Study On The Induction Of Tumor Cell Apoptosis And The Relationship Of D-mitosis And M-slippage By Evodiamine

Objective:Evodiamine is an indole quinazoline alkaloid,the main active substance of evodiae.Cell experiments show that evodiamine with antineoplastic activity, directly induce tumor cell apoptosis,inhibit tumor invasion and metastasis, inhibit tumor angiogenesis,inhibit activity of multidrug resistance cell line and so on.Preliminary studies have confirmed that evodiamine could induce tumor cells apoptosis and induce tumor cells arresting in G2/M phase,there is the phenomenon of D-mitosis.D-mitosis and M-slippage,these two events have taken place,apoptosis would be induced only.The current view consider that apoptosis is closely linked to the cell cycle as activities of life.Apoptosis induced by different factors,can occur in different cell cycle.The study was to elucidate the mechanisms of apoptosis induced by evodiamine and the relationship between apoptosis and cell cycle regulation,as well as the further development and application of evodiamine.At the same time,it was provided foundation of more effective Chinese herbal against the cell cycle and developed complex prescription according to the mechanism of Chinese herbal.Methods:2.1 Evodiamine could inhibit the growth of tumor cells in vitro.We selected SGC-7901,HepG2,CBRH7919,primary Kidney,primary Liver cells,used MTT assay to test above 5 cells antipoliferative effect after being treated with evodiamine.The cell cycle and apoptosis index of SGC-7901 were examined using FACScan flow cytometry,the colone assay was used to detect the colone inhibition ratio,microscope was used to observe the changes in cell morphology. 2.2 In order to reproduct the best method of SGC-7901 cell cycle synchronization,we used two methods,one was double TD blocking,the other was serum starvation.2.3 We used FACScan flow cytometry to look for reversible drug detection time after being treated with evodiamine.Accordance with irreversible point in time,after being treated with evodiamine the morphology of apoptosis and chemical detection were done;The cell cycle and apoptosis index of SGC-7901 were examined using FACScan flow cytometry;Expression of CyclinB1 and CyclinE were examined using Western blotting analysis;Comet assay and colone assay were used to prove hypothesis.2.4Construction of gene ND-cyclinB1 with GFP fusion gene expression systems, as well as FL-cyclinB1 gene fusion with the GFP gene expression system to confirm hypothesis by FACScan flow cytometry and Quantitative analysis of fluorescence.2.5 The initial mechanisms of Evodiamine induced apoptosis requires both activation of D-mitosis and M-slippage:through the JC-1 staining of mitochondrial membrane potential change,α-tubulin changes,Western blotting analysis of anti-apoptosis gene Survivin expression.2.6 Produce the transplanted liver cancer(H22) in Kunming mice,the general situation,tumor inhibition rate,tumor volume,survival period were determed after being treated with Evodiae and Indigo naturalis.Results:3.1Evodiamine in vitro cell growth inhibition results show:â‘ SGC-7901,HepG2,CBRH7919 three tumor cells respectively increased 0.9,1.13,1.39 times after 48hr,Kidney and Liver cells respectively increased 0.24,0.18 times after 48hr;Simultaneously cell acute toxicity experiments reveaIed that the evodiamine sensitivity of three types of tumor cells were significantly higher than the primary cells.â‘¡MTT assay confirmed growth inhibition of evodiamine was time and dose-dependent,IC₅₀ was1.36μmol.â‘¢FACScan flow cytometry showed 1μmol·L^-1 evodiamine for 12hr the G2/M phase cells account for 78.8%,but the apoptotic index was only 4.1%and negative was 4.4%in the control group no significant difference,1μmol·L^-1 evodiamine for 24hr the G2/M phase declineed in the proportion,the apoptotic index increased to 34.0%,evodiamine for 36hr the G2/M phase ceils accounted for 0.7%,the apoptotic index was 52.5%.â‘£Colone assay we found 2×10³cells/hole was the best experiment cell density in 6-well plates,evodiamine had significant inhibition of colony formation.⑤Experimental observation of cell morphology,we saw typical morphological changes of apoptotic cells,such as smaller cell size,vacuolization, chromatin cohesion,karyopyknosis,the emergence of apoptotic bodies.The results suggested that Evodiamine could inhibit the growth and division of tumor cells,and this inhibition was selective,that was,the faster growth cells were stronger sensitive to evodiamine.3.2 To product SGC-7901 cell cycle synchronization.