Interleukin-2 cDNA Research Articles

Molecular characterization and expression profiles of two interleukin genes IL-8 and IL-10 in Pacific cod (Gadus macrocephalus)

During the early development of the Pacific cod, high mortality due to viral infection is observed. To demonstrate the immune responses activated in Pacific cod during this critical period, two cytokines, the proinflammatory cytokine interleukin-8 (IL-8) and the anti-inflammatory cytokine interleukin-10 (IL-10), were identified and characterized in this study. The cDNA of IL-8 was 1047 bp in length, containing an ORF of 297 bp that encoded a 98 aa protein, whereas the IL-10 cDNA sequence consisted of 856 bp that harbored a 537 bp ORF encoding a 178 aa protein. Pairwise comparison and phylogenetic analysis of the two genes clearly demonstrated their close relationships with their counterparts in the Atlantic cod. Constitutive expression was detected in eight tested tissues; the transcription of both genes was observed during the critical period of the early developmental stage in Pacific cod, and their expression levels dramatically increased following poly I:C stimulation of larvae at 12 days post-hatching (dph). Taken together, these results suggested that IL-8 and IL-10 may be involved in the inflammatory responses of Pacific cod.

Molecular Cloning and Expression Analysis of Interleukin-8 and -10 in Yellow Catfish and in Response to Bacterial Pathogen Infection.

The yellow catfish (Pelteobagrus fulvidraco) is an important economic freshwater aquaculture species in Asia. However, little is known about its immune response to bacterial pathogen infection. Here, two cytokines, the proinflammatory cytokine interleukin-8 (IL-8) and the anti-inflammatory cytokine interleukin-10 (IL-10), were identified and characterized in the yellow catfish for the first time. We found that the full length of the IL-8 cDNA was 784 bp and contained an open reading frame (ORF) of 336 bp, while the IL-10 gene was 973 bp in length with a 549 bp of ORF. In addition, both the IL-8 and the IL-10 had similar tissue-specific expression patterns. They were more abundant in the spleen and lowest expressed in the liver. Furthermore, IL-10 but not IL-8 was significantly upregulated in the intestine of yellow catfish by feed supplementation of Clostridium butyricum (CB). More importantly, the expression levels of intestinal IL-10 and IL-8 were up- and downregulated by pathogen Aeromonas punctata stimuli with the presence of CB, respectively. Collectively, these results suggest that IL-10 and IL-8 mediate important roles in the immunity of yellow catfish, and feed supplementation of CB may able to reduce the intestinal inflammation caused by bacteria infections through regulating the expression of IL-10 and IL-8.

Protective efficacy of an IL-12-expressing baculoviral malaria vaccine.

Interleukin-12 (IL-12) plays an important role in antigen-specific adaptive immunity against Plasmodium sporozoites, and this requirement allows for a new approach to developing an effective malaria vaccine. In this study, we examined whether IL-12 could enhance protective efficacy of a baculovirus-based malaria vaccine. For this aim, a baculoviral vector expressing murine IL-12 (mIL-12) under the control of CMV promoter (BES-mIL-12-Spider) and a baculoviral vector expressing Plasmodium falciparum circumsporozoite protein (PfCSP) with post-transcriptional regulatory element of woodchuck hepatitis virus (BDES-sPfCSP2-WPRE-Spider) were generated. BES-mIL-12-Spider produced bioactive IL-12 which activates splenocytes, resulting in induction of IFN-γ. When co-immunized with BES-mIL-12-Spider and BDES-sPfCSP2-WPRE-Spider, the mouse number for high IgG2a/IgG1 ratios and the geometric mean in this group were both increased as compared with those of the other groups, indicating a shift towards a Th1-type response following immunization with BES-mIL-12-Spider. Finally, immunization with BDES-sPfCSP2-WPRE-Spider plus BES-mIL-12-Spider had a higher protective efficacy (73%) than immunization with BDES-sPfCSP2-WPRE-Spider alone (30%) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. These results suggest that co-administration of IL-12 expressing baculoviral vector, instead of IL-12 cDNA, with viral-vectored vaccines provides a new feasible vaccine platform to enhance Th1-type cellular immune responses against Plasmodium parasites.

