Shen Li, Lou Zhaohuan, Zhang Guangshun, Xu Guanhua, Zhang Guangji
J Tradit Chin Med. 2016 Aug;36(4):514-21. doi: 10.1016/s0254-6272(16)30069-3.
To identify the active anti-tumor constituents in the extract from Danshen (Radix Salviae Miltiorrhizae) and investigate the mechanisms underlying the actions.
First, we introduced a two-step counter- current chromatography to extract the therapeutically active diterpenoid, tanshinone from Danshen (Radix Salviae Miltiorrhizae). The cholecystokinin (CCK-8) method was used to evaluate the inhibitory effect of diterpenoid tanshinone in liver cancer QGY-7703, lung cancer PC9, lung cancer A549, gastric cancer MKN-45, gastric cancer HGC-27, colon cancer HCT116, myeloma cellU266/ RPMI8226, and human breast cancer MCF-7 in vitro. Fluorescence staining was used to observe the cytotoxicity ofditerpenoid tanshinone on PC9 cells. The Western blot was used to detect apoptosis- related protein poly ADP-ribose polymerase (PARP), cysteinyl aspartate specific proteinase3/9 (caspase3/9), and cleaved-cysteinyl aspartate specific proteinase3/9 (cleaved-caspase3/9). The endoplasmic reticulum stress-related activating transcription factor 4 (ATF4), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and phosphorylated jun amino-terminal kinase (p-JNK), and caspase- 12 were also analyzed using the Western blot.
Diterpenoid tanshinone inhibited the nine human tumor cell lines, with an IC50 of 4.37-29 μg/mL, with the PC9 and MCF-7 displaying the lowest values. Fluorescence staining showed a lethal effect of diterpenoid tanshinone on PC9 cells. The Western blot showed that the expression of caspase3/9 protein and ATF-4 protein decreased gradually. However, the PARP, cleaved-caspase 3/9 and the expression of p-eIF2 α, P-JNK, and caspase- 12 increased gradually, in a dose-dependent fashion.
We successfully introduced a two-step counter-current chromatography method to extract diterpenoid tanshinone, and demonstrated its antitumor activity. Diterpenoid tanshinone can induce apoptosis in nine human cancer cell lines.
鉴定丹参提取物中的活性抗肿瘤成分，并探讨其作用机制。
首先，采用两步逆流色谱法从丹参中提取具有治疗活性的二萜类化合物丹参酮。采用胆囊收缩素（CCK-8）法评价二萜类丹参酮对肝癌QGY-7703、肺癌PC9、肺癌A549、胃癌MKN-45、胃癌HGC-27、结肠癌HCT116、骨髓瘤细胞U266/RPMI8226和人乳腺癌MCF-7的体外抑制作用。采用荧光染色观察二萜类丹参酮对PC9细胞的细胞毒性。采用蛋白质免疫印迹法检测凋亡相关蛋白聚ADP-核糖聚合酶（PARP）、半胱氨酸天冬氨酸特异性蛋白酶3/9（caspase3/9）和裂解的半胱氨酸天冬氨酸特异性蛋白酶3/9（裂解的caspase3/9）。还采用蛋白质免疫印迹法分析内质网应激相关的激活转录因子4（ATF4）、磷酸化的真核起始因子2α（p-eIF2α）、磷酸化的Jun氨基末端激酶（p-JNK）和caspase-12。
二萜类丹参酮对9种人肿瘤细胞系均有抑制作用，IC50为4.37-29μg/mL，其中PC9和MCF-7的IC50值最低。荧光染色显示二萜类丹参酮对PC9细胞有致死作用。蛋白质免疫印迹法显示，caspase3/9蛋白和ATF-4蛋白的表达逐渐降低。然而，PARP、裂解的caspase 3/9以及p-eIF2α、P-JNK和caspase-12的表达呈剂量依赖性逐渐增加。
我们成功引入两步逆流色谱法提取二萜类丹参酮，并证明了其抗肿瘤活性。二萜类丹参酮可诱导9种人癌细胞系凋亡。