â‘ we used double TD blocking method.TD final concentration of 2mM,4mM,8mM after,G0/G1 phase cells were77.3%,77.5%and 77.0%,while G2/M phase cells were 2.0%,2.1%and 2.1%.Another large group in the above mentioned double TD blocking then released 12hr,TD final concentration of 2mM,4mM,8mM of G0/G1 phase cells decreased to 44.0%,38.0%and 36.6%,and G2/M phase cells increased to39.9%,46.2%and 49.1%.â‘¡The other method-serum starvation synchronization,flow cytometry indicated that the serum-free culture medium,respectively,24hr,30hr,36hr,G0/G1 phase cells were 57.4%,59.9%and 68.4%,and G2/M phase cells were11.4%,5.0% and 10.1%.Another large group in the above mentioned serum starvation then released 12hr,respectively,24hr,30hr,36hr of G0/G1 phase cells decreased to 50.3%,51.4%and 51.4%,while G2/M phase cells increased to 13.8%,22.7% and 22.7%,the period of cell number was not change significantly.Serum-free culture medium for 24hr,30hr and 36hr,both appeared typical hypodiploid apoptotic peak.The results suggested that Double TD blocking cell cycle synchronization method was an ideal access to gain a large number of ground state cells,and didn't rely on the TD concentration.Treatment by evodiamine for 16-20hr, SGC-7901 switched on apoptotic process,resulting in irreversible death.3.3 Explore the impaction in M phase of SGC-7901 by evodiamine.â‘ About the reversible point in time after experiment we found, 1μmol·L^-1evodiamine for12hr the apoptotic index was 4.1%.For16hr,the apoptotic index was 4.0%,then released the drug apoptotic index was 8.5%. For 20hr two different types had significant difference,the released group apoptotic index increased to40.4%,was significantly higher than non-released group-16.0%.For 24hr,28hr,32hr and 36hr the apoptotic index of the released group were significantly higher than non-released group.According to evodiamine-induced apoptosis irreversible point in time between 16-20hr,we used 1μmol·L^-1 evodiamine respectively for 16hr,20hr,24hr and 36hr.â‘¡Western Blot analysis of the level of CyclinB1:Treated by evodiamine for 16hr the level was significantly higher than negative cells,but the level of evodiamine for 20hr was lower than 16hr,while 24hr and 36hr with no protein expression.Western Blot analysis of the expression of CyclinE,negative cells, 1μmol·L^-1 evodiamine for16hr,20hr and 24hr were both no specific bands,and evodiamine for 36hr appeared band.The expression of CyclinE tangled to CyclinB1,suggesting that after treating by evodiamine for 36hr cells arrested in M phase,then went into G1 phase.â‘¢The growth type of SGC-7901 was polygonal or diamond.PI staining was observed under fluorescence microscope,chromosomes can be seen shrinkaged assuming trigeminal after treating evodiamine for 16hr;After treatment for 24hr,could not see the obvious apoptotic bodies,the majority of cells wore still in a state of chromosome condensation;After treatment for 36hr,we could see the obvious apoptotic bodies,at the same time could see the multinucleated cells.â‘£The comet assay,we found the damaged DNA swam,a long tail appeared behind the cell.The average optic density was lower than the negative cells after treating with statistical significance.The comet tail was longer than the negative cells after treating with statistical significance,both two changes were related to time.⑤FACScan flow cytometry showed:apoptosis index of 1μmol·L^-1 CDK1I was 7.6%, the negative cell was 3.5%,no significant difference.After treating by evodiamine forl8hr added CDK1I for 6hr the apoptotic index significantly increased.0.5μmol·L^-1 evodiamine for 24hr the apoptotic index was 8.9%,then another group added CDK1I the apoptotic index increased to 28.2%.1μmol·L^-1evodiamine for 24hr the apoptotic index was 20.0%,then another group added CDK1I the apoptotic index increased to 32.0%.â‘¥The colone assay,we found the colone inhibition index was 24.84%after treating by 7.5μM CDK1I,and added CDK1I combinding to evodiamine the colone inhibition index significantly increased.0.5μmol·L^-1 evodiamine for 24hr the colone inhibition index was 38.01%,then another group added CDK1I the colone inhibition index increased to 65.34%.1μmol·L^-1 evodiamine for 24hr the colone inhibition index was 49.81%,then another group added CDK1I the colone inhibition index increased to 70.32%.The colone Inhibition was dose-dependent.