Preclinical validation: LV/IL-12 transduction of patient leukemia cells for immunotherapy of AML

Interleukin-12 (IL-12) is a potent cytokine that may be harnessed to treat cancer. To date, nearly 100 IL-12-based clinical trials have been initiated worldwide. Yet systemic administration of IL-12 is toxic. Different strategies are being developed to reduce such toxicities by restricting IL-12 distribution. Our previous studies employed lentivector-mediated expression of murine IL-12 in tumor cells and demonstrated effective protection in both mouse leukemia and solid tumor challenge models. In this study, we carried out preclinical validation studies using a novel lentivector to engineer expression of human IL-12 in acute myeloid leukemia blast cells isolated from 21 patients. Acute myeloid leukemia cells were transduced with a bicistronic lentivector that encodes the human IL-12 cDNA as a fusion, as well as a LNGFR (ΔLNGFR)/mutant thymidylate kinase cassette as a marking and cell-fate control element. A range of 20–70% functional transduction efficiencies was achieved. Transduced acute myeloid leukemia cells produced bioactive IL-12 protein and displayed dose-dependent sensitivity to the prodrug 3′-azido-3′-deoxythymidine. In vitro immortalization assays using transduced mouse hematopoietic stem cells demonstrated minimal genotoxic risk from our IL-12 vector. Scale-up transduction and cell processing was subsequently validated in a GMP facility to support our (now approved) Clinical Trial Application (CTA).

Characterization of buffalo interleukin 8 (IL-8) and its expression in endometritis.

River buffalo (Bubalus bubalis bubalis) with a population over 135 million heads is an important livestock. Interleukin 8 (IL-8) is a member of the chemokine family and is an important chemoattractant for neutrophils associated with a wide variety of inflammatory diseases such as endometritis. Tissue samples from the mammary gland, uterus and ovary were obtained from river buffalo (Mediterranean type) with and without endometritis. Bacteriological examination showed the presence of both gram positive and negative in all buffalo with endometritis. RNA extraction and complementary DNA (cDNA) synthesis were conducted from all tissues. Specific primer for IL8 full coding regions was designed using known cDNA sequences of Bubalus bubalis, Genbank accession number AY952930.1. IL-8 gene expression was investigated in buffalo tissues. Expression of IL-8 in buffalo with endometritis was found to increase significantly over buffalo without endometritis only in the uterus (P = 0.0159). PCR products from uterus tissues (target organs) of buffalo with and without endometritis, were purified and sequenced. No polymorphic sites were detected in the investigated samples. IL-8 cDNA nucleotide sequences of buffalo with and without endometritis were 100% identical (accession number JX413057). Buffalo IL8 cDNAs were compared with corresponding sequences of member of subfamily Bovinae (buffalo and cattle) and subfamily Caprinae (sheep and goat). IL-8 species specific differences were identified.

Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3β signaling.

The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-γ and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3β protein in CRL1623 cells, whereas the selective GSK-3β inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC.

Matrix‐assisted refolding and purification of placenta‐derived recombinant human interleukin‐6 produced in Escherichia coli

Biological activity of human interleukin-6 (IL-6) is associated with a vast number of diseases such as rheumatoid arthritis, sepsis, and severe inflammatory diseases. In this study, human IL-6 cDNA was isolated from a cDNA library that was constructed with mRNA derived from human placental tissues. Subsequently, the complete human IL-6 cDNA was cloned and expressed in BL21DE3 cells. The recombinant human IL-6 (rhIL-6) protein was expressed in a form of an insoluble inclusion body. Inclusion bodies were solubilized under denaturing conditions and purified by immobilized metal affinity chromatography with gradual on-column refolding by the gradient elution method (from 6 to 0 M urea). The protein was purified to apparent homogeneity of about 99% with a yield of 50 mg/L. The purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion high-performance liquid chromatography, and Western blotting analysis. The bioactivity was assessed by proliferation assay of TF-1 cells in a dose-dependent manner. The present study confirms the expression of the placenta-derived IL-6 gene in a prokaryotic expression system and matrix-assisted on-column refolding and purification of rhIL-6 by immobilized metal affinity chromatography.

