Study On The Underlying Mechanisms Of Apoptosis Inhibited By FGF-2 In Small Cell Lung Cancer

Preface:Lung cancer remains the leading cause of cancer death. Small cell lung cancer(SCLC)represents 15-20%of all lung tumors. Despite initial sensitivity to therapy,relapse with chemoresistant disease is rapid and overall survival is very poor.It has reported that FGF-2 (Fibroblast Growth Factor-2)could induce broad-spectrum drug resistance via inhibiting apoptosis in various tumor types,but the molecular process is not clear.It is well known that the level of FGF-2 in blood is up-regulated in lung cancer.The report on inhibiting apoptosis induced by FGF-2 in SCLC is little.The present study is to investigate the mechanism that FGF-2 induced apoptosis inhibition in small cell lung cancer.Objective:(1)To study the effect of basic Fibroblastic Growth Factor(FGF-2)on apoptosis in human small cell lung cancer cells NCI-H446.(2)To elucidate partial mechanism of FGF-2 inhibited apoptosis,we investigate the effect of FGF-2 on the expression of Survivin and the subcellar location of Smac.Also,we determine the expression of FGF-2,Survivin and Smac in SCLC tissues and analyse the correlation between them.(3)To invest the role of PKCαin FGF-2-induced apoptosis inhibition and survivin expression.(4)To analyze the proteomics profiles of NCI-H446 cells with or without pretreatment by FGF-2 and search the candidate proteins induced by FGF-2 in NCI-H446 cells.Methods:(1)The effect of FGF-2 on apoptosis in NCI-H446 cells was evaluated by Flow cytometry,Hoechst 33258 staining and TUNEL assay.Western blot assay detected the expression of Survivin protein induced by FGF-2.The expression of Survivin protein was monitored by western blotting inhibited by neutralizing anti-FGF-2 antibody.The release of Smac from mitochondria to cytoplasm effected by FGF-2 was observed by western blotting and immunofluorescence.(2) Immunohisto—chemical SP method was used to detect the expression of FGF-2,Survivin and Smac in 20 SCLC tissues and 15 normal epithelial tissues of lung.(3)PKCαin the whole protein and the membrane protein of FGF-2-stimulated NCI-H446 cells were detected by Western blot. NCI-H446 cells were pretreated with special PKC inhibitor Calphostin C and then were treated with FGF-2:the expression of PKCαand survivin were detected by Western blot,and the apoptosis was deteced by flow and Hoechst fluorescein stain.(4)The total proteins of NCI-H446 Cells with or without pretreatment by FGF-2 were separated by two-dimensional gel electrophoresis(2-DE),respectively.PDQuest software was applied to analyze 2-DE images,the differentially expressed protein spots of NCI-H446 Cells between with and without pre-treat groups were identified by matrix-assisted laser desorption/ ionization time of flight mass spectrometry(MALDI-TOF-MS),and the expression levels of partial identified proteins were determined by western blotting and immunohistochemistry.Results:(1)FGF-2 increased the expression of Survivin in a concentration- dependent manner when the concentration of FGF-2 was from 0 to 75ng/ml,the expression of Survivin decreased after reaching peak.Survivin in all experiment groups expressed highlier than that in control group(P＜0.05).(2)The expression of Survivin protein rised after administration of FGF-2 1～6h and peaked at 4h in NCI-H446,the expression of Survivin protein in all experiment groups is higher than that in control group(P＜0.05).12.Sμg/ml and 25μg/ml neutralizing antibody to FGF-2 efficiently attenuated the 20%,34%expression level of survivin protein induced by FGF-2(P＜0.05).(3)75ng/ml FGF-2 inhibited serum starvation-induced apoptosis in NCI-H446 cells.After exposure to serum starving for 36h,cells showed typical morphologic changes of apopstosis, which was attenuated by FGF-2 stimulation.Percentages of apoptotic nuclei were 2.42±0.85(control),14.51±2.68(starving group)and 6.13±1.89(FGF-2 stimulation group)respectively(P＜0.05).the TUNEL.The number of apoptotic cells/HP were 2.52±1.15(control), 7.65±2.21(starving group)and 5.32±1.25(FGF-2 stimulation group) respectively.(4)Preincubating NCI-H446 cells with FGF-2 4h of which concentration was from 25 to 100ng/ml could inhibit the release of Smac protein from mitochondria to cytoplasm at 36h by serum starvation,the degree of inhibition was 53%～62%.The lever of Smac protein in cytoplasm in experiment groups was lower than that in serum stavation group(P＜0.05).(5)Cancer cells and endothelial cells in 20 SCLC tissues widely expressed FGF-2 and Survivin protein.The positive expression of FGF-2(95%)and Survivin(100%)protein in 20 SCLC tissues was significantly higher than that in 15 normal epithelial tissues of lung(6.7% and 0%separately)(P＜0.05).While the expression of Smac protein was obviously lower than that in normal epithelial tissues of lung(P＜0.05).A positive correlation was present between the expression of Survivin and FGF-2 protein(r=0.557,P＜0.05),while there were no correlations between Smac protein and FGF-2(r=-0.133,P＞0.05),Survivin protein(r=-0.024, P＞0.05).(6)The NCI-H446 cells were treated with FGF-2,the expression of PKCαin total protein did not change and that in membrane protein increased.Special PKC inhibitor Calphostin C decreased the PKCαexpression in cell membrane.Calphostin C reduced the survivin expression and increased the apoptosis rate from 3.8%to 6.32%.(7)Well-resolved, reproducible 2-DE maps of NCI-H446 Cells were established,24 different protein spots were isolated,9 proteins up-expression and 15 proteins down-expression in FGF-2 pre-treated NCI-H446 cell,4 proteins were identified by MALDI-TOF-MS.In FGF-2-treated NCI-H446 cell and SCLC tissue,the expression of Thioredoxin tissue upregulated and the expression of CuZn-SOD downregulated.Conclusion:(1)FGF-2 upregulats the expression of Survivin protein,and blocks the release of Smac from mitochondria to cytoplasm in SCLC cell line NCI-H446.Survivin and Smac may play important roles in the apoptosis inhibited by FGF-2.(2)The low expression of Smac and the disturbed balance between Survivin and Smac expression in SCLC may be a reason for apoptosis resistance.(3)FGF-2 could activate the PKCαin small lung cancer cells.PKCαmay play an important role in FGF-induced survivin expression and apoptosis inhibition.(4)up-regulated Thioredoxin may be involved in FGF-2 induced chemoresistance and apoptosis inhibition.