ABSTRACT
Objective To investigate the inhibitive effects of viral protein r (Vpr) of human immunodeficiency virus 1 ( HIV-1 ) on human colorectal cancer cell line HCT-8,and to find the possible mechanisms.Methods The HCT-8 cells were divided into the control group,adv group and adv-Vpr group.HCT-8 cells were not treated in the control group; HCT-8 cells were treated with Adv or Adv-Vpr at different multiplicity of infection (MOI) in the Adv group or Adv-Vpr group,respectively.Cell proliferation was detected by MTT assay.Cell cycle,apoptosis and mitochondrial membrane potential were detected by flow cytometry.The expression of apoptosisrelated proteins was detected by Western blot.All data were analyzed by using the q test,t test and one-way or two-way analysis of variance.Results The proliferation of HCT-8 cells was significantly inhibited by Vpr.The MTT value of HCT- 8 cells in the Adv-Vpr group was 1.03 ± 0.04,which was significantly lower than 2.46 ± 0.15 in the Adv group and 2.51 ± 0.14 in the control group at 72 hours after Adv-Vpr transfection ( MOI =200) ( F =144.6,P ＜ 0.05).The ratio of HCT-8 cells in the G2/M phase was 37.31％ ± 5.90％ in the Adv-Vpr group,which was significantly higher than 18.30％ ± 6.04％ in the Adv group and 16.66％ ± 3.51％ in the control group ( F =10.08,P ＜ 0.05 ).The ratio of HCT-8 cells with decreased mitochondrial membrane potential was 32.07％ ±5.64％ in the Adv-Vpr group,which was significantly higher than 3.32％ ±0.79％ in the Adv group and 2.76％ ±1.43 ％ in the control group at 48 hours after Adv-Vpr transfection ( MOI =200) ( F =64.45,P ＜ 0.05).The apoptosis rate of HCT-8 cells was 37.62％ ±6.48％ in the Adv-Vpr group,which was significantly higher than 3.44％ ± 1.11％ in the Adv group and 2.93％ ± 1.07％ in the control group at 72 hours after Adv-Vpr transfection ( MOI =200) ( F =122.4,P ＜ 0.05 ).The results of Western blot showed that Vpr induced cleavage and activation of Caspase-9 and Caspase-3 and phosphorylation of Chk1-S345,while the expression levels of Fas,Fas-L,ERK1,ERK2 remained the same at 48 hours after Adv-Vpr treatment ( MOI =200).Conclusions Vpr inhibits the proliferation of the HCT-8 cells in vitro through G2/M phase arrest and apoptosis.Vpr plays its role by activating DNA damaging pathway and initiating mitochondria apoptotic pathway.Vpr is a potential therapeutic agent for colorectal cancer.
ABSTRACT
Objective To unravel the relationship between Toll-like receptor 4 (TLR4) expression in platelets and the activation of platelet in thrombocytopenia (TCP) in case of sepsis. Method A total of 64 patients with sepsis were prospectively studied with control. Platelet count (PC), mean platelet volume (MPV),platelet distribution width (PDW),platelet TLR4 expression, platelet PAC-1 expression, sCD40L and TNF-α concentrations were compared between healthy control group (15 cases) and sepsis group on admission and 3 d,Sd,and 9 days after admission. The changes of MPV and PDW in TCP and non-TCP subgroups of sepsis before and after treatment were recorded. Prognostic index was analyzed. Results The PC was lower in sepsis group (P=0.006), and MPV and PDW were higher in sepsis group than those in healthy control group (P = 0.046, P =0.001). Platelet TLR4 and PAC-1 expressions, and sCD40L and TNF-α levels also increased more significantly in sepsis group (P ＜ 0.001). PAC-1 expression and TNF- level were higher in TCP group than those in non-TCP group before and after treatment (P = 0.023, P = 0.011). The sCD40L concentration and platelet TLR4 expression were significantly higher in treated TCP groups than in non-TCP group (P = 0.047, P = 0.001). Compared with non-TCP,the rate of bleeding was higher (P = 0.024) and the length of ICU stay was longer (P = 0.013).The APACHE Ⅱ score and the 28-day mortality were higher in TCP group (P ＜ 0.01, P = 0.048). Conclusions The elevation of platelet TLR4 expression in sepsis along with platelet activation is closely related to the incidence of thrombocytopenia. The occurrence of TCP is a poor prognosis sign of sepsis.
ABSTRACT
Objective To affirm the expression of Toll like receptor 4 (TLR4) on the surface membrane of platelet and to explore the immunomodulatory factors[(interlukine-8(IL-8),β-thromboglobulin(β-TG), soluble CD40 ligand(sCD40L)] released by platelets after platelets stimulated by TLR4 ligand.Methods TLR4 expressed on the platelet was detected by flow cytometry. Monoclonal anti-human FcγRⅡantibody(Ⅳ.3)-treated human platelets were cultured with LPS in the presence or absence of blocking monoclonal antibody to human TLR4. The release of IL-8, β-TG, sCD40L were measured by specific enzymelinked immunosorbent assay. Results Human platelets could express functional TLR4. The detection rate of TLR4 on platelets were decreased after LPS involvement(P<0.01). It was noted that sCD40L and β-TG were present in large concentration in the release of platelets stimulated by TLR4 ligand but the release of IL-8 was independent of platelet activation after TLR4 engagement. The concentration of sCD40L and β-TG had no statistical difference between 1-5 μg/ml LPS. The effects of LPS on the modulation of secretory factors were attenuated by preincubation of platelets with an anti-TLR4 monoclonal antibody. Conclusion The TLR4 on platelet could recognize and link LPS, induce the release of sCD40L, β-TG by platelet, but could not influence IL-8.