Abrogation of TNFα Production during Cancer Immunotherapy Is Crucial for Suppressing Side Effects Due to the Systemic Expression of IL-12

For more than a decade, the cytokine interleukin-12 (IL-12) has been utilized, either alone or in combination with other drugs, as a treatment for cancer. The numerous anti-tumor properties of IL-12 still generate interest in the clinical use of this cytokine, even though it has demonstrated toxicity when administrated systemically. As an approach to overcome this toxicity, numerous laboratories have attempted to induce IL-12 expression at the site of the tumor. However for tumors that are difficult to remove surgically or for the treatment of disseminated metastases, systemic expression of this cytokine still remains as the most efficient method of administration. Nevertheless, finding alternative approaches for the use of IL-12 in the treatment of cancer and unraveling the basis of IL-12-side effects remain a challenge. In the present work we demonstrate that systemic expression of IL-12 through hydrodynamic injection of IL-12 cDNA is able to induce different types of liver lesions associated with a toxic pathology. However we report here that hepatic toxicity is diminished and survival of mice enhanced in the absence of tumor necrosis factor alpha (TNFα). This observation is in contrast to several murine models and clinical trials that postulate interferon gamma (IFNγ) as the main cytokine responsible for IL-12 toxicity. Moreover, our work demonstrates that when IL-12 cDNA is co-injected with IL-18 cDNA or when mice are pre-treated with a low dose of IL-12 cDNA prior to receiving a high dose of IL-12 cDNA, systemic levels of TNFα are almost completely abrogated, resulting in improved survival and less hepatic damage. Importantly, abrogation of TNFα signaling does not affect the strong anti-tumor activity of IL-12. Thus, neutralizing TNFα with antagonists already approved for human use offers a promising approach to abrogate IL-12 side effects during the use of this cytokine for the treatment of cancer.

Effects of Interleukin-4 or Interleukin-10 gene therapy on trinitrobenzenesulfonic acid-induced murine colitis

BackgroundInflammatory bowel disease (IBD) is characterized by disturbance of pro-inflammatory cytokines and anti-inflammatory cytokines. Previous studies have demonstrated the effect of anti-inflammatory cytokines, such as interleukin-10 (IL-10) or IL-4 on IBD, but their data were controversial. This study further investigated the effect of IL-4 (IL-4), IL-10 and their combination on treatment of trinitrobenzenesulfonic acid (TNBS)-induced murine colitis.MethodspcDNA3.0 carrying murine IL-4 or IL-10 cDNA was encapsulated with LipofectAMINE 2000 and intraperitoneally injected into mice with TNBS-induced colitis. The levels of intestinal IL-4 and IL-10 mRNA were confirmed by quantitative-RT-PCR. Inflamed tissues were assessed by histology and expression of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-6.ResultsThe data confirmed that IL-4 or IL-10 over-expression was successfully induced in murine colon tissues after intraperitoneal injection. Injections of IL-4 or IL-10 significantly inhibited TNBS-induced colon tissue damage, disease activity index (DAI) and body weight loss compared to the control mice. Furthermore, expression of IFN-γ, TNF-α and IL-6 was markedly blocked by injections of IL-4 or IL-10 plasmid. However, there was less therapeutic effect in mice injected with the combination of IL-4 and IL-10.ConclusionsThese data suggest that intraperitoneal injection of IL-4 or IL-10 plasmid was a potential strategy in control of TNBS-induced murine colitis, but their combination had less effect.

Identification and expression profiles of IL-8 in bighead carp (Aristichthys nobilis) in response to microcystin-LR.

Microcystin-LR (MCLR) is a widespread cyanotoxin and has immunotoxicity to animals, including fish. Chemokines are considered to play important roles in inflammatory response induced by MCLR. In this study, we cloned the full-length cDNA of interleukin-8 (IL-8) from bighead carp (Aristichthys nobilis) for the first time. The full-length IL-8 cDNA was 552 bp and contained a 297-bp open-reading frame that encoded for a 98-amino acid protein. The deduced IL-8 protein had a typical aspartic acid (D)-leucine (L)-arginine (R) and a CXC motif at the N-terminal, which were conserved in most fish species. Phylogenetic analysis showed that bighead carp IL-8 protein was grouped in the teleost IL-8 lineage 2. Under normal conditions, the expression of IL-8 is constitutive and weak in all tested tissues. However, MCLR treatment could significantly increase the transcription of IL-8 in bighead carp in a temporal- and dose-dependent pattern. The present study will help us to understand more about the evolution of IL-8 and its function in the MCLR induced proinflammatory response in bighead carp.

