从自噬和副凋亡途径初探桔梗皂苷-D抗乳腺癌作用与机制

Preliminary study on the anti-tumor effect and mechanism of platycodin D through autophagy and paraptosis pathways

目的从自噬和副凋亡途径探讨桔梗皂苷-D(PD)抗乳腺癌的作用和潜在机制。方法结晶紫法检测不同浓度PD(10、20、40μmol/L)对三种乳腺癌细胞株4T1、MDA-MB-231、MCF-7细胞存活率的影响;MDC荧光标记法与LC3B荧光染色激光共聚焦显微镜观察适宜浓度PD作用乳腺癌细胞后自噬发生的情况;PD联合自噬抑制剂氯喹作用三种乳腺癌细胞株,明确PD诱导自噬在细胞死亡中的作用;光镜与电镜下观察适宜浓度PD作用后乳腺癌细胞形态学的变化。结果与PD 0μmol/L组比较、PD20、40μmol/L组4T1[(63.10±2.15)%、(32.28±1.19)比(99.75±4.20),P<0.01]、MDA-MB-231[(63.10±2.15)%、(52.87±3.46)%和(82.04±5.27)%,P均<0.01]、MCF-7[(32.38±1.20)%、(3.25±0.627)%、(59.30±5.72)%,P均<0.01]细胞存活率均明显降低。MDC法与LC3B荧光染色法发现经PD处理的三种乳腺癌细胞后发生自噬现象。与PD组比较,PD联合氯喹组可进一步降低4T1、MDA-MB-231和MCF-7三种乳腺癌细胞株的存活率[4T1:(23.31±1.07)%比(40.35±2.94)%,P<0.01;MDA-MB-231:(31.40±2.73)%比(56.69±4.02)%,P<0.01;MCF-7:(52.72±4.50)%比(92.24±7.85)%,P<0.001]。光镜与电镜观察经PD处理的三种细胞质内出现大量空泡结构,并伴有线粒体肿胀,类似副凋亡的形态学改变。结论PD具有明确的抑制乳腺癌细胞株4T1、MDA-MB-231和MCF-7存活率的效应,该效应伴随自噬现象的发生,但自噬抑制剂的加入提示PD诱导的细胞自噬是对自身的保护作用。经PD处理的三种乳腺癌细胞出现类似副凋亡的形态学改变,初步推测PD对乳腺癌细胞的抑制作用可能与副凋亡相关。

Objective To explore the anti-breast cancer effects and underlying mechanism of platycodin D(PD)through autophagy and paraptosis pathway.Methods The effect of PD at different concentrations(10,20,and 40μmol/L)on the viability of three breast cancer cell lines(4T1,MDA-MB-231,and MCF-7)was detected using crystal violet assay.Autophagy was assessed in breast cancer cells treated with PD using MDC fluorescence labeling and LC3B fluorescence staining under laser confocal microscopy.The role of PD-induced autophagy in cell death was investigated in three breast cancer cell lines treated with PD combined with autophagy inhibitor chloroquine.Morphological changes in breast cancer cells treated with PD were observed under a light microscope and electron microscopy.Results Compared with the blank control group,PD at 20 and 40μmol/L significantly reduced the viability rates of 4T1[(63.10±2.15)%,(32.28±1.19)%vs.(99.75±4.20)%,both P<0.01],MDA-MB-231[(63.10±2.15)%,(52.87±3.46)%,vs.(82.04±5.27)%,both P<0.01],and MCF-7 cells[(32.38±1.20)%,(3.25±0.627)%,vs.(59.30±5.72)%,both P<0.01].MDC assay and LC3B fluorescence staining revealed autophagy in the three breast cancer cells after PD treatment.Compared with the PD group,PD combined with the autophagy inhibitor chloroquine further reduced the viability rates of 4T1[(23.31±1.07)%vs.(40.35±2.94)%,P<0.001],MDA-MB-231[(31.40±2.73)%vs.(56.69±4.02)%,P<0.01],and MCF-7 cells[(52.72±4.50)%vs.(92.24±7.85)%,P<0.001].Under light and electron microscopy,a large number of vacuolar structures were observed in the cytoplasm of the three cells treated with PD,accompanied by mitochondrial swelling and paraptosis-like morphological changes.Conclusion PD inhibits the viability rates of breast cancer cell lines 4T1,MDA-MB-231,and MCF-7,which was accompanied by the occurrence of autophagy.However,the addition of an autophagy inhibitor suggests that PD-induced autophagy may serve a protective role.The observed paraptosis-like morphological changes in three breast cancer cells treated with PD indicate the preliminary hypothesis that the inhibitory effect of PD on breast cancer cells may be related to paraptosis.