| ObjectivesAs the natural bridge of innate and acquired immunity, Toll-like receptors (TLRs) play a crucial role in anti-tumor activities of BCG and PPS. But there are few research reports on the effects of PPS and BCG in vivo. We investigated the immune effects of Polyporus umbellatus and Polyporus polysaccharide on toll-like receptor mediating anti-tumor Activity of BCG in BBN-induced rat bladder cancer; To explore whether PPARγis involved in the inhibition of T24 bladder cancer cell survival by PPS in vitro; the expression of TLR4 and NF-kB was observed on J774A.1 cell line.Methods1 BBN induced rat bladder cancer model and all animal treatments were done. Blood was collected from anaesthetized rats by orbital and cardiac puncture every six weeks. The flow cytometry was used to detect CD3+, CD4+, CD8+ and TCRγδ+T cells in peripheral blood of different groups.2 Peritoneal macrophages were collected from the peritoneal cavity by lavage of killed rats. The percentage of phagocytosis and the expression of surface markers such as TLR4/CD14, co-stimulation CD86, CD40 were detected by flow cytometer and were analyzed using CXP software. Nitric oxide (NO) production in peritoneal macrophages supernatants was determined by using Griess reagent。J774A.1 macrophage cell line was treated with murine rHSP70 24h,48h, followed by the detection of the expression of TLR4 with FCM. The expression of NF-kB was evaluated by immunocytochemistry.3 Bladders were fixed, and embedded in paraffin. Serial tissue sections (5μm) were cut, and stained with H&E for histopathologic evaluation. The expressions of CD86, CD40, HSP70, NF-kB and PPARγwere examined by immunohistochemistry. Real-time PCR was carried out to determine the mRNA expression of PPARγ, TLR2 and TLR4.4 In vitro cell viability was evaluated by using MTT. Peroxisome proliferator-activated receptor gamma (PPARγ) expression in human T24 bladder cancer cells was evaluated employing immunocytochemistry; Real-time PCR and Western blot techniques.Part oneResults1 The results of CD3+, CD4+, CD8+ in peripheral blood of BBN group was significantly lower than those in the control group. After treatment with different does of Polyporus umbellatus and PPS six weeks, the levels of CD8+ CD3+ and CD28+ were markedly increased. After treatment with PPS twelve weeks, the levels of CD4+ CD3+,TCRγδ+were significantly increased (P<0.05)2 FCM analysis showed that the phagocytosis of BBN group was significantly decreased compared with that of control group (P<0.05). PPS significantly inceased the phagocytosis and the expression of surface markers TLR4/CD14, co-stimulation CD86, CD40. Polyporus umbellatus at the concentration of 50mg/kg,250mg/kg and 500mg/kg promoted the phagocytosis of peritoneal macrophages, and significantly increased the expression of CD86,but the expression of CD40, TLR4/CD14 were significantly decreased at the concentration of 250mg/kg and 500mg/kg. Polyporus umbellatus at the different concentration and PPS can decrease the production of NO induced by LPS.3 The expression of CD86 was mainly detected in the epithelial cells near the cancer tissue, but little even none expressed in cancer cells. CD40 was highly expressed in bladder transitional cell cancer. Significant differences of CD40 staining were observed between normal bladder tissues and bladder transitional cell cancer. The expression of NF-kB after PPS treatment was weaker than that of BBN group, but the expression of PPARγis stronger staining. The increased expression of PPARγmRNA, TLR2 mRNA, and TLR4mRNA were detected by using RT-qPCR after PPS treatment.4 PPARγmRNA expression were increased (Ratio>2.0) after 48h treatment with lmg/ml PPS. BADGE can abrogated the effect of PPS increasing expression of PPARγmRNA (Ratio<0.5). Addition of PPS to cultured T24 cells increased cytoplasmatic and nucleus PPARγexpression compared with control group. Furthermore, we confirmed the effect of PPS on increase in PPARγexpression by Western blot analysis in T24 cells lysates from control and PPS with or without BADGE groups which showed an increased level of PPARγprotein in PPS group.Conclusions1 In the present study show that Polyporus umbellatus and Polyporus polysaccharide (PPS) inhibit bladder tumor growth and progression through different stages of dysplasia and carcinoma, as well as the severity of the cancerous lesions in the BBN induced rat bladder tumor genesis model. We suppose that PPS may down-regulate NF-kB signaling, as well as up-regulate PPARγthrough TLR4 pathway. These effects were accompanied by activating macrophage and releasing co-stimulation markers.2 PPS may function via PPAR-dependent pathways to exert direct effect on T24 bladder cancer cells.Part TwoResults1 After treatment with BCG twelve weeks, the levels of CD3+ and CD4+ CD3+ were increased. The levels of CD3+,CD4+CD3+,CD8+CD3+,CD28+ were higher with the combination of BCG plus PPS or Polyporus umbellatus treatment than these of BCG group.2 FCM analysis showed that the expression of co-stimulation CD86, CD40 significantly increased with the combination of BCG plus PPS or Polyporus umbellatus treatment(P<0.05). BCG significantly inceased the the expression of surface markers TLR4/CD14, but a markedly decreased in the expression of TLR4/CD14 in PPS+ BCG group compared with BCG alone.3 Immunohistochemical analysis showed that the expression of CD86 and CD40 was stronger staining and NF-kB was prominent expressed in mesenchymal cells in PPS+BCG group. BCG significantly increased the PPARγmRNA, TLR4mRNA expression (P<0.05).TLR2 mRNA, TLR4mRNA expression levels were significantly up-regulated in PPS+BCG group (P<0.05).4 The expression level of TLR4 was significantly increased after 48h treatment with rHSP70 compared with that of control group. Addition of PPS to cultured J774A.1 cells increased nucleus NF-kB expression.ConclusionThe cooperation of PPS and BCG to produce a combined effect better than the separate effect of BCG in vitro experiments. As the natural bridge of innate and acquired immunity, Toll-like receptors(TLRs) play a crucial role in anti-tumor mechanisms of BCG and PPS.
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