ABSTRACT
CXCL3 belongs to the CXC-type chemokine family and is known to play a multifaceted role in various human malignancies. While its clinical significance and mechanisms of action in uterine cervical cancer (UCC) remain unclear. This investigation demonstrated that the UCC cell line HeLa expressed CXCL3, and strong expression of CXCL3 was detected in UCC tissues relative to nontumor tissues. In addition, CXCL3 expression was strongly correlated with CXCL5 expression in UCC tissues. In vitro, HeLa cells overexpressing CXCL3, HeLa cells treated with exogenous CXCL3 or treated with conditioned medium from WPMY cells overexpressing CXCL3, exhibited enhanced proliferation and migration activities. In agreement with these findings, CXCL3 overexpression was also associated with the generation of HeLa cell tumor xenografts in athymic nude mice. Subsequent mechanistic studies demonstrated that CXCL3 overexpressing influenced the expression of extracellular signal-regulated kinase (ERK) signaling pathway associated genes, including ERK1/2, Bcl-2, and Bax, whereas the CXCL3-induced proliferation and migration effects were attenuated by exogenous administration of the ERK1/2 blocker PD98059. The data of the current investigation support that CXCL3 appears to hold promise as a potential tumor marker and interference target for UCC.
Subject(s)
Chemokines, CXC/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Uterine Cervical Neoplasms/enzymology , Adult , Aged , Animals , Apoptosis , Cell Movement , Cell Proliferation , Chemokine CXCL5/genetics , Chemokine CXCL5/metabolism , Chemokines, CXC/genetics , Female , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Mice, Nude , Middle Aged , Neoplasm Invasiveness , Paracrine Communication , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction , Up-Regulation , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
PURPOSE: CXCL3 and its receptor CXCR2 were considered to play particularly important roles in the progression of malignancies. However, the investigations about CXCL3/CXCR2 axis in prostate cancer have been poorly involved. Herein we firstly reported our studies on the expression and biological roles of CXCL3 and CXCR2 in prostate cancer. METHODS: Expression levels of CXCL3 and CXCR2 in prostate cancer cell lines (PC-3, DU145 and LNCaP), immortalized prostate stromal cell line (WPMY-1) and immortalized prostate epithelial cell line (RWPE-1) were investigated by RT-PCR, ELISA and western blot, whereas expression levels of CXCL3 in a prostate tissue microarray were detected by immunohistochemistry. Cell counting kit-8 and transwell assays were, respectively, utilized to determine the effects of exogenous CXCL3 on the cell proliferation and migration. We further examined whether CXCL3 could regulate the expression of genes correlated with prostate tumorigenesis by RT- PCR. RESULTS: Elevated expression of CXCR2 was detected in DU145, LNCaP and RWPE-1. Moreover, high-level CXCL3 can be secreted by PC-3 and RWPE-1, and CXCL3 protein expression level in tissue microarray is concordant with prostate cancer metastasis. Exogenous CXCL3 does not contribute to proliferation, but has a significant effect on migration of prostate cancer cells and RWPE-1. Finally, our data showed that exogenous CXCL3 can regulate the expression of genes including ERK, TP73, NUMB, BAX and NDRG3. CONCLUSION: Our findings suggest that CXCL3 and its receptor CXCR2 are overexpressed in prostate cancer cells, prostate epithelial cells and prostate cancer tissues, which may play multiple roles in prostate cancer progression and metastasis.
Subject(s)
Chemokines, CXC/genetics , Gene Expression Regulation, Neoplastic/drug effects , Prostatic Neoplasms/genetics , RNA, Messenger/metabolism , Receptors, Interleukin-8B/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Chemokines, CXC/metabolism , Chemokines, CXC/pharmacology , Epithelial Cells/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Prostate/cytology , Prostate/metabolism , Prostatic Neoplasms/metabolism , Receptors, Interleukin-8B/metabolism , Stromal Cells/metabolism , Tumor Protein p73/genetics , bcl-2-Associated X Protein/geneticsABSTRACT
Water chestnut is one of the most popular vegetables in Asian countries that grows in shallow water. Eighteen water chestnut samples were collected from Lake Tai and six samples were bought at markets in Wuxi, China, in October 2007. Extraction solution of water chestnut was cleaned up with a solid phase extraction column and immunoaffinity chromatography cartridges, then the microcystin (MC) level was detected by indirect competitive enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). The results of ELISA showed that there were six samples collected from Lake Tai which contained MCs; the highest level of total MCs was 7.02 ng/g. The results of LC-MS confirmed that MC-LR and MC-RR were present in five samples. The highest level of MC-LR was 1.02 ng/g and that of MC-RR was 4.44 ng/g. Heavy cyanobacterial blooms had occurred, and MCs were detected in water at the points in Lake Tai where MCs occurred in water chestnuts collected in 2007. MCs were not detected in the six samples bought at Wuxi markets. The results suggest that MCs can accumulate in water chestnuts, which is a potential hazard for human health.
