SPL-A Sensitized Human Endometrial Cancer Cell Line To TRAIL-induced Apoptosis And Its Related Mechanism

Endometrial carcinoma is one of the three malignant tumors of the female genital tract that accounts for 20-30% of the gynecological malignancies.In recent years,owing to aging population,obesity and the popularization of hormone replacement therapy,the incidence of endometrial carcinoma is increasing year by year,with a younger age of onset in the world.Although most endometrial carcinomas are diagnosed at an early stage and have a favorable prognosis,advanced and recurrent endometrial carcinoma is generally associated with poor prognosis.At present,the initiation and progression of endometrial carcinoma remain poorly understood.Thus,it is of great significance to investigate molecular mechanism of endometrial carcinoma and develop novel targeted therapies against this cancer.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)could promote apoptosis in various cancer cells other than in normal cells.However,TRAIL resistance is found in a number of cancers,including endometrial carcinoma.This finding greatly limits the wide application of TRAIL in cancer treatment.How to enhance the TRAIL-induced apoptosis in tumor cells is the focus of current research.Spindlactone A(SPL-A)is a novel small molecule inhibitor of transforming acidic coiled-coil-containing protein 3(TACC3)that selectively inhibits the nucleation of centrosome microtubules and induces mitotic arrest in ovarian cancer cells.Dicoumarol has been shown to enhance pro-apoptotic effect of chemotherapy agents in various carcinoma cells.The structure of SPL-A is similar to that of dicoumarol,nevertheless,the efficacy of SPL-A on TRAIL-induced apoptosis remains unclear.In this study,the apoptosis of endometrial carcinoma cell lines treated by TRAIL was detected by flow cytometry,caspase-3 activity assay and DNA fragment analysis,in order to analyze the role of TRAIL in induction of apoptosis of endometrial carcinoma cells.SPL-A was added to the endometrial cancer cell lines treated with TRAIL,endometrial stromal cell lines and endometrial epithelial cell lines,respectively.Then the same techniques as described above were used to analyze the apoptosis of each cell line to explore the effect of SPL-A on TRAIL-induced apoptosis and to further analyze the potential mechanism by which SPL-A enhances TRAIL-induced apoptosis in endometrial carcinoma cells.Part One The effect of TRAIL on the apoptosis of endometrial carcinoma cellsObjective: To investigate the effect of TRAIL on the apoptosis of endometrial carcinoma cells.Methods: Endometrial carcinoma cell lines Ishikawa,HEC-1A and RL-952,endometrial stromal cell line HESCs and endometrial epithelial cell line HEECs were treated with TRAIL.Then the cell viability were measured by MTT.The apoptosis of each cell was analyzed by detecting the percentage of sub-G1 phase cells,caspase-3 activity assay and DNA fragment analysis.Therefore,the effects of TRAIL on apoptosis of endometrial cancer cells,endometrial stromal cells and endometrial epithelial cells were studied.Results:1.Effects of TRAIL on the viability of the endometrial carcinoma cell line IshikawaEndometrial carcinoma cell line Ishikawa was treated with different doses(0,20,40,and 80ng/ml)of TRAIL.The viability of the cells in each group was(99.97±0.025)%,(97.53±0.134)%,(94.21±0.639)% and(90.04± 1.237)%,respectively,and there was no significant difference in each group(P>0.05).2.Effects of TRAIL on the apoptosis of endometrial carcinoma cell line IshikawaEndometrial carcinoma cell line Ishikawa was treated with TRAIL(40ng/ml).Compared with the control group(5.23±0.69)%,the