title: Proteomic and functional analyses demonstrate the involvement of oxidative stress in the anticancer activities of oridonin in HepG2 cells.
abstract: Oridonin exhibits a curative effect on liver carcinoma in patients and experimental animals. In the present study, we performed proteomic and functional analyses to explore the mechanism involved in the anticancer activity of oridonin. Oridonin treatment for 24 h resulted in a dose-dependent decrease in cell viability with an IC50 value of 37.90 µM. Treatment with 40 µM oridonin for 24 h induced apoptosis as typical apoptotic nuclear alterations were observed following DAPI staining. Using a 2-DE-based proteomic approach, 3 upregulated oxidative stress markers, Hsp70-1, Hop and Prdx2, were identified in the HepG2 cells treated with 40 µM oridonin for 24 h. A pattern of alteration in Hsp70-1 was verified by western blotting. The mRNA expression patterns of Hsp70-1 and Hop as determined by qPCR were comparable to their protein expression patterns. Further investigations showed that oridonin treatment for 24 h resulted in reactive oxygen species (ROS) generation, and ROS scavenger N-acetylcysteine (NAC) completely inhibited ROS production and restored cell viability, suggesting that oxidative stress contributed to oridonin-induced HepG2 cell death. Western blot analysis of oxidative stress pathway-related proteins demonstrated that oridonin treatment increased p-JNK, p-p38 and p-p53, and decreased Bcl-2 protein expression levels, promoted cytochrome c release, decreased mitochondrial membrane potential, and activated caspase-9 and caspase-3. Furthermore, knockdown of Hsp70-1 expression with specific shRNA significantly decreased the viability of the cells treated with oridonin, suggesting a protective role of Hsp70-1 in oridonin-mediated oxidative stress. The results of the present study provide evidence for a link between oxidative stress and oridonin-induced apoptosis in HepG2 cells.
authors.affiliation: Guangdong Provincial Key Laboratory of Biotechnology Candidate Drug Research, School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, P.R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, SAR, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ye
authors.affiliation: Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, SAR, P.R. China.
authors.forename: Zhi-Ling
authors.initials: ZL
authors.lastname: Yu
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000096:biosynthesis; D053453:Caspase 9 / Q000096:biosynthesis; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D045786:Diterpenes, Kaurane / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D056945:Hep G2 Cells; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000096:biosynthesis; D008113:Liver Neoplasms / Q000188:drug therapy*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D054464:Peroxiredoxins / Q000096:biosynthesis; D040901:Proteomics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D034622:RNA Interference; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis; D048051:p38 Mitogen-Activated Protein Kinases / Q000096:biosynthesis
chemical_list: D045786:Diterpenes, Kaurane; C476884:HOPX protein, human; D018840:HSP70 Heat-Shock Proteins; D018398:Homeodomain Proteins; C483693:Hspa14 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C011959:oridonin; C086065:PRDX2 protein, human; D054464:Peroxiredoxins; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; D053453:Caspase 9; D000111:Acetylcysteine