RA-XII, a bicyclic hexapeptidic glucoside isolated from Rubia yunnanensis Diels, exerts antitumor activity by inhibiting protective autophagy and activating Akt-mTOR pathway in colorectal cancer cells

• Published in: Journal of ethnopharmacology Vol. 266; no. NA; p. 113438
• Main Authors: Wang, Jing, Wang, Jia, Li, Ling, Feng, Li, Wang, Yu-Rong, Wang, Zhe, Tan, Ning-Hua
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier B.V 10.02.2021
• Subjects: active ingredients; Animals; antineoplastic activity; Antineoplastic Agents, Phytogenic - isolation & purification; Antineoplastic Agents, Phytogenic - pharmacology; Antitumor mechanism; apoptosis; Apoptosis - drug effects; autophagosomes; autophagy; Autophagy - drug effects; cancer therapy; cathepsins; cell cycle checkpoints; Cell Line, Tumor; China; Colorectal cancer; colorectal neoplasms; Colorectal Neoplasms - drug therapy; Cyclopeptide; cytotoxicity; Female; fluorescence; glucosides; HT29 Cells; Humans; membrane potential; Mice; Mice, Inbred BALB C; Mice, Nude; mitochondrial membrane; Oriental traditional medicine; Peptides, Cyclic - isolation & purification; Peptides, Cyclic - pharmacology; plasmids; protein synthesis; Proto-Oncogene Proteins c-akt - metabolism; RA-XII; rheumatoid arthritis; Rubia; Rubia - chemistry; Rubia yunnanensis Diels; Signal Transduction - drug effects; TOR Serine-Threonine Kinases - metabolism; transfection; tuberculosis; Xenograft Model Antitumor Assays; China; ATGs; Antitumor mechanism; DMSO; Colorectal cancer; siRNA; RA-XII; MDC; CQ; Rubia yunnanensis Diels; Cyclopeptide; FBS; mTOR; CTSD; CTSB; JC-1
• DOI: 10.1016/j.jep.2020.113438
• ISSN: 0378-8741; 1872-7573

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. [Display omitted]