Synergistic Interactions Of TRAIL, Chemotherapeutic Agents And Radiation On Apoptosis Of Laryngeal Squamous Cell Carcinoma Cell Line Hep-2 In Vitro

Synergistic interactions of TRAIL, chemotherapeutic agents and radiationon apoptosis of laryngeal squamous cell carcinoma cell line Hep-2 in vitroObjective Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL/Apo2L) could specifically induce apoptosis in various tumor cellsand transformed cells by integrated with its special receptors, but itwas nontoxic to normal cells. It had demonstrated that TRAIL inducedapoptosis in tumor cells with different sensitivity, and somechemotherapeutic agents and radiation had synergistic effects with TRAILin inducing cell death because they could overcome resistance to TRAILor enhance sensitivity to TRAIL in TRAIL-resistant tumor cells. The aimof this study was to investigate the sensitivity of TRAIL inducedapoptosis in laryngeal squmous cell carcinoma cell line and its mechanism.Moreover, we wished to determine the synergistic killing effects of TRAILand cisplatin, paclitaxel, radiation and the possible mechanisms throughevaluating the expression of TRAIL receptors and the activity of caspase-8,caspase-9.Methods (1) Hep-2 cells were exposed to various concentrations of TRAIL,cisplatin or paclitaxel for 12h, 24h, 48h or 72h respectively. Theanti-proliferation effect was measured by CCK-8 assay. (2) After Hep-2cells were exposed to 5μg/ml cisplatin, 12nmol/L paclitaxel or 8Gyradiation for 24h, the expression of TRAIL receptors protein and mRNA wereassayed by flow cytometry and semiquantitive RT-PCR respectively. (3)After Hep-2 cells were exposed to 600ng/ml TRAIL, 5μg/ml cisplatin,12nmol/L paclitaxel, 8Gy radiation, 600ng/ml TRAIL+5μg/ml cisplatin,600ng/ml TRAIL+12nmol/L paclitaxel or 600ng/ml TRAIL+8Gy radiation for24h, morphological changes were observed by phase contrast microscopy,apoptosis was measured by flow cytometry and expressions of TRAILreceptors, caspase-3, caspase-8 and caspase-9 were determined by WesternBlot analysis. (4) After the activities of caspase-8 and caspase-9 wereinhibited by Z-IETD-FMK and Z-LETD-FMK respectively, Hep-2 cells wereexposed to various concentrations of TRAIL, cisplatin or paclitaxel for24h, and then, CCK-8 assay was used to determine the anti-proliferation effect.Results (1) TRAIL, cisplatin and paclitaxel inhibited Hep-2 cells growthin a dose- and time-dependent manner within a certain concentration, forTRAIL 24h IC50 was 596.92 ng/ml and 48h IC50 was 367.03ng/ml, for cisplatin24h IC50 was 5.09μg/ml and for paclitaxel 24h IC50 was 12.12nmol/L and48h IC50 was 7.13 nmol/L. Hep-2 cells was resisitant to TRAIL inducingapoptosis. (2) Apoptosis of Hep-2 cells induced by combined cisplatin,paclitaxel, radiation and TRAIL administration was significantly higherthan those of each one administration. They had synergistic killing effectto Hep-2 cells. (3) In the control Hep-2 cells, the expression of mRNAand protein of decoy receptors (DcR1, DcR2) was significantly higher thandeath receptors (DR4, DR5). After Hep-2 cells were exposed to 5μg/mlcisplatin, 12nmol/L paclitaxel and 8Gy radiation, the expression of deathreceptors increased, especially the DRS, the expression of DcR1 decreasedand the expression of DcR2 had no significant change. The changes ofexpression of TRAIL receptors enhanced the sensitivity of Hep-2 cells toTRAIL. (4) After the activity of caspase-8 was inhibited by Z-IETD-FMK,the inhibition effects of TRAIL, cisplatin and paclitaxel to Hep-2 cellsdecreased slightly (P＞0.05). After the activity of caspase-9 wasinhibited by Z-LETD-FMK, the inhibition effects of TRAIL, cisplatin andpaclitaxel to Hep-2 cells decreased significantly (P＜0.05). So TRAIL,cisplatin and paclitaxel could induce apoptosis of Hep-2 cells throughthe endogenous apoptotic pathway. (5) After Hep-2 cells were exposed to600ng/ml TRAIL 5μg/ml cisplatin, 12nmol/L paclitaxel and 8Gy radiation,the expression of caspase-8 increased slightly (P＞0.05) and caspase-9increased significantly (P＜0.05), but the expression of caspase-8increased significantly (P＜0.05) after Hep-2 cells exposed to combinedadministration. It suggested that TRAIL, cisplatin and paclitaxel inducedapoptosis of Hep-2 cells all through the endogenous apoptotic pathway,while casepase-8 was activated by activated casepase-9 and casepase-3through feedback loop in the combined administration. The activation ofcasepase-8 sensitized the Hep-2 cells to TRAIL and exerted the synergistickilling effects.Conclusion (1) The laryngeal squamous cell carcinoma cell line Hep-2 was resistant to TRAIL. TRAIL, cisplatin and paclitaxel inhibited the growthof Hep-2 cell in a dose- and time-dependent manner within a certainconcentration. Combined TRAIL with cisplatin, paclitaxel, radiation hadsynergistic killing effects to Hep-2 cells. (2) Cisplatin, paclitaxel andradiation could enhance the expression of death receptors, especially theDR5, decrease the expression of DcRl of Hep-2 cells. The changes ofexpression of TRAIL receptors might be responsible to overcome resistanceto TRAIL in Hep-2 and exert the synergistic killing effects. (3) Afterthe activity of caspase-9 was inhibited by Z-LETD-FMK, the inhibition ofTRAIL, cisplatin and paclitaxel to Hep-2 cells decreased significantly,it suggested that TRAIL, cisplatin and paclitaxel induced apoptosis ofHep-2 cells all through the endogenous apoptotic pathway. (4) Caspase-8could be activated by activated caspase-9 and caspase-3 through feedbackloop in the combined administration, which enhanced the sensitivity ofHep-2 cells to TRAIL and might be responsible to exert the synergistickilling effects.