| Treatment and prognosis is very poor for ovarian carcinoma which emerges secretly and examine late, so it is much more important to investigate positive etiology and mechanism of the disease.The balance of water is the key of live cell, and water transmembrane is important for maintaining normal metabolism. Aquaporins (AQPs) are a family of homologous water channels expressed in many epithelial and endothelial cell types, which selectively allow the passage of water through the plasma membrane, take part in secretion and absorption of water. Following ovarian cancer progresses, the peritoneal cavity of patients frequently accumulates malignant ascites, which the reason is unclear. There is no related report until now.AQP1 protein is not only widespread expressed in most secreted and absorbing epithelia, but abundant in endothelial cells of microvessel. Previous study showed that AQP1 expression was contributed to water vascular permeability. AQP5 distribute mainly in apical aspect of the plasma membrane of the salivary gland, respiratory and alimentary tract, which was thought to play a fundamental role in the secretions of gland. AQP1 gene deletion in mouse could reduce alveolar-capillary water exchange, and water permeability of microvessel decreased 10-fold. AQP5 gene deletion could reduce 90% lung water transport depending on osmotic pressure. Recent report had shown that the over-expression of AQPs was contributed to development of colon cancer and brain edema resulting from glial tumors.NF-κB is a nuclear factor of transcription, and distributed widely in various cells. It combines DNA of related gene promoter or enhancer and regulates multiple gene expression, which devoted to carcinogenesis, invasion and metastasis. The previous study showed that NF-κB in ovarian carcinoma was activated. Whether the AQPs expression regulated by NF-κB or not is unclear by now.The present study aims at investigating the role of aquaporin-1 and aquaporin-5 in the epithelial ovarian carcinoma and its mechanism of regulation. Cells cultures, immunohistochemical technique, Western blotting and RT-PCR are used in this study.The study was divided into two parts: 1. the expression of AQPl and AQP5 in epithelial ovarian tumors and its significance;2. the regulation mechanism of AQP5 protein.Part one The expression of AQP-1 and AQP-5 in epithelial ovariancarcinoma and its significanceMethods: The expression of AQPl and AQP5 protein and mRNA in 65 cases epithelial ovarian tumors and 13 cases normal tissue were measured by immunohistochemical technique, Western blotting and RT-PCR, respectively.Results: 1.Immunohistochemical study showed that AQPl protein was strongly expressed in the membrane of microvessels and small vessels, AQP5 protein is mainly localized in the basolateral membranes of benign tumor cells, the apical and basolateral membrane of borderline cells, and scattered in the membrane of malignant cells, and almost no or weak staining in normal ovarian epithelium;2.The protein and mRNA expression of AQPl and AQP5 in ovarian malignant and borderline tumors were significantly higher than that of benign tumors (P<0.05) and normal tissue (P<0.05). There are no difference between ovarian carcinoma and borderline tumors, benign tumors and normal tissues (P>0.05);3. Of all the epithelial ovarian malignant tumors, The protein and mRNA expression of AQPl and AQP5 in cases with ascites volume more than 1000ml was higher than that of ascites volume less than 500ml (P<0.05). Increased AQP5 protein level was associated with lymph node metastasis (P<0.05);4. There is a significant relationship between AQPl protein and mRNA expression and ascites volume (r=0.423, 0.429;P < 0.05, P < 0.05). There is a significant relationship between AQP5 protein and mRNA expression and ascites volume (r=0.441, 0.427\ P<0.01, P<0.05) as well as lymph node metastasis (r=0.410, 0.361;P<0.05, P<0.05) .Part two The regulation mechanism of AQP-5 protein in SKOV3Methods: The effect of ovarian carcinoma ascites, Cisplatinum and PDTC on the protein expression of AQP5, NF-kB p65 and IkBo: in SKOV3 was measured by cells culture, MTT, immunohistochemical technique and Western blotting, respectively.1. The effect of ovarian carcinoma ascites on the expression of AQP5 , NF-kB p65 and IkBcc protein in SKOV3Results: 1. The growth rate of SKOV3 in the malignant group is significant higher than the borderline group (P<0.001) , the borderline group higher than the benign group(P<0.001) and the control group (P<0.01);2. The expression NF-kB p65 karyon /cytoplasm protein rate and NF-kB p65/lKBot protein rate of SKOV3 cytoplasm in the malignant group is significant higher than the borderline group, the benign group and the control group (P<0.01), but there are no difference among the borderline group, the benign group and the control group (P>0.05). 