ABSTRACT
Objective To observe the relationship between Toll-like receptor 4 (TLR4) and the sensitivity of PANC1 cells to gemcitabine (GEM),and to analyze the potential mechanism.Methods PANC1 cells were divided into GEM group,lipopolysaccharide (LPS) + GEM group and TLR4-siRNA + GEM group.GEM group was treated by GEM alone.LPS + GEM group was pretreated with 1 mg/L LPS for 4 h and then treated by GEM.TLR4-siRNA + GEM group was transfected with 100 pmol/mL TLR4-siRNA for 4 h and then treated by GEM.The untreated cells were used as the control group.MTT method was used to detect the cell proliferation.Morphological changes and apoptosis rate of the cells were examined by Hoechst33258 staining and flow cytometry,respectively.The protein expression of TLR4,phosphorylated AKT (p-AKT) and activated Caspase-3 were detected by Western blot.Results The median inhibition concentration (ICs0) of GEM in the GEM group,LPS + GEM group and TLR4-siRNA + GEM group was (8.9 ± 0.32),(14.21 ±0.95),(3.96 ± 0.27) mg/L,respectively.The IC50 in LPS + GEM group was significantly higher than that in GEM group (P ＜ 0.01),and the IC50 of GEM in TLR4-siRNA + GEM group was significantly lower than that in GEM group (P ＜0.01).Compared with that in GEM group,the cells with typical apoptotic morphological changes were decreased in LPS + GEM group,which was increased in TLR4-siRNA + GEM group.The apoptotic rate in control group,GEM group,LPS + GEM group,TLR4-siRNA + GEM group was (2.1 ± 0.3) ％,(15.1 ± 2.3) ％,(9.8 ± 1.5) ％,(22.9 ± 3.1) ％,respectively.Compared with that in GEM group,the cells apoptotic rate was significantly reduced in LPS + GEM group (P ＜0.01),which was significantly increased in TLR4-siRNA + GEM group (P ＜0.01).TLR4 protein level in the 4 groups was 0.83 ±0.08,0.81 ±0.07,0.85 ±0.07 and 0.16 ±0.03;p-AKT protein level 0.61 ±0.05,0.36 ±0.03,0.73 ± 0.07 and 0.21 ± 0.02;activated Caspase-3 protein level was 0.66 ± 0.05,0.73 ± 0.07,0.45 ± 0.04 and 0.91 ± 0.07,respectively.The expression of TLR4 and p-AKT in TLR4-siRNA + GEM group was significantly lower than that in GEM group (P ＜0.01),while the expression of activated Caspase-3 protein was increased significantly (P ＜ 0.05).Compared with the GEM group,the expression of p-AKT protein in LPS + GEM group was significantly increased (P＜0.01),and the expression of activated Caspase-3 protein was significantly decreased (P＜0.01).Conclusions TLR4 can inhibit the sensitivity of pancreatic cancer PNAC1 cells to GEM,and the mechanism is related to the activation of PI3K/AKT pathway and downregulation of activated Caspase-3.
ABSTRACT
Objective To investigate the role of TLR4/NF-kB signaling pathway in inhibited invasion ability of pancreatic cancer cells caused by triptolide (TP).Methods PANC1 cells were divided into parental cells group,TP group,lipopolysaccharide (LPS) group and TP + LPS group.50 ng/ml of TP was added in culture medium in TP group,and 1 μg/ml of LPS was added in culture medium in LPS group,while 50 ng/ml of TP was pretreated for 2 h and 1 μg/ml of LPS was added in culture medium in TP + LPS group.All the ceils were cultured for 24 h.The TLR4 and matrix metalloproteinase-9 (MMP-9) mRNA and protein expression were evaluated by real-time PCR and Western blot.The NF-kB activity was determined by dual-luciferase reporter assay system.The invasion ability of pancreatic cancer cells was evaluated by transwell invasion chamberassay.Results The TLR4 mRNA expressions in parental cells group,TP group,LPS group and TP + LPS group were 0.41 ± 0.06,0.46 ± 0.10,0.20 ± 0.04,0.25 ± 0.06 ; the TLR4 protein expressions were 0.55 ±0.06,0.55 ±0.06,0.18 ±0.04,0.13 ±0.00; the activities of NF-kB were 13.0 ±3.0,31.6 ±4.3,7.3 ±1.5 and 10.8 ± 2.1,and the numbers of invasion cell were (56.8 ± 8.6),(104.5 ± 12.8),(32.0 ± 5.7) and (46.8 ± 7.0) ; the MMP-9 mRNA expressions were 0.36 ± 0.05,0.58 ± 0.07,0.18 ± 0.03,0.30 ± 0.004 ;the MMP-9 protein expressions were 0.31 ± 0.04,0.53 ± 0.08,0.11 ± 0.02,0.15 ± 0.00.In LPS group,TLR4 mRNA and protein expressions were not statistic significant when compared with those in parental cells group,but the activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions were statistically increased when compared with those in parental cells group (t =8.654,7.593,6.655,4.982,P ＜0.01).TLR4 mRNA and protein expressions,activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions in TP group were significantly lower than those in parental cells group (t =-7.609,-9.948,-4.176,-5.915,-8.179,-9.948,P＜ 0.01).TLR4 mRNA and protein expressions,activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions in TP +LPS group were significantly lower than those in LPS group (t =-4.437,-14.805,-10.506,-9.700,-9.055,-8.932,P＜ 0.01).Conclusions TP can inhibit pancreatic cancer cell invasion,and the mechanism is related to the inhibition of TLR4/NF-kB signaling pathway and down-regulation of MMP-9 expression.