Mechanisms Of Chelidonine Inducing Mitotic Catastrophe And Apoptosis In MCF-7 And MCF-7/DOX Cells

Chelidonium majus L.is rich in alkaloids Chelidonine,and its anti-tumor effect is significant.Studies have shown that Chelidonine has significant anti-tumor effect in bladder cancer,liver cancer,cervical cancer,pancreatic cancer,rectal cancer,colorectal cancer,etc.and it has arisen a wide concern among researchers at home and abroad in recent years.Our previous research found that Chelidonine not only has obvious antitumor effect on human gastric cancer SGC-7901 cells and human breast cancer MCF-7 cells,but also could significantly inhibit the proliferation of MCF-7/DOX human breast cancer cells resistant to adriamycin,which induces tumor cell death mechanism probably caused by tumor cells undergoing mitotic catastrophe.In this paper,MCF-7 cells and MCF-7/DOX cells were used as the object to further investigate the effects and possible mechanism of these two kinds of cells undergoing mitotic catastrophe death induced by Chelidonine,and ultimately determine the experience of mitotic catastrophe after the final way of cell death.The results of the dissertation will provide the basis for the development of new anti-breast cancer drugs that contain Chelidonine as the main component.Mitotic catastrophe is the phenomenon of cell death that occurs when the mitotic process is disordered,which is a true endogenous tumor suppressing mechanism.The results of this study will also provide new ideas for the treatment of resistant tumors.In this paper,Giemsa staining was used to observe the effects of Chelidonine(12?mol/L)on human breast cancer MCF-7 cells and their drug-resistant cells MCF-7/DOX 24,48,72 h and calculate the mitotic index(mitoticindex,MI).Using Western Blot method to further study Chelidonine on the expression of Chkl,p-Chk1,Plk-1,Aurora-A.Cdc25C,Securin.P21 in both cell lines to investigate the Chelidonine induced MCF-7 and MCF-7/DOX cells and molecular mechanism of mitotic catastrophe.The results showed that with the prolonging of Chelidonine treatment time,the above two kinds of cell edge shape fuzzy,cell volume become larger and the flat cell spacing increased gradually,large nucleus,multinucleated cells appeared and increased significantly;compared with the negative control group,MI increased significantly(P<0.05 or P<0.01);on the whole,with the prolongation of Chelidonine in cells after 48 h,the expression level of Chkl,Plk-1,Aurora-A,Cdc25C in MCF-7,MCF-7/DOX cells,reduced significantly(P<0.01).After 24 h,the expression of p-Chkl,Securin.P21 in MCF-7.MCF-7/DOX were up-regulated(P<0.01 or P<0.05).In order to study the final cell death pathway of MCF-7 and MCF-7/DOX.which occurs mitotic catastrophe induced by Chelidonine,DAPI staining was used to observe the effects of Chelidonine(12 ?mol/L)on cell morphology at different time points;and Annexin V-FITC/PI double staining flow cytometry was used to detect the change of apoptosis rate of both cells after Chelidonine.At the same time,using AnnexinV-FITC/PI double staining immunofluorescence microscope to observe cell morphology,and using Western Blot method to study Chelidonine(12 ?mol/L)on cell apoptosis related proteins expression of P53,Caspase-3,Caspase-9 and Survivin in two cell lines.The results showed that with the increase of Chelidonine administration time,the multi-nucleus and micronuclei of both cells increased obviously,accompanied with the condensation of nuclei and the deepening of staining.Some of the cells showed the shape of nuclear fragmentation into circular Small body,surrounded by the cell membrane;the total apoptosis rate of two cells increased significantly with time(P<0.05 or P<0.01);Under the fluorescence microscope,the number of apoptotic cells in red and green fluorescence increased gradually,indicating that Chelidonine induced breast cancer and drug-resistant cells after mitotic catastrophe with some cells apoptosis eventually.The results of Western Blot showed that the expression of Survivin protein in both cell lines was significantly decreased and the expression of Caspase-9,Caspase-3 and P53 increased significantly with the increase of Chelidonine activity(P<0.01).Overall,Chelidonine can induce mitotic catastrophe in MCF-7 and MCF-7/DOX cells by regulating the expression of Chkl,p-Chk1,Plk-1,Aurora-A,Cdc25C,Securin,P21 protein expression level.After Chelidonine induce mitotic catastrophe in MCF-7 and MCF-7/DOX cells,some cells eventually undergo apoptosis.The mechanism may be that Chelidonine up-regulated P53 protein,inhibits the expression of Survivin protein and causes anti-microtubule polymerization.It interferes with the activation of upstream Caspase-9 and activates the downstream Caspases pathway,which further activates the cascade to activate Caspase-3 downstream of the apoptotic pathway and promotes cell apoptosis.