从罗氏（Baker）N. Tanaka（同义词 Tupistra chinensis Baker）中分离得到的螺旋甾烷醇皂苷诱导 HL-60 细胞凋亡和促死亡自噬。

Apoptosis and pro-death autophagy induced by a spirostanol saponin isolated from Rohdea chinensis (Baker) N. Tanaka (synonym Tupistra chinensis Baker) on HL-60 cells.

机构信息

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China; Guangdong Engineering Research Center for Lead Compounds & Drug Discovery, Guangzhou 510006, China.

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China.

出版信息

Phytomedicine. 2018 Mar 15;42:83-89. doi: 10.1016/j.phymed.2018.03.030.

DOI:10.1016/j.phymed.2018.03.030

PMID:29655701

Abstract

BACKGROUND

Our previous study has revealed that the spirostanol saponins isolated from the rhizomes of Rohdea chinensis (Baker) N. Tanaka (synonym Tupistra chinensis Baker) (Convallariaceae) (a reputed folk medicine) exhibited potent antiproliferative activity. However, the underlying mechanism of purified saponins remains unclear. More studies are necessary to assess the apoptosis and autophagy activities of the saponins from R. chinensis and clarify their antiproliferative mechanisms.

PURPOSE

The present study certificated the potential antiproliferative activity and mechanism of 5β-spirost-25(27)-en-1β,3β-diol-1-O-α-L-rhamnopyranosyl-(1→2)- β-D-xylopyranosyl-3-O-α-L-rhamnopyranoside (SPD), a spirostanol saponin from R. chinensis, against human acute promyelocytic leukemia cells (HL-60).

METHODS

The antiproliferative activity of SPD in vitro was evaluated by MTT assay compared with cis-dichlorodiammineplatinum (II). The autophagic activity was assessed using MDC staining and western blot, cell apoptosis inspection was detected by Annexin V-FITC/PI double staining and the mitochondrial membrane potential was detected by JC-1 fluorescence dye combined with flow cytometry. The potential mechanisms for protein levels of apoptosis and autophagy were evaluated by western blot.

RESULTS

Treatment of HL-60 cells with SPD resulted in growth inhibition (IC value of 2.0 ± 0.2 µM, after 48 h treatment) and induction of apoptosis and autophagy. Results from Annexin V-FITC/PI double-staining assay and mitochondrial membrane potential detection showed that apoptosis was happened after SPD treatment. The regulation of caspase-3, Bax, Bcl-2, PARP following SPD treatment contributed to the induction of mitochondria-dependent apoptosis. Meanwhile, SPD induced autophagy related with Akt/mTOR/p70S6K signaling and activated of AMPK signaling pathway. Furthermore, blocking autophagy with bafilomycin A1 reduced the cytotoxicity of SPD in HL-60 cells.

CONCLUSION

The antiproliferative, apoptosis and pro-death autophagy activities of SPD suggested that spirostanol saponins from R. chinensis would be a potential cytotoxic candidate against acute promyelocytic leukemia.

摘要

背景

我们之前的研究表明，从钩吻根茎中分离得到的螺旋甾烷醇皂苷（钩吻根茎，（钩吻）（天门冬科）（一种著名的民间药物））具有很强的抗增殖活性。然而，纯化皂苷的作用机制尚不清楚。需要进一步研究来评估钩吻皂苷的诱导细胞凋亡和自噬活性，并阐明其抗增殖机制。

目的

本研究旨在验证 5β-螺旋甾-25（27）-烯-1β,3β-二醇-1-O-α-L-鼠李吡喃糖基-（1→2）-β-D-木吡喃糖基-3-O-α-L-鼠李吡喃糖苷（SPD）作为钩吻中的一种螺旋甾烷醇皂苷对人急性早幼粒细胞白血病细胞（HL-60）的潜在抗增殖活性及机制。

方法

通过 MTT 法与顺铂（Ⅱ）比较，评价 SPD 在体外的抗增殖活性。通过 MDC 染色和 Western blot 检测自噬活性，通过 Annexin V-FITC/PI 双染色和 JC-1 荧光染料结合流式细胞术检测细胞凋亡，通过 Western blot 检测凋亡和自噬相关蛋白水平的变化，探讨其作用机制。

结果

SPD 处理 HL-60 细胞导致生长抑制（IC 值为 2.0±0.2μM，48 小时后）和诱导细胞凋亡和自噬。Annexin V-FITC/PI 双染色和线粒体膜电位检测结果表明，SPD 处理后发生了细胞凋亡。SPD 处理后，caspase-3、Bax、Bcl-2、PARP 的调节导致线粒体依赖性凋亡的诱导。同时，SPD 诱导的自噬与 Akt/mTOR/p70S6K 信号通路有关，并激活了 AMPK 信号通路。此外，用巴弗洛霉素 A1 阻断自噬可降低 SPD 在 HL-60 细胞中的细胞毒性。