percentage of sub-G1 phase cells(6.81±1.02)% showed no significant difference(P>0.05).The activities of caspase-3 were(0.28±0.078)in the control group and(0.37±0.051)in TRAIL group,and there was no significant difference between the two groups(P>0.05).The contents of DNA fragments were 2.73±0.95 in the control group and 3.12±1.04 in the TRAIL group.No significant difference were noted between the two groups(P>0.05).3.Effects of TRAIL on the apoptosis of endometrial carcinoma cell lines HEC-1A and RL-952Endometrial carcinoma cell lines HEC-1A and RL-952 were treated with TRAIL(40ng/ml).The percentages of sub-G1 phase cells in each cell line were(9.37±0.85)% and(8.07±0.74)% respectively,and the percentages in each responding control group were(7.89±0.96)% and(6.31±0.78)% respectively,suggesting no significant difference(P>0.05).4.Effects of TRAIL on the apoptosis of endometrial stromal cell line HESCs and endometrial epithelial cell line HEECs.Endometrial stromal cell line HESCs and endometrial epithelial cell line HEECs were treated with TRAIL(40ng/ml).The percentages of sub-G1 phase cells in each cell line were(5.13±0.84)% and(5.01±1.14)% respectively,and the percentages in each responding control group were(4.96±0.95)% and(5.67 ± 0.89)% respectively.Thus,no significant difference was found(P>0.05).Conclusions:1.There are no significant effects of TRAIL on the viability of endometrial carcinoma cell line Ishikawa.2.TRAIL exerts no remarkable effects on apoptosis induction in endometrial carcinoma cell lines,normal endometrial stromal cell lines and endometrial epithelial cell lines in vitro.Part Two SPL-A sensitized human endometrial cancer cells to TRAIL-induced apoptosisObjective: To investigate the effects of SPL-A on TRAIL-induced apoptosis in endometrial carcinoma cells.Methods: SPL-A was synthesized using dicoumarol as substrate.The endometrial carcinoma cell lines Ishikawa,HEC-1A,and RL-952,and normal endometrium cell lines HESCs and HEECs pretreated with TRAIL were treated with SPL-A.The apoptosis of each cell group was analyzed by detecting the percentage of sub-G1 and PARP cleavage,the assay of Caspase-3 viability and DNA fragmentation analysis.Then the effects of SPL-A on TRAIL-induced apoptosis in endometrial carcinoma cells,endometrial stromal cells and endometrial epithelial cells were investigated.Results:1.Ishikawa endometrial carcinoma cells were treated with SPL-A(20μM and 40μM)alone,TRAIL(40ng/ml)alone and SPL-A(20μM and 40μM)combined with TRAIL(40ng/ml)respectively for 24 h.The percentages of sub-G1 phase cells were(5.12±0.77)% in control group,(7.31±0.98)% in SPL-A 20μM group,(8.23±1.04)% in SPL-A 40μM group,(25.5±.82)% in SPL-A 20μM+TRAIL 40ng/ml group,(39.23±2.67)% in SPL-A 40μM+TRAIL 40ng/ml group,and(7.06±1.01)% in TRAIL 40ng/ml group.Compared with the control group,SPL-A alone and TRAIL alone group,combination of SPL-A and TRAIL could remarkably increase the percentage of sub-G1 phase cell(P<0.05),and significantly enhance the expression of PARP cleavage.2.After detection of the percentage of sub-G1 phase cells and the expression of PARP Cleavage in each group,SPL-A 40μM+TRAIL 40ng/ml group was found to have the highest percentage of sub-G1 phase cell(39.23±2.67)%,and there was significant difference(P<0.05).3.Ishikawa was treated with SPL-A(40μM)alone,TRAIL(40ng/ml)alone,and SPL-A(40μM)combined with TRAIL(40ng/ml)for 24 h.The activity of Caspase-3 was 0.32±0.089 in control group,0.38±0.081 in SPL-A group,0.97±0.092 in SPL-A+TRAIL group and 0.42±0.058 in TRAIL group.Compared with the control group,SPL-A group and TRAIL group,the activity of Caspase-3 was significantly enhanced in SPL-A+TRAIL group,and the difference