Abstract：
DMC, a kind of compound derived from the dry flower buds of Cleistocalyx operculatus, has been shown to inhibit the growth of various cancer cells, but research on triple-negative breast cancer cells remains scarce. To explore this issue, MDA-MB-231 cells were selected, and the results showed that DMC has strong proliferation inhibit effects on this kind of cells. The inhibit rate of 30 μM DMC incubated for 24 h was 56.25%, and 40.6% cells were arrested under the G2/M phase. The levels of pro-apoptosis protein Bax and active caspase-3, cleaved PARP and cell cycle related proteins, such as p21 and p27 increased, but apoptosis regulators, like Bcl-2, Cdc 2, Cyclin B1, and LC3 II decreased dramatically. In addition, DMC induced the accumulation of autophagosomes and autophagic substrates, and the combination of DMC with CQ promoted apoptosis of MDA-MB-231 cells, which suggested that DMC induced apoptosis partly by blocking autophagy flow. Moreover, the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and its mechanistic target of rapamycin kinase (mTOR) were also decreased after 30 μM DMC incubating for 24 h. The proteins play a critical role in cell proliferation, apoptosis, and autophagy modulation. The inhibition of autophagy flow and PI3K/AKT/mTOR pathway could be reversed after being treated with ROS scavenger NAC. Altogether, the results of the present study suggest that DMC effectively induces apoptosis and growth inhibition in MDA-MB-231 cells through blocking autophagy flow and regulating the PI3K/AKT/mTOR pathway by increasing ROS level.
摘要：
DMC,一种来自白虱干花蕾的化合物，已经被证明可以抑制各种癌细胞的生长，但是关于三阴性乳腺癌细胞的研究仍然很少。为了探讨这个问题，选择MDA-MB-231细胞，结果表明，DMC对这类细胞具有很强的增殖抑制作用。30μMDMC孵育24h的抑制率为56.25%,40.6%的细胞被阻滞在G2/M期。促凋亡蛋白Bax和活性caspase-3，裂解PARP和细胞周期相关蛋白的水平，如p21和p27增加，但是细胞凋亡调节剂，如Bcl-2，Cdc2，细胞周期蛋白B1和LC3II急剧下降。此外,DMC诱导自噬体和自噬底物的积累，DMC与CQ联合促进MDA-MB-231细胞凋亡,提示DMC部分通过阻断自噬流诱导细胞凋亡。此外，磷脂酰肌醇3-激酶（PI3K）的磷酸化水平，蛋白激酶B（AKT），30μMDMC孵育24h后，其机制靶标雷帕霉素激酶（mTOR）也降低。该蛋白在细胞增殖中起关键作用，凋亡，和自噬调节。用ROS清除剂NAC处理后，自噬流和PI3K/AKT/mTOR通路的抑制作用可以逆转。总之,本研究结果表明，DMC通过阻断自噬流和通过增加ROS水平调节PI3K/AKT/mTOR通路，有效诱导MDA-MB-231细胞凋亡和生长抑制。
