The Signal Mechanism Underlying Fas-AD-induced Apoptosis In Bel-7402 Cells

Fas(CD95/APO-1)is one of the representatives of the death receptor subgroup of the tumor necrosis factor(TNF)receptor/nerve growth factor receptor(NGFR) superfamily and has been implicated in a wide range of physiological and pathophysiological apoptosis-related processes,including tumor surveillance,immune privilege,fulminant hepatitis,and neurodegeneration.Fas has previously been used in anticancer therapy,however,the therapeutic application of Fas was largely limited due to its general toxicity and the fact that it activates the NF-κB-family transcription factors,which are proinflammatory and antiapoptotic.To overcome this problem in vitro,specific NF-κB inhibitors or transcription or protein synthesis inhibitors such as actinomycin D are usually used in combination to increase Fas killing of tumor cells.Apoptosis or programmed cell death is an essential phenomenon during embryogenesis and development,and it maintains tissue homeostasis of the adult. Among these members,the Fas antigen(Fas)is a next cell surface receptor that triggers apoptosis in sensitive cells when it binds to Fas ligand(FasL)or agonistic anti-Fas antibody,and induce receptor trimerization to transduce signaling pathways.Molecule mechanism of Fas-induced cells apoptosis has been extensively researched.Multysignaling pathways can be triggered in the process of Fas-AD-induced cells apoptosis, in which caspase is known for executing programmed cell death.Apoptosis normally occurs in mammalian cells by one of two pathwathes: mitochondrial-dependent pathway or mitochondrial-independent pathway.In particular, the Bcl-2 family proteins play a central role in the regulation of mitochondrial apoptosis pathway.Some studies have demonstrated the Bcl-2 family stimulates mitochondrial cytochrome c release,the cytochrome c together with Apaf-1 activates caspase-9,and the latter activates caspase-3 and consequently resulting in cell apoptosis.For mitochondrial-independent pathway,induction of apoptosis via death receptors typically results in the activation of an initiator caspase such as caspase-8. These caspases can then activate other caspases in a cascade.This cascade eventually leads to the activation of the effector caspases,such as caspase-3 and caspase-6. Caspase-8 can activate caspase-3 directly,besides,it can activate caspase-3 indirectly by endocytoplasmic reticulum(ER).In both pathways,the final step involves activation of the caspase proteins from their inactive forms by means of large multi-protein complexes.Once activated,the caspases act as proteases,cleaving various substrates that lead to the death of the cell, in which caspase-8 is the apical caspase essential for triggering Fas-induced apoptosis. Among caspase subfamily,caspase-3 is especially important for the understanding of apoptotic cell death because of its variant substrate-specificity.The retinoblastoma(Rb)tumor suppressor protein plays a vital role in regulating the proliferation of normal mammalian cells by maintaining the integrity of the G₁/S checkpoint.It is generally accepted that inactivation of Rb at the G₁/S boundary by phosphorylation releases transcriptionally active E2F which can induce the cell cycle genes,facilitation S-phase entry and cell proliferation.Mitogen-activated protein(MAPK)have been implicated as an upstream signal for the initiation of apoptosis.MAPK include extracellular signal-regulated kinase (ERK1/2),NH₂-terminal protein kinase(JNK),p38 kinase and ERK5.They are members of separated modules regulated by distinct extracellular stimuli.In mammalian cells,MAPKs can transduce a diverse extracellular stimuli(such as UV radiation and osmotic stress)to the nucleus via kinase cascades to regulate cell survival, differentiation and apoptosis.The regulation and role of this pathway in death receptor-induced apoptosis remain unclear and may depend on the specific death receptor and cell types.But,in the process of Fas-AD-induced human hepatoma cells apoptosis,it remains to be unclear whether P38MAPK and caspase-8 are involved in together,the relation between P38MAPK and caspase-8,the relation between P38MAPK,caspase and Bcl-2, RB.Therefore,in this study,we focused on the involvement of P38MAPK and caspase-8 in the apoptosis of Bel-7402 cells,how P38MAPK and caspase-8 activations participate in the apoptosis of Bel-7402 cells by the role of P38MAPK inhibitor SB203580 and caspase-8 inhibitor Ac-IEFD-cho,to reveal the signal transducation mechanism of apoptotic Bel-7402 cells.Methods1.Fas-AD-treated Bel-7402 