抑制蛋白磷酸酶2A对鼻咽癌放射敏感性的影响

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。

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

OBJECTIVE: To investigate the radiosensitizing effect of protein phosphatase 2A(PP2A) inhibition on nasopharyngeal carcinoma cell line CNE2 and its xenografts. METHODS: The cells and subcutaneous xenografts in BALB/c nude mice were randomly divided into control, norcantharidin (40 μmmol/L in vitro, 27 μmol/kg in vitro), irradiation (8 Gy in vitro, 20 Gy in vitro), and combination of norcantharidin and irradiation groups. MTT, Co-immunoprecipitation and flow cytometry were used to examine the effect of PP2A inhibition, cell cycle analysis, apoptosis of CNE2 and its xenografts by norcantharidin and (or) irradiation. RESULTS: We measured PP2A activity in CNE2 cells and xenografts 5 hours after exposure to 8 Gy radiation in vitro and 20 Gy radiation in vivo, with and without prior exposure to NCTD. Compared with control group, NCTD alone was associated with decreasedin PP2A activity of 70% in vitro and in vivo both. Radiation alone was associated with increased in PP2A activity of 210% and 165% in vitro and in vivo, respectively. The cell cycle arrested in G2/M and increasing of apoptotic ratio in CNE2 cells were significantly different between the control and the both two treatment alone groups (P<0.05). Compared with control group, the combination of 8 Gy and 40 μmol/L norcantharidin produced significant inhibition of PP2A(80% in vitro and 72% 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 combination with radiation produced the greatest effects on tumor growth slowing and decreasing tumor weight by 87.98% relative to control in CNE2 xenografts(compared to control, P<0.05). Furthermore, more apoptosis, necrotic cells and formation of apoptotic bodies were found in pathological section in norcantharidin plus radiotherapy group. CONCLUSION: PP2A might be a potential target for sensitization of nasopharyngeal carcinoma to radiatotherapy.