An Experimental Study Of The Synergistic Effect Of TRAIL Combined With Cisplatin In NSCLC Cell Lines

[Objectives] Currently, the clinical application of chemotherapeutic agents has been severely restricted by their severe toxic side effects. Thus, searching for novel treatment strategies with high specificity to tumor cells and less side effects is urgently required. Apo2 Ligand/Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) being a new member of TNF superfamily was discovered by its sequence homology to TNF and CD95L. TRAIL is a candidate for clinical investigation in cancer therapy because it can trigger apoptosis in a broad spetrum of human cancer cell lines but not in many normal cells, and exhibits potent anti-tumor activity without normal tissue toxicity in various cancer xenograft models. Therefore, this study was to investigate the cytotoxicity of TRAIL in NSCLC cell line NCI-H460 and A549, its synergistic effect with cisplatin, and the possible mechanisms.[Methods] The cytotoxicity was observed by Sulforhodamine B (SRB) assay after treated with TRAIL or/and DDP at different density for 48 hours on NCI-H460 and A549 cells. Growth inhibitory curves were delineated and half inhibitory concentrations (IC₅₀) were analyzed. Cell apoptosis stained by Annexin V-FITC/PI was detected by flow cytometry. RT-PCR was applied to assay mRNA expression of cell-surface receptors DR4,DR5,DcR1 and DcR2 among different groups. The changes of Caspase-8,Caspase-9,Bcl-2 and Bax in protein level were quantified by Western blot analysis.[Results] NCI-H460 cells were sensitive to TRAIL with IC₅₀=6.119ng/ml and present with obviously dose-dependent cytotoxicity, and A549 cells were resistant to TRAIL with IC₅₀=82.822μg/ml but also present with dose-dependent cytotoxicity. Both cells were sensitive to cisplatin with dose-dependent cytotoxicity, and IC₅₀=0.407μg/ml, 0.519μg/ml respectively. TRAIL and cisplatin showed synergistic cytotoxicity in combined treatment. The synergistic effect was mostly due to apoptosis according to FCM, and typical apoptotic morphology obviously observed through fluorescence staining. The expression levels of DR4 and DR5 were not significantly different and the expression of DcR2 was slightly downregulated in combined treatment group of NCI-H460 cells, DcR1 could not been detected. The expression level of Caspase-8, Caspase-9, Bax protein was upregulated in group of TRAIL combined with sub-toxic dose cisplatin, and the level of Bcl-2 was not significant changed among groups.[Conclusion] TRAIL can inhibit both NSCLC cell lines NCI-H460 and A549 by inducing apoptosis, and synergize with sub-toxic dose cisplatin to induce apoptosis effectively not only on TRAIL sensitive cell NCI-H460 but also on TRAIL resistant cell A549. TRAIL and cisplatin don't affect the mRNA expression of DR4 and DR5, The level of DcR2 mRNA, Caspase-8, Caspase-9, Bax protein may involved in the mechanism of synergistic effect. It suggests that the combination of TRAIL and lower dose cisplatin could be the potential strategy against NSCLC.