Research Of Treament For Human Tongue Squamous Cell Carcinoma By Interleukin18

Tongue squamous cell carcinoma(TSCC) is the most common malignanttumor in oral and maxillofacial region. A higher incidence of tongue cancer,and increased year by year, the prevalence of age is getting younger. Due torich blood supply and lymphatic of the tongue, particularly genioglossusfrequent extrusion tongue cancer more likely to early transfer. Tongue cancerwith poor prognosis, postoperative lifequality has declined markedly. Therefore,tongue cancer is a disease of a serious threating to human health.Interleukin-18is a pleiotropic inflammatory cytokine. In the year of1995,Okamura et al purified a new protein. Because it could significantly stimulateThl cell to produce IFN-γ,it was named IGIF (IFN-γ-inducing factor). Due toIGIF possessing many kinds of biological function, it was formally namedIL-18in1996. IL-18, a member of the IL-1superfamily of cytokines and likeIL-1β, is produced by several cell types, including macrophages, dendritic cells,microglial cells, and keratinocytes. IL-18is initially produced as pro-IL-18(24kDa), which encodes a193–amino acid biologically inactive precursorprotein. It is then processed by caspase-1, which is an IL-1β-converting enzyme(ICE), to a mature form with a molecular weight of18kDa. IL-18affects theimmune system through:1.inducing IFN-γ secretion by T, NK, or B cells;2.enhancing proliferation of anti-CD3monoclonal antibody, IL-2, orconcanavalin A-stimulated T cells;3.augmenting Fas ligand-mediated NK cellcytotoxic activity;4. inhibiting osteoclast formation in vitro;5. reinforceing theactivity of NK cell, regulating the activation of CD8~+T and CD4~+T cell, andCTL giving free rein of cytolytic function. Moreover, IL-18has a synergisticeffect with IL-12for IFN-γ production. Markedly retarded blood vessel growth are likely the result of more effective inhibition of angiogenesis by thecombination of the two cell types. Because of the above biologic activity,IL-18is probably becoming an antineoplastic cytokine. It has exhibited significantantitumor activities in multiple animal models and patients with advancedcancer. However, an antitumor property of IL-18on tongues quamous cellcarcinoma has not been directly shown.At present, a large volume of research has been performed regarding theprevention and treatment of TSCC, including targeting tumour cell-basedintegrins to enable optical imaging of oral SCC, and the use of curcumin toinduce apoptosis via endoplasmic reticulum stress and mitochondria-dependentpathways. In addition, tongue cancer resistance-associated protein1(TCRP1)has been implicated in the radiation sensitivity of oral squamous cell carcinomacells through the Akt signaling pathway. There is, however, limited dataregarding immunotherapy in TSCC.Hypothesis of the research:The introduction of cytokine genes into tumor cells has become one of themain techniques in cancer gene therapy. We transfected into human tonguesquamous cell carcinoma cell line CRL-1623with pcDNA3.1(+) vectorcontaining the human interleukin18gene. Because of the immunoregulationand antitumor property of IL-18, we presume that IL-18probably inhibitgrowth of CRL1623. It may be a critical problem of approaching a newantineoplastic pathway in our study.Our research is divided into two parts. In the first part we construct andidentify the eukaryotic expression vector of human Interleukin-18. In thesecond part, we use pcDNA3.1(+) vector containing the human interleukin18gene and transfected into human tongues quamous cell carcinoma cell lineCRL-1623. Analysis of cell growth, detection of apoptosis, assessment ofcaspase3/7activity, and real-time reverse transcription–polymerase chain reaction were performed to assess the related genes expression of correlatedcytokine.Part one Construction and Identification of EukaryoticExpression Vector of Human Interleukin-18ObjectThe eukaryotic expression vector of Interleukin-18was constructedaccording to the Interleukin-18gene of human．