Role Of Caspase8 In The Induced Apoptosis Of Neuroblastoma Cells Through TRAIL-death Receptor Pathway

Introduction and objectiveNeuroblastoma ( NB) is the most common extracranial solid tumor in children. High risk prognostic makers include > lyear of age, loss of heterogygosity on IP, amplification of N -myc. What's more, some present therapeutics such as surgery, chemotherapy and stem cell transplantation are not very effective. Chemoresistance and metastatic spread are main difficult questions in clinic. So new anticancer methods are continuously explored. Inducing apoptosis in tumor cells through activating TRAIL (tumor necrosis factor related apoptosis inducing ligand ) pathway may be a hot point in NB therapy. TRAIL can selectively induce apoptosis in tumor cells and this makes it have a good clinical application prospect. It has been reported that combination of TRAIL, IFN-γ and cytotoxic drugs could enhance the TRAIL - sensitivity of tumor cells.TRAIL is a member of the tumor necrosis factor super - family discovered recently. Different from TNF and FASL, TRAIL can induce apoptosis in tumor cells and transformed cells specially but has no toxicity to the normal cells. 5 kinds of membrane receptors are identified to bind TRAIL, they are death receptors DR4 and DR5, decoy receptors DCR1, DCR2 and OPG (osteoprotegerin). The two decoy receptors are homogeneous to the death receptors but lack functional intracellular death domain, so they cant induce apoptosis. TRAIL can initiate the intracellular signal transduction by binding to death receptors and initiate Caspase (cysteine containing asparate specific protease) cascade reactionthat leads to the programmed cell death. In this process, death receptor activates Caspase 8 through intracellular Fas related protein, the active Caspase 8 cleaves and activates downstream Caspase - Caspase3 and then induces apopto-sis of NB cells.Most NB cell lines are TRAIL resistant for their loss of Caspase 8 expression. The silence of Caspase 8 gene in NB is the relult of hypermethylation of promoter region in the Caspase 8 gene. How to restore Caspase 8 expression and TRAIL sensitivity in NB cells becomes a hot point in NB studies. We used 5 -Azacytidine (5AZA) , IFN7 and chemotherapeutic drugs combined with TRAIL to treat the NB cell lines and studied the molecular mechanisms of TRAIL - resistance of NB cells, and whether combination of these drugs could overcome the resistance in order to provide theoretical basis for the clinical application of TRAIL.Methods1. To detect the level of Caspase 8, TRAIL, TRAIL receptor DR5 and other apoptosis - related genes before and after treatment of 5 AZA or IFN7 by RT -PCR.2. To detect the expression of Caspase 8 protein before and after treatment of 5 AZA or IFN-y by cell immunochemistry and Western blot analysis. To detect the expression of DR5 protein of CHP212, SH - SY5Y and SKNDZ cell lines and the expression of DR5 protein of SKNDZ cells after treatment of chemotherapeutic drugs by Western blot analysis.3. MTT analysis;(1) SY5Y cells treated by 5AZA + TRAIL (0,50,100, 200 g/L) and different concentration of TRAIL alone for 12, 24, 48 hours;Caspase 8 inhibitor group that 5 ujnol/L inhibitors were added 1 hour prior to different concentration of TRAIL treatment for 24 hours. (2)There were 3 groups for CHP212 cells;group treated by different concentration of TRAIL for 24 hours;group treated by lOOjjug/L TRAIL for different time;Caspase 8 inhibitor group . There were 4 groups for SY5 Y cells: group treated by different concentration of IFN-y for 48 hours;group treated by different concentration of TRAILfor 24 hours after cells were pretreated by IFN-y for 48 hours;group treated by different concentration of TRAIL for 24 hours;Caspase 8 inhibitor group. ( 3 ) There were 3 groups for SKNDZ cells: control group ( not treated by chemothera-peutic drugs);group treated by doxorubicin;group treated by etoposide . Each group was treated by TRAIL, IFN7, IFN7 + TRAIL for 48 hours.4. To detect apoptosis rate by flow cytometer (FCM).5. Measurement