Conjunction of glutathione level, NAD(P)H/FAD redox status and hypericin content as a potential factor affecting colon cancer cell resistance to photodynamic therapy with hypericin

SummaryBackgroundPhotodynamic therapy (PDT) is a highly efficient approach for tumour therapy, though it also has its drawbacks, too. There are multiple mechanisms involved in cell death regulation that can be successfully targeted for improvement of PDT in particular cases. We assumed, however, that the potential to manage radical stress might be the primary factor responsible for resistance to hypericin-mediated PDT (HY-PDT).MethodsWe compared the sensitivity of six colon-derived cancer cell lines to HY-PDT at IC50 equitoxic doses acquired by formazan-based (MTT) assay. Intracellular hypericin content, cell survival/metabolic activity, caspase-3 activation/mitochondrial membrane potential dissipation, apoptosis, glutathione level, redox status (NAD(P)H/oxidized flavins ratio) and Western blot analyses of proteins relevant in apoptosis regulation were measured to demonstrate differences between tested cell lines.ResultsAnalyses revealed a whole spectrum of responses from insignificant to high cytotoxicity, despite the MTT-based “equitoxicity”. Further critical evaluation of multiple parameters linked to cell physiology and proteomics proved that intracellular hypericin content, glutathione level or redox status demonstrate partial but not direct correlation with resistance to HY-PDT, when considered separately. However, their logical conjunction did copy the trend of cellular resistance.ConclusionsWe may conclude that intracellular level of hypericin and glutathione together with redox state of the target cell represent a potential combination of parameters responsible for the primary cytotoxicity of HY-PDT. We also present evidence that cytotoxic assays, such as the MTT, should be accompanied with other tests of cell survival/cytotoxicity in order to avoid incorrect conclusions.