Characterization of human hepatocellular carcinoma [HepG2] cell death following combined treatment of adriamycin and mild hyperthermia

ABSTRACT

A huge number of investigations confirmed the role of hyperthermia at 42-45°C in the enhancement of tumor cell killing, however, the effect of hyperthermia within the physiological range 38 to 40°C, requires more investigations. The aim of this study is to evaluate the efficacy of mild hyperthermia [MHT] at 40°C in increasing the intracellular uptake and cytotoxicity of adriamycin against hepatocellular carcinoma cells with a special focus on the mechanism of cell death following exposure to separate treatments of mild hyperthermia, adriamycin, or a combination of both. An experimental in vitro study. National institute of laser enhanced science, Cairo University. The antiproliferative effect or adriamycin [ADR], mild hyperthermia [MHT] at 40°C and 42°C and a combined treatment of adriamycin [ADR] and mild hyperthermia [MHT] was studied on hepatocellular carcinoma cell line [HepG2] at 4 and 24h post treatment[s] using trypan blue exclusion assay. A set of HepG2 cells in tissue culture plates were divided into 5 groups [a] cells incubated with ADR at 37°C for different time intervals, [b] cells exposed to MHT at 40°C and/or 42°C for different heating durations, [c] cells treated with ADR and exposed to MHT [ADR+MHT] and [d] control group of cells neither incubated with ADR nor exposed to MHT. The intracellular uptake of ADR was assessed as a function of incubation time in absence and in presence of mild hyperthermia and the amount of adriamycin recovered from cells lysates was measured by spectrofluorimetry. Malondialdehyde levels [MDA] were measured in control and treated groups at 4 h post treatment [s] as an index of lipid peroxidation and generation of reactive oxygen species [ROS]. Transmission electron microscopy [TEM] and light microscopy were performed for control and treated groups. Viable cell counts at 24h post- treatment [s] revealed that MHT at 40°C and 42°C equally induced a modest lethal effect against HepG2 cells after a heating duration of 20 min. Both temperatures were equally synergestic on enhancing the cytotoxicity of adriamycin against HepG2 cells as the cellular uptake of ADR increased by 1.5 folds in presence of MHT. Assessment of MDA levels in treated cells revealed that the generation of reactive oxygen species in cells exposed to [ADR+MHT] increased significantly, compared to cells treated with ADR or MHT separately. Analysis of cell morphological changes by light microscopy, indicated an evident increase In apoptotic cell death. Transmission electron microscopy confirmed our findings, Mild hyperthermia at 40°C is physiologically tolerable and could be a useful adjunct treatment in cancer therapy as it can increase the effectiveness and decrease the toxicity of currently available cancer treatments such as chemotherapy and radiation