Effect of bufalin on proliferation and apoptosis through ERK/RSK2 pathway in human esophageal ;carcinoma cell line xenografts in nude mice
10.3760/cma.j.issn.0253-3766.2016.05.002
- VernacularTitle:Bufalin通过细胞外信号调节激酶／P 90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
- Author:
Meng YUE
1
;
Xuejiao LIU
;
Yan DING
;
Xiaoling WANG
;
Huichai YANG
;
Yueping LIU
Author Information
1. 河北医科大学第四医院病理科
- Keywords:
Subject words] Esophageal neoplasms;
Bufalin;
Extracellular-signal regulated kinase;
Ribosomal S6 kinase 2;
Xenograft model antitumor assays;
Mice,nude
- From:
Chinese Journal of Oncology
2016;38(5):325-332
- CountryChina
- Language:Chinese
-
Abstract:
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, GSK3β, p?GSK3β, 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) cm3, (1.680±0.150) cm3, (1.285±0.134) cm3, (0.873±0.095) cm3, (0.815±0.108) cm3 and (0.530±0.104) cm3, 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 ΔCT 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 ΔCT 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 immunoreactivity 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.