Mosquitocidal Immune Response in BALB/c Mice Is Enhanced When Anopheles gambiae Mucin-1 cDNA Is Co-Administered with Interleukin-12 cDNA

The midgut of the malaria - transmitting mosquito, Anopheles gambiae, can be targeted by vaccine-induced host immune factors that kill the mosquito after it ingests immunized host blood. The An. gambiae mucin 1 protein (AgMuc1) is expressed on the mosquito midgut where it likely functions in protecting the midgut epithelium from its own secreted digestive enzymes, toxic substances and pathogenic microbes taken in with the blood meal. Immunization of mice with plasmid containing the AgMuc1 gene has been shown to induce mosquitocidal immune responses. In this paper, we co-immunized mice with AgMuc1 cDNA and plasmid containing Murine granulocyte-macrophage stimulating factor (GM-CSF) or Interleukin 12 (IL-12) cytokine cDNA in order to further potentiate the mosquitocidal immune response and better define the nature of this mosquitocidal immunity. While co-immunization with GM-CSF cDNA failed to increase anti-mosquito immunity (Chisq = 3.3 on 1 degree of freedom, p = 0.068), a significantly enhanced mosquitocidal effect was observed from mice co-immunized with AgMuc1 and IL-12 cDNA (Chisq = 39.1 on I degree of freedom, p = 4.06e-10). Furthermore, the cumulative survival of the blood fed mosquitoes surviving to day 7 in the AgMuc1/IL-12 co-immunized group highly correlated negatively with the anti-mucin IgG1 antibody subtype levels (Pearson correlation coefficient r = -0.782) suggesting that the mosquitocidal immunity induced by AgMuc1 cDNA immunization could be IgG1 antibody subtype mediated.

Immunopathological Features Developing in the Mosquito Midgut after Feeding on Anopheles gambiae Mucin-1 / Interleukin-12 cDNA Immunized Mice

El intestino medio es el organo al cual pasa la sangre consumida por el mosquito y donde, mediante una membrana peritrofica secretada por el epitelio, esta sangre es mantenida durante la digestion y absorcion. El intestino del mosquito esta revestido por microvellosidades llenas de actina que son expuestas a las complejas condiciones en torno a la luz intestinal, tales como la abrasion producida por particulas de alimentos, hidrolasas digestivas y el ataque de patogenos y parasitos que son tomados en la sangre consumida. Estas microvellosidades se protegen de estos efectos mediante la matriz peritrofica, el glicocalix y las proteinas de mucina que revisten las superficies epiteliales. La inmunizacion con AgMUC1/IL-12 ADNc en ratones BALB/c ha demostrado ser util para matar los mosquitos cuando se alimentan de estos ratones. La mucina es una de las proteinas producidas en el intestino medio del mosquito despues de consumir sangre. La fina estructura del epitelio del intestino interactua con factores inmunes tales como anticuerpos o celulas inmunes es de especial importancia para interpretar los eventos tempranos en la interaccion entre el revestimiento del intestino medio y los componentes inmunologicos especificos presentes en la sangre de ratones BALB/c inmunizados con AgMUC1/IL-12 cDNA. Despues de observar mediante microscopias de luz, electronica de barrido y de transmision las caracteristicas de secciones del intestino medio del mosquito Anopheles gambiae alimentado de ratones BALB/c inmunizados con AgMUC1/IL-12 cDNA, mecanismos inmunes mediados por citotoxicidad celular dependiente de anticuerpos (ADCC) podrian ser los responsables de matar a los mosquitos. Los procesos necroticos y apoptoticos que pueden ser la causa de la muerte del mosquito tienen lugar en las celulas que recubren el epitelio del intestino medio.

Scientific Scholarship and Impact Factors

Scientific Scholarship and Impact Factors

Analysis of the molecular characteristics and cloning of full-length coding sequence of Interleukin-2 in tree shrews

While the tree shrew (Tupaia belangeri chinensis) is an excellent animal model for studying the mechanisms of human diseases, but few studies examine interleukin-2 (IL-2), an important immune factor in disease model evaluation. In this study, a 465 bp of the full-length IL-2 cDNA encoding sequence was cloned from the RNA of tree shrew spleen lymphocytes, which were then cultivated and stimulated with ConA (concanavalin). Clustal W 2.0 was used to compare and analyze the sequence and molecular characteristics, and establish the similarity of the overall structure of IL-2 between tree shrews and other mammals. The homology of the IL-2 nucleotide sequence between tree shrews and humans was 93%, and the amino acid homology was 80%. The phylogenetic tree results, derived through the Neighbour-Joining method using MEGA5.0, indicated a close genetic relationship between tree shrews, Homo sapiens, and Macaca mulatta. The three-dimensional structure analysis showed that the surface charges in most regions of tree shrew IL-2 were similar to between tree shrews and humans; however, the N-glycosylation sites and local structures were different, which may affect antibody binding. These results provide a fundamental basis for the future study of IL-2 monoclonal antibody in tree shrews, thereby improving their utility as a model.

