Effects Of3-Methyladenine And Chloroquine On The Apoptosis Of Human Cervical Cancer Cells Induced By VK3

Aim:We used Human cervical cancer cells as the object of thisstudy,toinvestigate the effects and mechanisms of the autophagic specificinhibitor3-Methyladenine and Chloroquine on the apoptosis induced byVK3.Methods:The following groups were setd up in this study:control group,VK3group,3-MA group,VK3+3-MA group,CQ group,VK3+CQ group.(1)The expression of beclin1in Hela cells were detected by Westernblot;The accumulation of LC3were observed by fluorescencemicroscope;Fluorescence microscopic analysis for acridine orangestaining in Hela cells.(2)The survival rate of each group was detected byMTT method,and the apoptosis of Hela cells were observed byHoechst33258staining;The effect of caspase inhibitor ZVAD on thesurvival rate of Hela cells in each group was detected by MTTassay.(3)Cells of each group were labeled with Rhodamine123,the lossof ΔΨm was determined by flow cytometric analysis; The mRNAexpression levels of mitochondrial fusion and fission genes weredetected by RT–PCR.Results:(1)Western blot results showed the increased expression of Beclin1protein in VK3group,and the decreased expression level in3-MA and VK3+3-MA group;The expression of Beclin1was also markedlyincreased in CQ group,the level of that in VK3+CQ group was moreincreased.（2）The immunofluorescence results showed the increasedexpression of LC3in VK3group compared with control,the decreasedexpression in3-MA and VK3+3-MA group;In contrast,the expression ofLC3was significantly increased in CQ group,and appearing punctateaggregation,this phenomenon was more pronounced in VK3+CQ group.（3）Compared with control group,VK3made the integrity of lysosomemembrane decrease,the addition of3-MA resulted in the development ofdecrease,there was no differencebe observed between3-MA and controlgroup;In contrast,the intensity of the red fluorescence in CQ group isweaker than control,the red intensity of VK3+CQ is more weaker thanVK3or CQ group.(4) The proliferation of Hela cells was inhibited in adose-and time-dependent manner after VK3treatment;In VK3+CQ andVK3+3-MA group,the cell viability decreased compared with VK3group,no effect of3-MA or CQ on the viability of Hela cells wasobserved.(5)Hoechst33342staining results suggest that VK3made Helacells apoptosis,the addition of3-MA and CQ increased the apoptosisrate.(6)The addition of Z-VAD increased the cell viability ofVK3,VK3+3-MA and VK3+CQ group.(7)No effect of CQ onmitochondrial membrane potential(ΔΨm) was detected compared withcontrol,however,the loss ofΔΨm in3-MA group was detected.The ΔΨmof VK3group decreased compared with control,the addition of3-MA orCQ cause no significant change compared with VK3.(8)The expressionlevels of Mfn1,Mfn2,Opa1and MTP18mRNA decreased in VK3groupcompared with control,the addition of3-MA or CQ made the mRNAlevel of Mfn1,Mfn2and Opa1more dereased; The expression levels ofDrp1and Fis1mRNA increased in VK3group compared with control,the addition of CQ made the mRNA level of Fis1remarkedlyincreased compared with VK3group,VK3+3-MA caused an increasedexpression level of Drp1and Fis1mRNA compared with VK3group.Conclusion:(1)Hela cell death induced by VK3is associated withcaspase-dependent cell death,and caspase independent cell death.(2)Inthe process of VK3induced Hela cell death,the level of autophagy wasimproved. In VK3+CQ and VK3+3-MA group,the cell viabilitydecreased compared with VK3group,indicating that autophagy play aprotective role.(3)In the process of VK3induced Hela celldamage,mitochondrial membrane potential declined,mitochondrialfusion protein decreased and fission protein increased,indicating thatmitochondrial fusion function decline and/or fission functionincrease,resulting in decreased mitochondrial membrane integrity,maycause an increased release of ROS,make a vicious circle.(4)The differenteffect of3-MA and CQ in VK3induced mitochondrial fission-fusionimbalance may be associated with mitochondrial exposed to differentenvironments.