⑦Dapi staining was observed under fluorescence microscope,negative cells were firmly adherent,bright,extended,uniform.While SGC-7901 after treating by CDK1I were bigger and stronger.0.5μmol·L^-1and 1μmol·L^-1evodiamine group could be seen larger sizes,condensed chromatin status,apoptotic bodies.Added CDK1I for 3hr cells re-adherent and at the same time the combined effect for 6hr,multinucleated cells appeared concording with PI staining.The results suggested that Evodiamine induced SGC-7901 apoptosis,and signal apoptosis related on the generation and maintenance of D-mitosis and Mitotic-slippage event.3.4 Gene ND-CyclinB1,FL-CyclinB1 have successfully connected with the GFP gene.â‘ Recombinant plasmid pEGFP-N1-ND CyclinB1,pEGFP-N1-FL CyclinB1 transfected SGC-7901,ND-CyclinB1,FL-CyclinB1 gene were expressed in SGC-7901.â‘¡Recombinant plasmid transfected SGC-7901,after treating by evodiamine, FACScan flow cytometry showed that the apoptotic index of negative cell was 4.1%,pEGFP-N1-ND CyclinB1,pEGFP-N1-FL CyclinB1 respectively was 38.2%,41.3%, and without the role of evodiamine the apoptotic index respectively was14.5%,10.7%,with significant difference.â‘¢Recombinant plasmid transfected SGC-7901 cells,1μmol·L^-1evodiamine for 36hr the blind score of pEGFP-N1-ND CyclinB1was 3.60,were higher than pEGFP-N1-FL CyclinB1 and empty pEGFP-N1 as the role of evodiamine 2.50 and 1.40.The results suggested that Recombinant plasmid of pEGFP-N1-ND and pEGFP-N1-FL were success,they could applicated to research cell cycle-related study in the future.Method of making cells arrest in M phase,we can get the same conclusion,that is evodiamine induced SGC-7901 apoptosis relatde on D-mitosis event.3.5 Explore the possible mechanisms of cell cycle regulation inducting apoptosis â‘ Application JC-1 to observe mitochondrial membrane potential changes,result showed:red,green fluorescence dispersed uniformly in the negative cells, red fluorescent spread in cells.After treatment for 16hr,the red fluorescence intensity decreased,while the green fluorescence intensity enhanced,indicating that mitochondrial membrane potential damaged.After treatment for 20hr,the red fluorescence intensity decreased significantly, while the green fluorescence intensity increased significantly.After treatment for 24hr,red fluorescence scattered,while for 36hr almost no red fluorescence.â‘¡Then we Observedα-tubulin,the negative cells with clearly visible fibers, showing fine filaments.After treatment for 16hr,tubulin fibers could be seen beginning with disorders,shorter fracture.After treatment for 20hr,tubulin fibers could be seen bending happened,changing the status polymerization.Treatment lasted 36hr,tubulin polymerized in the side, fluorescence intensity increased,the changes similar to taxol-tubulin.â‘¢Western Blot analysis Survivin expression,negative cell was in a high expression,after treatment for 16hr evodiamine,Survivin begen to decline.After treatment for 20hr,24hr and 36hr then had no Survivin specific bands appeare.The results suggested that Evodiamine induced SGC-7901 apoptosis might be related to the loss of mitochondrial transmembrane potential,the impact of tubulin polymerization and inhibit the expression of Survivin gene,was a multi-target process.3.6 Rutaecarpa combined Qingdai anti-tumor experiments in vivo.â‘ Transplanted subcutaneously mice of liver cancer model could formed tumor 100%.â‘¡Experiment I,we found that Cyclophosphamide group mice was in best general state of health,but only five mice survived.The tumor inhibition rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 43.9%,35.6%27.7%and 37.2%.Tumor size results showed that the treatment groups' were smaller than control group and combinded group compared with cyclophosphamide group had statistical significace(p=0.048).â‘¢Experimentâ…¡,general state of health was similar to the experimentâ… .The tumor inhibition rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 50.1%,44.9%,45.9%and 51.9%.Tumor size results showed that the treatment groups' were smaller than control group. The life extension rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 40.1%,36.04%,18.78%,70.05%,the combinded group was significant higher than cyclophosphamide group.Conclusion:Evodiamine could inhibit the growth and division of tumor cells,and this inhibition is selective,that is,the faster growth cells are stronger sensitive to evodiamine.Treatment by evodiamine for 16-20hr,SGC-7901 switches on apoptotic process,resulting in irreversible death.Evodiamine induces SGC-7901 apoptosis,and signal apoptosis relats to the generation and maintenance of D-mitosis and Mitotic-slippage event,Evodiamine induces SGC-7901 apoptosis may be related to the loss of mitochondrial transmembrane potential,the impact of tubulin polymerization and inhibit the expression of Survivin gene,is a multi-target process.