积雪草苷通过阻断p65和p38丝裂原活化蛋白激酶（p38MAPK）的激活来抑制胰腺癌PANC-1细胞的上皮-间质转化和干细胞样特性。
Asiaticoside inhibits epithelial-mesenchymal transition and stem cell-like properties of pancreatic cancer PANC-1 cells by blocking the activation of p65 and p38MAPK.
作者信息
He Yonggang, Peng Xuehui, Zheng Lu, Tang Yichen, Li Jing, Huang Xiaobing
机构信息
Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Army Medical University, Chongqing, China.
出版信息
J Gastrointest Oncol. 2021 Feb;12(1):196-206. doi: 10.21037/jgo-20-533.
BACKGROUND
To analyze the inhibitory effects of Asiaticoside (ATS) on the epithelial-mesenchymal transition (EMT) and stem cell-like properties of a pancreatic cancer cell line (PANC-1) by blocking the activation of p65 and p38 mitogen-activated protein kinase (p38MAPK).
METHODS
ATS concentrations were set at 0, 10, 25, and 50 µmol/L. The survival rate of PANC-1 cells in each group was detected by CCK-8, and CD133 and CD44 positive cells were detected by flow cytometry. The levels of Ki67 and proliferating cell nuclear antigen (PCNA) mRNA were detected by RT-PCR. The expression of E-cadherin, N-cadherin, vimentin, sex-determining region Y-box2 (SOX2), and octamer-binding transcription factor 4 (OCT4) proteins, and the phosphorylation levels of p65 and p38MAPK were detected by western blot. Nude mouse xenograft models of the tumor were established by subcutaneous injection of PANC-1 cells (1×10-1×10/mL), and they were randomly divided into the control group (0 mg/kg), and low-dose, medium-dose, and high-dose ATS groups (2.5, 5, 10 mg/kg). Apoptosis in xenograft tissue was detected by TUNEL, and the expression of vimentin and SOX2 proteins was detected by immunohistochemistry.
RESULTS
As the ATS concentration increased to 25 µmol/L, cell survival rate, levels of Ki67 and PCNA mRNA, expression of N-cadherin, vimentin, SOX2, OCT4, p-p65/p65, and p-p38MAPK/p38MAPK proteins, and the proportions of CD44 and CD133 positive cells significantly decreased (P<0.05), while the expression of E-cadherin protein significantly increased (P<0.05). The results of tumor formation in nude mice showed that with the increase of ATS concentration, at 5 mg/kg the volume of the xenograft significantly decreased (P<0.05), the apoptosis rate significantly increased (P<0.05), and positive expression rates of vimentin and SOX2 proteins significantly decreased (P<0.05).
CONCLUSIONS
ATS may inhibit the proliferation, EMT, and stem cell-like properties of pancreatic cancer cells by blocking the phosphorylation of p38MAPK and nuclear factor-κB (NF-κB)/p65 in PANC-1 cells.
背景
通过阻断p65和p38丝裂原活化蛋白激酶（p38MAPK）的激活，分析积雪草苷（ATS）对胰腺癌细胞系（PANC-1）上皮-间质转化（EMT）和干细胞样特性的抑制作用。
方法
将ATS浓度设置为0、10、25和50 μmol/L。采用CCK-8检测各组PANC-1细胞的存活率，采用流式细胞术检测CD133和CD44阳性细胞。采用RT-PCR检测Ki67和增殖细胞核抗原（PCNA）mRNA水平。采用蛋白质免疫印迹法检测E-钙黏蛋白、N-钙黏蛋白、波形蛋白、性别决定区Y框蛋白2（SOX2）和八聚体结合转录因子4（OCT4）蛋白的表达，以及p65和p38MAPK的磷酸化水平。通过皮下注射PANC-1细胞（1×10⁶-1×10⁷/mL）建立裸鼠肿瘤异种移植模型，并将其随机分为对照组（0 mg/kg）、低剂量、中剂量和高剂量ATS组（2.5、5、10 mg/kg）。采用TUNEL检测异种移植组织中的细胞凋亡，采用免疫组织化学检测波形蛋白和SOX2蛋白的表达。
结果
随着ATS浓度增加至25 μmol/L，细胞存活率、Ki67和PCNA mRNA水平、N-钙黏蛋白、波形蛋白、SOX2、OCT4、p-p65/p65和p-p38MAPK/p38MAPK蛋白的表达以及CD44和CD133阳性细胞比例均显著降低（P<0.05），而E-钙黏蛋白的表达显著增加（P<0.05）。裸鼠成瘤结果显示，随着ATS浓度增加，在5 mg/kg时异种移植瘤体积显著减小（P<0.05），凋亡率显著增加（P<0.05），波形蛋白和SOX2蛋白的阳性表达率显著降低（P<0.05）。
结论
ATS可能通过阻断PANC-1细胞中p38MAPK和核因子κB（NF-κB）/p65的磷酸化来抑制胰腺癌细胞的增殖、EMT和干细胞样特性。
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