The Mechanism Of Lysosomal-Mitochondraial Pathway On Cell Death Of Human Cervical Carcinoma Induced By Oxidative Stress

Programmed cell death was divided by Apoptosis,Autophgy and Necroptosis. Genes controlling programmed cell death were two sorts, in which one is inhibiting cell death, the other is promoting cell death.The two sorts of genes interact with each other in order to control cell normal death.Vitamin K is a kind of necessary vitamin. Study on Vitamin K suggested that it may have anti-tumor activity. At present, VK3 anti-tumor may be related to oxidative stress.ROS is increased in oxidative stress,which results in injury widely.Lately,study on apoptosis suggested that there was intracelluar abnormal signaling transduction. Intracellular acidification in these abnormal signaling, is a character of apoptosis. Intracellular pH adjust maybe tightly related to apoptosis.In order to investigate the roles of oxidative stress in inducing cell death, we make use of VK3 to induce Hela oxidative stress and cytosolic acidification, to observe mechanisms of oxidative stress in cell death.Methods:(1)To detect the apoptosis and autophagy mechanisms of VK3-induced Hela cells injury, by MTT,Hoechst33342,DCFH-DA immunofluorescent,RT-PCR,Western blot and transmission Electron Microscope.(2)To investigate the effect of oxydative stress througy lysosome on mitochondria in Hela cells death by AO, Rh123 immunofluorescent,MTT,RT-PCR and Western blot analysis and transmission Electron Microscope.(3)To observe role of intracellular acidification in Hela cells injuride by oxydative stress by BCECF-AM Flowcytometry analysis.Results:(1)During the VK3-induced Hela cells injuries, the survival rate was decreased and apoptosis appeared,with the increased level of ROS.The Caspase3 and cytC proteins expression were increased significantly. The ratio of Bax/Bcl-2 mRNA and protein was increased obviously. The expression of autophagy-associated Beclin 1 protein and LC3-II was up-regulated. Autophagic vacuoles was observed.(2)During the VK3-induced Hela cells injuries,lysosome was instabilized at 3h and mitochondrial△Ψm was decreased at 6h. Akt,mTOR and p70S6K mRNA expression and phospho-Akt/mTOR were all decreased.Mfn1,Opa1,MTP18 mRNA were all decreased and Mfn2,Drp1及Fist1 mRNA appeared no obvious change. In the VK3 combined with NAC group,lysosome stabilization and mitochondrial△Ψm were all not decreased. Akt,mTOR and p70S6K mRNA expression and phospho-Akt/mTOR were all not decreased.Mfn1,Opa1,MTP18 mRNA were all not decreased.In the VK3 combined with NH4Cl group,lysosome stabilization and mitochondrial△Ψm were all decreased obviously.Mfn1 mRNA expression was not decreased and Drp1,MTP18 mRNA expression was decreased.(3)BCECF-AM staining appeared cytoplasmic acidification in VK3 group. Compared to VK3 group,cytoplasmic acidification in VK3 combined with NAC group was decreased,while that in VK3 combined with NH4Cl group was increased severely.Conclusions:(1)By observing that cleavaged caspase3 and cytosolic CytC protein level were inceresed, Bax mRNA level was increased, Bcl-2 mRNA level was decreased,apoptosis cellular nuclear morphology of VK3-induced Hela oxydative stress,as well as Beclin1 and LC3-II protein expressions were increased with autophgosomes formation , while ROS level decresed,apoptosis nuclear appearance and autophagosome formation decreased induced by combinated with VK3 and NAC,suggests that ROS was related to programme I cell death(apoptosis) and also autophagy in Hela cells injuries.(2)By observing the death process of VK3-induced Hela cells in different time point,it is appeared that decreasing in red fluroscence and increasing in green fluroscence in the cytoplasm,it suggestes that lysosomal appeares instability.Moreover, lysosomal instability began at 3h induced by VK3, and mitochondrial clapse began at 6h,while lysosomal instability and mitochondrial△Ψm clapse not obvious induced by combinated with VK3 and NAC.It suggests that lysosomal stability was ahead of mitochondrial clapse. VK3 maybe injury mitochondria through lysosome.(3) By observing that the Akt,mTOR and p70S6K mRNA expressions obviously decreased,phospho-Akt at Ser-473 with phospho-mTOR at Ser-2481 decreased induced by VK3,then phospho-Akt,phospho-mTOR protein expressions decreased in combined with VK3 and NH4Cl,while Akt,mTOR, p70S6K mRNA expressions and phospho-Akt,phospho-mTOR were not decresed in combined with VK3 and NAC,it indicates that Akt/mTOR signal pathway may be involved in programme I cell death(apoptosis) and also autophagy in Hela cells injuries induced by VK3.(4)Mitochondrial mission genes Mfn1 and Opa1 as well as fussion genes MTP18 mRNA expressions were obviously decreased. Mfn1,Opa1 and MTP18 mRNA expressions were increase in combined with VK3 and NAC. It suggests that mitochondrial mission genes and fussion genes abnormal expressions may be involved in mitochondrial pathway death mechanism induced by oxydative stress.(5)Compared with VK3, cell viability by MTT,lysosomal stability and mitochondrial△Ψm were all decreased,as well as mitochondrial mission Mfn1,Opa1 genes espressions increasing and fussion gene Drp1 and MTP18 mRNA expressions decreasing in VK3 combined with NH4Cl group Hela cells. It suggests that lysosome through affecting mitochondrial death pathway maybe the main mechanism in ROS-induced Hela cells death.The balance between lysosome and mitochondria is important to cell survival.(6)By observing increase in autuphagosome and caspase3 protein expression in VK3 combined with NH4Cl group Hela cells,suggests that VK3 combined with NH4Cl may inhibit fussion process between autophagosome and lysosome,in turn strongly induces apoptosis.(7)FL1/FL2 ratio and intracellular pH were all decreased,and cell number with acidic intracellular pH increase(13.6%)induced by VK3. FL1/FL2 ratio and intracellular pH were all increased,and cell number with acidic intracellular pH decreased(5.4%)induced by combined with VK3 and NAC. FL1/FL2 ratio and intracellular pH were all obvious decreased and cell number with acidic intracellular pH increase(43.8%)induced by combined with VK3 and NH4Cl.It suggests that oxydative stress induces Hela cells death may be related to cytosolic acidification. Cytosolic acidification maybe along with apoptosis, moreover also result in lysosomal and mitochondrial injury aggravating lysosome-mitochondrial cell death pathway,which mechanism remain be further investigated.