| Objectives: Toxicarioside H(ToxH)is a new type of cardiac glycoside.In our previous study,we found that a variety of cardiac glycoside compounds can induce tumor cell damage,and induce apoptosis by mitochondrial pathway.In addition,other studies have found that cardiac glycosides can induce autophagy through various signaling pathways.Mitophagy is a mechanism to protect mitochondria from injury.Our previous studies found that cardiac glycosides could increase mitochondrial membrane potential and promote apoptosis of tumor cells through mitochondrial pathway.Therefor,we supposed that ToxH may protect cells damage by mitophagy.In the present study,we use the methods of molecular biology and cell biology to further understand whether ToxH can induce mitophagy in A549 and H460 lung cancer cells,and to study its molecular mechanism preliminarily.Methods: Different doses of ToxH were treated A549 and H460 lung cancer cells,and the cells were observed and detected at different time.The cellular morphology was observed directly by microscope.MTT and EDU studies were used to detected the influence of ToxH on lung cancer cells growth and proliferation.The changes of mitochondrial membrane potential were analyzed by JC-1 staining,the results of direct observation by microscopes,and combined with flow cytometry.Apoptosis,mitophagy(including autophagy)and the expression of signal pathway related protein molecules were detected by Western Blotting.At the same time,the copolymerization of autophagy markers(LC3,P62,etc.)and mitochondrial autophagy markers(Mito-Red)was observed by laser confocal microscopy in order to understand whether mitophagy was emerged.RNA interference(siRNA)or autophagic blockers were used to interfere the related molecules,and the relationship between ToxH induced mitophagy and related molecules autophagy were analyzed by all the above methods.Co-immunoprecipitation combined with Western Blotting research were used to analysis the relationship between mitophagy and corresponding molecules.Results: Through direct observation of the microscope were found that the number of cells decreased gradually as the ToxH dose increased,the connection between cells were disappeared,the cells gradually fell off,the shape of the cells were changed,and the number of the cells death were increased.The results of MTT and EDU showed that ToxH could significantly inhibited the growth and proliferation of lung cancer cells.After JC-1 staining,it was observed directly under the fluorescence microscope and found that the cells without ToxH treatment showed a significant red change,but the green part in the cells increased with the increase of ToxH dose.The quantitative analysis of the membrane potential with the flow cytometry showed the same changes.Those results indicated that the mitochondrial membrane potential or mitochondrial damage increased with the increase of the dose of ToxH.The expression of apoptosis related expression molecules was detected by Western Blotting.It was found that the expression of cytochrome C(Cyt C)declined in the mitochondria of the lung cancer cells with the prolongation of the ToxH processing time,while the expression of Cyt C in the cytoplasm increased correspondingly.Moreover,the expression of PARP,Caspase 3 and Caspase 9 was also increased,but Caspase 8 haven't changed,which indicated that ToxH could induce apoptosis of lung cancer cells by damaging the mitochondrial pathway.Western Blotting was also used to detected the expression of mitochondrial membrane protein.After treated by ToxH,the expression of Tom20,Tim23 and Hsp60 in cells were down regulation obviously.The number of LC3 spots in ToxH treated cells was significantly increased was found by confocal microscopy,and there were obvious co localization of these LC3 spots and Mito-Red labeled mitochondria and P62 molecules.The autophagy markers in cytoplasm and mitochondria were detected by Western blotting.It was found that the mitochondria of ToxH treated cells contain more P62,LC3-II and Parkin,indicating that ToxH can induce mitophagy through Parkin pathway in lung cancer cells.In order to further understand the molecular mechanism of ToxH induced mitophagy in lung cancer cells,we detected the expression of Sirt3 and Tom20 by Western blotting firstly.It was found that the expression of Sirt 3 in the ToxH treated cells was significantly higher,but the expression of Tom20 decreased obviously.And the expression of Tom20 was restored after RNA interfered Sirt3(siSirt3)expression.LC3 spots were not been found and Mito-Red has obvious copolymerization phenomenon in lung cancer cells which treated with siSirt3 and ToxH.Besides,it was found that no obvious P62,LC3-II and Parkin were detected in the mitochondria of lung cancer cells treated by ToxH after RNA interfered Parkin(siParkin)or autophagic blocker inhibited mitophagy.These results suggest that ToxH promotes mitophagy by inducing Sirt3 high expression in lung cancer cells.In order to clarify the molecular mechanism of ToxH induced high expression of Sirt3 in lung cancer cells,we detected the relationship between Sirt3,hexokinase II(Hex II),Parkin and voltage dependent anion channel protein 1(VDAC1)by Western blotting,and found that the expression of Sirt 3 was increased in the To H treated cells,and there was no obvious difference of total Hex II concentration in the control cells expression.The experiments detected the Hex II in the cytoplasm and mitochondria,respectively.It was found that the Hex II in the cytoplasm was obviously increased after ToxH treatment,which was not be found in the siStirt3 cells.The results suggested that Tox H couldn't promote re-synthetic expression of Hex II,but to urged the Hex II which mainly distributed in mitochondria transfer into cytoplasm.The interaction of VDAC1,Parkin and Hex II in the lung cancer cells which over expressing Parkin was detected by immunoprecipitation.In the control group(siCtrl),there were obvious Parkin and VDAC1 co precipitation,and Hex II and VDAC1 co precipitation were not be found in the ToxH treated cells.However,Hex II and VDAC1 co precipitation were not be found clearly in the without ToxH treated cells(UT),which contrary to ToxH group.After interfering with the expression of Sirt 3,the co precipitation of Parkin and DVAC1 was disappeared in siSirt3-ToxH group,while Hex II and VDAC1 co precipitated has been found.There was no significantly changes in UT group.These results suggest that ToxH induced mitophagy in lung cancer cells through high expression of Sirt3 to reduces the binding of Hex II to VDAC1,thereby promoting the binding of VDAC1 to Parkin and thus activating the Parkin pathway of autophagy.Finally,we interfered Sirt3 expression and autophagic blocker 3-MA inhibition or blocking-up ToxH induced autophagic,and then repeat the above methods to detect cell growth,proliferation and apoptosis.It was found that ToxH inhibited the growth and proliferation of lung cancer cells after interfered or blocked mitochondrial autophagy,and the apoptotic cells through mitochondrial pathway were significantly increased,indicated that ToxH induced autophagy is a protective autophagy.Conclusions: ToxH can inhibits the growth and proliferation of lung cancer cells,induces apoptosis through mitochondrial pathway,and promotes the mitophagy of lung cancer cells.ToxH induced mitophagy by up-regulation the expression of Sirt3 and down regulation the binding of Hex II to VDAC1,and then promoting the binding of VDAC1 to Parkin,which may be one of the mechanism for activating the classical Parkin pathway of mitophagy.ToxH induced autophagy is a protective autophagy,which suggested tha t combined with Blocking mitophagy drugs and new autophagy blocking techniques may be an important strategy to enhance the efficacy of ToxH antitumor drugs. |
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