Subject(s)
Cyanobacteria/chemistry , Eleocharis/chemistry , Eutrophication , Microcystins/analysis , Antibodies, Monoclonal/chemistry , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Mass Spectrometry , Plant Extracts/analysisABSTRACT
Microcystin-LR (MC-LR) is a heptapeptide hepatotoxin produced by cyanobacteria. Immunoaffinity chromatography (IAC) column was prepared with CNBr-activated Sepharose 4B and monoclonal antibody of MC-LR. Water sample was cleaned up by IAC column and the content of MC-LR in water was determined by liquid chromatography-mass spectrometry (LC-MS). The results suggested that the IAC column exhibited highly selective specificity for MC-LR and selective removed interference from complex water sample. Water sample was concentrated for 2,000-fold through once purification. Cyanobacterial blooms had broken out in 2007 in Lake Tai, the third largest freshwater lake in China. Water samples from two parts of Lake Tai had been analyzed. The highest concentration of MC-LR in water from Lake Tai was 0.522 microg/L.
Subject(s)
Microcystins/analysis , Antibodies, Monoclonal/immunology , China , Chromatography, Liquid , Cyanobacteria/growth & development , Marine Toxins , Mass Spectrometry , Water MicrobiologyABSTRACT
OBJECTIVE: Production of the gold labeled monoclonal antibody against MC-LR. Study the simple mechanism of the conjugation by the spectral analysis. METHODS: Production and purification monoclonal antibodies against MC-LR by hybridoma technology. Production the 20nm colloidal gold and label the monoclonal antibody. Study the gold--McAb conjugate by the spectroanalysis. RESULTS: Monoclonal antibody produced by the hybridoma cells were tested for subtypes and designated as IgG2a for 8C11. The titers of antibody in ascites was 108.The IC50 for MC-LR was around 3ng/ml. The IgG molecule weight was 1.5 x 10(5). The structure of the antibody was altered to more tighter and stabler. CONCLUSION: Product the gold labeled monoclonalantibody against MC-LR conjugate successfully and keep the antige-binding determinant.
Subject(s)
Antibodies, Monoclonal , Gold Colloid , Microcystins/analysis , Microcystins/immunology , Reagent Strips , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Hybridomas , Immunoassay/methods , Marine Toxins , Mice , Mice, Inbred BALB CABSTRACT
OBJECTIVE: Production and characterization the polyantibodies against MCYST-LR. METHODS: microcystin-LR (MC-LR) was conjugated to KLH, then immune the rabbit by the routine method. Purify the antiserum by centrifugal and saturated sulfuric ammonium precipitated methods. Count the affinity constant and measure the titers, IC50 Value and the sensitivity. RESULTS: The titers of the antiserum is over 25600. IC50 value is about from 0.1 ng/ml to 1 ng/ml. The across-reactivity ratio to MC-LW and MC-LR is about from 0.1% to 20%.
Subject(s)
Antibodies, Bacterial/biosynthesis , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Microcystins/immunology , Animals , Antibodies, Bacterial/immunology , Antibodies, Bacterial/isolation & purification , Antibodies, Monoclonal/immunology , Male , Marine Toxins , RabbitsABSTRACT
OBJECTIVE: To prepare Microcystins-LR (MC-LR) conjugates with the carrier protein for developing a new immune assay for MC-LR. METHODS: MC-LR was coupled to BSA or KLH by "activated ester" and "water-soluble carbodimide". Measure the combine ratio of MC-LR with KLH or BSA by the UV scanning or the Bradford's method. RESULTS: The results are 9.1 and 9.4. New Zealand rabbits was immunized by MC-LR-KLH. The titer of the polyantibodies was over 25,600 measured by id ELISA. CONCLUSION: MC-LR-KLH was proved the suitable immunogen against MC-LR.
