Study On The Mechanism Of Mitotic Catastrophe Induced By Oxocrebanine From Stephania Hainannensis H.S.Lo Et Y.Tsoong In MCF-7 Cells

Stephania Hainannensis H.S.Lo et Y.Tsoong is a species of genus Stephania(Menispermacae),mainly distributed in Baisha County,Hainan,which is an important traditional medicine of the Li nationality in Hainan.It’s breathless,bitter taste,slightly cold and slightly toxicity.Based on the previous research,oxocrebanine was the active alkaloids in Stephania Hainannensis H.S.Lo et Y.Tsoong which exhibited a best inhibition effect on MCF7 cells with IC50 values of 16.66 μmol/L.Oxocrebanine inhibited the activity of topoisomeraseⅠ and Ⅱ a result in DNA damage in MCF-7 cells.Oxocrebanine had the potential to induce the cycle arrest in MCF-7 breast tumour cells thanks to the up-regulated in the level of p-H3.Based on the results that oxocrebanine induced up-regulated p-H3 protein level and DNA damage in MCF-7 cells,the mechanisms of suppressing proliferation of oxocrebanine were further studied by observing the cell cycle.This study demonstrated the role of oxocrebanine in cell proliferation and mitotic catastrophe in MCF-7 cells,suggesting an alternative therapeutic strategy for breast treatment.In order to observe the effect of oxocrebanine on the cell cycle distribution,the flow cytometry were flow cytometry analysis was performed.The percentage in G2/M phase was increased in MC F-7 cells treated with 8.33,16.66 μmol/mL oxocrebanine for 48 h.The experiment continued by staining MCF-7 cells with DAPI,observing the changes of nucleus in MCF-7 cells with the administration of oxocrebanine for 48 h by fluorescence microscope.And detecting the presence of multinucleated cells and abnormal mitotic cells by Fluorescence microscope after treatment with oxocrebanine.And multinucleated cells were counted to calculate the proportion of mitotic cells and multinucleated cells,and to discuss whether oxocrebanine can inhibit the proliferation of MCF-7 cells by affecting the mitosis of MCF-7 cells.Cytomorphology with DAPI staining demonstrated the enlargement of cell volume and multinucleation in MCF-7 cells treated with oxocrebanine.That suggested the extensive mitotic-arresting effects of oxocrebanine.Percentage of mitosis and multinucleated cells in MCF-7 cells treated with oxocrebanine were increased.The results suggest that oxocrebanine could cause human breast cancer MCF-7 cells to show morphological changes typical of mitotic catastrophe.The experiment further studied the mitotic catastrophe-related pathways(p53-dependent and p53-independent pathway)of human breast cancer MCF-7 induced by oxocrebanine.Next studied the expression of proteins in p53-dependent pathways(p53 and p21)and p53independent pathway(Cdc25C,Cdc2 and p-Cdc2)after treatment with oxocrebanine.Western blot results showed that increased p53,p21 and Cdc2 phosphorylation,and decreased Cdc2 kinase and Cdc25C acitivity suggesting that oxocrebanine induced mitotic catastrophe by p53dependent and p53-independent pathway.To investigate the mitotic phase marker protein Cyclin B1,the immunofluorescence staining was observed the localization of Cyclin B1 in MCF-7 cells treated with oxocrebanine.Western blot assays were applied to evaluate the effects of oxocrebanine on the expression of Cyclin B1,Aurora A and PLK1.The Cyclin B1 distributed in cytoplasmic of MCF-7 cells treated with oxocrebanine under fluorescence microscope.The results of Western blot showed that oxocrebanine up-regulated the expression level of Cyclin B1 protein,leading to the accumulation of Cyclin B1 protein.The experiment then detected the protein levels of Aurora A and PLK1 in MCF-7 cells treated with oxocrebanine to verify whether the up-regulation of Cyclin B1 protein levels was caused by oxocrebanine inhibiting the hydrolysis of Cyclin B1 in MCF-7 cells.The results showed that oxocrebanine could further down-regulate the level of PLK1 protein by reducing Aurora A,leading to the inhibition of Cyclin B1 proteolysis and accumulation in MCF-7 cells,leading to MCF-7 cell mitotic catastrophe.The immunofluorescence analysis through specific antibodies to α-tubulin was applied to observe the change of microtubule in MCF-7 cells after oxocrebanine treatment.The results revealed that oxocrebanine induced microtubule depolymerization with scattered and short microtubule fragments in cytoplasm of MCF-7 cells.To further investigate whether oxocrebanine destroyed microtubule assembly dynamics,the cellular tubulin levels were determined through Western blot.Oxocrebanine caused the increased of depolymerized(soluble)form of α-tubulin,and the polymerized(insoluble)form of α-tubulin was decreased.In a word,these findings demonstrated that oxocrebanine disturbed tubulin polymerization at both molecular and cellular levels,which induced mitotic catastrophe in MCF-7 cells.In summary,oxocrebanine induced MCF-7 cell mitosis arrest through p53-dependent and p53-independent pathways by up-regulated p53,p21 and Cdc2 phosphorylation and downregulated Cdc2.And oxocrebanine down-regulated the protein expression levels of Aurora A and PLK1,it leads to multinucleated MCF-7 cells and the accumulation of Cyclin B1 protein.Oxocrebanine changed the distribution of Cyclin B1 in MCF-7 cells.Then,oxocrebanine increased the depolymerized(soluble)form of α-tubulin,indicating abnormal mitosis,multinucleated cells,and lead to mitotic catastrophe in MCF-7 cells that induced mitotic catastrophe in MCF-7 cells.