was statistically significant(P<0.05).The content of DNA fragments was 2.46±0.73 in control group,3.43±0.96 in SPL-A group,11.39±1.11 in SPL-A +TRAIL group and 3.18±1.02 in TRAIL group.As compared with the control group,SPL-A group and TRAIL group,the DNA fragmentation of SPL-A+TRAIL group was increased significantly,and there were significant differences(P<0.05).4.SPL-A(40μM)alone,TRAIL(40ng/ml)alone and combination of SPL-A and TRAIL were used for incubation of endometrial stromal cell line HESCs and endometrial epithelial cell line HEECs for 24 h.The percentage of sub-G1 phase cells was(5.19±0.93)%/(5.82±0.93)% in control group,(6.27±0.79)%/(6.31±0.83)% in SPL-A group,(8.11±1.01)%/(6.58±0.89)% in SPL-A+TRAIL group,and(5.32±0.87)%/(5.13±1.09)% in TRAIL group.There was no significant difference between two groups(P>0.05).5.Endometrial carcinoma cell lines HEC-1A and RL-952 were treated with SPL-A(40μM)alone,TRAIL(40ng/ml)alone and SPL-A(40μM)combined with TRAIL(40ng/ml)for 24 h.The percentages of sub-G1 phase cells were(6.47±0.82)%/(5.88±0.65)% in control group,(8.82± 0.59)% /(9.12±0.76)% in SPL-A group,(35.94±1.94)%/(38.24±1.88)% in SPL-A +TRAIL group,and(8.88±0.83)/(10.06±0.88)% in TRAIL group.Compared with the control group,SPL-A group and TRAIL group,SPL-A combined with TRAIL could significantly increase the percentage of sub-G1 phase cell in both cell lines(P<0.05).Conclusions:1.SPL-A sensitizes human endometrial cancer cells to TRAIL-induced apoptosis,other than the normal cells.2.SPL-A exerts a sensitization effect on TRAIL-induced apoptosis of endometrial carcinoma cells in a dose-dependent manner.Part Three The effect of SPL-A on the expression of anti-apoptotic protein and NQO1 in endometrial carcinoma cellsObjective: To investigate the potential mechanism by which SPL-A enhances TRAIL-induced apoptosis via detecting the expression of a series of anti-apoptotic proteins(c-FLIP,Bcl-2,Bcl-xL,Mcl-1 cIAP1 and XIAP).The active oxygen ROS and NQO1 activity in each group was compared to confirm the role of SPL-A in sensitizing the apoptosis induced by TRAIL.Methods: SPL-A was used for incubation of endometrial carcinoma cell line Ishikawa pretreated with TRAIL and that transfected with Bcl-2,Mcl-1,and c-FLIP,respectively.The Bcl-2 mRNA expression of each group was detected by real-time quantitative RT-PCR,whereas the expression levels of anti-apoptotic proteins c-FLIP,Bcl-2,Bcl-xL,Mcl-1,cIAP1 and XIAP were detected by Western blot.The generation of intracellular ROS was observed by fluorescence microscope,and the activity of NQO1 was measured by the enzyme labeling instrument.Subsequently,the potential pathway of SPL-A sensitization of TRAIL-induced apoptosis was studied.Results:1.Effects of SPL-A on the expression of anti-apoptotic proteins Bcl-2,Bcl-xl,Mcl-1,c-FLIP,cIAP1 and XIAP in endometrial carcinoma cell line Ishikawa.SPL-A could downregulate the expression of Bcl-2,Bcl-xl,Mcl-1 and c-FLIP in Ishikawa cells in a dose-dependent manner.With the increase in the dose of SPL-A,its inhibitory effect on Bcl-2,Bcl-xl,Mcl-1 and c-FLIP was enhanced gradually,suggesting statistically significant differences(P<0.05).However,the expression of cIAP1 and XIAP did not differ significantly(P>0.05).Endometrial carcinoma cell line Ishikawa was treated with different doses of SPL-A(0μM,10μM,20μM and 40μM)for 24 h.The relative expressions of Bcl-2 mRNA in each group were 1.01±0.021,0.68±0.042,0.44±0.023,and 0.19±0.009,respectively.There were statistical differences in between-group comparison(P<0.05).Ishikawa cells were treated with the same dose of SPL-A(40μM)for different time(0h,6h,12 h,and 24h).The expressions of Bcl-2 mRNA in each group were 