The Study Of Interleukin-18, ICE Expression And Its Significance In Epithelial Ovarian Cancer

Objective :Ovarian cancer is one of the most common malignant tumor in the Gynecology tumor .The mortality of ovarian cancer is the highest among all the gynecologic tumors because of its indistinct clinical symptoms .And we can not make a diagnosis during the early stage .So the patients with ovarian cancer are poor prognosis .The occurrence of cancer is the result of complex biologic factors .The research about mechanism of tumor genesis will be known more at the molecular level .The malignant tumor is the disease caused by the polygenic abnormality .Ovarian cancer is regulated and controlled by the polygene too. IL-18 is the gene that was found recently year .It is important immune regulation factor, it has the function of inducing the generation of IFN-γand enforcing the activity of NK cell, enforcing Th1 type immune response .And it has very distinct function of anti-tumor .Study have approved the breast cancer cell which had been transfected into IL-18 was planted the hypodermic of naked –mice and they had not formed the tumor .Study indicate it is necessary that the homeostasis of the apoptosis and proliferation of cells. It is important for the multiple cell organism keeping its stable structure and function balance .IL-1β-converting enzyme (ICE ) has the important function during the cell apoptosis .The decrease of its expression is closely related to normal cell converting to the malignant cell. In our research ,we detect the expression and correlation of IL-18 and ICE in epithelial ovarian carcinoma at the transcript and protein level .As well as the relationship among the expression of IL-18 ,ICE and histological type ,stage ,age of ovarian cancer .we have investigated the function of IL-18 and ICE in the generation and development of ovarian cancer and offered experiment basis to immune gene therapy of ovarian cancer with IL-18 and ICE. Methods :All the specimens were recruited from the patients with ovarian cancer who were treated first in the gynecology and obstetrics department of the Fourth Affiliated Hospital of Hebei Medical University from February 2003 to April 2004. All the epithelial ovarian cancer were confirmed by pathologic observation . 32 epithelial ovarian cancer ,10 normal ovarian tissues and 6 benign ovarian tissue were recruited. All the tissue specimens were snap-frozen in liquid nitrogen and transfered to -80℃freezer until analysis .And another tissue specimens were taken and fixed by the 10% formalin ,embedded in paraffin ,cut for use .1. RT-PCR to examine the expression of IL-18,ICE gene ,Total RNA of ovarian cancer tissues and ovarian cancer cell lines were distilled .The RNA yield and purity were measured by the spectrophotometric determination at 260nm and 280nm .And it was visualized on formaldehyde denaturing agarose gel . 2ug RNA was subjected to RT-PCR .β-actin was the inner parameter . Products of RT-PCR wereelectrophoresed in 1.5% agarose .Then, IL-18 and ICE were evaluated by a Gel-analyzer software package respectively . 2. The protein expression of IL-18 and ICE were detected by the method of immunohistochemistry and immune cell chemistry . Tissue specimens were cut , Dewaxed in water routinely , endogenous peroxidase activity was removed using 3%H2O2 carbinol at room temperature for 15 minutes .The antigens were repaired by the hot citrate solution .The nonspecific antigen were blocked with normal serum .Then the section were incubated for one night at 4 ℃after were instilled 1:200 rabbit anti human IL-18 ,1:50 rabbit anti human ICE ;Ⅱantibody and Ⅲantibody were instilled ; The compound was visualized using DAB .PBS was substituted for the antibody as a negative control .3. Statistical method :statistic analysis was performed with t test ,chi-square test ,and linear regression and correlation .Differences were considered to be statistically significant when the probability value was less than 0.05. Results :1.The mRNA expression of IL-18,ICE in epithelial ovarian cancer ,normal ovarian tissue and benign ovarian tumor :IL-18 mRNA expressed in all the tissues .Its IL-18 mRNA expression level was 0.7746±0.3524 ,1.3015±0.4778 , 1.0157±0.1676 in epithelial ovarian cancer ,normal ovarian tissue and benign ovarian tumor respectively ;ICE mRNA expressed in 29 of 32 epithelial ovarian cancer tissues , expressed in all the normal ovarian tissues and benign ovarian tumor tissues . ICE mRNAexpression level was 0.6341±0.3420,0.9614±0.5141, 0.7349±0.3429.IL-18 and ICE mRNA expression level decreased in epithelial ovarian cancer than in normal ovarian tissues . IL-18 and ICE mRNA expression level in benign ovarian tumor tissues had no significantly differences with normal ovarian and epithelial ovarian cancer. IL-18 and ICE mRNA expression level was not related to age ,stage and pathology type .2. IL-18 mRNA expression was positive in human ovarian cancer cell lines SKOV3 ,3AO .While ICE mRNA expression was negative .3.The correlativity of IL-18 and ICE at transcript level :they were in a positive correlation ,correlation coefficient r was 0.372(P=0.036).4. Immunohistochemistry analysis of IL-18 in ovarian cancer and human ovarian cancer cell lines SKOV3 ,3AO:The staining of IL-18 was cytoplasm .Its positive rate was 31.3%,83.3%,80% in ovarian cancer tissues, benign ovarian tumor tissues and normal ovarian tissues respectively .The protein expression rate of IL-18 in epithelial ovarian cancer decreased significantly than in benign ovarian tumor and normal ovarian tissues .The differences was significant ;It had no difference between benign ovarian tumor and normal ovarian tissues(P=1.0). The protein expression of IL-18 had no significant difference in age ,stage and pathological type of epithelial ovarian cancer(P>0.05) . Human ovarian cancer cell lines SKOV3 ,3AO had the protein expression of IL-18,The staining was in the cytoplasm of cancer cell.5. The protein expression of ICE in ovarian cancer tissuesand ovarian cancer cell lines SKOV3 ,3AO :The staining of ICE was cytoplasmic .The expression rate of ICE was 37.5%,50%,80% in epithelial ovarian cancer ,benign ovarian tumor and normal ovarian tissues respectively . The protein expression of ICE decreased distinctively in ovarian cancer than normal ovarian tissues (P =0.047);It had no significant differences between benign ovarian tumor and normal ovarian tissues (P=0.299);And it also had no differences between benign and malignant ovarian tumor (P=0.633).The protein expression of ICE had no significant difference in age ,stage and pathology type of epithelial ovarian cancer (P>0.05). The protein expression of ICE was negative in human ovarian cancer cell lines SKOV3 ,3AO. 6.The correlativity of IL-18 and ICE at protein level: Their expression were in a positive correlation in ovarian cancer tissues. Correlative coefficient r =0.453(P=0.009) Conclusions : 1.The mRNA expression level of IL-18,ICE in epithelial ovarian cancer decreased significantly than normal ovarian tissue .They had no relation to age ,pathology type and stage of patients with ovarian cancer .Human ovarian cancer cell lines SKOV3 ,3AO had mRNA expression of IL-18,no mRNA expression of ICE .2.The protein expression of IL-18,ICE in epithelial ovarian cancer decreased significantly than normal ovarian tissues . The protein expression had no relation to age ,pathology type and stage of patients with ovarian cancer .But their protein expression had the decrease tendency in stage Ⅲ, Ⅳthan in stage Ⅰ, Ⅱof ovarian cancer .Human...