3. The expression AQP5 protein in the malignant group is significant higher than the benign group and the control group(P<0.05), the borderline group higher than the benign group (P<0.01);4. There is a positive correlation between the growth rate of SKOV3 and AQP5 protein (r =0.926, P<0.001) as well as NF-kB p65 karyon/cytoplasm protein rate(r=0.878,P<0.001), and NF-kB p65/lKBa protein rate (r=0.839, P<0.01) . There is a positive correlation between AQP5 protein and NF-kB p65 karyon/cytoplasm protein rate (r=0.658, P<0.05) as well as NF-kB p65/hcBa protein rate (r=0.622, P<0.05) . There is a positive correlation between NF-kB p65 protein and IkBci protein in SKOV3 cytoplasm(r=0.839, P<0.01) .2. The effect of Cisplatinum on the expression of AQP5 , NF-kB p65 and IkBgc protein in SKOV3Results: 1. Following the concentration of Cisplatinum and culture time increasing, the inhibiting rate of SK0V3 is increasing gradually (P<0.001), and there is a time-dependent and dose-dependent manner. The shape of SKOV3 cells become brim and round, and separated from other cells;2. Following the concentration of Cisplatinum and culture time increasing, the expression AQP5 protein in the SKOV3 cytoplasm is decreasing (P<0.001), and AQP5 protein expresses in the karyon membrane of SKOV3;3. Following the concentration of Cisplatinum and culture time increasing, the expression NF-kB p65 and IkBoi protein in SKOV3 cytoplasm and karyon as well as p65 karyon/cytoplasm protein rate is decreasing gradually (P<0.001);4. There is a negative correlation between the inhibiting rate of SKOV3 and AQP5 protein (r=-0.598, P<0.01) , and NF-kB p65 protein in the SKOV3 cytoplasm (r=-0.835, P<0.001) and karyon (r=-0.710, P<0.01) , and NF-kB p65 karyon/ cytoplasm protein rate (r=-0.815, P<0.001) . There is a positive correlation between AQP5 protein and NF-kB p65 karyon/cytoplasm protein rate, as well as NF-kB p65 and IkBcx protein in SKOV3 cytoplasm (r=0.883, 0.894, 0.857;P<0.001, 0.001, 0.001), There is a positive correlation between NF-kB p65 protein and IkBoi protein in SK.OV3 cytoplasm (r=0.934;P<0.001);.3. The effect of PDTC on the expression of AQP5 , NF-kB p65 and IkBoc protein in SKOV3Results: Results: 1. Following the concentration of PDTC and culture time increasing,the inhibiting rate of SKOV3 is increasing gradually (P<0.001), and there is a time-dependent and dose-dependent manner;2. Following the concentration of PDTC and culture time increasing, the expression AQP5 protein in the SKOV3 cytoplasm is decreasing (P<0.001);3. Following the concentration of PDTC and culture time increasing, The expression NF-kB p65 and IicBa protein in SKOV3 cytoplasm and karyon as well as NF-kB p65 karyon/cytoplasm protein rate is decreasing gradually (P<0.001);4. There is a negative correlation between the inhibiting rate of SKOV3 and AQP5 protein (r=-0.983, P<0.001), and NF-kB p65 cytoplasm protein as well as karyon protein (r=-0.716, -0.744;P<0.01,0.01). There is a positive correlation between AQP5 protein and NF-kB p65 protein in the cytoplasm and karyon of SK.OV3 as well as iKBa protein in the cytoplasm (r=0.668, 0.625, 0.675;P<0.05, 0.05, 0.05).Conclusion:1. AQP1 and AQP5 protein distribute in the different cells. The former localizes in endothelia of microvessel and small vessels and the latter in ovarian tumor cells, which suggest the AQP1 and AQP5 play a role in different cells.2. High AQP1 and AQP5 expression may play an important role in the ovarian carcinogenesis, progression and prognosis.3. AQP1 and AQP5 protein over-expression in ovarian epithelial carcinoma may be a main cause of ascites formation.4. Ascites of ovarian epithelial carcinoma activates NF-kB activity, up-regulates AQP5 protein and promote SKOV3 cells growth.5. Cisplatinum may activate NF-kB in the early period, which suggests that the dose and time of drug shoud be paid an attention, and reduce drug-resistance.6. Cisplatinum inhibits NF-kB activity in the later period, down-regulates AQP5 protein, and inhibits SKOV3 cells growth.7. PDTC down-regulates NF-kB p65 protein and IkB |
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