Research For Apoptosis Mechanism Of Colorectal Cancer Cell Induced By BH3 Domain

ObjiectiveCancer of colon is a frequent malignant tumor of alimentary tract.It threatens seriously human healthy.Its pathogenesis does not elucidate now.Many scholars consider that it relates not only with oncogene mutation and anti-oncogene inactivation but also with disequilibrium of cell proliferation and apoptosis control.Cell apoptosis is a programmed cell death in condition of physiology or pathology.It is a process of cell damage.It is controled by gene differently from cell necrosis.It is presently known that there are at least two main path to mediate and adjust apoptosis:â‘ Death receptor path;â‘¡Mitochondrion path.BH3-only protein is necessary condition of cell apoptosis by mitochondrion path. When the cells are stimulated by cytotoxic signal,BH3-only protein is activated and Bax or Bak are activated by some way that formed Bax and Bak oligomer on the mitochondrial membrane.Membrane permeability rises then apoptosis protein such as cytochrome C releases.Caspase is actived and cell apoptosis.Unnormal control of apoptosis can resulted in disequilibrium of cellular proliferation.It is considered a mechanism of tumor formation.Recent research indicate that apoptosis relatives closely with happening,development and transition of tumor.BH3 domain counterpart BH3I-2'(3- iodo - 5 - chloro - N -ï½›2 - chloro - 5 -[(4 - chlorophenyl ) sulphonyl]phenyl｝- 2 - hydroxybenzamide ) can induce cell apoptosis by interacting the balance of pro-apoptosis protein and anti-apoptosis protein.The research discussed whether BH3I-2' can induce HCT116 cell apoptosis and mechanism of it by detecting cell proliferation,cell apoptosis,Mitochondrion transmembrane potential and Pro-caspase-3 protein expression.Methods1,MTT method.2,Flow cytometry FITC/PI double staining method3,Flow cytometry JC-1 simple staining method4,Western Blot method.Using the four methods,we detect cytotoxicity,cell apoptosis,mitochondrion transmembrane potential and Pro-caspase-3 protein in HCT-116 cells treated by BH3I-2'.Results1,BH3I-2' influenced HCT116 cells multiplication:after 30,40,50,60,70,80,90μmol/L BH3I-2' treated HCT116 cells 24 hour,growth inhibiting ratio:10.08±1.12,23.13±1.46,34.37±1.61,43.36±1.92,67.79±1.66,76.53±1.89,86.69±1.52(mean±SD,n=3,P＜0.05).The IC₅₀ is 60μmol/L.2,BH3I-2' induced HCT116 cells apoptosis:after 60μmol/L BH3I-2' treated HCT116 cells 2,4,8,24 hour,early apoptosis ratio:12.91±1.88,8.49±0.77,5.32±1.27,3.59±1.30.Late apoptosis ratio:24.22±2.20,27.44±2.36,40.67±1.78,45.52±1.23(mean±SD,n=3,P＜0.05).As the time growing,early apoptosis cells reduced while late apoptosis cells increased.3,BH3I-2' can cause the damage of HCT116 cells mitochondrial inner membrane: after 60μmol/L BH3I-2'treated HCT116 cells 0,1,2,4,6,24 hour,cell apoptosis ratio of the first quadrant:91.32±2.18,89.76±1.12,89.67±1.46,89.45±1.90,84.60±1.49*,83.71±1.39*.Cell apoptosis ratio of the fourth quadrant:5.24±0.91,6.63±1.24,6.81±1.17,9.38±1.18*,10.14±1.23*,13.72±1.64*(mean±SD,n=3, *P＜0.05).△ψm of BH3I-2' treated HCT116 cells 6 hour reduced quickly.And the longer BH3I-2' treated,the more significant△ψm reduced.4,BH3I-2' inducing HCT116 apoptosis depended on activation of caspase-3:after 60μmol/L BH3I-2' treated HCT116 cells 0,2,4,6,8,12 hour,gray scale value of pro-caspase-3 protein expression:1.05±0.09,0.91±0.09,0.84±0.08,0.63±0.10*,0.56±0.10*,0.53±0.12*(mean±SD,n=3,*P＜0.05).Expression of pro-caspase-3 protein decreases after BH3I-2' treates HCT116 cells 6 hour.And the decreasing tendency strengthens as time increasing.Conclusion1,BH3I-2' can inhibite colorectal cancer cell HCT116 proliferation.2,BH3I-2' can promote colorectal cancer cell HCT116 apoptosis.3,The process that BH3I-2' induces Human cancer of colon HCT-116 cell apoptosis causes mitochondrion transmembrane potential cut down.4,The process that BH3I-2' induces Human cancer of colon HCT-116 cell apoptosis causes caspase-3 protein actived.