Caspase-8和DR5在TRAIL诱导神经母细胞瘤细胞凋亡中的作用

Role of caspase-8 and DR5 in TRAIL-induced apoptosis of neuroblastoma cells

摘要:

目的：肿瘤坏死因子相关凋亡诱导配体(TRAIL)能诱导多种肿瘤细胞凋亡而对正常细胞无诱导凋亡作用。大多数神经母细胞瘤细胞株对TRAIL的诱导凋亡作用耐受与其Caspase8表达缺失有关,也与细胞表面TRAIL受体的表达和分布有关。该文主要探讨Caspase8及TRAIL受体DR5的表达在TRAIL诱导神经母细胞瘤细胞株SKNDZ凋亡中的作用及其发生机制。方法：应用RTPCR方法检测IFNγ作用前后SKNDZ细胞Caspase8的表达;应用WesternBlot方法检测化疗药作用前后SKNDZ细胞DR5的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测TRAIL、IFNγ+TRAIL、化疗药+TRAIL及化疗药+IFNγ+TRAIL对SKNDZ细胞生长及凋亡的影响。结果：SKNDZ细胞不表达Caspase8,IFNγ作用后的SKNDZ细胞Caspase8表达明显增加。对照组未检测到DR5蛋白表达,而阿霉素和依托泊苷处理后检测到DR5蛋白表达。表达Caspase8的SKNDZ细胞对TRAIL的诱导凋亡作用仍不敏感,而同时表达Caspase8和DR5的SKNDZ细胞对TRAIL的诱导凋亡作用敏感。阿霉素/依托泊苷+IFNγ+TRAIL组早期凋亡率为:(17.9±3.6)%、(14.8±3.3)%,与IFNγ+TRAIL组(3.9±1.2)%比较,差异有显著性(F=26.233,P<0.01)。结论：同时表达Caspase8和DR5的SKNDZ细胞恢复了对TRAIL的敏感性,Caspase8和DR5在TRAIL诱导SKNDZ细胞凋亡中起着十分关键的作用。

Abstract:

OBJECTIVE: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cell death in a variety of tumors but not in normal cells. TRAIL- resistance of most neuroblastoma (NB) cell lines is related to the loss of caspase-8 expression and the expression and distribution of membrane TRAIL-receptors. This study investigated the role of caspase-8 and DR5 in TRAIL-induced apoptosis of NB cell line SKNDZ. METHODS: The expression of caspase-8 mRNA was detected by RT-PCR. The expression of DR5 protein was detected by Western Blot analysis. The effects of TRAIL, IFNγ+TRAIL, chemotherapeutic agent (adriamycin or etoposide) + TRAIL, and chemotherapeutic agent +TRAIL+ IFNγ on the growth and apoptosis of SKNDZ cells were detected by MTT assay and flow cytometry. RESULTS: caspase-8 was not expressed in SKNDZ cells but IFNγ treatment resulted in an increase of caspase-8 expression. Expression of DR5 protein was not detected in SKNDZ cells but an increased DR5 protein expression was found after treatment with adriamycin or etoposide. The SKNDZ cells expressing caspase-8 were not sensitive to TRAIL but those SKNDZ cells expressing both caspase-8 and DR5 were sensitive. The early apoptosis rates of the adriamycin /etoposide + IFNγ+TRAIL groups [( 17.9±3.6)%, (14.8±3.3)%] were higher than that of the IFNγ+TRAIL group [(3.9± 1.2)% ](F=26.233, P< 0.01). CONCLUSIONS: SKNDZ cells expressing both caspase-8 and DR5 restored the TRAIL sensitivity. caspase-8 and DR5 play a key role in TRAIL-induced apoptosis of NB cells.