Zhe Wu1, Yaqiong Zhan2, Li Wang3, Jiepeng Tong4, Li Zhang5, Mengjia Lin6, Xuehang Jin7, Lushun Jiang8, Yan Lou9, Yunqing Qiu10. 1. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: 11518159@zju.edu.cn. 2. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: 11818228@zju.edu.cn. 3. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: 11618298@zju.edu.cn. 4. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: 11718172@zju.edu.cn. 5. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: 11918223@zju.edu.cn. 6. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: linmengjia9@zju.edu.cn. 7. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: zyyyjxh@zju.edu.cn. 8. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: 21818032@zju.edu.cn. 9. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: yanlou@zju.edu.cn. 10. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310000, People's Republic of China. Electronic address: qiuyq@zju.edu.cn.
Abstract
BACKGROUNDS: Ribonucleotide reductase (RR) catalyzes the essential step in the formation of all four deoxynucleotides. Upregulated activity of RR plays an active role in tumor progression. As the regulatory subunit of RR, ribonucleotide reductase subunit M2 (RRM2) is regarded as one of the effective therapeutic targets for DNA replication-dependent diseases, such as cancers. Recent studies have revealed that osalmid significantly inhibits the activity of RRM2, but the metabolic profile of osalmid remains unknown. OBJECTIVE: The aim of this study was to clarify the metabolic profile including metabolites, isoenzymes and metabolic pathways of osalmid. The anti-human hepatocellular carcinoma activity and mechanism of metabolites were further investigated. MATERIALS AND METHODS: Ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was used for identifying metabolites and for characterizing phase I and phase II metabolic pathways with recombinant enzymes or in human liver microsomes of osalmid. The eHiTS docking system was used for potential RRM2 inhibitor screening among metabolites. Cytotoxicity assays were performed for evaluating cell proliferation inhibitory activity of metabolites. Cell cycle assays and cell apoptosis assays were assessed by flow cytometry. Western blotting analysis of RRM2, cyclin D1, p21, p53, phosphorylated p53, Bcl-2 and Bax was performed to explore the anti-hepatocellular carcinoma mechanism of the active metabolites. RESULTS: Ten metabolites of osalmid were identified, and none of them have been reported previously. Hydroxylation, glucuronidation, sulfonation, acetylation and degradation were recognized as the main metabolic processes of osalmid. Isozymes of CYP1A2, CYP2C9, UGT1A1, UGT1A6, UGT1A9, UGT2B7 and UGT2B15 were involved in phase I and phase II metabolism of osalmid. Metabolites M7, M8 and M10 showed higher binding affinities with the RRM2 active site than osalmid. Metabolite M7 exhibited potent inhibitory activity to hepatocellular carcinoma cell lines by both competitive inhibition and down-regulation of RRM2. Moreover, M7 significantly induced cell cycle arrest and apoptosis by activating p53-related pathways. CONCLUSIONS: The metabolic profile of osalmid was identified. M7 significantly inhibited human hepatocellular carcinoma progression by inhibiting RRM2 activity. Furthermore, M7 induced cell cycle arrest and apoptosis by activating p53-related signaling pathways.
BACKGROUNDS: Ribonucleotide reductase (RR) catalyzes the essential step in the formation of all four deoxynucleotides. Upregulated activity of RR plays an active role in tumor progression. As the regulatory subunit of RR, ribonucleotide reductase subunit M2 (RRM2) is regarded as one of the effective therapeutic targets for DNA replication-dependent diseases, such as cancers. Recent studies have revealed that osalmid significantly inhibits the activity of RRM2, but the metabolic profile of osalmid remains unknown. OBJECTIVE: The aim of this study was to clarify the metabolic profile including metabolites, isoenzymes and metabolic pathways of osalmid. The anti-humanhepatocellular carcinoma activity and mechanism of metabolites were further investigated. MATERIALS AND METHODS: Ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was used for identifying metabolites and for characterizing phase I and phase II metabolic pathways with recombinant enzymes or in human liver microsomes of osalmid. The eHiTS docking system was used for potential RRM2 inhibitor screening among metabolites. Cytotoxicity assays were performed for evaluating cell proliferation inhibitory activity of metabolites. Cell cycle assays and cell apoptosis assays were assessed by flow cytometry. Western blotting analysis of RRM2, cyclin D1, p21, p53, phosphorylated p53, Bcl-2 and Bax was performed to explore the anti-hepatocellular carcinoma mechanism of the active metabolites. RESULTS: Ten metabolites of osalmid were identified, and none of them have been reported previously. Hydroxylation, glucuronidation, sulfonation, acetylation and degradation were recognized as the main metabolic processes of osalmid. Isozymes of CYP1A2, CYP2C9, UGT1A1, UGT1A6, UGT1A9, UGT2B7 and UGT2B15 were involved in phase I and phase II metabolism of osalmid. Metabolites M7, M8 and M10 showed higher binding affinities with the RRM2 active site than osalmid. Metabolite M7 exhibited potent inhibitory activity to hepatocellular carcinoma cell lines by both competitive inhibition and down-regulation of RRM2. Moreover, M7 significantly induced cell cycle arrest and apoptosis by activating p53-related pathways. CONCLUSIONS: The metabolic profile of osalmid was identified. M7 significantly inhibited humanhepatocellular carcinoma progression by inhibiting RRM2 activity. Furthermore, M7 induced cell cycle arrest and apoptosis by activating p53-related signaling pathways.