Effect of oridonin-mediated hallmark changes on inflammatory pathways in human pancreatic cancer (BxPC-3) cells
Effect of oridonin-mediated hallmark changes on inflammatory pathways in human pancreatic cancer (BxPC-3) cells
复制标题
DOI：
10.3748/wjg.v20.i40.14895
复制
发表时间：
2014-10-28
影响因子：
4.3
通讯作者：
Xu, Jian
中科院分区：
文献类型：
--
作者：
Chen, Ru-Yi;Xu, Bin;Xu, Jian
目的：探讨冬凌草甲素对人胰腺癌（BxPC-3）细胞核转录因子的影响，并研究其生物学行为与炎症因子的关系。方法：用不同浓度的冬凌草甲素处理BxPC-3细胞，绘制细胞活力曲线，检测药物对细胞的抑制作用。使用酶联免疫吸附测定 (ELISA) 检测 BxPC-3 细胞上清液中白细胞介素 1 α (IL-1 β)、IL-6 或 IL-33 等细胞因子的表达，以及核转录因子的蛋白表达，包括核因子 kappa B、激活蛋白 1、信号转导子和转录激活子 3、骨形态发生蛋白 2、转化生长因子 β 1 以及 sma 和 mad 同源物。使用Western blot检测BxPC-3细胞。还使用免疫印迹法检测癌标志相关蛋白，如生存素、血管内皮生长因子和基质金属肽酶 2，并使用免疫荧光染色检测核内 IL-33 表达。 结果：冬凌草甲素处理以剂量依赖性方式降低 BxPC-3 细胞的活力。 8μg/mL冬凌草甲素处理后细胞生长减少（13.05%+/-3.21%，P<0.01），最高抑制率为90.64%+/-0.70%，冬凌草甲素剂量为32μg/mL时达到最高抑制率。冬凌草甲素在BxPC-3细胞中的IC50值为19.32 μg/mL。 ELISA 分析显示冬凌草甲素以剂量依赖性方式下调炎症因子 IL-1β、IL-6 和 IL-33。与对照组相比，16 和 32 μg/mL 治疗组的 IL-1 β 表达显着降低（12.97 +/- 0.45 pg/mL、11.17 +/- 0.63 pg/mL vs 14.40 +/- 0.38 pg/mL，P < 0.01）。 IL-6 表达也观察到类似的趋势，与对照组相比，16 和 32 μg/mL 治疗组显着降低（4.05 +/- 0.14 pg/mL 与 4.45 +/- 0.43 pg/mL，P < 0.05；3.95 +/- 0.13 pg/mL 与 4.45 +/- 0.43 pg/mL，P < 0.01）。与对照组相比，8、16和32 μg/mL治疗组的IL-33表达显着降低（911.05 +/- 14.18 pg/mL vs 945.25 +/- 12.09 pg/mL，P < 0.05；802.70 +/- 11.88 pg/mL，768.54 +/- 10.98 pg/mL 与 945.25 +/- 12.09 pg/mL，P < 0.01）。 Western blot和免疫荧光染色分析表明冬凌草甲素改变了胰腺癌细胞的标志并调节了多种核转录因子的表达。结论：所得结果表明冬凌草甲素通过调节核转录因子来改变胰腺癌细胞的标志。 (C) 2014 百事登出版集团有限公司版权所有。
AIM: To investigate the effect of oridonin on nuclear transcription factors and to study the relationship between biological behavior and inflammatory factors in human pancreatic cancer (BxPC-3) cells.METHODS: BxPC-3 cells were treated with various concentrations of oridonin, and viability curves were generated to test for inhibitory effects of the drug on cells. The expression of cytokines such as interleukin-1 alpha (IL-1 beta), IL-6, or IL-33 was detected in BxPC-3 cell supernatants using an enzyme-linked immunosorbent assay (ELISA), and the protein expression of nuclear transcription factors including nuclear factor kappa B, activating protein-1, signal transducer and activator of transcription 3, bone morphogenetic protein 2, trans-forming growth factor beta 1 and sma and mad homologues in BxPC-3 cells was detected using Western blot. Carcinoma hallmark-related proteins such as survivin, vascular endothelial growth factor, and matrix metallopeptidase 2 were also detected using immunoblotting, and intra-nuclear IL-33 expression was detected using immunofluorescent staining.RESULTS: Treatment with oridonin reduced the viability of BxPC-3 cells in a dose dependent manner. The cells exhibited reduced growth following treatment with 8 mu g/mL oridonin (13.05% +/- 3.21%, P < 0.01), and the highest inhibitory ratio was 90.64% +/- 0.70%, which was achieved with oridonin at a dose of 32 mu g/mL. The IC50 value of oridonin in BxPC-3 cells was 19.32 mu g/mL. ELISA analysis revealed that oridonin down-regulated the inflammatory factors IL-1 beta, IL-6, and IL-33 in a dose-dependent manner. IL-1 beta expression was significantly reduced in the 16 and 32 mu g/mL treatment groups compared to the control group (12.97 +/- 0.45 pg/mL, 11.17 +/- 0.63 pg/mL vs 14.40 +/- 0.38 pg/mL, P < 0.01). Similar trends were observed for IL-6 expression, which was significantly reduced in the 16 and 32 mu g/mL treatment groups compared to the control group (4.05 +/- 0.14 pg/mL vs 4.45 +/- 0.43 pg/mL, P < 0.05; 3.95 +/- 0.13 pg/mL vs 4.45 +/- 0.43 pg/mL, P < 0.01). IL-33 expression was significantly reduced in the 8, 16, and 32 mu g/mL treatment groups compared to the control group (911.05 +/- 14.18 pg/mL vs 945.25 +/- 12.09 pg/mL, P < 0.05; 802.70 +/- 11.88 pg/mL, 768.54 +/- 10.98 pg/mL vs 945.25 +/- 12.09 pg/mL, P < 0.01). Western blot and immunofluorescent staining analyses suggested that oridonin changed the hallmarks and regulated the expression of various nuclear transcription factors.CONCLUSION: The results obtained suggest that oridonin alters the hallmarks of pancreatic cancer cells through the regulation of nuclear transcription factors. (C) 2014 Baishideng Publishing Group Inc. All rights reserved.