The Effect Of TLR5 And NLRC4 Pathway On Proliferation Of Breast Cancer Cells And Partial Immune Cells

Objective: To investigate effect of TLR5 and NLRC4 pathway on proliferation of breast cancer cells and murine neutrophils, NK cells, DCs and regulatory T cells. Methods: There were four different recombinant flagellins:FliC(activating both TLR5 and NLRC4); FliCΔ90-97(activating NLRC4),FliC-L3A(activating TLR5); FliCΔ90-97:L3A(unable to activate TLR5 nor NLRC4).1. IPTG and nickel ion affinity chromatography was used to induce the expression of rFli C and to purify rFliC. IL-8 and IL-1βwere detected to reflect the activation of TLR5 and NLRC4 pathway activated by rFliC. 2. QRT-PCR was explored to detect the expression of TLR5 and NLRC4 in MCF-7 and MDA-MB-231 cells. We used CCK8 method to investigate the influence of rFli C on breast cancer cells and western-blot was used to detect the activation of TLR5 and NLRC4 in MCF-7 cells treated wih rFli C. 3. Flow cytometry was used to test the effect of rFliC on partial immune cells. Results: 1. The purity of rFliC was >95% and remaining endotoxin was under 0.01EU/mg protein. The four recombinant flagellins had corresponding bioactivatity. 2. All of the recombinant flagellins could inhibit the proliferation of MCF-7 cells and MDA-MB-231 cells. All groups had a >20% inhibition rate in MCF-7 cells on the concentration of 0.1μg/ml. Western-blot results indicated that NF-?B which transferred to the nucleus increased in group FliC and Fli C-L3 A compared with control group. Fragment P10 increased in group FliC and FliCΔ90-97.3. Flow cytometry results showed that the percentage of neutrophils and NK cells increased significantly in group FliC, FliCΔ90-97 and FliC-L3 A compared with PBS group. The percentage of neutrophils and NK cells in the four groups are(8.41±0.04)% and(9.9±2.6)%,(9.1±2.1)% and(10.3±0.3)%,(8.0±0.3)% and(11.0±2.6)%,(2.6±1.3) % and(3.2±0.2)% respectively. CD80 and CD86 are upregulated by FliC and Fli C-L3 A compared with the control group(P<0.05).Regulatory T cells increased in group Fli C-L3 A compared with other groups.Conclusion: 1. The inhibition of recombinant flagellins on MCF-7 cells was not fully dependent on TLR5 nor NLRC4 pathway.It is possible that there are some other mechanism. 2.TLR5 and NLRC4 pathways played profound roles in the chemotaxis of neutrophils and NK cells. 3. Upregulation of CD80 and CD86 by recombinant flagellin was mainly in a TLR5 dependent manner.