Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China.
J Ethnopharmacol. 2021 Feb 10;266:113438. doi: 10.1016/j.jep.2020.113438. Epub 2020 Oct 2.
The roots of Rubia yunnanensis Diels (Chinese name 'Xiao-Hong-Shen'), a traditional Chinese medicine native to Yunnan province (China), have a long history of use for treating several diseases, such as tuberculosis, rheumatism and cancers. A bicyclic hexapeptidic glucoside named RA-XII was isolated from R. yunnanensis, which has been reported to exert anti-inflammatory and antitumor activities.
This study was designed to investigate the antitumor activity and potential mechanism of RA-XII on colorectal cancer (CRC) cell lines.
Sulforhodamine B assay, clonogenic assay and cell cycle analysis were conducted to assess the anti-proliferative activity of RA-XII on CRC cells. GFP-LC3B plasmid transfection, MDC and AO staining assays, cathepsin activity assay, and siRNAs against several genes were used to investigate the effect of RA-XII on autophagy. Western blotting was used to examine the expression levels of proteins associated with cell cycle arrest, apoptosis and autophagy. Human CRC xenograft-bearing BALB/c nude mice were used to evaluate the antitumor effect of RA-XII in vivo.
RA-XII showed favorable antineoplastic activity in SW620 and HT29 cells in vitro and in vivo. RA-XII did not induce apoptosis indicated by no obvious changes on mitochondrial membrane potential and apoptosis-related marker proteins in SW620 or HT29 cells. Treatment of RA-XII inhibited the formation of autophagosomes, which is implied by the GFP-LC3 fluorescent dots, MDC-stained autophagic vesicles and LC3 protein expression. It was indicated that RA-XII suppressed autophagy by regulating several signaling pathways including mTOR and NF-κB pathways. Pharmacological or genetic inhibition of autophagy could enhance the cytotoxicity of RA-XII while autophagy inducer could rescue RA-XII-induced cell death. Besides, RA-XII could increase the susceptibility of CRC cells to bortezomib.
Our study demonstrated that RA-XII exerted antitumor activity independent of apoptosis, and suppressed protective autophagy by regulating mTOR and NF-κB pathways in SW620 and HT29 cell lines, which suggested that RA-XII is a key active ingredient for the cancer treatment of Rubia yunnanensis and possesses a promising prospect as an autophagy inhibitor for CRC therapy.
云南茜草（中国云南本土的一种传统中药）的根在治疗结核病、风湿病和癌症等多种疾病方面有着悠久的应用历史。一种名为 RA-XII 的双环六肽葡萄糖苷已从云南茜草中分离出来，据报道其具有抗炎和抗肿瘤活性。
本研究旨在研究 RA-XII 对结直肠癌细胞系的抗肿瘤活性及其潜在机制。
采用磺酰罗丹明 B 测定法、集落形成测定法和细胞周期分析评估 RA-XII 对 CRC 细胞的抗增殖活性。采用 GFP-LC3B 质粒转染、MDC 和 AO 染色测定、组织蛋白酶活性测定以及针对几种基因的 siRNAs 研究 RA-XII 对自噬的影响。采用 Western blotting 检测与细胞周期阻滞、细胞凋亡和自噬相关的蛋白表达水平。用人结直肠癌细胞异种移植裸鼠模型评估 RA-XII 的体内抗肿瘤作用。
RA-XII 在体外和体内对 SW620 和 HT29 细胞均表现出良好的抗肿瘤活性。RA-XII 并未诱导 SW620 或 HT29 细胞发生明显的线粒体膜电位变化和凋亡相关标志物蛋白变化，表明其未诱导细胞凋亡。RA-XII 处理抑制了自噬体的形成，这一点可从 GFP-LC3 荧光斑点、MDC 染色的自噬小泡和 LC3 蛋白表达中得到证实。结果表明，RA-XII 通过调节 mTOR 和 NF-κB 等信号通路抑制自噬。自噬的药理学或遗传学抑制可增强 RA-XII 的细胞毒性，而自噬诱导剂可挽救 RA-XII 诱导的细胞死亡。此外，RA-XII 可增加 CRC 细胞对硼替佐米的敏感性。
本研究表明，RA-XII 通过调节 mTOR 和 NF-κB 等信号通路发挥抗肿瘤活性，抑制 SW620 和 HT29 细胞系中的保护性自噬，这表明 RA-XII 是云南茜草治疗癌症的关键活性成分，有望作为 CRC 治疗的自噬抑制剂。
