425 Hyperthermia Induced Cell Death in Human Melanoma Cells

Abstract

PurposeTreating disease with hyperthermia dates back to 3000 BC when Egyptian physicians explored the therapeutic benefits of heating diseased tissues. There has been renewed interest in using hyperthermia to treat cancer over the last 30 years. Hyperthermia as an adjuvant anti-cancer therapy has demonstrated improved clinical outcomes and increased response rates when combined with conventional treatments of tumors. This project investigated the molecular mechanisms of hyperthermia induced cell death in human melanoma cells in-vitro. Data from these experiments may help in the generation of new treatment modalities for skin cancer.MethodsCopper blocks inside incubators delivered a uniform and reproducible hyperthermia treatment to human melanoma cells grown in 96 well plates. A sophisticated measurement device with fast-response thermocouples in a 96 well format measured the thermal dose delivered to the cells. The human melanoma cells received hyperthermia treatments ranging from 15-120 mins at 41, 45 and 48°C. The treated cells were analyzed for overall viability, the mechanism of cell death, and whether there was a relationship between the thermal-doses delivered and the cell death pathway induced. Cell viability was measured in each well post treatment using an MTS based assay (CellTiter 96 Aqueous One Promega). The activity of apoptosis specific caspases 3 and 7 were measured using a homogeneous luminescent assay (Caspase-Glo3/7 Promega). Nuclear condensation and fragmentation was assessed in an eloquent microscopic approach using a modified ethidium bromide and acridine orange staining assay (EB/AO) in a 96 well format.ResultsWe consistently demonstrated that hyperthermia decreased cell viability in human melanoma cells in-vitro, and that the diminished cell viability was proportional the thermal dose delivered. Hyperthermia also induced activation of caspase 3 and 7, and increased the population of cells with condensed or fragmented nuclei. This data suggest that hyperthermia induced apoptosis in human melanoma cells.ConclusionsHyperthermia induces dose-dependent apoptosis in human melanoma cells. Further elucidation of these pathways will help in the design and discovery of novel methods for treating skin cancers.