蜂胶乙醇提取物及其成分咖啡酸苯乙酯通过抑制 TLR4 信号通路和诱导细胞凋亡和自噬来抑制炎症微环境中的乳腺癌细胞增殖。
Ethanol extract of propolis and its constituent caffeic acid phenethyl ester inhibit breast cancer cells proliferation in inflammatory microenvironment by inhibiting TLR4 signal pathway and inducing apoptosis and autophagy.
机构信息
School of Life Science, Liaocheng University, Liaocheng, 252059, China.
Institute of Apiculture and bee product quality inspection of Shandong Province, Taian, 271000, China.
出版信息
BMC Complement Altern Med. 2017 Sep 26;17(1):471. doi: 10.1186/s12906-017-1984-9.
BACKGROUND
Propolis and its major constituent - caffeic acid phenethyl ester (CAPE) have good abilities on antitumor and anti-inflammation. However, little is known about the actions of propolis and CAPE on tumor in inflammatory microenvironment, and inflammatory responses play decisive roles at different stages of tumor development. To understand the effects and mechanisms of ethanol-extracted Chinese propolis (EECP) and its major constituent - CAPE in inflammation-stimulated tumor, we investigated their effects on Toll-like receptor 4 (TLR4) signaling pathway which plays a crucial role in breast cancer MDA-MB-231 cell line.
METHODS
80% confluent breast cancer MDA-MB-231 cells were stimulated with 1 μg/mL lipopolysaccaride (LPS). Then the cells were divided for treatment by CAPE (25 μg/mL) and EECP (25, 50 and 100 μg/mL), respectively. Cell viability, nitric oxide (NO) production and cell migration were measured by sulforhodamine B assay, chemical method and scratch assay. The levels of TLR4, MyD88, IRAK4, TRIF, caspase 3, PARP, LC3B and p62 were investigated through western blotting. The expression of TLR4, LC3B and nuclear factor-κB p65 (NF-κB p65) were tested by immunofluorescence microscopy assay.
RESULTS
Treatment of different concentrations of EECP (25, 50 and 100 μg/mL) and CAPE (25 μg/mL) significantly inhibited LPS-stimulated MDA-MB-231 cell line proliferation, migration and NO production. Furthermore, EECP and CAPE activated caspase3 and PARP to induce cell apoptosis, and also upregulated LC3-II and decreased p62 level to induce autophagy during the process. TLR4 signaling pathway molecules such as TLR4, MyD88, IRAK4, TRIF and NF-κB p65 were all down-regulated after EECP and CAPE treatment in LPS-stimulated MDA-MB-231 cells.
CONCLUSIONS
These findings indicated that EECP and its major constituent - CAPE inhibited breast cancer MDA-MB-231 cells proliferation in inflammatory microenvironment through activating apoptosis, autophagy and inhibiting TLR4 signaling pathway. EECP and CAPE may hold promising prospects in treating inflammation-induced tumor.
背景
蜂胶及其主要成分咖啡酸苯乙酯（CAPE）具有良好的抗肿瘤和抗炎作用。然而，人们对蜂胶和 CAPE 在炎症微环境中对肿瘤的作用知之甚少，而炎症反应在肿瘤的不同发展阶段起着决定性的作用。为了了解乙醇提取的中国蜂胶（EECP）及其主要成分 CAPE 在炎症刺激的肿瘤中的作用及其机制，我们研究了它们对 Toll 样受体 4（TLR4）信号通路的影响，该通路在乳腺癌 MDA-MB-231 细胞系中起着至关重要的作用。
方法
将 80%汇合的乳腺癌 MDA-MB-231 细胞用 1μg/mL 脂多糖（LPS）刺激。然后将细胞分为 CAPE（25μg/mL）和 EECP（25、50 和 100μg/mL）处理组。通过磺酰罗丹明 B 测定法、化学方法和划痕试验分别测量细胞活力、一氧化氮（NO）产生和细胞迁移。通过 Western blot 法检测 TLR4、MyD88、IRAK4、TRIF、caspase 3、PARP、LC3B 和 p62 的水平。通过免疫荧光显微镜法检测 TLR4、LC3B 和核因子-κB p65（NF-κB p65）的表达。
结果
不同浓度的 EECP（25、50 和 100μg/mL）和 CAPE（25μg/mL）处理均显著抑制 LPS 刺激的 MDA-MB-231 细胞系增殖、迁移和 NO 产生。此外，EECP 和 CAPE 通过激活 caspase3 和 PARP 诱导细胞凋亡，并在该过程中上调 LC3-II 并降低 p62 水平诱导自噬。在 LPS 刺激的 MDA-MB-231 细胞中，EECP 和 CAPE 处理后 TLR4 信号通路分子如 TLR4、MyD88、IRAK4、TRIF 和 NF-κB p65 均被下调。
结论
这些发现表明，EECP 及其主要成分 CAPE 通过激活细胞凋亡、自噬和抑制 TLR4 信号通路抑制炎症微环境中的乳腺癌 MDA-MB-231 细胞增殖。EECP 和 CAPE 可能在治疗炎症诱导的肿瘤方面具有广阔的前景。
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