Characterisation andIn SilicoAnalysis of Interleukin-4 cDNA of Nilgai (Boselaphus tragocamelus) and Indian Buffalo (Bubalus bubalis)

Interleukin-4 (IL-4) produced from Th2 cells modulates both innate and adaptive immune responses. It is a common belief that wild animals possess better immunity against diseases than domestic and laboratory animals; however, the immune system of wild animals is not fully explored yet. Therefore, a comparative study was designed to explore the wildlife immunity through characterisation of IL-4 cDNA of nilgai, a wild ruminant, and Indian buffalo, a domestic ruminant. Total RNA was extracted from peripheral blood mononuclear cells of nilgai and Indian buffalo and reverse transcribed into cDNA. Respective cDNA was further cloned and sequenced. Sequences were analysed in silico and compared with their homologues available at GenBank. The deduced 135 amino acid protein of nilgai IL-4 is 95.6% similar to that of Indian buffalo. N-linked glycosylation sequence, leader sequence, Cysteine residues in the signal peptide region, and 3′ UTR of IL-4 were found to be conserved across species. Six nonsynonymous nucleotide substitutions were found in Indian buffalo compared to nilgai amino acid sequence. Tertiary structure of this protein in both species was modeled, and it was found that this protein falls under 4-helical cytokines superfamily and short chain cytokine family. Phylogenetic analysis revealed a single cluster of ruminants including both nilgai and Indian buffalo that was placed distinct from other nonruminant mammals.

Molecular cloning and expression of the IL-10 gene from guinea pigs

The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.

Interleukin-8 associates with adhesion, migration, invasion and chemosensitivity of human gastric cancer cells

To investigate the relationship between Interleukin-8 (IL-8) and proliferation, adhesion, migration, invasion and chemosensitivity of gastric cancer (GC) cells. The IL-8 cDNA was stably transfected into human GC cell line MKN-45 and selected IL-8-secreting transfectants. The expression of IL-8 in human GC cell line KATO-III was inhibited by RNA interference. The expressions of mRNA and protein of IL-8 in GC cells were detected by real-time reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay (ELISA). The overexpression of IL-8 resulted in an increased cell adhesion, migration and invasion, and a significant resistance to oxaliplatin in MKN-45 cells. Inhibition of IL-8 expression with small interfering RNA decreased the adhesion, migration and invasion functions and oxaliplatin resistance in KATO-III cells. IL-8 increased NF-κB and Akt activities and adhesion molecules ICAM-1, VCAM-1, and CD44 expression in GC cells. Overexpression of IL-8 promotes the adhesion, migration, invasion, and chemoresistance of GC cells, indicating that IL-8 is an important therapeutic target in GC.

Interleukin‐8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL-8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL-8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL-8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL-8-secreting transfectants. Real-time RT-PCR confirmed that IL-8 mRNA was overexpressed in IL-8 transfectants with 45- to 85-fold higher than parental cells. The IL-8-transfected clones secreted 19- to 28-fold more IL-8 protein than control and parental cells as detected by ELISA. The IL-8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL-8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL-8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL-8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL-8-expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL-8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL-8 to be an important therapeutic target in CRC.

