核因子κB抑制剂对^131Ⅰ导致甲状腺癌细胞凋亡的协同作用

Synergistic effects of nuclear factor-kappa B inhibitor on^131Ⅰ induced apoptosis in differentiated thy- roid cancer cells

目的研究核因子κB（NF-κB）抑制剂Bay11-7082和^131Ⅰ导致DTC细胞凋亡的效果，并探讨二者的协同作用。方法用Westernblot鉴定^131Ⅰ、Bay11-7082以及两者联合处理DTC细胞24h后细胞内NF-κB调控的凋亡抑制因子[x染色体相关凋亡抑制蛋白（XIAP）和生存素蛋白（stir-vivin）]以及凋亡关键因子[天冬氨酸特异性半胱氨酸蛋白酶（caspase3）和多聚腺苷二磷酸核糖聚合酶（PARP）]相对表达水平的变化，以β-actin作内参，进行半定量分析。用膜黏连蛋白-5-异硫氰酸荧光磺／碘化丙啶双标记染色流式细胞技术鉴定不同方式处理后癌细胞的凋亡变化。多组间均数比较采用单因素方差分析，两两比较采用q检验进行统计分析。结果Westernblot证实对照组DTC癌细胞内XIAP和survivin相对表达水平为（16．62±0．73）％和（15．20±0．53）％，^131Ⅰ作用24h后两者增至（95．22±3．27）％和（71．80±2．76）％，而^131Ⅰ联合Bay11-7082作用后两者分别被抑制至（8．29±0．35）％和（6．87±0．28）％。不同处理方式作用后XIAP和survivin表达水平差异均有统计学意义（F=1823．47和1406．12，P均〈0．01），联合处理组与^131Ⅰ组，对照组两两比较q值13．37至45．38（P均〈0．01）。对照组easpase3的2个亚基p19和p17以及PARP失活水解产物p89相对表达水平为（4．93±0．49）％、（4．67±0．34）％和（4．87±0．64）％，^131Ⅰ作用24h后增高至（25．07±1．26）％、（18．29±1．14）％和（34．97±1．90）％，Bay11-7082作用后增至（60．32±3．59）％、（41．29±3．23）％和（66．49±2．96）％；联合用药后增高更为明显，分别为（104．62±5．02）％、（94．72±4．28）％和（101．59±4．04）％。不同处理方式间三者差异均有统计学意义（F=575．13、625．95和712．87，P均〈0．01），联合处理组与单独用药组比较g值15．95～86．01（P均〈0．01）。流式细胞技术同样证明联合用药导致凋亡细胞比例增高的幅度较单独用药更为明显，^131Ⅰ和Bay11-7082单独作用后凋亡早期细胞比例分别为（9．44±0．66）％和（18．92±1．84）％，联合用药后增至（47．02±4．53）％，差异有统计学意义（F=201．12，P〈0．01），联合处理组与^131Ⅰ组和Bay11-7082组两两比较q值为13．86和17.13，P均〈0．01。结论^131Ⅰ通过活化NF-κB途径导致DTC细胞内凋亡抑制因子表达升高，而联合使用Bay11-7082可以明显抑制这种变化。联合用药对^131Ⅰ导致DTC细胞的凋亡可产生协同效应。

Objective To study pro-apoptotic effects of a nuclear factor-kappa B （NF-κB） inhibi- tor （ Bay 11-7082） and ^131Ⅰ on DTC cells and investigate the synergistic effects of the combined treatments. Methods Western blot was used to detect relative protein expression changes of NF-κB regulated to anti- apoptotic factors （ X-chromosome-linked inhibitor of apoptosis （XIAP） and survivin） as well as key apoptot- ic factors （caspase 3 and poly-adenosine diphosphate-ribose polymerase （PAP, P）） after either mono-treat- ment and combined treatment for 24 h. β-actin was used as control and semi-quantitative analysis was per- formed. Flow cytnmetry with Annexin V-fluoreseein isothiocyanate/propidium iodide double staining wasused to analyze the induction of apoptosis after different treatments as well. One-way analysis of variance and q test were used for statistical analysis. Results Basic expressive levels of XIAP and survivin were （ 16.62 ± 0.73） % and （ 15.20 ± 0.53 ） % , respectively. The levels increased to （95.22 ± 3.27） % and （71.80 ±2.76）% after ^131Ⅰ treatment, respectively. In the combined treatment group with ^131Ⅰ and Bay 11- 7082, XIAP and survivin expressions were （ 8.29 ± 0.35 ） % and （6.87 ± 0.28 ） %, respectively. The differences of XIAP and survivin with different treatments were statistically significant （ F = 1823.47 and 1406.12, both P 〈 0.01 ）. The expression levels in the combined treatment group showed significant differ- ences compared with that in ^131Ⅰ mono-treatment or the control group （ q = 13.37 - 45.38, all P 〈 0.01 ）. Basic levels of caspase 3 subunits p19 and p17, as well as degraded PARP protein p89 were （4.93 ± 0. 49） %, （4.67 ± 0.34） % and （4.87 ± 0.64） %, respectively. After 131i treatment they displayed ex- pressive enhancement as （25.07 ±1.26）%, （18.29 ± 1.14）% and （34.97 ± 1.90）%, and after Bay 11-7082 treatment their levels increased to （60.32 ±3.59）%, （41.29 ±3.23）% and （66.49 ± 2. 96 ） %, respectively. In the combination treatment group the above parameters were （ 104.62 ± 5.02） %, （94.72 ± 4.28 ） % and （ 101.59 ± 4.04） % and the differences were statistically significant （ F = 575.13, 625.95 and 712.87, all P 〈 0.01 ）. Compared with the mono-treatment group, the combined treatment group showed significantly higher levels of p19, p17 and p89 （q = 15.95 - 86.01, all P 〈0.01 ）. Flow cy- tometry also showed significantly higher percentage of apoptotic cells in the combined treatment group （47.02± 4.53 ）% than that in ^131Ⅰ treatment group （9.44 ± 0.66）% or Bay 11-7082 treatment group （18.92±1.84）% （F=201.12, q =13. 86 and17.13, allP〈0.01）. Conclusions 131Imayinduceen- hancement of anti-apoptotic factor expressions by activation of NF-κB pathway in DTC cells. This enhance- ment may be inhibited by combination with NF-κB inhibitor Bay 11-7082. Thus NF-κB inhibitor may exert synergistic effects on^131Ⅰ induced apoptosis in DTC cells.