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Objective:To investigate the regulation effect of miR-125b in the gastric cancer cell growth mediated by apoptosis related protein (Fas)/apoptosis related protein ligand (FasL) signal.Methods:Gastric cancer SGC-7901 cells were cultured in vitro. MiR-125b inhibitor sequence, NC sequence and transfection reagent were transfected into SGC-7901 cells and divided into three groups: miR-125b inhibited group, NC group and control group. The expression of miR-125b in transfected cells was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The colony formation was detected by plate cell clone formation assay. Cell apoptosis and cycle were detected by flow cytometry. The protein expression of Fas and FasL was detected by Western blot. The targeted regulation of Fas by miR-125b was detected by luciferase activity assay. Results:The expression level of miR-125 and the number of cell colony in miR-125b inhibited group was significantly lower than those in control group and NC group, and the inhibition rate of cell proliferation and apoptosis rate were significantly higher than that in control group and NC group (all P<0.05). The DNA content in G 1 phase in miR-125b inhibited group was significantly higher than that in control group and NC group, and the DNA content in S phase in miR-125b inhibited group was significantly lower than that in control group and NC group (all P<0.05). The expression of Fas and FasL protein in miR-125b inhibited group was significantly higher than that in control group and NC group (all P<0.05). The target site of miR-125b was found in 3′-UTR of Fas mRNA, and compared with the NC+ Fas 3′UTR-Wt group, the activity of luciferase in the miR-125b inhibited group+ Fas 3′-UTR-Wt group decreased significantly ( P<0.05). Conclusions:Inhibition of miR-125b expression can activate Fas/FasL signal and inhibit SGC-7901 cell proliferation, induce G 1 phase arrest of cell cycle and promote apoptosis.
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Objective: To explore the possibility of promoting tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis in prostate cancer PC-3 cell by inhibiting Krüppel-like factor 5 (KLF5). Methods: MTT assay, flow cytometry, Western blot assay and qRT-PCR assay were deployed to detect the cell viability, apoptosis and apoptotic markers in KLF5-inhibited and TRAIL-induced PC-3 cells. Results: After KLF5 was inhibited in TRAIL-induced PC-3 cells, cell viability reduced, apoptosis enhanced, the expressions of DR4 and DR5 increased while the expression of cellular fas-associated death domain-like interleukin-1β converting enzyme inhibitory protein (c-FLIP) decreased. Conclusion: Inhibiting KLF5 suppresses cell viability by promoting TRAIL-induced apoptosis in prostate cancer cell PC-3. It may be a potential means to treat hormone-insensitive prostate cancer.
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Objective To explore the influence of down-regulating Daxx on cell cycle and chemotherapeutic drug resistance in human ovarian cancer cells.Methods SiRNA and NC negative control(NC) RNA of Daxx were constructed and divided into the NC group and silencing Daxx(siDaxx) group after transfecting to ovarian cancer cell line C13k.The doxorubicin concentration gradient of 0,0.15,0.30,0.60 μmol/L was set.The cellular cycle changes and apoptosis changes of these two groups were detected by using the flow cytometry.Western blot was used to detect the expression changes of apoptosis related protein cyclinB1 and cleavedparp.Results 0.30 μmol/L doxorubicin down-regulated Daxx to result in significant G2/M arrest(P＜0.05).Down-regulating Daxx resulted in doxorubicin resistance in C13k cells.Conclusion The effect of Daxx on ovarian cancer chemotherapy might be related to its regulation on cell cycle.
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Objective:To investigate the damage effect of bisphenol A (BPA)on the testis tissue of adult mice, and to reveal the reproductive toxicity of BPA in the body of animal and mechanism.Methods:40 KM mice aged 8 weeks were randomly divided into control group (according to the weight ratio of corn oil gavage), low dose of BPA group (100 mg·kg-1BPA),moderate dose of BPA group (200 mg·kg-1 BPA),and high dose of BPA group (400 mg·kg-1 BPA).4 weeks laster,the testis tissue was taken.The apoptotic rates in the testis tissue were detected by flow cytometry;the distribution and expression of Fas and FADD were measured by immunohistochemistry.Results:Compared with control group,the apoptotic rate,the expression rates of Fas and FADD in testis tissue of the mice in low dose of BAP group had no changes (P>0.05),while the apoptotic rates in the testis tissue and the positive expressions rates of Fas and FADD in moderate and high doses of BPA groups were increased (P<0.05).Compared with low dose of BPA group,the apoptotic rates and the positive expression rates of FAS and FADD in tests tissue of the mice in moderate and high doses of BPA groups were significantly increased (P<0.05).Compared with moderate dose of BPA group,the apoptotic rate and the positive expression rates of FAS and FADD in testis tissue of the mice in high dose of BPA group was significantly increased (P<0.05).The overexpression of Fas and FADD was positively correlated to the apoptotic rate in testis tissue in moderate dose of BPA group (r=0.430,P<0.05;r=0.238,P<0.01)and high dose of BPA group (r=0.637,P<0.01;r=0.359,P<0.01).Conclusion:BPA with content dose can increase the apoptotic rates of cells in testis tissue and the expressions of Fas and FADD.BPA’s reproductive toxicity may be closely associated with the activation of Fas signal pathway and resulting in massive apoptosis.
