[關(guān)鍵詞]
[摘要]
目的 探討香貝散體外抗乳腺癌作用及其分子機(jī)制。方法 體外培養(yǎng)人乳腺癌細(xì)胞 MCF-7、MDA-MB-231,給予香貝散 1、2、3、4、5 mg·mL-1加藥處理,對(duì)照組不加藥。利用 MTT 法和克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞增殖,利用流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡,細(xì)胞劃痕和細(xì)胞侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞遷移和侵襲能力,借助吖啶橙染色法觀察細(xì)胞自噬,借助轉(zhuǎn)錄組測(cè)序技術(shù)探索香貝散誘導(dǎo)乳腺癌細(xì)胞死亡的作用機(jī)制,并采用 Western blotting 法檢測(cè) AMP 依賴的蛋白激酶(AMPK)、哺乳動(dòng)物雷帕霉素靶蛋白(mTOR)、LC-3、Beclin-1 蛋白表達(dá)水平。結(jié)果 與對(duì)照組比較,香貝散顯著抑制人乳腺癌 MCF-7、MDA-MB-231 細(xì)胞活力(P<0.05、0.001),48 h 半數(shù)抑制濃度(IC50)分別為 2.344、1.961 mg·mL-1,且具有時(shí)間、濃度相關(guān)性;顯著抑制 MCF-7、MDA-MB-231 細(xì)胞的集落形成能力;顯著誘導(dǎo) MCF-7、MDA-MB-231 細(xì)胞凋亡(P<0.001);明顯抑制人乳腺癌 MCF-7、MDA-MB-231 細(xì)胞的體外遷移能力和侵襲能力;吖啶橙染色實(shí)驗(yàn)結(jié)果提示香貝散能夠誘導(dǎo) MCF-7、MDA-MB-231 細(xì)胞發(fā)生自噬,Western blotting 實(shí)驗(yàn)結(jié)果顯示香貝散能夠顯著上調(diào) MCF-7、MDA-MB-231 細(xì) 胞 中 自 噬 關(guān) 鍵 蛋 白 Beclin-1 和 LC3-Ⅱ的表達(dá)(P<0.01、0.001);轉(zhuǎn)錄組測(cè)序技術(shù)發(fā)現(xiàn)差異基因變化最明顯的通路包括 mTOR 信號(hào)通路、自噬信號(hào)通路等 ,Western blotting 實(shí)驗(yàn)結(jié)果顯示香貝散顯著下調(diào) mTOR 的磷酸化水平(P<0.001),顯著上調(diào) AMPK 的磷酸化水平(P<0.001)。結(jié)論 香貝散能夠顯著抑制人乳腺癌細(xì)胞的增殖、遷移、侵襲能力,能夠誘導(dǎo)凋亡和自噬的發(fā)生,激活 AMPK/mTOR 信號(hào)通路可能是其重要機(jī)制。
[Key word]
[Abstract]
Objective To investigate the in vitro anti-breast cancer effects of the Chinese herbal prescription Xiangbeisan and its molecular mechanisms. Results Human breast cancer MCF-7 and MDA-MB-231 cells were treated with Xiangbeisan 1, 2, 3, 4, or 5 mg·mL-1, with a control group not receiving any drug. MTT assay and clone formation assay were used to detect the effects of Xiangbeisan on the proliferation of human breast cancer MCF-7 and MDA-MB-231 cells, and flow cytometry was used to detect the changes in apoptosis of human breast cancer MCF-7 and MDA-MB-231 cells after the administration of Xiangbeisan. Subsequently, the effects of Xiangbeisan on the migration and invasion ability of human breast cancer MCF-7 and MDA-MB-231 cells were detected using cell scratch and cell invasion assays. Through acridine orange staining, the effect of Xiangbeisan on autophagy in human breast cancer cells was explored. Finally, the transcriptome sequencing technique was used to explore the mechanism of Xiangbeisan-induced breast cancer cell death, and the western blotting experiment was used to detect the protein levels of AMPK, mTOR, LC3, and Beclin-1. Results Compared with the control group, Xiangbeisan significantly inhibited the viability of MCF-7 and MDA-MB-231 cells (P<0.05, 0.001), and the median inhibitory concentration (IC50) at 48 h were 2.344 and 1.961 mg·mL-1, respectively, with time and concentration correlation. The colony formation ability of MCF-7 and MDA-MB-231 cells was significantly inhibited. Apoptosis of MCF-7 and MDA-MB-231 cells was significantly induced (P<0.001). Xiangbeisan obviously inhibited the migration and invasion ability of MCF-7 and MDA-MB-231 cells in vitro. The results of acridine orange staining indicated that Xiangbeisan induced autophagy in MCF-7 and MDA-MB-231 cells. Western blotting experiments showed that Xiangbeisan significantly up-regulated the expressions of Beclin-1 and LC3-Ⅱ, the key autophagy proteins, in MCF-7 and MDAMB-231 cells (P<0.01, 0.001). Transcriptome sequencing assay revealed that the most significant pathways with differential gene changes included the mTOR signaling pathway, autophagy pathway, etc. Western blotting experiment results showed that Xiangbeisan significantly down-regulated the phosphorylation level of mTOR (P<0.001), and significantly up-regulated the phosphorylation level of AMPK (P<0.001). Conclusion Xiangbeisan significantly inhibited the proliferation, migration and invasion of human breast cancer cells, induced apoptosis and autophagy, and the activation of AMPK/mTOR signaling pathway may be an important mechanism.
[中圖分類號(hào)]
285.5
[基金項(xiàng)目]
中央高?;究蒲袠I(yè)務(wù)費(fèi)專項(xiàng)資金項(xiàng)目(2023-JYB-JBQN-051);北京中醫(yī)藥大學(xué)國(guó)家級(jí)人才精準(zhǔn)培育計(jì)劃項(xiàng)目(JZ‐PY202206);教育部雙一流引導(dǎo)專項(xiàng)經(jīng)費(fèi)-國(guó)際合作平臺(tái)項(xiàng)目(90020361020006)