Effect Of BCG And Polyporus Polysaccharide On TLR2/4-NF-κB Signal Pathway In Bladder Cancer Cell

Intravesical instillation of Bacillus Calmette-Guerin(BCG) is currently a comparatively effective immunotherapy for treating and preventing superficial bladder cancer and it is considered that the mechanism of the effect lies in the immune response induced and enhanced by BCG and especially its action on local immune cells.Whereas, the above mechanism is unable to completely explain the fact that the BCG is rarely applied and has much less effect on other types of cancer than on superficial bladder cancer.Thus it is highly probable that apart from activation of local natural immune cells by BCG in cancer tissue,there are some other underlying pathways and mechanisms which are still uncovered involved in its antitumor activity.In the recent years,it is reported that TLR2 and TLR4 were found in epithelial cells in urine and the expression can be induced by BCG. Considering bladder cancer cells also express TLRs,possibly,it is BCG that directly stimulates TLRs levels in the bladder transitional epithelial cells and subsequently causes the release immune activating factors via TLR-NF-κB signaling pathway,thereby strengthens the immunogenicity of tumor and promotes the antitumor immune reaction. The current study was designed to determine the role of BCG in regulating the genes expression via TLR2/4—NF-κB signaling pathway in mouse bladder cancer cell line T739.First,the changes of mRNA expressionin of NF-κB signaling pathway associated-genes in BCG-treated bladder cancer cells T739 were analyzed by real-time PCR and some of the genes such as TLR2,TLR4,CD14,MD-2,CHUK,MAP3K1, LKBKB,NFKB1(NF-κB p50),Rel A(NF-κB p65),CCL2 and ICAM1 significantly increased after BCG treatment.The expression of the suppressor genes in NF-κB pathway Nfkbia and Nfkbibof could not be detected.Through the NF-κB p65 DNA-binding activity assay in the following experiment,it was revealed the fact that NF-κB p65 in T739 cells was constitutively activated,which corresponded to the common feature of NF-κB molecules in major mammalian tumor cells.BCG treatment had an effect on the expression of NFKB1 and RelA but not on the expression of NFKB2,thus,it seems reasonable to conjecture that the molecular form of NF-κB is Rel A/NFKB1.Using the antibody blocking assay,we discovered that the levels of MAP3K1,IKBKB,Rel A(NF-κB p65), CCL2 and ICAM1 mRNA rose after TLR2 and TLR4 were blocked. Secondly,we applied vector based RNAi technology for investigating the effect of the expression of TLR2 and TLR4 on the BCG-mediated treatment for bladder cancer.Three pairs of miRNA precursor sequences were designed based on the different target sequences in mice coding region(ORF) and we constructed three miRNA expression vectors rom which the vector pcDNA^TM6.2-GW/EmGFPmiR- TLR2.949 were selected for its best transient interference effect.The vector was transfected into the cells and the TLR2-silencing stable cell line T739-TLR2Δwere selected by Blasticidin-resistant selection and passaged continually for a month.The TLRs expression maintained at a low level in T739-TLR2Δcells for the future experiments.Another stable cell line T739-TLR4Δ, silenced mouse gene TLR4,was established in the same way.By using the two stale cell lines mentioned above,it was shown that BCG increased MAP3K1,IKBKB mRNAs via TLR2,up-modulated ICAM1 mRNAs via TLR4, up-regulated mRNA levels of Rel A and CCL2 via either TLR2 or TLR4.In a further study,the protein expression of IKKβ,CCL2 and ICAM1 were measured by western blot and flow cytometry to determine their relation with TLR2 and TLR4 following the BCG treatment.The results indicated that,in consistent with its mRNA expression,the increase in CCL2 protein levels associated both with TLR2 or TLR4.Up-regulation of IKKβprotein level mediated by TLR2 also matched up with the result of its mRNA.While inconsistent with its regulation of mRNA expression,ICAM1 protein level up-modulation mainly related with TLR2.The enhancement effect of BCG on NF-κB p65 activity and nuclear translocation mostly mediated by TLR2 and correlated with TLR4 to a certain extent.Polyporus polysaccharide(PPS),as a main active component of polyporus,is a polymer of glucose and lactose.It has been applied clinically on the treatment of type B hepatitis and immunity enhancement.It can also inhibit the growth of carcinoma cells, modulate the immune function of tumor animals and patients,and reduce toxicity as well as side effect of chemotherapeutics.However,the mechanism of its anti-tumor activity is still unclear.NF-κB signaling pathway plays important roles in regulation of virus replication, autoimmune diseases,inflammatory response,tumorigenesis and apoptosis etc,and closely relates to the tumor development and metastasis.NF-κB signaling pathway in most tumor cells is activated constitutively or continuously.The inhibitor of NF-κB has been used as an adjuvant therapy for tumor.The finding of Toll-like receptor(TLRs) in