cells viability rate was measured by a MTT assay. Bel-7402 cells(1×10⁴/well)were incubated with different concentrations of Fas antibody in 96-well plates for 24 h in the presence of AD(20μM),and measured at different time.2.The effects of Fas-AD,SB20358 and Ac-IEFD-cho on apoptosis in Bel-7402 cells were determined by the inverted research microscope,electron microscopy and flowcytometer respectively.3.The activity effect of Fas-AD SB20358 and Ac-IEFD-cho on the P38MAPK, caspase-8 and caspase-3 of Bel-7402 cells and the effect of them on protein expression of FasR,Bcl-2 and Rb induced by Fas-AD were estimated by Western-blot and RT-PCR.4.The location of P38MAPK and caspase-3 in cells was observed with a confocal laster scanning microscope by immunofluorescence.5.The relation between P38MAPK and caspase-3,caspase-3 and Rb was identified by immunoprecipitation. Results1.Fas-AD could significantly suppress Bel-7402 cells proliferation and had does-depending and time-depending relationship with Fas anyibody consentration.2.Fas-AD could significantly induce Bel-7402 cells apoptosis,which could be suppressed by SB203580 and Ac-IEFD-cho.It was showed under inverted research microscope that after being treated with Fas-AD for 24 h,Bel-7402 cells showed the apoptosis character.Lots of vescile was observed in cytoplasm,the number of nucleolus decreased and the number of dying cell increased.It was showed under the electron microscopy that after being treated with Fas-AD for 24 h,the typical character of apoptotic cell was observed,such as cell shrinkage, chromatin condensation,nucleus,cytoplasm fragment and apoptotic body.The Annexin-FITC/PI flowcytometer asssay showed Fas-AD significantly induced Bel-7402 cells apoptosis compared with the control group,the apoptotic rate of Bel-7402 cells was 50.55±3.25%after being treated with Fas-AD for 24 h.3.Fas-AD could significantly induce FasR expression after Bel-7402 cells were treated with Fas-AD for 24 h.The western-blot data showed FasR expression increased with Fas antibody consentration increasing,however,the tendence of increase slowed down when Fas antibody consentration is 10μM.4.P38MAPK and caspase-8 were involved in Fas-AD apoptotic pathway and interacted after Bel-7402 cells were treated with Fas-AD for 24 h.That MAPK inhibitors and caspase-8 inhibitors interfered with Fas-AD-induced apoptosis strongly suggested that P38MAPK and caspase-8 played an important role in Fas-AD induced apoptosis.5.The effect of SB203580 and Ac-IEFD-cho on the process of Fas-AD-induced apoptosis indicated P38MAPK and caspase-8 regulated the expression of Bcl-2 and RB after Bel-7402 cells were treated with Fas-AD for 24 h.P38MAPK and caspase-8 could decrease the expression of Bcl-2 and increase the expression of RB significantly.6.P38MAPK and caspase-8 induced the expression of caspase-3 and activated caspse-3 after Bel-7402 cells were treated with Fas-AD for 24 h.The activated caspase-3 increased with Fas antibody consentration increasing after Bel-7402 cells were treated with Fas-AD for 24 h.P38MAPK inhibitors and caspase-8 inhibitors could significantly surpess caspase-3 expressien and activation.7.In the process of Bel-7402 cells apoptosis induced by Fas-AD,P38MAPK and caspase-3 translocated from cytosol to nucleus and temporary P38MAPK/caspase-3 complex was formed after Bel-7402 cells were treated with Fas-AD for 24 h,but SB203580 and Ac-IEFD-cho can significantly prevent P38MAPK and caspase-3 translocating.8.In the process of Bel-7402 cells apoptosis induced by Fas-AD,Caspase-3 interacted with RB and formed the caspase-3/RB complex after Bel-7402 cells treated with Fas-AD for 48 h.Conclusion1.Fas-AD can suppress Bel-7402 cells proliferation and induce Bel-7402 cells apoptosis.2.Fas-AD induce Bel-7402 cells apoptosis through P38MAPK,caspase-8 signal transduction pathway.3.In the process of Bel-7402 cells apoptosis induced by Fas-AD,P38MAPK and caspase-8 regulate mutully not only through the protein level but also through mRNA level.4.Fas-AD activates caspase-3 by activating P38MAPK and caspase-8,and caspase-3 actes on its downstream RB and regulates RB expression.P38MAPK and caspase-8 can surpress Bcl-2 expression and accelerate apoptosis.5.In the process of Bel-7402 cells apoptosis induced by Fas-AD,P38MAPK and caspase-3 translocates from cytosol to nucleus and temporary P38MAPK/caspase-3 complex is formed which further regulates caspase-3 expression.When caspase-3 disassociates from P38MAPK/caspase-3 complex,it interactes with RB and regulates RB expression,then results in Bel-7402 cells apoptosis.