The Role Of C Reactive Protein In Tongue Squamous Cell Carcinoma And Prelinary Study Of Its Mechnism

Objective:Tongue squamous cell carcinoma(TSCC)is one malignant tumor with high invasiveness and early metastasis of lymph nodes.Despite advances in treatment strategies involving surgery,chemotherapy and radiotherapy,many patients may still suffer from eating disorders,impaired language functions and cancer-related death.Recently,more and more researchers are pursuing for new methods such as gene therapy,targeted therapy and biological therapy,to treat TSCC in order to get better results.Tumorigenesis and tumor development are related to not only the biological characteristics of the tumor cells themselves,but also many elements in tumor microenvironment.Exploring cancer-associated factors and genes is important for the development of cancer treatments.The relationship between inflammation and tumor has been paid more and more attention in the study of oncology.Inflammation factors in tumor microenvironment have a variety of tumor-promoting effects.They can promote tumor cells proliferation,tumor angiogenesis and metastasis and weaken the immune response to hormones and chemotherapy drugs.Many inflammatory factors in tumor microenvironment,such as tumor necrosis factor(TNF),interleukin-6(IL-6),and interferon,play important roles in tumor cells proliferation,invasion,migration and apoptosis.Just as the other inflammatory mediators,C-reactive protein(CRP)is a classical acute-phase reactant protein.It is synthesized by hepatocytes and secreted into the tumor microenvironment via the blood circulation.By binding to a variety of autologous and extrinsic ligands exposed on the membrane of injured,necrotic and apoptotic cells,CRP serves several functions.Serum CRP levels are closely related with infective inflammation and cardiovascular disease.Recent studies showed that the serum CRP level was significantly higher in patients with tumors.The serum CRP level was associated with tumor size,clinicopathological features and lymph node metastasis.Furthermore,CRP was also an important biomarker of tumor prognosis and treatment response.However,to the best of our knowledge,the present attentions are mainly paid to diagnostic and prognostic values of CRP,while the influence of CRP on the development of cancers including TSCC has seldom been investigated.Therefore,the purpose of tns study was to investigate the correlation between CRP expression and TSCC and preliminarily explore the effects of CRP on TSCC cells,as well as potentially related signaling molecules.Materials and Methods:This study mainly contains three parts:Part Ⅰ:Expression patterns of CRP in TSCC tissues and cellsExperiment 1:Expression patterns of CRP in TSCC tissuesTongue tissue samples from 42 TSCC patients were collected with definitive demographic and clinicopathological records.The sections were deparaffinized in xylene,hydrated through a graded alcohol series.Antigen retrieval was performed by treating the samples with 0.1%(w/v)trypsin.The activity of endogenous tissue peroxidase was blocked with 3%H2O2.Normal goat serum was used to block non-specific binding.Then,the sections were incubated with anti-CRP antibody at 4℃ overnight.Biotinylated goat anti-rabbit immunoglobulin G(IgG)and streptavidin-peroxidase conjugate were respectively incubated the sections.A diaminobenzidine solution was used to visualize localization.Finally,the sections were lightly counterstained with haematoxylin.Image-Pro Plus 6.0 software was used to determine the integrated optical density(IOD)of CRP staining quantitatively.Experiment 2:Expression patterns of CRP in TSCC cells 1.ELISA analysis for the secreted CRP of TSCC cellsCAL27,SCC4 and SCC25 cells were cultured in DMEM for 24 h.The medium were collected for ELISA analysis.2.Western blot analysis for the expression of CRP in TSCC cellsCAL27,SCC4 and SCC25 cells were seeded in 6-well plates.The cells were lysed in ice-cold lysis buffer.Protein concentrations were measured using a Bicinchoninic Acid Protein Assay Kit.A total of 20 μg of protein was loaded for separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and were then electrotransferred to polyvinylidene fluoride membranes.After being blocked,the membranes were incubated with primary antibody.Protein bands were visualized on Canon films.Part Ⅱ:The role of CRP on the