226 ENHANCED SENSITIVITY TO ANDROGEN WITHDRAWAL DUE TO OVEREXPRESSION OF INTERLEUKIN-6 IN ANDROGEN-DEPENDENT HUMAN PROSTATE CANCER LNCAP CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2010226 ENHANCED SENSITIVITY TO ANDROGEN WITHDRAWAL DUE TO OVEREXPRESSION OF INTERLEUKIN-6 IN ANDROGEN-DEPENDENT HUMAN PROSTATE CANCER LNCAP CELLS Tomoaki Terakawa, Hideaki Miyake, Junya Furukawa, Martin Gleave, and Masato Fujisawa Tomoaki TerakawaTomoaki Terakawa Kobe, Japan More articles by this author , Hideaki MiyakeHideaki Miyake Kobe, Japan More articles by this author , Junya FurukawaJunya Furukawa Kobe, Japan More articles by this author , Martin GleaveMartin Gleave Vancouver, Canada More articles by this author , and Masato FujisawaMasato Fujisawa Kobe, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.284AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of multiple pathophysiological functions. It has been suggested that crosstalk between androgen receptor (AR) and IL-6 may play an important role in the activation of AR signaling in a ligand-independent manner; however, it remains controversial whether IL-6 confers a malignant phenotype on prostate cancer cells, particularly under an androgen-deprived condition. The objective of this study was to investigate the effects of IL-6 overexpression in androgen-dependent prostate cancer LNCaP cells on their phenotype under an androgen-deprived condition. METHODS We established IL-6-overexpressing LNCaP (LNCaP/IL-6) by introducing the expression vector containing IL-6 cDNA. Changes in the phenotype in LNCaP/IL-6 before and after androgen withdrawal were compared with those in LNCaP transfected with control vector alone (LNCaP/Co). RESULTS In vitro, the growth of LNCaP/IL-6 was significantly inferior to that of LNCaP/Co under an androgen-deprived condition. Similarly, LNCaP/IL-6 tumor in nude mice rapidly regressed after castration; however, LNCaP/Co tumor growth was transiently inhibited after castration and then continuously accelerated. After androgen withdrawal, expression levels of phosphorylated p44/42 mitogen activated protein kinase (MAPK) and Akt in LNCaP/IL-6 were markedly upregulated compared with those in LNCaP/Co; however, additional treatment with specific inhibitor of the MAPK or Akt signaling pathway significantly inhibited the growth of LNCaP/IL-6 compared with that of LNCaP/Co, which might be due to the response to proapototic stimuli representing an adaptive cell survival mechanism. Furthermore, gene microarray analyses showed that androgen deprivation resulted in differential expression of genes which are known to have functions mediating cell growth, apoptotsis, and tumorigenesis, represeting expression profiles that are reasonable for theoretically elucidating the molecular mechanism underlying enhanced sensitivity to androgen withdrawal by overexpression of IL-6. CONCLUSIONS These findings suggest that excessive secretion of IL-6 by LNCaP cells in an autocrine manner may play a suppressive role in their growth and acquisition of androgen-independent phenotype under an androgen-deprived condition. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e88-e89 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomoaki Terakawa Kobe, Japan More articles by this author Hideaki Miyake Kobe, Japan More articles by this author Junya Furukawa Kobe, Japan More articles by this author Martin Gleave Vancouver, Canada More articles by this author Masato Fujisawa Kobe, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Enhanced sensitivity to androgen withdrawal due to overexpression of interleukin-6 in androgen-dependent human prostate cancer LNCaP cells

Background:The objective of this study was to investigate the effects of interleukin-6 (IL-6) overexpression in androgen-dependent prostate cancer LNCaP cells on their phenotype under an androgen-deprived condition.Methods:We established IL-6-overexpressing LNCaP (LNCaP/IL-6) by introducing the expression vector containing IL-6 cDNA. Changes in the phenotype in LNCaP/IL-6 were compared with that in LNCaP transfected with control vector alone (LNCaP/Co).Results:In vitro, the growth of LNCaP/IL-6 was significantly inferior to that of LNCaP/Co under an androgen-deprived condition. Similarly, LNCaP/IL-6 tumour in nude mice rapidly regressed after castration; however, LNCaP/Co tumour growth was transiently inhibited after castration and then continuously accelerated. After androgen withdrawal, expression levels of phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and Akt in LNCaP/IL-6 were markedly upregulated compared with those in LNCaP/Co; however, additional treatment with specific inhibitor of the MAPK or Akt signalling pathway significantly inhibited the growth of LNCaP/IL-6 compared with that of LNCaP/Co. Furthermore, gene microarray analyses showed that androgen deprivation resulted in differential expression of genes involved in growth, apoptotsis and tumorigenesis between LNCaP/Co and LNCaP/IL-6.Conclusion:Excessive secretion of IL-6 by LNCaP cells in an autocrine manner may have a suppressive function in their growth and acquisition of androgen-independent phenotype under an androgen-deprived condition.