| Background：Medullary thyroid cancer (MTC) is a malignant neuroendocrine tumor originatingfrom parafollicular C cells of thyroid gland and accounting for about8%of all thyroidcancers．Currently, surgery performed at an early stage-preferably after the early diagnosis, is themain treatment of MTC. Due to its early stage transfer and the little efficacy of radioiodineablation, distant metastases are the main cause of MTC-related death which accounts for up to13.4%death caused by all thyroid cancers. Thus, more efforts are needed for the novel strategiesof MTC.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can preferentially induceapoptosis in transformed or malignant cells but not in normal cells demonstrating potential as atumor-selective apoptosis-inducing cytokine for cancer treatment.However,many type of cancer,including medullary thyroid cancer,are resistant to TRAIL-induced apoptosis which limits thepotential of TRAIL for cancer treatment. Cyclooxygenase-2(COX-2) highly expressed in variouscancer cells including MTC, has been described an important role in carcinogenesis. Which maysuggests a close link between COX-2and the progression of MTC. It’s noteworthy that certaincancer therapeutic agents including COX-2inhibitor, can sensitize various type of cancer cells toTRAIL-induced cancer cells. And celecoxib, a specific COX-2inhibitor, can reverse thechemoresistance in medullary thyroid cancer. For the reasons above, the co-administration ofTRAIL and celecoxib may be a promising method of improving symptoms and enhancing theprognosis of patients with MTC.Objective: In this study, we investigated the synergistic effects of celecoxib on TRAIL-inducedapoptosis of TT cell line and the related mechanism.Methods: we analyzed the combination effect of TRAIL and celecoxib on the apoptosis inductionof TT cells. Cell growth inhibition was determined by MTT assay. PI staining was used for thedetection of cell cycle distribution. Apoptosis was evaluated by Hochest33258staining、DNAladder and flow cytometry. Gen expression of DR4、DR5and c-FLIP was measured by real-time PCR. The cell cycle and apoptosis associated proteins were measured by Western blot.Results:1)、The growth inhibition on TT cell induced by TRAIL at low concentration was weak,and TRAIL at the concentration of2000ng/ml had a15.77±1.42%growth inhibition on TT cell.TT cell was insensitive to TRAIL. The growth inhibition of celecoxib on TT cell was time-anddose-dependent. The inhibition rate of the combination treatment of50uM celecoxib and1000ng/ml TRAIL at24h and48h was13.40±2.40%、24.49±1.57%respectively, which issignificantly higher than celecoxib(13.40±2.40%、24.49±1.57%) and TRAIL (2.15±1.12%、7.75±3.84%) alone (P<0.01).2)、the G0/G1phase cell distribution of TT cell induced by celecoxibgroup and the combination group treatment for48h was82.82±0.38%and81.76±0.64%respectively, which was significantly higher than control group(72.35±0.51%) and TRAILgroup(72.08±0.74%), P<0.05. And the level of cell cycle regulating protein cdk2and cycling Awas very low in celecoxib group and the combination group, but there was no significant change incontrol group and TRAIL group.3)、The nucleus fragmentation level of combination group wassignificantly higher than it was in TRAIL group and celecoxib group. And the nucleusfragmentation and concentration in combination group was significantly higher than TRAIL groupand celecoxib group. And the apoptosis rate of combination group was31.92±1.35%wassignificantly higher than it was in TRAIL group (6.32±0.83%) and celecoxib group(15.29±2.15%) alone.4)、The activation level of caspase-8was significantly higher than that of TRAILgroup and celecoxib group. And the relative expression of DR5mRNA was significantly higherthan that of TRAIL group and celecoxib group (P<0.05), which was similar to the expression ofDR5protein, but there was no significant change in the expression of DR4mRNA and DR4protein. The relative expression of c-FLIP mRNA was significantly higher than that of TRAILgroup and celecoxib group (P<0.05), which was similar to the expression of c-FLIP protein. Theexpression of RIP was significant low in celecoxib group and the combination group.Conclusion:1、TT cells was insensitive to TRAIL and the treatment of celecoxib could reverse theresistance of TT cells to TRAIL.2、Down-regulation of cyclin A and Cdk2accompanied G0/G1arrest caused by celecoxibcontributed to the growth inhibition of TT cells induced by TRAIL.3、Celecoxib synergistically activated the cleavage of caspase-8and increased the apoptosis level induced by TRAIL through the up-regulation of DR5and down-regulation ofc-FLIP in TT cells. |
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