抑制蛋白磷酸酶2A对鼻咽癌放射敏感性的影响

Effects of protein phosphatase 2A inhibition on radiosensitivity of nasopharyngeal carcinoma

目的:研究放射处理条件下蛋白磷酸酶2A(PP2A)的抑制对鼻咽癌CNE2细胞及其裸鼠移植瘤放射增敏的效果。方法:建立鼻咽癌细胞CNE2体外实验与裸鼠体内移植瘤模型,随机分为空白对照组(PBS)、单纯去甲斑蝥素药物组(NCTD,体外40μmol/L,体内27μmol/kg)、单纯放射组(体外8 Gy,体内20 Gy)及联合处理组。采用四甲基噻唑蓝(MTT)、免疫共沉淀、流式细胞术等方法研究去甲斑蝥素和或放射处理对鼻咽癌CNE2细胞PP2A活性、细胞周期、细胞凋亡及裸鼠体内移植瘤的影响。结果:体外和体内实验均显示,单纯NCTD处理组5 h后PP2A活性均被抑制,约为对照组的70%,而单纯放射组处理5 h后PP2A蛋白活性明显升高,分别约为对照组的210%(体外)和165%(体内),差异均具有统计学意义(P<0.05)。单纯NCTD处理组、单纯放射组均可不同程度的引起CNE2细胞G2/M期阻滞及细胞凋亡率的增加,与对照组间的差异均具有统计学意义(P<0.05)。联合处理组与对照组比较,PP2A的活性明显降低,分别约为对照组的80%(体外细胞实验)和72%(体内裸鼠实验);同时G2/M期细胞显著增多(87.88%±2.10%),细胞凋亡率也明显提高(77.15%±7.62%);裸鼠移植瘤抑瘤率达到87.98%,其差异均具有统计学意义(P均<0.05)。移植瘤组织病理学观察发现联合处理组大量细胞坏死,部分融合成片,核膜核仁破碎,在肿瘤组织细胞非死亡区可见到被瘤细胞吞噬形成的凋亡小体。结论:PP2A很有可能成为提高鼻咽癌临床放射治疗作用的增敏新靶点。

OBJECTIVE： To investigate the radiosensitizing effect of protein phosphatase 2A（PP2A） inhibition onnasopharyngeal carcinoma cell line CNE2 and its xenografts. METHODS：The cells and subcutaneous xenografts inBALB/c nude mice were randomly divided into control,norcantharidin （40 μmmol/L in vitro,27 μmol/kg invitro）,irradiation （8 Gy in vitro,20 Gy in vitro）,and combination of norcantharidin and irradiation groups. MTT,Coimmunoprecipitationand flow cytometry were used to examine the effect of PP2A inhibition,cell cycleanalysis,apoptosis of CNE2 and its xenografts by norcantharidin and （or） irradiation. RESULTS：We measured PP2Aactivity in CNE2 cells and xenografts 5 hours after exposure to 8 Gy radiation in vitro and 20 Gy radiation in vivo,withand without prior exposure to NCTD. Compared with control group,NCTD alone was associated with decreasedin PP2Aactivity of 70% in vitro and in vivo both. Radiation alone was associated with increased in PP2A activity of 210% and165% in vitro and in vivo,respectively. The cell cycle arrested in G2/M and increasing of apoptotic ratio in CNE2 cellswere significantly different between the control and the both two treatment alone groups （P〈0.05）. Compared with controlgroup,the combination of 8 Gy and 40 μmol/L norcantharidin produced significant inhibition of PP2A（80% in vitro and72% in vitro compared to control）,accumulation of cells in G2/M-phase （87.88%±2.10%） and more apoptosis（77.15%±7.62%） in CNE2 cell lines compared to norcantharidin alone and radiation alone. Norcantharidin in combinationwith radiation produced the greatest effects on tumor growth slowing and decreasing tumor weight by 87.98% relative tocontrol in CNE2 xenografts（compared to control,P〈0.05）. Furthermore,more apoptosis,necrotic cells and formation ofapoptotic bodies were found in pathological section in norcantharidin plus radiotherapy group. CONCLUSION：PP2Amight be a potential target for sensitization of nasopharyngeal carcinoma to radiatotherapy.