The Effect Of Apoptosis Related Protein In Bufalin-Induced Apoptosis Of Colorectal Cancer Cell HT-29

ObjectiveColorectal cancer was still one of the most common malignancies inhuman，it was estimated that1.2million global new cases of the2008, thedeath of608000at the same year, so it was still a major threat to humanhealth[1]; about20%-25%of new cases of colorectal cancer in newlydiagnosed had staged IV metastatic colorectal cancer, One-third of patientswho received early radical treatment had recurrenced and metastasis andbecame late,so about half of patients ultimately belonged to the advancedmetastatic colorectal cancer in all colorectal cancer（Metastatic ColorectalCancer mCRC）[2].Treated by surgery radiotherapy and chemotherapy andtargeted therapy but had poor efficacy. Researched and developed efficiencyand low toxicity of new drugs became popular current research. Bufalin wasone of toxicity ligand of the Chinese medicine Bufalin, research studyed athome and abroad confirmed Bufalin can inhibit leukemia cells and gastriccancer、ovarian cancer、lung cancer and other solid tumors cell proliferationand induce apoptosis[3-6]. But about Bufalin-induced apoptosis in colon cancercells and livin、Bcl-2、Bax and Caspase-3related basic research had not beenreported.This study applied different concentrations of Bufalin for colonadenocarcinoma cell HT-29, explored the apoptosis of Bufalin-induced coloncancer cell HT-29and the changes in protein of livin、Bax、Bcl-2and caspase-3in the apoptotic process. Provided a theoretical basis for further research. Methods(1) MTT assayed Bufalin had inhibition of proliferation in colonadenocarcinoma cell HT-29;(2) Wright-Giemsa staining observed the changes of morphological;(3) Flow cytometric analyzed apoptosis;(4) Western-bolt assayed the expression of protein of Capase-3、livin、Bcl-2and Bax;(5) Statistical methods:All data from three independent experimentalresults,expressed with x±s, applied SPSS16.0software t-test and one-wayaNOVA analysis,P<0.05was considered statistically significant;Results(1) Bufalin inhibited HT-29cell proliferation had relation of dose and timedependencies,24h,48h,72h IC50for90.29nmol/L、50.1nmol/L and19.9nmol/L.(2) Bufalin-induced apoptosis in HT-29observed typical apoptotic bodies in themorphology.(3) Flow cytometry detected significantly sub-G1(subdiploid-G1), sub-G1percentage from the control group (0.54±0.31)%to (3.68±1.63)%、(18.31±1.82)%and (22.41±3.81)%, the difference compared with the controlgroup was statistically significant (P<0.05).(4) Western-bolt detection of Bcl-2expression were decreased to the controlgroup (88.51±7.71)%(62.53±6.51)%,(45.70±5.18)%. Bax protein expressionwere raised to the control group,1.50times and1.74times,2.05times. Eachset of data with the control group the difference was statistically significant(P<0.01).The expression of protein of livin was reduced to the control group(81.31±1.12)%、(52.42±3.28）%and (29.10±5.62）%. The difference of eachset of data compared with the control group was statistically significant(P<0.01),20nmol/L Bufalin in colon cancer cell HT-29could not detect theexpression of protein of Caspase-3,but40nmol/L and80nmol/L group coulddetect the activity of17KD subunit. Conclusions(1) The bufalin inhibit colon adenocarcinoma cell line HT-29proliferation wasdose and time dependencies.(2) Bufalin induced apoptosis of colon cancer cells HT-29cells.(3) Bufalin induced during apoptosis in HT-29colon cancer cells cansignificantly increase the ratio of Bcl-2/Bax down livin protein expression andenhanced activation of Caspase-3protein.(4) Increase the ratio of Bcl-2/Bax, down to livin protein expression andenhanced activation of Caspase-3protein, bufalin induced apoptosis in coloncancer cells HT-29cells pathway.