癌性腹水激活核因子刺激卵巢癌SKOV3细胞增生

　　1 Introduction
　　
　　Ovarian carcinoma is the most lethal gynecological malignancy. Due to the lack of reliabletumor markers and vague or absent symptoms in the early stages of the disease, 70% of patientsinitially present with advanced disease. Ovarian carcinoma is characterized by rapid growth ofsolid tumors and production of large volumes of ascites. A progressive relationship between stageof malignancy and incidence as well as volume of ascites was also observed and rapidlyaccumulating ascitic fluid is associated with poor prognosis . Malignant ascitic fluid has beenshown to contain a number of physiologically active soluble constituents including molecules suchas growth factors that can potentially promote tumor growth and metastasis formation, therebyaccelerating disease progression. However, its molecule mechanisms are not yet fully understood.
　　The NF-κB/Rel signal transduction pathway operates at the center of a variety of differentsignaling events in the living cell. Nuclear factor kappaB (NF-κB) is an inducible dimerictranscription factor that belongs to the Rel/NF-κB family of transcription factors, the prototype ofwhich in most non lymphoid cells is a heterodimer composed of the RelA (p65) and NF-κB (p50)subunits. NF-κB normally resides in the cytoplasm as an inactivated form in a complex withinhibitor of NF-κB (IκBa). Phosphorylation of IκBa by IKK promotes its degradation, allowingNF-κB to translocate to the nucleus and induce target genes. The transcriptional activation ofgenes associated with cell proliferation, angiogenesis, metastasis, and suppression of apoptosis [1, 2,3, 4, 5]
　　In light of our observation and reported findings, we hypothesized that NF-κB may play animportant role in the malignant ascites promoting ovarian carcinoma growth and metastasisformation. Thus, we investigated the effect of malignant ascites on NF-κB p65 and IκBa proteinof human ovarian carcinoma (SKOV-3) cells. We found the malignant ascites may activate NF-κBand promote proliferation of SKOV-3 cells.
　　
　　2 Materials and Methods
　　
　　2.1 Malignant ascites and cystic fluid
　　Malignant ascites and cystic fluid were collected from patients with primary mucous ovariancarcinoma (malignant ascites), ovarian benign mucous tumor (benign fluid) and borderlinemucous tumors (borderline fluid), respectively. Those patients underwent surgical resection of theovaries at Womens Hospital of Zhejiang University, Hangzhou, China. All patients signedinformed consent letters, and methods for experimenting were approved by the local InstitutionalReview Boards. Ascites and cystic fluid were centrifuged at 3000 rpm for 15 min at the time ofcollection to remove debris, including blood cells. The samples were pooled and filtered through asterile 0.22-Am cellulose acetate filter and stored at -70°C before use. During the collection andhandling of ascites and fluid in this study, samples that clearly contained blood were discarded.
　　
　　2.2 Cell culture
　　The human ovarian carcinoma (SKOV-3) cells were obtained from American Type CultureCollection (ATCC, Manassas, VA). SKOV3 cells were cultured in RPMI 1640 medium (GiBCO)supplemented with 15% fetal bovine serum and maintained at 37°C and 5% CO2 in a humidenvironment. SKOV-3 cells seeded at 2×103 cells per 5-cm plate were incubated in medium. Cellswere grown to confluence and then washed with phosphate-buffered saline (PBS) prior to 48hincubation with one of the following solutions: (1) RPMI 1640 medium alone (the control group),(2) benign cystic fluid (the benign group), (3) borderline cystic fluid (the borderline group), (4)malignant ascites (the malignant group). Cell growth was determined by counting the number ofcells with coulter counter and expressed with the following formula:
　　Growth rate (%) = cell number (experiment) /cell number (control) ×100
　　
　　2.3 Immunocytochemistry
　　The localization of NF-κB p65 and IκBa in SKOV-3 was examined by immun ocytochemistry. Cells were grown on a chamber culture slide. After incubated with ascites or fluid, thecells were washed twice with cold PBS (10 mM, pH 7.4). The endogenous peroxidase wasblocked by 10% hydrogen peroxidase and nonspecific binding reduced by normal non-immuneserum. they were incubated at 4°C overnight with primary antibody (Santa Cruz Biotechnology,Santa Cruz, CA, USA; 1:50 dilution), and then biotin-conjugeted secondary antibody andStreptavidin-peroxidase at 37°C for 30 min, respectively (UltraSensitiveTM kits, ZymedLaboratories Inc., South San Francisco, CA). The reaction visualization was made with DAB, andthe slides counterstained with hematoxylin.
　　
　　2.4 Western blotting analysis
　　Following treatment, the cells were washed twice with cold phosphate-buffered saline (PBS,Ph7.2) and scraped. Cytosol and nuclear protein were extracted using Nuclear/CytosolFractionation Kit (Pierce Biotechnology, USA). Protein concentrations were measured by theBio-Rad protein test kit (Bio-Rad Laboratories Inc., Hercules, CA). Equal volume of protein(50μg) was subjected to 12% SDS-polyacrylamide gel eletrophoresis (SDS-PAGE) and then theproteins were transferred onto nitrocellulose membranes. After 1h blocking with 5% non-fat milkpowder in Tween-20 TBS (TBST) at room temperature, the membranes were incubated with1:400 dilution of NF-κB p65 and IκBa and 1:1000 dilution of β-actin primary antibody (SantaCruz) at 4°C overnight. After being washed three times for 10 min each with TBST, Themembranes were incubated 1h at room temperature with 1:5000 dilution of the second antibodylabeled horseradish peroxidase (HRP) (Santa