MethodWe isolated leukocyte from fresh blood of one healthy donor withdensity gradient centrifugation,then extracted and assessed IL-18total RNAusing TRIzol reagent according to manufacturer's instruction. Aftersynthetized primer for PCR, interleukin-18gene was obtained from humangenome DNA by PCR. Vector pMD18-T/Interleukin-18was cloned andtransformed into Escherichia coli,and the recombinant pCDNA3.1/Interleukin-18was constructed and identified．ResultThe result of agarose gel electrophoresis showed that an expected580bpband was amplified. The result of enzyme-disgetion identification indicated aband was in accordance with the expected length. Sequencing result ofpCDNA3.1/Interleukin-18showed that the reading frame was not changedexcept two base compared with the GeneBank, and amino acid sequence wasthe same.ConclusionThe eukaryotic expression vector pCDNA3.1/IL-18was constructedsuccessfully. Part two Construction of human tongue squamous cellcarcinoma cells transducted by IL-18and study of itsantitumor effect in vitroObjectiveThis study investigated the role of interleukin-18(IL-18) in regulating thegrowth of the human tongue squamous cell carcinoma cell line CRL-1623.Methods:1Constructing the eukaryotic expression vector pCDNA3.1/IL-18;2Culturing of tongue carcinoma cell line CRL1623;3The human IL18gene was cloned and transfected into CRL-1623cellsusing the transfection vector pcDNA3.1(+), then real-time reversetranscription–polymerase chain reaction was performed;4Analysis of cell growth using MTS method, detection of apoptosis usingannexin V–fluorescein isothiocyanate,5Assessment of caspase3/7activity using Apo-ONE Homogeneous Kit6Real-time reverse transcription–polymerase chain reaction to assessexpression of the IL18, CCND1(cyclin D1), CCNA1(cyclin A1) and IFNG(interferon-γ) genes.7Statistical analyses were carried out using the SPSS statistical package,version16.0for Windows. The Student's t-test was used to compare thedifferences between groups. A P-value <0.05was considered to be statisticallysignificant.Result1Constructing the eukaryotic expression vector successfully.2Transfection of CRL-1623cells with pRFP appeared qualitatively toresult in high transfection efficiency at48h after transfection, as determined by fluorescence microscopy.3The number of apoptosis and dead cells increased from24to72h,following transfection with pIL18.4.Transfection with pIL18reduced cell viability compared withuntransfected cells and this difference was statistically significant at48h (P=0.031) and72h (P=0.007); Cell viability was also reduced slightly bytransfection with empty vector pcDNA3.1(+) from24h to72h compared withuntransfected cells, although this difference was not statistically significant.The number of apoptotic cells in the pIL18transfected group also increasedsignificantly between0and72h compared with untransfected cells (P=0.046at24h; P=0.037at48h; P=0.016at72h). Transfection with pIL18but notpcDNA3.1(+) resulted in increased caspase-3/7activity between0and72h.although this difference was only statistically significant at72h.5.The levels of both IL18and IFNγ mRNA were increased in cellstransfected with pIL18compared with untransfected cells (P=0.009and P=0.014, respectively). Cyclin D1mRNA was reduced in pIL18-transfected cellscompared with untransfected cells (P=0.024). Transfection with pIL18did notinduce any change in the level of cyclin A1mRNA.Conclusion1Over-expression of an exogenous IL18gene directly inhibited thegrowth of, and induced apoptosis in the human tongue squamous cell carcin-oma cell line CRL1623；2The induction of apoptosis might occurr via a caspase-dependentpathway；3IL-18stimulates Th1-mediated immune responses, which play a criticalrole in host defence against intracellular microbes through the induction ofIFN-γ；4IL-18signal transduction may result in interference with the regulation of the cyclin proteins and/or the replication of chromosome during S phase,leading to a slow proliferative rate。These findings suggest that IL-18plays a role in the regulation of tonguesquamous cell carcinoma and may represent a potential therapeutic target inthis cancer.