Bufalin通过细胞外信号调节激酶／P90核糖体S6激酶2通路对人食管癌细胞裸鼠移植瘤增殖及凋亡的影响

Effect of bufalin on proliferation and apoptosis through ERK/RSK2 pathway in human esophageal carcinoma cell line xenografts in nude mice

目的探讨bufalin通过细胞外信号调节激酶（ERK）／P90核糖体s6激酶2（RSK2）通路对裸鼠移植瘤增殖及凋亡的影响。方法建立裸鼠食管癌细胞移植瘤模型，并分为模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组，观察bufalin对裸鼠食管癌移植瘤的影响，显微镜下观察各组移植瘤的组织学表现。采用原位末端转移酶标记技术（TUNEL）检测各组移植瘤的凋亡指数，采用实时定量PCR法检测各组移植瘤组织中ERK、RSK2mRNA的表达，采用Westernblot和免疫组化法检测移植瘤组织中ERK、RSK2、糖原合成激酶3β（GSK313）、Bad及其磷酸化水平。结果模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的裸鼠肿瘤体积分别为（1．758±0．181）cm3、（1．680±0．150）cm3、（1．285±0．134）cm3、（0．873±0．095）cm3、（0．815±0．108）cm3和（0．530±0．104）cm3。HE染色显示，各组移植瘤组织均有不同程度的坏死，以联合用药组最为明显。TUNEL法显示，模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的凋亡指数分别为（6．0±0．6）％、（11．0±0．7）％、（19．1±0．9）％、（25．1±1．4）％、（20．0±1．2）％和（17．1±0．7）％，以bufalin高剂量组凋亡指数最高。实时定量PCR检测显示，模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的ERKmRNA的Act值分别为0．270±0．084、0．293±0．081、0．596±0．224、0．857±0．183、0．868±0．187和1．313±0．282；RSK2mRNA的Act值分别为0．340±0．062、0．337±0．071、0．642±0．226、0．915±0．170、0．923±0．176和1．413±0．269，ERK和RSK2mRNA表达均呈降低趋势。Westernblot和免疫组化检测显示，各组ERK、RSK2、Bad蛋白水平的差异均无统计学意义（均P〉0．05）；模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的P-ERK蛋白相对表达量分别为0．721±0．094、0．695±0．095、0．555±0．080、0．388±0．052、0．341±0．060和0．235±0．056，免疫反应评分中位数分别为8、8、6、4、5和3分。模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的p-RSK2蛋白相对表达量分别为0．613±0．085、0．612±0．084、0．427±0．089、0．305±0．056、0．258±0．051和0．158±0．058，免疫反应评分中位数分别为8、8、5、3、3和1分。模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的GSK313蛋白相对表达量逐渐升高，p-GSK313和p-Bad蛋白相对表达量均呈降低趋势，bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组与模型组比较，差异均有统计学意义（均P〈0．05）。结论bufalin对裸鼠食管癌移植瘤有明显的抑制作用。Bufalin可通过抑制ERK、RSK2的磷酸化水平抑制移植瘤的生长，通过抑制GSK3β的失活而抑制肿瘤的增殖，通过下调p-Bad的表达而发挥促凋亡作用。

Objective To investigate the effect of bufalin on proliferation and apoptosis through ERK/RSK2 pathway in esophageal squamous cell carcinoma xenografts in nude mice. Methods The subcutaneous xenograft model of esophageal cancer ECA109 cells in nude mice was established. The mice were divided into the model group, low-dose bufalin group, medium-dose bufalin group, high-dose bufalin group, PD98059 group and combination group to evaluate the effect of bufalin on the xenografts. The morphology of xenografts was observed by microscopy. The cell apoptosis index of xenografts was detected by TUNEL assay. The expression of ERK and RSK2 mRNA of human ECA109 cell transplantation tumor in nude mice was examined by real-time quantitative PCR. The protein levels of ERK, p-ERK, RSK2, p-RSK2, GSK313, p-GSK313, Bad and p-Bad in the xenografts were examined by Western blot and Immunohistochemistry. Results The tumor size of nude mice in the model group, low-dose bufalin group （BL）, medium -dose bufalin group （BM）, high-dose bufalin group （BH）, PD98059 group and combined therapy group （BP） was （1.758±0.181） em3, （1.680±0.150） em3, （1.285±0.134） em3, （0.873±0.095） em3, （ 0.815-1-0.108） em3 and （0.530±0.104） em3, respectively. Histological examination showed that the xenografts of each group had varying degrees of necrosis, and the most extensive necrosis was observed in the BP group. The TUNEL assay showed that the cell apoptosis index of xenografts in the model, BL, BM, BH, PD98059 and BP groups was （6.0±0.6）%, （11.0±0.7）%, （19.1±0.9）%, （25.1±1.4）%, （20.0±1.2）% and （17.1±0.7）%, respectively, which is highest in the BH group. The real-time quantitative PCR results showed that the ACT values of ERK mRNA in the model, BL, BM, BH, PD98059 and BP groups were 0.270± 0.084, 0.293±0.081, 0.596±0.224, 0.857±0.183, 0.868±0.187 and 1.313±0.282, respectively. The ACT values of RSK2 mRNA in the model, BL, BM, BH, PD98059 and BP groups were 0.340±0.062, 0.337± 0.071, 0.642±0.226, 0.915±0.170, 0.923±0.176 and 1.413±0.269, respectively. The relative expression of ERK and RSK2 mRNA was gradually decreased. Western blot and immunohistochemistry results showed that the protein levels of ERK, RSK2 and Bad in each group were not significantly different （P〉0.05）. The protein levels of p-ERK in the model, BL, BM, BH, PD98059 and BP groups were 0.721±0.094, 0.695± 0.095, 0.555±0.080, 0.388±0.052, 0.341±0.060, 0.235± 0.056, respectively. The median immunoreactivity scores of p-ERK in each group were 8, 8, 6, 4, 5 and 3. The protein levels of p-RSK2 in the model, BL, BM, BH, PD98059 and BP groups were 0.613±0.085, 0.612±0.084, 0.427±0.089, 0.305±0.056, 0.258±0.051, 0.158±0.058, respectively. The median immunoreaetivity scores of p-RSK in each group were 8, 8, 5, 3, 3 and 1. The protein level of GSK3β in the model, BL, BM, BH, PD98059 and BP groups were increased gradually, while the protein level of p-GSK3β and p-Bad were decreased gradually. Conclusions Bufalin exerts significant inhibitory effect on the esophageal squamous cell carcinoma xenogragts in nude mice. Bufalin may suppress the growth of xenogragts in nude mice by down-regulating the level of ERK and RSK2 phosphorylation, inhibit the proliferation of xenogragts via inactivating GSK3βand promote apoptosis through down-regulation of p-Bad.