Mechanisms Of Chelidonine Inducing Mitotic Catastrophe And Apoptot-Like Death In SGC-7901 Human Gastric Carcinoma Cells

Mitotic Catastrophe(MC)is a type of cell death takes place during mitosis,which is induced by abnormal mitosis.When MC occurs,the cell went through polyploidy,plurality of micronuclei,aging,necrosis,apoptosis,and finally,death.In recent years,inducing tumor cells into MC has become a new way of anti-tumor therapy.Chelidonine is the main effective alkaloids constituents of Chelidonium majus L.known for antitumor activity.Our previous expperiments have proved that Chelidonine can induce MC in SGC-7901 cells.In this current study,we explored the mechanism of MC induced SGC-7901 cells and Apoptosis-like cell death,from the perspective of ultrastructure,apoptosis rate and expression of apoptosis related proteins,hoping to provide a scientific basis for the development and application of anticancer drugs.First,we studied the effect of 10μmol/L Chelidonine on cell ultrastructure by electron microscopy observation.The results showed that,after Chelidonine treatment for 24h,the cells become larger,get irregular shapes and more micronucleus,As the time of the treatment gets longer,we observed a reduction in microvilli,cells become multinucleated,chromatin condensation,nuclear fragmentation and formation of apoptotic body,which are all signatures of cell apoptosis.Then we use Western Blot method to detect the effect of Chelidonine on the expression of MC related kinases Aurora-A protein.The results showed that after Chelidonine treatment for 24h,compared with the negative control group,the expression of Aurora-A is reduced,similar as the VCR(positive control)group.Andas the time of the treatment gets longer,the expression of Aurora-A decreased significantly(P<0.01).In order to study the effect of Chelidonine on Apoptosis-like MC,we using AnnexinV-FITC/PI double staining flow cytometry to detect cell apoptosis rate.Results showed that,in gourps with Chelidonine treatment of 24,48,and 72 h,respectively,all group showed apoptosis and apoptotic rateis positively related with time of treatment.We also use AnnexinV-FITC/PI double dye laser confocal microscope for morphology observation.The results showed that,after 24h of treatment,cell showed fluorescent color of red and green,indicating cells are experiencing late apoptotic stage,and as the time of treatment increases,the number of late apoptotic cells gradually increased,which further confirmed that tumor cells went to MC,and some of the cells went into apoptosis.To explore the mechanism of Apoptosis-like death further,we use Western blot method to detect the effect of Chelidonine on the expression of apoptosis inhibitory protein survivin and apoptosis promoting proteins Csapase-9 and Caspase-3.Results showed that after Chelidonine treatment for 24h,the expression of survivin and caspase-9 and caspase-3 differ significantly with the negative control group,and as the time of expression gets longer,the expression of survivin protein was significantly decreased,and the expression of caspase-9 and caspase-3 were significantly increased(P<0.01).In summary,Chelidonine induced MC is related with the inhibition of expression of Aurora-A protein.Some cells went into apoptosis after Chelidonine treatment,and the late apoptosis rate increased as treatment time increases.The mechanism of Chelidonine induced Apoptosis-like death could be the following:Chelidonine inhibits Survivin expression,and then interfere with microtubule polymerization,which lead to M-phase arrest,and at the same time activate Caspase-9,which activate a cascade that lead to the activation of Caspase-3,finally leads to Apoptotic-like cell death.