ABSTRACT
Background Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction,so they remain a close association.However,the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear.Objective This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs.Methods This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours,and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells,including autophagy related gene 5 (ATG-5),Beclin-1,light chain 3 (LC-3).Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups.Results The gray scales for the expressions of Beclin 1,ATG-5,LC-3-Ⅰ and LC-3-Ⅱ proteins in HTFs were 0.365:±0.045,0.765 ±0.055,0.120±0.030 and 0.215 ±0.035 in the control group,and those in the hydroxycamptothecin treated group were 0.980±0.070,1.495±0.095,0.585±0.025 and 0.785±0.055,showing a significant decline in the hydroxycamptothecin treated group(P=0.018,0.022,0.007,0.013).The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group,with a significant reduce in comparison with 0.765 ±0.055 of the control group (P =0.010).However,no obvious distinctions were seen in the band intensities of the expressions of Beclin-1,ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790± 0.060 in the 0.10 g/L hydroxycamptothecin group,which was significantly higher than 0.880 ± 0.070 of the control group (P =0.010).Expression levels (gray scales) of Beclin-1,ATG-5,LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475 ± 0.045,0.390 ± 0.040,0.055 ± 0.015 and 0.075 ± 0.025,which were significantly lowed in comparison with 0.955 ± 0.065,0.765 ± 0.055,0.155 ± 0.015 and 0.280 ± 0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P =0.026,0.031,0.042,0.034).In addition,the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+0.10 g/L hydroxycamptothecin group compared with the control+0.10 g/L hydroxycamptothecin group.Conclusions Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway.
ABSTRACT
Background Previous study showed that both hydroxycamptothecin (HCPT) and etoposide (VP-16) can induce the apoptosis of human Tenon capsule fibroblasts (HTFs).However,whether the combination of HCPT with VP-16 enhance the efficacy of drugs is unknown.Olbjeetive This study was to investigate the synergistic effect and its mechanism of HCPT combined with VP-16 on apoptosis of HTFs.Methods Human Tenon capsule tissue was obtained from the eye bank of Jiangsu Province People's Hospital.HTFs were cultured in vitro using explant method and identified by immunofluorescence with vimentin.The fourth generation of cells were incubated in 96-well plate,and different concentrations of HCPT (1,5,10,50,100 mg/L),VP-16 (0.6,2.5,5.0,25.0,50.0 mg/L) and HCPT+VP-16 (2:1,final concentrations 0.80,3.75,7.50,37.50,75.00 mg/L) were added for 24 hours.The inhibiting rate of drugs to HTFs growth was detected using CCK-8 kit.The HTFs were divided into blank control group,HCPT (50 ng/L) treated group,VP-16 (25 mg/L) treated group and HCPT+ VP-16 (37.5 mg/L) treated group,and the apoptosis rates of HTFs in various groups were assayed by flow cytometry.The expressions of caspase-3,cleaved caspase-3,bax,bcl-2,JNK,p-JNK,Akt,p-Akt in the cells were detected by Western blot assay.Results Cultured cells grew well with the polygon shape and positive response for vimentin.The inhibiting rate was elevated with the increase of drug dosage 24 hours after addition of drugs (HCPT:F=41.34,P=0.00 ; VP-16:F =62.60,P =0.00 ; HCPT+VP-16:F =46.77,P =0.00).The half maximal inhibitory concentrations (IC50) of HCPT,VP-16,HCPT+VP-16 were 80.99,27.93,19.81 mg/L,respectively,and the combined index (CI) of HCPT with VP-16 was 0.399,showing a stronger synergistic action.The apoptotic rates of HTFs were (4.87±0.78) ％,(11.20± 1.94)％,(12.67±1.51)％ and (19.77±2.01)％ in the blank control group,HCPT treated group,VP-16 treated group and HCPT+VP-16 treated group,respectively,with a significant difference among them (F=18.23,P ＜ 0.01),and the apoptotic rate was significantly raised in the HCPT + VP-16 treated group,HCPT treated group and VP-16 treated group compared with the blank control group (q'=15.67,16.32,26.88,all at P＜0.01).Compared with the blank control group,the grey scale values of cleaved caspase-3,bax,p-JNK in the cells of HCPT+VP-16 treated group,HCPT treated group and VP-16 treated group were significantly increased (all at P＜0.01),and those in the HCPT+VP-16 treated group significant ascent in comparison with the HCPT treated group and VP-16 treated group (all at P＜0.01).However,the changes of caspase-3,JNK and Akt expression were insignificant.The grey scale values of bcl-2 and p-Akt in the HTFs of the HCPT,VP-16 and HCPT+VP-16 treated groups were significantly lower than those of the blank control group,with a dominant reducing in the HCPT+VP-16 treated group (all at P＜0.01).Conclusions HCPT and VP-16 induce the apoptosis of HTFs in vitro at a dose-dependent manner.The combination of HCPT with VP-16 has a stronger synergistic efficacy.The up-regulation of p-JNK and bax as well as the down-regulation of p-Akt and bcl-2 in HTFs are involved in the coaction of HCPT and VP-16.