Cellular And Molecular Mechanisms Of Biological Function Of 6-Gingerol

Phytochemicals derived from fruits and vegetables are generally regarded as nontoxic based on the fact of daily consumption. They are known to exhibit a variety of biological and pharmacological activities. According to their structure and function, they can be divided into plants estrogen, carotenoids,phytosterols,porphyrin,compound containing sulphur, protein inhibitors, spice, phytic acid, saponins, etc. In recent years, public and scientists put more and more concerns on the treatment of human disease, using phytochemicals, especially for cancer and cardiovascular disease.Ginger is an important cultivated plant in Asia, especially in South-eastern Asia regions. In China, the ministry of health named ginger as the plant resource for medicine and diet. One of the gingerols, 6-gingerol, is a major pharmacologically active component of ginger. It has anti-inflammatory, antioxidant and anticancer activities. However, the mechanisms of action for 6-gingerol are not well understood.In this study, we used HepG2 cells to examine the genotoxicity, anticancer, and antioxidant activities of 6-gingerol.Comet assay and micronuclei test (MNT) were used to test the genotoxicity of 6-gingerol in HepG2 cells. Exposure of the cells to 6-gingerol caused significant increase of DNA migration in comet assay, increase of micronuclei frequencies at high concentrations at 20μM-80μM and 20μM-40μM, respectively. These results indicate that 6-gingerol caused DNA strand breaks and chromosome damage. To further elucidate the underlying mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH). In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis on 8-hydroxydeoxyguanosine (8-OHdG). The results showed that lysosomal membrane stability was reduced after treatment by 6-gingerol (20μM-80μM) for 40 min and mitochondrial membrane potential decreased after treatment for 50 min as well as GSH level was decreased and ROS level was significantly increased after treatment for 60 min. Moreover, 8-OHdG level was significantly increased after treatment for 3 h. These suggest 6-gingerol induces genotoxicity probably by oxidative stress and lysosomal and mitochondrial damage were caused by 6-gingerol.The DNA damage induced by 6-gingerol is dose-dependent. This supports the hypothesis that 6-gingerol may play a conflicting dual role in carcinogenesis according to its doses, and 6-gingerol in low concentration might possess anti-oxidant effect. Patulin (PAT) is a mycotoxin produced by several Penicillium, Aspergillus and Byssochlamys species, with the most commonly encountered species being Penicillium expansum. PAT is a frequent contaminant of ripe apples and apple products such as apple juice and cider. Concerns about the toxin properties of patulin have led various countries and the World Health Organization to establish 50μg/L as the recommended limit in apple juice. The carcinogenic risk of PAT to humans is classified in Group 3 by the International Agency for Research on Cancer (IARC), since the evidence of carcinogenicity is inadequate in humans and in experimental animals. PAT is a potent genotoxic compound and PAT contamination is common in fruits and fruit products. Therefore, it is required to find newer and better agents for protection against genotoxicity of PAT. In this study, the chemoprotective effect of 6-gingerol against PAT-induced genotoxicity in HepG2 cells was investigated. The comet assay and MNT were used to monitor genotoxic effects. To further elucidate the underlying mechanisms, we tested the intracellular generation of ROS and level of reduced GSH. In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis on 8-OHdG. The results showed that 6-gingerol (10μM) significantly reduced the DNA strand breaks and micronuclei formation caused by PAT. Moreover, 6-gingerol effectively suppressed PAT-induced intracellular ROS formation and 8-OHdG level. The GSH depletion induced by PAT in HepG2 cells was also attenuated by 6-gingerol pretreatment. These findings suggest that 6-gingerol has a strong protective ability against the genotoxicity caused by PAT and the antioxidant activity of 6-gingerol may play an important part in attenuating the genotoxicity of PAT.6-Gingerol has been known to possess anti-carcinogenic and pro-apoptotic activities. However, the mechanisms are not well understood. In our study, we have demonstrated that lysosome and mitochondria may be the primary targets for 6-gingerol in HepG2 cells. Therefore, we set up some experiments to evaluate the lysosome-mitochondria cross-signaling in 6-gingerol-induced apoptosis. Apoptosis was detected by Hoechst 33342 and TUNEL assay after 24 h treatment. Lysosomal membrane stability, mitochondrial membrane potential, and the levels of GSH and ROS were tested using fluorescent method. Moreover, cathepsin D and cytochrome c release of cytosolic protein samples were detected by Western blot. Here, we showed apoptosis was induced by 6-gingerol in HepG2 cells, and the destabilization of lysosome and mitochondria were early upstream initiating events. GSH level was decreased and ROS level was significantly increased. These findings suggest that the apoptosis may be related to oxidative stress.Here, we showed that cathepsin D played a crucial role in the process of apoptosis. The release of cathepsin D to the cytosol appeared to be an early event that preceded the release of cytochrome c from mitochondria. Moreover, inhibition of cathepsin D activity by pepstatin A resulted in suppressed the release of cytochrome c. In a word, the cells apoptosis is a“cascade model”activation process induced by endogenous gene, enzyme, and signal transduction pathway. Taken together, these results implicate that cathepsin D is a positive mediator of 6-gingerol-induced apoptosis in HepG2 cells, which acts upstream of cytochrome c release. The apoptosis of HepG2 cells induced by 6-gingerol may be associated with oxidative stress. However, we need to investigate how 6-gingerol caused the change of lysosomal membrane stability in our further study.Above all, the innovation of our study is as follows. First, we are the first to find that 6-gingerol imposed oxidative stress and caused the genotoxicity in HepG2 cells. Second, 6-gingerol at the concerntratrion of 10μM reduces the cytotoxicity and genotoxicity induced by PAT through decreasing the PAT-induced oxidative stress. Finally, 6-gingerol might induce apoptosis through“lysosome-mitochondria cross-signaling”in HepG2 cells.