1.01±0.002,0.76±0.013,0.33±0.024,and 0.19±0.009,respectively.There were statistical differences between the two groups(P<0.05).Different doses of SPL-A(0μM,20μM and 40μM)were added to Ishikawa cells transfected with the luciferase reporter gene containing Bcl-2 promoter,and the activity of Bcl-2 promoter in each group was 1.0±0.041,0.73±0.038 and 0.46±0.022,respectively.There were statistical differences between two groups(P<0.05).2.The effect of SPL-A on the expression of tumor suppressor gene p53 in endometrial carcinoma cell line IshikawaDifferent doses of SPL-A(0,10μM,20μM,and 40μM)were added for incubation of endometrial carcinoma cell line Ishikawa for 24 h.The expression of p53 was gradually increased.However,the inhibitory effect of SPL-A on Bcl-2 was weakened after adding PFT-α(p53 inhibitor).3.Endometrial carcinoma cell line Ishikawa was treated with SPL-A(40μM)alone,TRAIL(40ng/ml)alone,and SPL-A(40μM)combined with TRAIL(40ng/ml)respectively for 24 h.The percentages of sub-G1 phase cells were(5.12±0.77)% in control group,(8.23±1.04)% in SPL-A group,(39.23±2.67)% in SPL-A+TRAIL group,and(7.06±1.01)% in TRAIL group.The percentage of sub-G1 phase cells was the highest in SPL-A+TRAIL group,indicating significant differences(P<0.05).After transfection with Bcl-2,Mcl-1 and c-FLIP plasmid,the role of SPL-A in enhancement of TRAIL-induced apoptosis was significantly decreased.The percentages of sub-G1 phase cells were(11.13±0.56)%,(12.93±0.98)% and(19.25±2.06)%,respectively,which were higher than those in SPL-A and TRAIL group,revealing significant differences(P<0.05).4.The correlation between SPL-A and ROSEndometrial carcinoma cell line Ishikawa was treated with SPL-A(40μM)for 2h,and ROS production was increased significantly.Morover,Ishikawa was treated with SPL-A combined with TRAIL for 24 h after adding antioxidants(NAC or Trolox).The percentages of sub-G1 phase cells were(16.25±1.19)% and(19.06±1.13)% respectively,which were decreased significantly compared with(43.75±2.25)% in non-antioxidant group.No significant differences were observed(P<0.05).5.The correlation between SPL-A and NQO-1Compared with the control group,both SPL-A and ES936(NQO-1 inhibitor)could significantly inhibit the activity of NQO-1,with 1.01±0.031 in the control group,0.34±0.037 in SPL-A group,and 0.39±0.051 in ES936 group,suggesting significant differences(P<0.05).Endometrial carcinoma cell line Ishikawa was treated with TRAIL for 24 h after ES936 was added.The percentage of sub-G1 phase cells was(40.03±1.47)%,which was significantly higher than(8.73±0.66)% in the control group(P<0.05).Conclusions:1.SPL-A inhibits the activity of anti-apoptotic molecule Bcl-2 by up-regulating the expression of P53.2.SPL-A downregulates Bcl-2 family members Mcl-1 and c-FLIP,which contributed to sensitivity of endometrial cancer cells to TRAIL-induced apoptosis.3.The effect of SPL-A on TRAIL-induced apoptosis depends on the generation of ROS in cells and inhibition of NQO1 activity.Conclusions:1.TRAIL has no obvious effects on induction of apoptosis in endometrial carcinoma cell lines and normal endometrial cell lines in vitro,suggesting potential TRAIL resistance in endometrial carcinoma cells.2.Combination of SPL-A and TRAIL can significantly induce apoptosis of various human endometrial carcinoma cells,but has no effect on apoptosis of normal human endometrial stromal cells and endometrial epithelial cells.SPL-A as TRAIL sensitizer is expected to bring new prospects for the adjuvant treatment of endometrial carcinoma.3.SPL-A increases the sensitivity of endometrial carcinoma cells to TRAIL-induced apoptosis by regulating apoptosis-related proteins and inhibiting NQO1 activity.