Oxidative stress evaluation in hepatocellular carcinoma: therapeutic implications

Abstract

Hepatocellular carcinoma is one of the most frequent cancers worldwide and effective therapy is currently lacking. Several known environmental risk factors for hepatocellular carcinoma development lead to generation of reactive oxygen species promoting oxidative stress. On the other hand, since mitochondria is the main site for reactive oxygen species production, it may have a relevant role in hepatocarcinogenesis. Moreover, neoplastic cells have a higher mitochondrial membrane potential than normal cells, which may be explored in the development of new approaches to treat hepatocellular carcinoma. The aim of this work is to evaluate the therapeutic efficacy of new compounds targeting the mitochondria, such as Dequalinium, a lypophilic cation, and the natural bioactive compounds, vitamin C (ascorbic acid and dehydroascorbic acid), and epigallocatechin-3-gallate, a green tea polyphenol, both in monotherapy and in association with each other and with conventional anticarcinogenic drugs (5-fluorouracil and doxorubicin) in order to identify which of them may be a useful therapeutic approach in hepatocellular carcinoma. We also intended to clarify the molecular mechanisms involved in the cytotoxicity induced by these new drugs, including the influence of oxidative stress, mitochondrial function and the expression levels of proteins involved in apoptosis mitochondrial pathway. For this purpose, we use the HUH-7 cells, an hepatocellular carcinoma cell line, maintained in culture in absence and presence of increasing concentrations of Dequalinium, epigallocatechin-3-gallate, ascorbic acid and dehydroascorbic acid, in monotherapy or in combination with each other and with conventional the anticarcinogenic drugs, 5-fluorouracil and doxorubicin, during 96 hours. The antiproliferative effect was assessed by the Alamar Blue assay and cell death by optic microscopy and flow cytometry upon staining cells with Oxidative Stress Evaluation in Hepatocellular Carcinoma – therapeutic implications 3 Annexin V and propidium iodide. The expression of the apoptosis-regulating molecules, BAX and BCL-2, was assessed using monoclonal antibodies labelled with fluorescent probes. Oxidative stress was evaluated through the intracellular reactive oxygen species accumulation, peroxides and superoxide anion, using the fluorescent probes DCFH2-DA and DHE, respectively. The mitochondrial function was analysed through the determination of the mitochondrial transmembrane potential using the fluorescent probe JC1. All these parameters were analysed by flow cytometry. The results obtained suggest that dequalinium, epigallocatechin-3-gallate and vitamin C, as single agents, have an antiproliferative and cytotoxic effect in a dose and time dependent manner. This effect increases when these compounds are used in a daily administration scheme with a lower total dosage. On the other hand, when used in association, a synergistic antiproliferative and cytotoxic effect is observed with dequalinium and epigallocatechin-3-gallate that may be mediated mainly by apoptosis. In opposite when cells are treated with DHA associated with 5-FU an antagonistic effect is observed. When cells are incubated with Dequalinium, mitochondria seems to play an important role in HUH-7 cell death. Besides we observed a cytotoxic effect upon incubation of cells with natural bioactive compounds, a pro-oxidant effect wasn’t evident, suggesting other mechanisms involved in cell death. This study suggests that dequalinium, epigallocatechin-3-gallate and vitamin C may constitute new therapeutic options for hepatocellular carcinoma both in monotherapy and in association. However, as the schedule of drug administration schemes and new drugs associations could interfere with drug efficacy, they should be tested in order to improve the therapeutic potential in hepatocellular carcinoma.