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Objective To investegate the effect of ginsenoside Rg1 on the apoptosis related protein FLICE-inhibitory protein(FLIP),Fas-associated death domain protein (FADD)and Caspase-3 in the subatania nigra(SN)of 1-methyl-4-phenyl-1,2,3,6-tetrahyd-ropyridine (MPTP)-induced mouse models of Parkinson’s disease(PD), and to investigate the role of FADD and FLIP in the pathogenesis of PD and the protective effect of ginsenosides Rg1 on dopaminergic neurons.Methods 45 C57BL/6N mice were randomly divided into control group,model group and ginsenoside Rg1 group (n=15).The mice in model group were injected with MPTP by intraperitoneal,the mice in Rg1 group were injected with ginsenoside Rg1 before injecting MPTP,and the mice in control group were injected with normal saline by intraperitoneal. The behavioral changes of the mice in various groups were observed, and immunohistochemistry and Western blotting methods were used to observe the expressions of tyrosine hydroxylase (TH),FADD,FLIP and Caspase-3 in substantia nigra of the mice.Results Compared with control group,the mice in model group presented with typical symptoms of PD, the TH-positive neurons in the subatania nigra was significantly reduced (P<0.01 ), the number of FADD, FLIP and Caspase-3 positive cells was significantly increased(P<0.01),and the cytoplasm was deeply stained;the protein expression levels of FADD,FLIP and Caspase-3 were significantly increased (P<0.01).Compared with model group,the PD symptoms of the mice in ginsenoside Rg1 group reduced, the number of TH-positive neurons was significantly increased, the number of positive cells of FLIP,FADD and Caspase-3 were significantly reduced(P<0.01),and the cytoplasm was lightly stained;the protein expression levels of FADD, FLIP and Caspase-3 were significantly reduced (P<0.01 ). Nonlinear correlation analysis found that there was a positive relationship between the number of FADD and Caspase-3 positive cells (r=0.791,P<0.05).Conclusion Ginsenoside Rg1 may play a neural protective effect dopaminergic on neurons by modulating the FADD and FLIP expressions in SN of PD model mice.
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Objective To investigate the effects of gene silencing of Fas-associated death domain (FADD) with synthetic small interfering RNA (siRNA) on apomorphine-induced contralateral rotation,and the expression of Fas and caspase-8 in rat models of parkinsonism. Methods Sprague-Dawley rats were randomly divided into 5 groups:control group,Parkinson' s disease (PD) group,FADD siRNA group,FADD siRNA positive control group and FADD siRNA negative control group. Synthetic FADD siRNA sequences,siRNA positive sequences or siRNA negative sequences were infused into right substantianigra of midbrain using RNA interference and stereotactic techniques before parkinsonian rat model establishment.Apomorphine-induced contralateral rotations of the rats were observed after the injection.The protein and mRNA expression levels of FADD,Fas and caspase-8 were measured by Western blot and RT-PCR.Results In the control group,no rotation was observed after injecting apomorphine; however,in the rest groups,the number of rats respectively was 12 (12/14),3 (3/13),4 (4/15) and 11 ( 11/14 ) in apomorphine-induced contralateral rotation,which had significant statistical differences ( x2 ＝ 18.56,P =0.000).In parkinsonian substantia nigra,marked increases in the protein and mRNA levels of FADD,Fas and caspase-8 were observed,compared with control group.Furthermore,compared with PD group,FADD protein and mRNA levels were strongly suppressed by administration of FADD siRNA in FADD siRNA group. FADD siRNA administration also resulted in great attenuation of 6-hydroxydopamine-induced increases in expression and activation of caspase-8.However,no decrease in expression of Fas was observed in FADD siRNA group and FADD siRNA positive control group,compared with PD group.Conclusion Our results suggest that death receptor signaling pathway plays a critical role in the pathogenesis of PD.FADD siRNA is effective against this pathway and it may,at least in part,provide a potential neuroprotective effect.
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Objective To examine the sensitivity of human colorectal carcinoma cells to 5-fluorouracil treatment by stable transfection of extrinsic Fas-associated death domain protein(FADD) gene，both in vitro and in vivo，so as to investigate the feasibility of combination therapy of FADD gene and 5-fluorouracil in human colorectal carcinoma.Methods①RT-PCR and Western blotting were used to detect the expressions of both mRNA and protein of FADD gene in SW480／FADD (stably transfected with FADD)，SW480／neo and SW480 cells.②After treatment with 5-fluorouracil as an apoptotic inducer，in vitro cell growth activities were investigated by MTT assay.Cell apoptosis and its rates were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assay and flow cytometry of annexin V-FITC／PI staining.The expressions of caspase-8 and caspase-3 were examined by Western blotting.③To examine the inhibitory effect of FADD gene combined with 5-fluorouracil, tumor xenograft model was prepared for in vivo study.Results ① Compared with SW480 and SW480/neo cells, FADD mRNA and protein levels of SW480/FADD cells were higher (P<0.05). ② Inhibitory rate of SW480/FADD cells was remarkably higher than SW480 and SW480/neo cells (P＜0.05 ). ③ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L), the apoptotic rate of SW480/FADD cells was (33.3 ± 4.5)%, which was higher than SW480 [(13. 9 ± 3. 2)%3 and SW480/neo [(14. 1 ± 3. 4)%], with significant difference (P< 0.05).④ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L),procaspase-8 and procaspase-3 expressions of SW480 and SW480/neo cells were higher than SW480/FADD cells, whereas their cleaved caspase-8 and cleaved caspase-3 expressions were lower than SW480/FADD cells (P＜0. 05).⑤ In in vivo study, SW480/FADD cells increased the efficacy of fluorouracil-induced inhibition of tumor growth in nude mice. ConclusionsStable overexpression of FADD increases sensitivity of the cells to 5-fluorouracil and combination of FADD with 5-fluorouracil will he a promising alternative in colorectal cancer treatment.
