,Purificantion and Gene Alterations in Tumor-associated Human Endometrial Endothelial CellsOBJECTIVE:Endometrial cancer is one of the most common malignancies of the female genital tract.Although early-stage endometrial cancer can be treated surgically,mortality in women with advanced disease has remained largely unchanged despite improvements in surgical technique,chemotherapy regimens,and radiation protocols.Therefore,novel therapeutic strategies are highly needed.It is now well known that tumor growth and proliferation are associated with angiogenesis. The growth of malignant tumor can be reduced through inhibiting angiogenesis.The therapeutic effect of antiangiogenesis is limited by deficiency of specific target for endometrial cancer.In the present study,we aim to immunopurify endothelial cells from freshly resected specimens of endometrial cancers and normal endometrial tissues and investigate the gene expression profile using microarrays.The growth characteristics and the capacity of immigration,invasiveness,and tube formation of isolated human endometrial endothelial cells(HEECs) are examined additionally.The alterations in gene expression profile in tumor-associated human endometrial endothelial cells(HEECs) may allow opportunities for developing new therapeutic approaches to inhibit angiogenesis in endometrial cancer.METHODS:Endothelial cells were isolated from 3 freshly collected endometrial cancer tissues and 3 normal endometria tissues with anti-CD31 conjugated magnetic microbeads.Global expression patterns of purified HEECs were analyzed using 22K Human genome oligonucleotide microarrays.All differentially expressed genes were analyzed using Molecular Annotation System 5.0 which integrates three pathway resources—KEGG,BioCarta and GenMAPP.Real-time RT-PCR and Western blot were used to validate the reproducibility of microarray analysis.We also performed in vitro culture and identified the endothelial origin,as well as observed the functional characteristics in angiogenesis by MTT,Wound Healing Assays,Transwell Cell Invasion Assay and Tube Formation Assay of HEECs from the two different sources.RESULTS:Flow cytometry revealed that the immunopurification technique yielded endothelial cell purity of＞95%in all samples.HEECs separated from normal and malignant endometrial tissue presented similar appearance in culture medium under phase contrast microscope.Cell cultures presented flattened monolayers,with typical cobblestone morphology.All purified cells were characterized as endothelial cells on the basis of expression of the classical endothelial markers vWF,CD31,and CD34 as shown by immunofluorescence examination.In addition,the cells were negative when stained with antibodies recognizing the epithelial cell markers(keratin 8 and 6A),hematopoietic cells(CD14 and CD45),and smooth muscle cell actin.Microarray analyses revealed distinct gene expression pattems and consistent up-regulation of certain endometrial endothelial marker genes across patient samples. 317 genes that exhibited≥2-fold differences,including 191 up-regulated genes and 126 down-regulated one,were identified in tumor-associated HEECs.Several proteins involved in extracellular matrix function,such as MMP10,MMP9,LAMB1,SPP1, LOX,and LGALS3BP,had increased expression in tumor vasculature.Several proteins responsible for actin cytoskeleton organization and regulation,such as CAPG, CAPZA1 and TMSB10,were also up-regulated.MAPRE1,CDC25B and CCNB2, genes correlated with regulation of cell cycle,were also elevated in tumor endothelium.There were 25 genes with≥5-fold decrease in tumor endothelium, including several genes with potential antiangiogenic or antiproliferative roles,such as Cyclin-dependent kinase inhibitor 2A(CDKN2A) and Solute cartier family 29 member 2(SLC29A2),nerve growth factor receptor associated protein 1(NGFRAP1), apoptosis-associated speck-like protein containing a CARD(PYCARD),and cellular adhesion,molecule Catenin beta 1(CTNNB1).Pathway analysis showed that pathways of Cell cycle,Cell adhesion molecules (CAMs),focal adhesion,and extracellular matrix(ECM)-receptor interaction were obviously predominant.The results of the microarray analysis were confirmed by quantitative real-time PCR,immunohistochemistry,and/or western blotting. After cells were cultured for 24,48,72 and 96 h,there was no significant difference in proliferation rate of tumor-associated HEECs and normal HEECs (P=0.173).Moreover,although the tumor-associated HEECs didn't show faster proliferation than normal HEECs,they exhibited enhanced migration ability (P=0.006),potent invasiveness(P=0.033),and elevated tube formation in vitro (P=0.026).CONCLUSION:HEECs can be efficiently isolated from endometrial cancer and normal endometrial tissues by immunomagnetic methods and these cells were verified for angiogenesis research in endometrial cancer.The present study shows that the tumor-associated HEECs exhibited enhanced migration ability,potent invasiveness,and elevated tube formation in vitro.Microarray analysis show that tumor and normal endothelium differ at molecular level,and additional characterization of this gene expression database will provide insights into the angiogenesis of endometrial cancers and might be of great benefit for finding potential therapeutic targets.