of relative Caspase 8 activity by colorimetric assay.6. To detect the morphous of the apoptosis cells by transmission electron mocroscope (TEM) .7. Statistical analysis SPSS 11.5 software was used to analyze the data. Values of mean and standard deviation from the repeat of independent experiments were calculated. ANOVA was used to test whether the treated cultures were significant from the controls. P <0.05 was considered significant in all cases.Results1. The expression of Caspase 8, TRAIL, TRAIL receptor DR5 and other apoptosis -related genes.The expression of Caspase 8 mRNA was undetectable in SY5Y cells but the level of Caspase 8 mRNA increased with the prolongation of 5 AZA action time. The expression of Caspase 8 mRNA was undetectable in SKNDZ cells. The expression of Caspase 8 mRNA of SY5Y and SKNDZ cells also increased with the prolongation of IFN7 action time. The expression of Caspase 8 mRNA was detectable in CHP212 cells with increased expression after treatment with IFN7.IFN-y treatment didn' t affect the TRAIL receptor DR5 but induced TRAIL mRNA expression. NB cells expressed detectable basal level of cFLIP mRNA that was enhanced by IFN7 treatment. BCL - 2 was detected but not affected by IFN7 treatment , IFN7 treatment also increased BAK mRNA expression.2. To detect the expression of Caspase 8 protein by cell immunochemistry. The expression of Caspase 8 protein was undetectable in SY5Y cells and itwas positive after treatment of 5|xmol/L 5AZA for 5 days. TTie expression ofCaspase 8 protein was undetectable in SKNDZ cells and it was positive after treatment of lOOjig/L IFN-y for 48 hours.3. To detect the expression of Caspase 8 protein by Western blot analysis.The expression of Caspase 8 protein was detectable in CHP212 cells and increased after treatment of 50,100,200(ig/L IFN-y for 48 hours. The expression of Caspase 8 protein was undetectable in SY5Y cells and the level of Caspase 8 protein increased with the adding of the concentration of IFN7.4. To detect the expression of DR5 protein by Western blot analysis.The expression of DR5 protein was detectable in CHP212 and SY5Y cells. The expression of DR5 protein was undetectable in SKNDZ cells and it was positive after treatment of chemotherapeutic drugs for 24 hours.5. To detect the survival rate by MTT.The killing effect of TRAIL in SY5 Y cells increased slightly with the adding of the concentration of TRAIL and the action time and the survival rate of 200|Ag/L TRAIL group was still 88.73%. There was no marked variation among different concentration of TRAIL groups. It suggested that SY5Y cells were not sensitive to TRAIL. The survival rates of 5AZA + TRAIL groups were greatly lower than those of TRAIL groups( P <0.05 ). There was great significance between 5AZA + TRAIL group and Caspase 8 inhibitor group(P <0.05).CHP212 cells were sensitive to TRAIL and had time and dose dependency. The survival rates decreased with the increase of TRAIL concentration and action time. There was great significance between each experimental group and control group(P <0.05). The survival rates of Caspase 8 inhibitor groups were greatly higher than those of TRAIL groups and there was great significance from 25/xg/ L TRAIL and 8h TRAIL action time( P <0.01). There was no marked effect on SY5Y cells when we used IFN7 or TRAIL alone. The survival rates of IFN7 + TRAIL groups were of great significance compared with IFN7 or TRAIL groups (P <0.01). The survival rates of Caspase 8 inhibitor groups were much higher than those of IFN7 + TRAIL groups ( P <0.01). It suggested that Caspase 8 inhibitor could markedly inhibit the killing effect of TRAIL on NB cells expressing Caspase 8.SKNDZ cells were not sensitive to TRAIL and SKNDZ cells expressingCaspase 8 after treatment with IFN-y were not sensitive to TRAIL too. TTie difference of the inhibition rates between TRAIL group and IFN-y + TRAIL group was not significant( P > 0.05 ). SKNDZ cells pretreated with adriamycin or etoposide were not sensitive to TRAIL but sensitive to IFN7 + TRAIL, and the difference