innate immune system turns out to be a new way to study the mechanism of anti-tumor effect of polysaccharide.Recently,it has been reported that the plant polysaccharide is one of the ligands of TLRs.Bladder epithelial cells expressed TLR2 and TLR4 that can respond to corresponding ligands.Our previous research indicates that PPS can significantly increase p53 expression in the bladder cancer cells which culture in vitro and inhibit their proliferation by blocking the S-G2 phase transition in cell cycle.Other researcher reported that Polyporus could reduce the incidence of BBN-induced bladder cancer and the recurrence rate of bladder cancer can drop from 65.1%to 33.3%in contrast to the control group after administration of Polyporus.Therefore,according to the previous study about the role of Polyporus in the treatment of bladder cancer,we suppose that PPS may regulate some genes expression in proliferation,differentiation,cell cycle and apoptosis by means of direct inhibitation of the NF-κB signaling pathway in bladder cancer cells and excessive activation of some kinases or signal transduction molecules.Based on the assumption above,we utilized the RNAi technology to silence the genes of TLR2 and TLR4 so that whether the regulation of the PPS to NF-κB pathway in bladder cancer cells was mediated by TLR2 of TLR4 could be tested.The increased expression of some membrane molecules such as TLR2,TLR.4,CD14 and MD-2 was detected by using qRT-PCR to study the effect of the PPS on the NF-κB signaling pathway-associated genes in bladder cancer cell T739,thus we think that PPS might transfer signals into cells through TLRs.As the adaptor molecules in Toll-like signaling pathway,TRAF6,TIRAP and IRAK1 had varying changes in their gene expression.The mRNA of IKBKB(IKKβ) decreased greatly and progressively over time and the ration declined from 0.32 to 0.07.Abnormal expression of IKKs is reported to have an important role in the tumorigenesis and tumor progression,its overexpression and overactivity gives a rise to malignant growth characteristic and anti-apoptosis capability of tumor.The decreased expression of IKKβdue to PPS could be explained by the our previous finding that PPS had an inhibitory effect on the proliferation of bladder cancer cells and S-G2 phase transition in cell cycle.NF-κB p65 mRNA in T739 cells decrease after PPS treatment.ICAM1 and CCL2 act as downstream genes of the NF-κB pathway.CCL2 is a secretory protein involving in monocyto chemotaxis.ICAM1 is an intercellular adhesion molecule that can promote adhesion between inflammatory cells and target cells.The decreased expression of ICAM1 and CCL2 mRNA suggests that PPS has the ability to down-regulate some of the gene expression in NF-κB pathway and inhibit the activity of NF-κB,however,the inhibitory effect PPS on NF-κB pathway in T739 cells have not been studied yet at biological functional level.Our further experiment demonstrated that the IKBKB mRNA and protein could be down-regulated by PPS via TLR4,but TLR2 only mediated the down-regulation of IKKβprotein.CCL2 mRNA and protein are down-modulated respectively via TLR4 and TLR2.PPS decreased the ICAM1 mRNA expression via TLR2 while had no significant effect on ICAM1 at protein level.The silencing of TLR2 or TLR4 gene can reduce the mRNA expression of Rel A,it shows that TLR2 and TLR4 are involved in the procedure of PPS down-regulating Rel A mRNA.The evidence above indicates that the genes we mentioned here are mainly mediated by TLR4.TLR2 also works on it in some way.The DNA-binding activity of NF-κB p65 and its nuclear expression in T739 cells can be reduced by PPS.The DNA-binding activity of NF-κB p65 in T739-TLR2Δcells could be brought down by PPS,but the counterpart in T739-TLR4Δhad no significant response to this.Moreover,through nuclear expression assay,the effect of PPS on reducing NF-κB p65 nuclear expression in T739-TLR2Δcells but not in T739-TLR4Δwas exhibited.The results above show that the PPS reduce both DNA-binding activity and nuclear expression of NF-κB p65 in T739 cells mainly via TLR4.Both BCG and PPS have the effect against bladder cancer and they regulate NF-κB pathway in different ways,so we focus on determining the effect of the both chemicals on the expression of main NF-κB pathway-related genes in bladder cancer cells.Our data show that PPS can antagonize the action of BCG on increasing the levels of IKBKB,ICAM1,CCL2 and NF-κB p65 and the DNA-binding activity of NF-κB p65,the antagonism is extremely significant to the latter one,DNA-bind activity of NF-κB p65.BCG works well on against bladder cancer but it has some side effects,such as haematuria and pollakisuria,which are correlated with hyperimmunization.PPS can attenuate the above immune reaction and toxicity reaction when it is added to the treatment. Considering the combined medication with PPS can reduce the toxicity and increase the efficacy of BCG,this research will provide a crucial and helpful experimental and theoretical basis for the future clinical practice.