Jing Wang1, Jia Wang2, Ling Li3, Li Feng4, Yu-Rong Wang5, Zhe Wang6, Ning-Hua Tan7. 1. Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China. Electronic address: 18851107621@163.com. 2. Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China. Electronic address: 1731020094@stu.cpu.edu.cn. 3. Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China. Electronic address: 1821020435@stu.cpu.edu.cn. 4. Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China. Electronic address: cpu_fengli2012@163.com. 5. Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China. Electronic address: yurong1987213@163.com. 6. Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China. Electronic address: wangzhe@cpu.edu.cn. 7. Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China. Electronic address: nhtan@cpu.edu.cn.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: This study was designed to investigate the antitumor activity and potential mechanism of RA-XII on colorectal cancer (CRC) cell lines. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: This study was designed to investigate the antitumor activity and potential mechanism of RA-XII on colorectal cancer (CRC) cell lines. MATERIALS AND METHODS: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. RESULTS: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. CONCLUSION: 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.