TSCC biological behaviors including cell proliferation,invasion,migration and apoptosis1.CCK-8 proliferation assayThe suspended CAL27 and SCC4 cells(3.5×104/ml)were seeded in 96-well plates,and then cultured in DMEM containing different concentrations of human recombinant CRP(0,5,10,20,and 30 μg/ml)for 24 h.In addition,the cells were also stimulated with various concentrations of human recombinant CRP(5,10,and 20μg/ml)for different time(48,36,24,12,6,and 0 h).Subsequently,the cells were treated with a 10%CCK-8 solution.The absorbance was measured at 450 nm.2.Cell invasion assayAfter starvation,the CAL27 cells(4×105/ml)in serum-free DMEM were seeded on the upper side of the transwell membrane plates.Then,different culture mediums(DMEM without FBS,serum-free DMEM with 10 μg/ml CRP,and DMEM-with 10%FBS)were added to the lower chamber and incubated for 24 h.Migrated cells remaining on the transwell membrane were fixed and then stained with 10%crystal-violet.Cells were counted under a microscope.3.Cell migration assayThe CAL27 cells were seeded in 6-well plates and cultured till cell monolayer was formed.A sterile micropipette tip was then used to create a scratch on the cell monolayer.The cells in the plates were further incubated with different culture mediums(DMEM without FBS,serum-free DMEM with 10 μg/ml CRP,and DMEM with 10%FBS)for up to 18 h,and the wound width was then measured by microscopy at 0,6,12 and 18 h to assess cell migration.4.Cell apoptosis assayThe CAL27 and SCC4 cells were seeded in 6-well plates and cultured in different mediums[serum-free DMEM,serum-free DMEM + CRP(10 μg/ml);complete medium,complete medium + CRP,complete medium + DDP(2 μg/ml),complete medium + DDP + CRP]for 24 h.Then,the number of apoptotic cells was quantified by flow cytometry.5.Western blot analysis for the expression of PCNAThe CAL27 cells were seeded in 6-well plates and cultured with CRP(10 μg/ml)stimulation for different time(48,36,24,12,6,and 0 h).The procedure for Western blot analysis was performed as described.Part Ⅲ:The study of the receptor of CAL27 cells interacted with CRP,and related signaling pathways in TSCC cells with the CRP stimulationExperiment 1:The receptor of CAL27 cells interacted with CRP1.Immunofluorescence(IF)stainingCAL27 cells were growing on the glass slides and then fixed.The serum was used to block nonspecific binding.The sections were incubated with primary antibodies(FcγⅠ,FcγⅡ,FcγⅢ)at 4℃ overnight and then incubated with secondary antibody.DAPI staining was applied to visualize the nuclei.2.Immunoprecipitation assayCAL27 cells were incubated in DMEM with or without CRP(25 μg/ml)for 30 min.The cells were lysed in ice-cold lysis buffer.The supernatant with antibody was shaken overnight and then incubated with agarose beads.The procedure for Western blot analysis was performed as described.Experiment 2:The study of AKT/mTOR/S6 signaling pathway in TSCC cells with the CRP stimulation1.Western blot analysis for the expression of pAKT,pmTOR and pS6 in TSCC cells with the CRP stimulationCAL27 cells were cultured in serum-free DMEM containing CRP(10 μg/ml)for 120,60,30,15 and 0 min.The procedure for Western blot analysis was performed as described.2.CCK-8 assay for cell proliferation with CRP and PARP stimulationCAL27 cells were seeded in 96-well plates and cultured in different mediums(DMEM+DMSO,DMEM+CRP,DMEM+CRP+RAPA(2 μm/L),DMEM+RAPA)for 48,36,24,12,6,0 h.The procedure for CCK-8 analysis was performed as described.3.Western blot analysis for the expression of PCNA with CRP and PARP stimulationCAL27 cells were seeded in 6-well plates and cultured in different mediums(DMEM+DMSO,DMEM+CRP,DMEM+CRP+RAPA,DMEM+RAPA)for 24 h.The procedure for Western blot analysis was performed as described.Experiment 3:The study of Caspase 3/9 signaling pathway in TSCC cells with the CRP stimulationCAL27 cells were seeded in 6-well plates and cultured in different mediums(DMEM+DDP+PBS,DMEM+DDP,DMEM+DDP+CRP)for 24 h.The procedure for Western blot analysis was performed as described.Results:1.CRP was expressed in TSCC tissues,and TSCC cells might produce and secrete CRP1.1.CRP was expressed in TSCC tissuesThe results of immunohistochemical staining showed positive staining for CRP in TSCC tissue,which was primarily localized in the cytosol of tumor cells.Furthermore,the CRP staining in moderately and poorly differentiated