of the inhibition rates between adriamycin /etoposide + IFN7 + TRAIL group and adriamycin /etoposide + TRAIL group was significant ( P < 0.01). Inhibition rates of IFN7 + TRAIL group in SKNDZ cells pretreated with adriamycin or etoposide were much higher than those of control groups( P <0.01).6. To detect the apoptosis rate by FCM.The apoptosis rate of 5AZA + TRAIL group in SY5Y cells was greatly higher than that of TRAIL group( P <0.01). CHP212 cells were sensitive to TRAIL and the early stage apoptosis rate of the 10u,g/L ^SOjig/L^OOu^/L TRAIL group was (6.27 ±0.36)% , (29.44 ±5.02)% and( 40.12±3.98 )% ,there was great significance among the groups (P <0. 05). The early stage apoptosis rate of IFN7 + TRAIL group in SY5 Y cells was higher than that of TRAIL group (P < 0.01). The early stage apoptosis rate of adriamycin or etoposide + IFN7+ TRAIL group was higher than that of IFN7 + TRAIL group in SKNDZ cells (P<0.05).7. To detect the relative Caspase 8 activity by colorimetric assay.The relative Caspase 8 activity of SY5Y cells in 5AZA + TRAIL group was much higher than those of control group, 5AZA group, TRAIL group and inhibitor group(P <0. 01) . There was no obvious difference among 5AZA group, TRAIL group and control group (P >0.05). The relative Caspase 8 activity of CHP212 cells increased with the prolongation of TRAIL action time with a peak time at 16h. There was marked difference between 4h and 16h /24h(P <0. 01);8h and 16h( P < 0.05 ). The relative Caspase 8 activity decreased when u-sing Caspase 8 inhibitor zIETD - FMK, and there was great significance between TRAIL group and inhibitor group /control group {P < 0. 01). The relative Caspase 8 activity of SY5 Y cells in IFN7 + TRAIL group was much higher than those of control group, IFN-ygroup, TRAIL group and inhibitor group (P < 0. 01) . There was no obvious difference among other groups ( P > 0. 05 ). The relative Caspase 8 activity of SKNDZ cells in chemotherapeutic drugs + IFN7 +TRAIL group was much higher than those of control group, IFN7 group, TRAIL group and IFN7 + TRAIL group(P <0.01). There was no marked difference a-mong other groups ( P > 0.05 ).8. To survey the shape of the cell apoptosis by TEM.The shape of CHP212 cells in control group , SY5Y cells in the TRAIL group was as follow lower tracing: the intranuclear chromatin was well - distributed , the nuclear membrane was clear and the mitochondria ,ribosome and lyso-some could be seen. The shape of CHP212 cells in the TRAIL group , SY5Y cells in the 5AZA + TRAIL and IFN7 + TRAIL group was as follow lower tracing: the cells were shrinkaged but the cell membranes were intact, the nucleus were pycnosis, chromatic agglutination in the nucleus, the chromatin were side, looking like crescent, and the intermembrance space was not identical. Those were typical characters of cell apoptosis.Conclusions1. 5AZA could induce expression of Caspase 8 mRNA and protein in SY5Y cells and the expression of Caspase 8 mRNA increased with the prolongation of 5AZA action time. IFN-y could induce expression of Caspase 8 mRNA and protein in NB cells and the expression of Caspase 8 mRNA increased with the prolongation of IFN-y action time.2. CHP212 cells were sensitive to TRAIL with time and dose dependency. SY5Y cells expressing Caspase 8 after treatment with 5AZA or IFN7 were sensitive to TRAIL with an increase of relative Caspase 8 activity. 5AZA or IFN7 could enhance the TRAIL sensitivity of SY5Y cells and this may be realized by upregulation of Caspase 8.3. DR5 protein was not detected in SKNDZ cells but an increased expression of DR5 protein was found after treatment with adriamycin or etoposide for 24h. SKNDZ cells expressing Caspase 8 after treatment with IFN7 were not sensitive to TRAIL but SKNDZ cells expressing Caspase 8 and DRS simultaneously were sensitive to TRAIL with an increase of relative Caspase 8 activity. IFN'y and chemotherapeutic drugs could reverse the TRAIL - resistance of SKNDZcells and this may be realized by upregulation of Caspase 8 with IFN7 and DR5 with chemotherapeutic drugs.