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Objective:To imestigate the effect of Shenxiong injection on neuronal aooptosis and Fasassociated death dormin protein(FADD)and its mRNA expression after ischernia-reperfusion in rats.Methods:Atotal of 100 SD rats wgre randomly allocated into normal(n=10),sham-operation(n=10),cerebral ischemia-reperfusion(n=40),and Shenxiong injection(n=40)groups.A model of middle cerebral wtery occlusion was induced by suture method.The neuronal apoptosis was detected by the TUNEL assay.The expressions of FADD protein and mRNA were detected by inmmnohistochemical staining and reverse tramcription-polymerase chain reaction(RT-PCR),respectively.Results:As compared with the normal and sham-operation groups.the numbers of apoptotic cell in the cerebral ischemia-reperfusion group were increased significantly(P＜0.01),and the expressions of FADD protein and mRNA were enhanced significantly(all P＜0.01).As compared with the ceretral ischemia-reperfusion group,the numbers of apoptotic cell were decreased significantly (P＜0.05,P＜0.001),and the expressiom of FADD protein and mRNA were reduced significantly in the Shenxiong group(P＜0.05,P＜o.001).Convlusions:Shertxiong injection may inhibit the neuronal apoptosis after ischemia-reprfusion in r-cas by down-regulaftng the expression of FADD.
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Objective To investigate the dose and time kinetics of induction of apoptosis induced by doxorubicin in J urkat leukemiacells, and to explore its pertinent molecular mechanisms. Methods Human Jurkat leukemia T - cells were treated with doxorubicin at theconcentration of 0.1 mg/L, 0.2 mg/L, 0.5 mg/L and 1.0 mg/L for 6,12,24 and 36 hours, respectively, of which one sample was pre-treated with zVAD- fmk (benzyloxycarbonyl - Val -Ala - Asp - fluoromethylketone) prior to addition of doxorubicin 0.2 mg/L. Apop-tosis was detected with both annexin V - FITC and propidium iodide ( PI ) staining and annexin V FITC and PI double positive cellswere analyzed by flow cytometry. Western blot was used to evaluate the level of Fas ligand (FasL) and FADD (Fas - associated death do-main) expression. Results The differences of apoptotic cells induced by all dose of doxorubicin were not significant (P＞0.05 ) at 6hour;at 12 hour, only the highest dose, 1 mg/L, significantly induced cell apoptosis;while the lowest dose,0.1 mg/L, did not significantlycaused cell apoptosis for all time points. After exposure to the doses of 0.2 and 0.5 mg/L for 24 or 36 hours,a significant increase in per-centage of apoptotic cells was observed (P ＜ 0.001 ). Apoptosis induced by doxorubicin was completely inhibited when the cells were incu-bated with doxorubicin in the presence of zVAD - fmk (P ＜ 0. 001 ). The level of FasL and FADD expression correspondingly increasedwith exposure time to doxorubicin. Conclusions Doxorubicin induces apoptosis in a dose - and time - dependent manner; upregulatedFasL may initiate the activation of the Fas signaling pathway and caspases are the ultimate executioner in the induction of leukemia cellapoptosis by doxorubicin.
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Objective: To investigate the induction of Tca8113 cells to apoptosis by fadd gene.Methods: RT PCR and recombinant PCR were used to amplify human fadd gene and cloned into expression vector pcDNA3 and pIRES2 EGFP, then transfered into Tca8113 cells. The growth and apoptosis of the cells were tested by cell counting,fluorescent microscopy, electron microscopy and flow cytometery. Results: fadd gene was obtained and transfered into Tca8113 cells. After transfection of the gene the growth of the cells was inhibited by 25%～52%, cell number in G 1 phase increased and that in S phase decreased. Apoptosis of the cells was observed. Conclusion: fadd gene can effectively inhibite cell growth and induce Tca8113 cells to apoptosis.
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0.05) at 6 hour;at 12 hour,only the highest dose,1 mg/L,significantly induced cell apoptosis;while the lowest dose,0.1 mg/L,did not significantly caused cell apoptosis for all time points.After exposure to the doses of 0.2 and 0.5 mg/L for 24 or 36 hours,a significant increase in percentage of apoptotic cells was observed(P