tumor tissues was stronger than that in well-differentiated tumor tissues.The statistical analysis showed that the tumor CRP expression level was positively associated with both tumor size and pathological differentiation,whereas no significant relationship was found between the CRP expression level and patient gender or age.Moreover,CRP was strongly expressed in those patients with lymph node metastasis.1.2.Tongue cancer cells might produce and secrete CRPThe results of ELISA showed that all tongue cancer cells could secrete different amounts of CRP.Western Blot showed that CRP was expressed in different tongue cancer cells.2.CRP enhanced TSCC cell proliferation,invasion and migration and protected TSCC cells from apoptosisThe CCK-8 assay showed that CRP significantly enhanced TSCC cell proliferation in a dose-and time-dependent manner.In addition,10 μg/ml and 24 h were found to be the significantly effective concentration and time of CRP on CAL27 and SCC4 cells.However,increasing the CRP concentration from 10 to 30 μg/ml did not result in greater proliferation effects and the growth ability of TSCC cells was inhibited after 24 h.The highest expression of PCNA was found with the CRP stimulation at a concentration of 10 μg/ml for 24 h.Transwell chamber and wound-healing assays showed CRP could increase TSCC cell invasion and migration.Compared with the negative control group,more cells were found on the lower surface of the membrane after treatment with CRP(10 μg/ml)for 24 h.In addition,the wound-healing assay showed that the distance migrated by cells after CRP treatment for 6,12 and 18 h was approximately 0.3-,1-and 0.5-fold greater than that migrated by cells in the negative control group.The results of Annexin V-FITC/PI assay showed CRP could protect TSCC cells from starvation-and drug-induced apoptosis.With the addition of CRP(10 μg/ml),there was about a 1-fold decrease in apoptotic cells compared to the control group treated with DMEM or DDP only.3.CRP could interact with Fey I R of CAL27 cells and up-regulate the expression levels of pAkt,pmTOR and pS6 while down-regulate the expression levels of cleaved Caspase-3/93.1.CRP could interact with Fey I receptor of CAL27 cellsFcγ ⅠR,FcγⅡR and FcyⅢR were expressed on the surface of CAL27 cells revealed by immunofluorescence.Immunoprecipitation showed CRP could interact with FcγⅠ receptor of CAL27 cells.3.2.CRP could up-regulate the expression levels of pAkt,pmTOR and pS6Western Blot showed the expression levels of pAkt and pmTOR were up-regulated after treatment with CRP at 10 μg/ml for 60 min,and the ratios of pAkt/Akt and pmTOR/mTOR were significantly increased.Moreover,after CRP stimulation for 30 min,the expression of pS6 was also significantly increased,and the ratio of pS6/S6 was increased by approximately 0.3-fold.However,when the treatment time was extended to 120 min,the protein expression levels of pAkt,pmTOR and pS6 were down-regulated and the ratios of pAkt/Akt,pmTOR/mTOR and pS6/S6 were also decreased.The expression of pNF-Kb presented no significant difference,and the ratio of pERK/ERK was decreased.To further investigate the mTOR signaling pathway induced by CRP,we added rapamycin(RAPA),an inhibitor of mTOR,to detect the proliferation of CAL27 cells.The results showed that RAPA could significantly decrease the proliferation activity of CRP-induced tongue cancer cells,and the expression of PCNA protein was significantly lower than that of CRP-induced group.3.3.CRP could down-regulate the expression levels of cleaved Caspase-3/9Western Blot showed the expression of cleaved Caspase-3 was decreased with the addition of CRP.The down-regulation of cleaved Caspase-3 indicated that the cell apoptosis was decreased with the CRP stimulation.Moreover,the expression of cleaved PARP and cleaved Caspase-9 protein was also decreased.Conclutions:1.CRP might play an important role in the development of TSCC.2.CRP stimulation significantly promoted the proliferation,invasion and migration of TSCC cells and could protect TSCC cells from starvation-and drug-induced apoptosis.3.CRP could interact with FcⅠγ receptor to infect the biological behaviors of CAL27 cells.4.AKT,mTOR and S6 might be important players in CRP-mediated TSCC cell proliferation,invasion and migration.5.CRP could protect TSCC cells from drug-induced apoptosis via reducing the activation of Caspase-3/9.