Breast cancer resistance protein is the enemy of hypericin accumulation and toxicity of hypericin-mediated photodynamic therapy.

Breast cancer resistance protein (BCRP) belongs to the family of ATP-binding cassette (ABC) transporters, overexpression of which can confer a multidrug-resistant phenotype in cancer cells and tumors. BCRP mediates efflux of numerous xenobiotics, including various chemotherapeutic agents and photosensitizers. Hypericin (HY) is a naturally-occurring photosensitizer synthesized by plants of the genus Hypericum. Our recently published results indicate that accumulation of HY in cancer cells of different tissue origin can be affected mostly by BCRP. Considering all known facts, the main goal of this study was to verify whether not only HY accumulation but also toxicity of HY-mediated photodynamic therapy (PDT) can be affected by the presence of some ABC transporters. To specifically prove our hypothesis, we used an experimental model of human leukemia cell lines differing in the expression level of the main drug efflux transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and BCRP. The lowest HY accumulation, and consequently the highest resistance to HY-PDT, was found in cells overexpressing BCRP. Moreover, pretreatment with BCRP inhibitor Ko143 significantly increased HY accumulation and sensitized cells to HY-PDT. Therefore, our findings represent direct evidence that BCRP is the nemesis of HY accumulation and toxicity of HY-PDT. Thus, we should emphasize that individualized screening for BCRP expression and activity may represent a useful tool for prediction of HY-mediated photodynamic diagnosis (PDD) or PDT effectiveness.

Hypericin affects cancer side populations via competitive inhibition of BCRP.

OBJECTIVE: Cancer stem-like cells (CSLCs) are considered a root of tumorigenicity and resistance. However, their identification remains challenging. The use of the side population (SP) assay as a credible marker of CSLCs remains controversial. The SP assay relies on the elevated activity of ABC transporters that, in turn, can be modulated by hypericin (HYP), a photosensitizer and bioactive compound of St. John's Wort (Hypericum perforatum), a popular over-the-counter antidepressant. Here we aimed to comprehensively characterize the SP phenotype of cancer cells and to determine the impact of HYP on these cells. METHODS: Flow cytometry and sorting-based assays were employed, including CD24-, CD44-, CD133-, and ALDH-positivity, clonogenicity, 3D-forming ability, ABC transporter expression and activity, and intracellular accumulation of HYP/Hoechst 33342. The tumorigenic ability of SP, nonSP, and HYP-treated cells was verified by xenotransplantation into immunodeficient mice. RESULTS: The SP phenotype was associated with elevated expression of several investigated transporters and more intensive growth in non-adherent conditions but not with higher clonogenicity, tumorigenicity or ALDH-positivity. Despite stimulated BCRP level and MRP1 activity, HYP reversibly decreased the SP proportion, presumably via competitive inhibition of BCRP. HYP-selected SP cells acquired additional traits of resistance and extensively eliminated HYP. CONCLUSIONS: Our results suggest that SP is not an unequivocal CSLC-marker. However, SP could play an important role in modulating HYP-treatment and serve as a negative predictive tool for HYP-based therapies. Moreover, the use of supplements containing HYP by cancer patients should be carefully considered, due to its proposed effect on drug efflux and complex impact on tumor cells, which have not yet been sufficiently characterized.

Hypericin in the Light and in the Dark: Two Sides of the Same Coin.

Hypericin (4,5,7,4',5',7'-hexahydroxy-2,2'-dimethylnaphtodianthrone) is a naturally occurring chromophore found in some species of the genus Hypericum, especially Hypericum perforatum L. (St. John's wort), and in some basidiomycetes (Dermocybe spp.) or endophytic fungi (Thielavia subthermophila). In recent decades, hypericin has been intensively studied for its broad pharmacological spectrum. Among its antidepressant and light-dependent antiviral actions, hypericin is a powerful natural photosensitizer that is applicable in the photodynamic therapy (PDT) of various oncological diseases. As the accumulation of hypericin is significantly higher in neoplastic tissue than in normal tissue, it can be used in photodynamic diagnosis (PDD) as an effective fluorescence marker for tumor detection and visualization. In addition, light-activated hypericin acts as a strong pro-oxidant agent with antineoplastic and antiangiogenic properties, since it effectively induces the apoptosis, necrosis or autophagy of cancer cells. Moreover, a strong affinity of hypericin for necrotic tissue was discovered. Thus, hypericin and its radiolabeled derivatives have been recently investigated as potential biomarkers for the non-invasive targeting of tissue necrosis in numerous disorders, including solid tumors. On the other hand, several light-independent actions of hypericin have also been described, even though its effects in the dark have not been studied as intensively as those of photoactivated hypericin. Various experimental studies have revealed no cytotoxicity of hypericin in the dark; however, it can serve as a potential antimetastatic and antiangiogenic agent. On the contrary, hypericin can induce the expression of some ABC transporters, which are often associated with the multidrug resistance (MDR) of cancer cells. Moreover, the hypericin-mediated attenuation of the cytotoxicity of some chemotherapeutics was revealed. Therefore, hypericin might represent another St. John's wort metabolite that is potentially responsible for negative herb-drug interactions. The main aim of this review is to summarize the benefits of photoactivated and non-activated hypericin, mainly in preclinical and clinical applications, and to uncover the "dark side" of this secondary metabolite, focusing on MDR mechanisms.

Potentiation of hypericin-mediated photodynamic therapy cytotoxicity by MK-886: focus on ABC transporters, GDF-15 and redox status.

BACKGROUND: Pretreatment with 5-LOX pathway inhibitor MK-886 potentiates cytotoxic effects of photodynamic therapy mediated by natural photosensitizer, hypericin. In this study, we focused on elucidating mechanisms beyond the increased efficacy of combined treatment. METHODS: Metabolic activity/viability, caspase-3 activation/mitochondrial membrane potential dissipation, intracellular hypericin level, glutathione level and redox status (NAD(P)H/oxidized flavins ratio) analyses, as well as drug efflux assays, were performed by flow cytometry. Changes in protein expression of ATP-binding cassette transporters, GDF-15 and other selected proteins were evaluated by Western blotting. Silencing of gdf-15 was carried out to verify its role in response to treatment. RESULTS: MK-886 pretreatment led to a concentration-dependent increase in intracellular hypericin content, accompanied by changes in ATP-binding cassette transporters levels and efflux efficiency. Intracellular accumulation of cytokine GDF-15 correlated with increased cell death markers; however, the impact of gdf-15 silencing on the evaluated markers was negligible. A marked decrease in the glutathione level of a majority of cells was observed after more toxic combination treatment. CONCLUSION: The significant increase in cell death markers after combination treatment confirms the potentiating effect of MK-886 on hypericin-mediated photodynamic therapy in HT-29 and MCF-7 cells. Although BCRP downregulation was not confirmed as leading mechanism responsible for elevated levels of hypericin content, changes in expression and efflux activity of ABC transporters caused by MK-886 suggest its potential in combination treatment with drugs that are substrates of these transporters, predominantly MRP1. However, complex cellular response to MK-886 pretreatment needs to be considered and further elucidated.