ABSTRACT
Orostachys japonicus shows various biological activities. However, the molecular mechanisms remain unknown in LPS-stimulated macrophages. Here, we investigated the anti-oxidizing effect of the dichloromethane (DCM) and hexane fractions from O. japonicus (OJD and OJH) against oxidative stress in RAW 264.7 cells stimulated by LPS. OJD and OJH significantly increased the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Additionally, it was found that the expression of HO-1 was stimulated by Nrf2 activated via degradation of Keap1. ERK and p38 inhibitors repressed HO-1 induced by OJD and OJH in LPS-stimulated cells, respectively. In conclusion, these results suggest that OJD and OJH may block oxidative damage stimulated by LPS, via increasing the expression of HO-1 and Nrf2, and MAPK signaling pathway.
Subject(s)
Antioxidants/chemistry , Crassulaceae/chemistry , Mitogen-Activated Protein Kinase Kinases/metabolism , NF-E2-Related Factor 2/metabolism , Plant Extracts/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antioxidants/isolation & purification , Antioxidants/pharmacology , Cell Line , Cell Survival/drug effects , Crassulaceae/metabolism , Cytoskeletal Proteins/metabolism , Heme Oxygenase-1/metabolism , Hexanes/chemistry , Kelch-Like ECH-Associated Protein 1 , Lipopolysaccharides/toxicity , Methylene Chloride/chemistry , Mice , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Plant Extracts/pharmacology , Signal Transduction/drug effects , Up-Regulation/drug effects , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Cancer rates are increasing dramatically, and there is currently a strong emphasis on identifying biologically active substances with anti-cancer activity from traditional herbs, as these are thought to have less adverse side-effects than conventional chemotherapy. Here, we examined the effects of extracts of Orostachys japonicus A. BERGER (O. japonicus) on cancer cell proliferation and apoptosis, and investigated the underlying signaling pathways. Dried powdered O. japonicus was extracted with 95% ethyl alcohol and fractionated with a series of organic solvents, including n-hexane (hexane), dichloromethane (DCM), ethylacetate (EtOAc), n-butanol (BuOH), and water (H(2)O). These extracts were tested for anti-cancer activity on a range of cancer cells; of all these, the EtOAc soluble fraction showed the highest anti-cancer activity, which was most marked in AGS human gastric cancer cells. The EtOAc fraction inhibited the proliferation of AGS cells in a dose-dependent and time-dependent manner, by inducing apoptosis and cell cycle arrest, as evidenced by 4,6-diamidino-2-phenylindole (DAPI) staining, annexin V-fluorescein isothiocyanate staining, propidium iodide-labeling, and DNA fragmentation assays. Western blot analysis revealed that p53 and cleaved caspase-3 proteins were up-regulated, and B cell lymphoma-2 (bcl-2) protein and pro-caspase-3 were down-regulated, but bcl-2 associated x protein (bax) protein was not regulated, in response to treatment of AGS cells with the EtOAc fraction. However, the changes of pro-caspase-3 and cleaved caspase-3 could be abolished by the pan-caspase inhibitor Z-VAD-FMK. These results suggest the EtOAc fraction from O. japonicus has substantial anti-cancer activity in human gastric cancer cells.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Apoptosis/drug effects , Crassulaceae/chemistry , Phytotherapy , Plant Extracts/therapeutic use , Stomach Neoplasms/drug therapy , Tumor Suppressor Protein p53/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Caspase 3/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Humans , Plant Extracts/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Stomach Neoplasms/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Orostachys japonicus A. Berger (O. japonicus) is known to reduce the risk of many diseases. AIM OF THE STUDY: We investigated the anti-inflammatory effects of the dichloromethane (DCM) fraction from O. japonicus (OJD) in LPS-stimulated RAW 264.7 cells. MATERIALS AND METHODS: NO was measured using the Griess method. Key pro-inflammatory cytokines and mediators including IL-1ß, TLR4, iNOS, and COX-2; 2 important pro-inflammatory transcription factors, NF-κB p65 and IκBα; and MAPKs such as ERK1/2, JNK, and p38 were analyzed by Western blotting. RESULTS: OJD significantly inhibited NO production, IL-1ß, TLR4, iNOS, and COX-2 expression in LPS-stimulated cells. Additionally, it inhibited LPS-induced NF-κB p65 activation via inhibition of IκBα phosphorylation. Furthermore, phosphorylation of p38 and JNK was suppressed by OJD in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. CONCLUSIONS: Our data suggest that OJD inhibits the inflammatory response via suppression of NF-κB activation and MAPK signaling.
