Effect And Mechanism Of Bufalin On Apoptosis In Human Colorectal Cancer SW620and Esophageal Cancer ECA109Cells In Vitro

ObjectiveTo assess the inhibition of proliferation and the induction of apoptosis ofbufalin on human colorectal cancer （SW620） and esophageal cancer （ECA109）cells.we investigated the effect of p-Stat3,Stat3,Survivin and Caspase-3and thepossible mechanism in bufalin-induced cells apoptosis.Materials and Methods1. Human colorectal cancer （SW620） and esophageal cancer （ECA109）cells were cultured with different concentrations and different time. The effect ofbufalin on cells proliferation was measured using MTT assay.2. Morphological analysis was performed by cytospin preparation withWright-Giemsa stain and the slides were observed under a light microscope.3. Cell cycle phase distribution and hypodiploid DNA was determined byFlow cytometry with PI.4. Expression of p-Stat3, Stat3, Survivin, PARP, and Caspase-3wasanalyzed by Western blot.5. Statistical analysis. All values are expressed as means±SEM. Thedifferences of the results between two groups were evaluated by Student’s t-test.The data from three or more groups were evaluated by one-way analysis ofvariance （ANOVA） followed by SNK test. P<0.05was considered to be statistically significant.ResultsOne, colon cancer SW620cell1. MTT assay showed that bufalin inhibited SW620cell proliferation in time-and dose-dependent manner. The inhibiting concentration of50%cell growth(IC₅₀) at24,48and72h was76.72±6.21,34.05±4.21and16.7±6.37nmol/L,respectively.2. The morphological analysis demonstrated that most of the cells were in abinucleate state without cell division and some were in metaphase afterexposure to20nmol/L Bufalin for24h. An increase in G₂/M phase was observedand the percentage of cells in G₂/M phase was from18.39±1.74%to36.29±2.11%（p<0.001） compare with the untreated control group by Flowcytometer. PARP uncleavage by Western blot.That little effect on apoptosis.When the cells were treated with80nmol/L bufalin for24h, the morphologicalanalysis demonstrated that the apoptotic cells became rounded in shape andtheir nuclei exhibited a fragmented morphology, forming apoptoticbodies,sub-G1peak （representing apoptosis） emerged and the percentage ofsub-G1phase was17.83±2.38%（vs control group，p<0.001）. PARP cleavage.3. After80nmol/L bufalin treatment for1-24h, bufalin did not affect totalStat3protein levels.Stat3phosphorylation increased initiation at1h and6h topeak, then was to descend at12h and inhibited obviously at24h comparedwith untreated control cells, that to hint bufalin inhibit Jak-Stat3signalpassageway. Pretreatment with20μmol/L AG490, a specific inhibitor of Jak, for1h before exposure to80nmol/l bufalin for1and6h completely blocked Stat3activation. SW620cells were pretreated with20μmol/L AG490and thenexposed to80nmol/L bufalin for24h, MTT assay demonstrated that the cellproliferation reduced from49.97±2.52%to38.61±2.9%（P=0.007）and thepercentage of apoptotic cells increased from19.69±1.63%to34.63±2.57% （P=0.002）and Western blot analysis showed PARP cleavage more obviouslycompare with treatment with bufalin alone. AG490treatment alone reduced cellproliferation and induced apoptosis very slightly.Two, esophageal cancer ECA109cell1. MTT assay showed that bufalin inhibited ECA109cell proliferation intime-and dose-dependent manner. The inhibiting concentration of50%cellgrowth （IC50） at24,48and72h was88.12±5.38,38.19±4.81,14.05±7.29nmol/L,respectively.2. Apoptosis of ECA109cell could be efficiently induced by bufalin, After20nmol/L，40nmol/L and80nmol/L bufalin treatment for24h，the percentage ofapoptosis cells were4.83%±0.47%，9.57%±0.56%and19.83%±2.38%（p<0.05）compare with the untreated control group.3. ECA109cell exposed to20nmol/L，40nmol/L and80nmol/L bufalin for24h, the expression of Survivin was decreased to （79.14±5.32）%，（59.03±7.21）%and（29.49±5.67）%（P<0.05） of the untreated control cells. The cleavage ofCaspase-3was observed.Conclusion1.Bufalin inhibited SW620, ECA109cell proliferation in time-anddose-dependent manner.2. It induced M phase arrest of cell cycle at low concentration, but inducedapoptosis at high concentration in SW620cells. Bufalin inhibited activation ofStat3during SW620cell apoptotic progression and AG490significantlyenhanced bufalin-induced proliferation inhibition and apoptosis in SW620cells.3．Bufalin downregulation of Survivin and the activation of Caspase-3during induced apoptosis in ECA109cells.