College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, China; The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, China; Cell Resource Bank and Integrated Cell Preparation Center of Xiaoshan District, Hangzhou Regional Cell Preparation Center (Shangyu Biotechnology Co., Ltd), Hangzhou, China.
J Ethnopharmacol. 2022 Jun 28;292:115214. doi: 10.1016/j.jep.2022.115214. Epub 2022 Mar 21.
Radix Salviae miltiorrhizae (also called Danshen in traditional Chinese medicine) is a famous herbal medicine, which has been frequently used to treat blood stasis syndrome including osteosarcoma (OS) in traditional Chinese medicine. Main components of Danshen have been assumed to exhibit anti-OS capacity. Nevertheless, tanshinol (TS, main component of Danshen)'s efficacy and mechanism in OS hasn't been clearly described ever since. This drew our attention, since OS is the most frequent primary bone carcinomas in children and adolescents, with a high incidence and fatality rate. Unfortunately, chemotherapy for OS has faced many clinical challenges due to the increasing chemoresistance and recurrence. This study was then designed to deeply explore TS's role in OS therapy.
To explore the anti-OS efficacy and mechanism of TS, we conducted in vivo and in vitro experiments by using a zebrafish xenograft model and U2-OS cells.
CCK-8 assay, DAPI and γ-H2A.X immunofluorescence staining, and flow cytometry (apoptosis verification) were employed to determine the anti-proliferative and pro-apoptotic effects of TS. qPCR and Western blot were used to examine TS's molecular actions and mechanism on apoptosis of U2-OS cells.
The in vivo data showed that TS significantly inhibited U2-OS tumor growth in larval zebrafish from 2 to 20 ng/mL. In vitro data indicated that TS exerted significant anti-proliferative and pro-apoptotic effects on U2-OS cells in a dose-dependent manner. Moreover, TS has no inhibitory effect on bMSCs, suggesting its safety on normal bone-forming cells. Molecular data illustrated that TS obviously activated the p53 signaling-related proteins (p-p53, Bax, CASP3, CASP9) and its upstream JNK (p-JNK, p-c-JUN) and ATM (p-ATM) signaling molecules through phosphorylation and cleavage, followed by up-regulation of the pro-apoptotic genes, NOXA, PUMA, TP53, BAX, and BIM, and down-regulation of Bcl-2 protein.
In sum, TS specifically induced apoptosis of U2-OS cells by activating p53 signaling pathways, indicating TS as a promising candidate for OS treatment.
丹参（中药名为丹参）是一种著名的草药，常用于治疗血瘀证，包括骨肉瘤（OS）。丹参的主要成分被认为具有抗 OS 能力。然而，丹参醇（TS，丹参的主要成分）在 OS 中的疗效和机制尚未得到明确描述。由于 OS 是儿童和青少年中最常见的原发性骨癌，发病率和死亡率都很高，因此引起了我们的关注。不幸的是，由于化疗耐药性和复发率的增加，化疗在 OS 中的应用面临许多临床挑战。因此，本研究旨在深入探讨 TS 在 OS 治疗中的作用。
通过使用斑马鱼异种移植模型和 U2-OS 细胞进行体内和体外实验，探讨 TS 对 OS 的治疗作用及其机制。
采用 CCK-8 法、DAPI 和 γ-H2A.X 免疫荧光染色、流式细胞术（凋亡验证）检测 TS 对 U2-OS 细胞增殖和促凋亡作用。qPCR 和 Western blot 检测 TS 对 U2-OS 细胞凋亡的分子作用及机制。
体内数据显示，TS 以 2 至 20ng/ml 的浓度显著抑制幼虫斑马鱼中的 U2-OS 肿瘤生长。体外数据表明，TS 以剂量依赖的方式对 U2-OS 细胞表现出显著的抗增殖和促凋亡作用。此外，TS 对 bMSCs 没有抑制作用，提示其对正常成骨细胞的安全性。分子数据表明，TS 通过磷酸化和切割明显激活了 p53 信号相关蛋白（p-p53、Bax、CASP3、CASP9）及其上游 JNK（p-JNK、p-c-JUN）和 ATM（p-ATM）信号分子，随后上调促凋亡基因 NOXA、PUMA、TP53、BAX 和 BIM，下调 Bcl-2 蛋白。
总之，TS 通过激活 p53 信号通路特异性诱导 U2-OS 细胞凋亡，表明 TS 是治疗 OS 的一种有前途的候选药物。
