The Relevance Between The Influence Of Human Recombinant Globular Adiponectin On Proliferation And Apoptosis Of MCF-7Cell And NF-kB

Abstract Objective To explore the effect of the globular adiponectin (g-APN)onproliferation and apoptosis of MCF-7cells and to research the relationship betweenthe effect and nuclear factor kappa B (NF-κB).Methods Developing breast cancer cells MCF-7in the high sugar environment, thecells were treated with different concentrations of globular adiponectin (0、10、20、40、80ng/ml) for24h and48h.The cell survivals were determined by MTT method. Theflow cytometry with Annexin-FITC/PI double staining detected apoptosis. Western blotdetected the expression of NF-κB protein. Selected40ng/ml g-APN as experimentconcentration and cultured respectively MCF-7cell for0,12,24,36,48h, westernblot measured NF-κB protein expression. Selected40ng/ml g-APN as experimentconcentration,10ng/ml recombinant human tumor necrosis factor (TNF-α) as theexperimental concentration, the experiment was divided into the normal group, TNF-αgroup, the TNF-α+g-APN group, g-APN group and every group intervened MCF-7cell for24h respectively. Western blot detected NF-κB protein expression. Selected40ng/ml g-APN as experiment concentration,50umol/l NF-κB activationinhibitors ammonium pyrrolidinedithiocarbamate (PDTC) as the experimentalconcentration, The experiment was divided into normal group, PDTC group, g-APNgroup, PDTC+g-APN group. The cell survivals were determined by MTT method.The flow cytometry with Annexin-FITC/PI double staining detected apoptosis. Westernblot detected NF-κB protein expression.Results:(1)After24h of g-APN intervening to MCF-7cells, the cell opticaldensity value (OD value) in40and80ng/ml groups were decreased significantly compared with0ng/ml group（P＜0.05).Compared the optical density value between10and20ng/ml groups and0ng/ml group, there was no statistically significant difference.After48h of g-APN intervention to MCF-7cells, the cell optical density value (ODvalue) in20、40、80ng/ml groups were decreased significantly comparing with0ng/mlgroup（P＜0.05); And the OD values among20ng/ml group、40ng/ml group and80ng/ml group were significantly gradually reduced（P＜0.05).(2) After24h of g-APNintervening MCF-7cells, the cell apoptosis rate in0、10、20、40and80ng/ml groupswas increased gradually, but there was no statistically significant difference betweenfive groups. After48h of g-APN intervening MCF-7cells, the cell apoptosis rate in0、10、20、40and80ng/ml groups was increased gradually.The cell apoptosis rate in40and80ng/ml groups were higher significantly compared with0ng/ml group（P＜0.05).The cell apoptosis rate in80ng/ml group was higher than40ng/ml group.(3) According to the results of Western blot, the NF-κB protein expression quantity in40ng/ml and80ng/ml groups decreased significantly than0ng/ml group; the NF-κBprotein expression quantity in10ng/ml and20ng/ml groups were less than0ng/mlgroup,but there was no statistically significant difference;(4) Selecting40ng/ml g-APN as experiment concentration, respectively interveningMCF-7cell for0、12、24、36and48hours, the NF-κB protein expression quantity wasless gradually and the difference was statistically significant (P <0.05).(5) Normal group, TNF-α group, the TNF-α+g-APN group, g-APN group interveningrespectively MCF-7cells for24h, the result of western blot indicated that the NF-κBprotein expression quantity declined more than normal group(P <0.01); The NF-κBprotein expression quantity was lower than normal group(P <0.01);The NF-κB proteinexpression quantity declined gradually and there was significant difference in TNF-αgroup, the TNF-α+av group, g-APN group (P <0.05). (6)Normal group, PDTC, g-APN, PDTC+g-APN group intervening respectivelyMCF-7cells for48h after PDTC, MTT results showed that the OD value of g-APNgroup was lower than normal group. The differences of OD value in normal group,PDTC group, PDTC+g APN group was no statistical significance; The cell apoptosisrate in g-APN group was higher than normal group, but there was no statisticallysignificant difference in normal group, PDTC group, PDTC+g APN group. Accordingto the results of Western blot, the NF-κB protein expression quantity in g-APN groupwas lower than normal group. The differences of NF-κB protein expression quantity innormal group, PDTC group, PDTC+g APN group was no statistical significanceConclusion (1) The g-APN can inhibit the proliferation of MCF-7and the effect has arelationship of concentration and time dependent;(2) The g-APN can promote the MCF-7cell apoptosis and the effect has a relationshipof concentration and time dependent;(3) The g-APN can inhibit the NF-κB protein expression and the effect has a relationship ofconcentration and time dependent;(4) The g-APN can suppress the NF-κB pathway activation which is activated byTNF-α;(5) After PDTC blocking the NF-κB pathway, the g-APN can’t display the effect ofinhibiting MCF-7growth and NF-κB expression.(6) The g-APN can inhibit the growth of MCF–7which may be has association with theNF-κB path way.