Malgorzata Firczuk

Aminolevulinic Acid (ALA) as a Prodrug in Photodynamic Therapy of Cancer

Aminolevulinic acid (ALA) is an endogenous metabolite normally formed in the mitochondria from succinyl-CoA and glycine. Conjugation of eight ALA molecules yields protoporphyrin IX (PpIX) and finally leads to formation of heme. Conversion of PpIX to its downstream substrates requires the activity of a rate-limiting enzyme ferrochelatase. When ALA is administered externally the abundantly produced PpIX cannot be quickly converted to its final product - heme by ferrochelatase and therefore accumulates within cells. Since PpIX is a potent photosensitizer this metabolic pathway can be exploited in photodynamic therapy (PDT). This is an already approved therapeutic strategy making ALA one of the most successful prodrugs used in cancer treatment.

Approaches to improve photodynamic therapy of cancer.

Photodynamic therapy (PDT) is a clinically approved method of tumor treatment. Its unique mechanism of action results from minimal invasiveness and high selectivity towards transformed cells. However, visible light used to excite most photosensitizers has rather limited ability to penetrate tissues resulting in insufficient destruction of deeply seated malignant cells. Therefore, novel strategies for further potentiation of the anticancer effectiveness of PDT have been developed. These include combined treatments with surgery, chemo- and radiotherapy, strategies targeting cytoprotective mechanisms induced in PDT-treated cells, as well as attempts aimed at enhancement of PDT-mediated antitumor immune response. Moreover, new photosensitizers and novel light sources are being developed. Impressive progress in nanotechnology and understanding of tumor cell biology rise hopes for further improvements in this elegant and promising method of cancer treatment.

Statins impair glucose uptake in human cells

Objective Considering the increasing number of clinical observations indicating hyperglycemic effects of statins, this study was designed to measure the influence of statins on the uptake of glucose analogs by human cells derived from liver, adipose tissue, and skeletal muscle. Design Flow cytometry and scintillation counting were used to measure the uptake of fluorescently labeled or tritiated glucose analogs by differentiated visceral preadipocytes, skeletal muscle cells, skeletal muscle myoblasts, and contact-inhibited human hepatocellular carcinoma cells. A bioinformatics approach was used to predict the structure of human glucose transporter 1 (GLUT1) and to identify the presence of putative cholesterol-binding (cholesterol recognition/interaction amino acid consensus (CRAC)) motifs within this transporter. Mutagenesis of CRAC motifs in SLC2A1 gene and limited proteolysis of membrane GLUT1 were used to determine the molecular effects of statins. Results Statins significantly inhibit the uptake of glucose analogs in all cell types. Similar effects are induced by methyl-β-cyclodextrin, which removes membrane cholesterol. Statin effects can be rescued by addition of mevalonic acid, or supplementation with exogenous cholesterol. Limited proteolysis of GLUT1 and mutagenesis of CRAC motifs revealed that statins induce conformational changes in GLUTs. Conclusions Statins impair glucose uptake by cells involved in regulation of glucose homeostasis by inducing cholesterol-dependent conformational changes in GLUTs. This molecular mechanism might explain hyperglycemic effects of statins observed in clinical trials.

Studies toward Novel Peptidomimetic Inhibitors of Thioredoxin–Thioredoxin Reductase System

Thioredoxins (Trx) are ubiquitous multifunctional low-molecular weight proteins that together with thioredoxin reductases (TrxR) participate in the maintenance of protein thiol homeostasis in NADPH-dependent reactions. An increasing number of data reveal that the Trx-TrxR system is an attractive target for anticancer therapies. In this work, we have elaborated a new and simple synthetic approach employing Ugi reaction to synthesize several new inhibitors of this system. The influence of various electrophilic fragments of this new class of compounds on the inhibition of the Trx-TrxR system was evaluated. As a result, a new compound 19a (SK053), which inhibits the activity of the Trx-TrxR system and exhibits antitumor activity, was obtained. Biologic analyses revealed that 19a inhibits induction of NF-κB and AP-1 and decreases H(2)O(2) scavenging capacity in tumor cells. Altogether, we show that 19a is a novel potential antitumor peptidomimetic inhibitor that can be used as a starting compound for further optimization.

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.

Adenanthin targets proteins involved in the regulation of disulphide bonds

Adenanthin has been recently shown to inhibit the enzymatic activities of peroxiredoxins (Prdx) I and II through its functional α,β-unsaturated ketone group serving as a Michael acceptor. A similar group is found in SK053, a compound recently developed by our group to target the thioredoxin-thioredoxin reductase (Trx-TrxR) system. This work provides evidence that next to Prdx I and II adenanthin targets additional proteins including thioredoxin-thioredoxin reductase system as well as protein disulfide isomerase (PDI) that contain a characteristic structural motif, referred to as a thioredoxin fold. Adenanthin inhibits the activity of Trx-TR system and PDI in vitro in the insulin reduction assay and decreases the activity of Trx in cultured cells. Moreover, we identified Trx-1 as an adenanthin binding protein in cells incubated with biotinylated adenanthin as an affinity probe. The results of our studies indicate that adenanthin is a mechanism-selective, rather than an enzyme-specific inhibitor of enzymes containing readily accessible, nucleophilic cysteines. This observation might be of importance in considering potential therapeutic applications of adenanthin to include a range of diseases, where aberrant activity of Prdx, Trx-TrxR and PDI is involved in their pathogenesis.

Prenyltransferases Regulate CD20 Protein Levels and Influence Anti-CD20 Monoclonal Antibody-mediated Activation of Complement-dependent Cytotoxicity

Background: The influence of farnesyltransferase inhibitors (FTIs) on CD20 levels is unknown. Results: FTIs increase CD20 expression and improve rituximab-mediated activation of complement-dependent cytotoxicity. Conclusion: FTIs sensitize tumor cells to anti-CD20 mAbs. Significance: The combination of FTIs with anti-CD20 mAbs seems to be a reasonable therapeutic approach worth to be tested in patients with B-cell tumors. Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.

SK053 triggers tumor cells apoptosis by oxidative stress-mediated endoplasmic reticulum stress

Thioredoxins (Trx) together with thioredoxin reductases (TrxR) participate in the maintenance of protein thiol homeostasis and play cytoprotective roles in tumor cells. Therefore, thioredoxin-thioredoxin reductase system is considered to be a promising therapeutic target in cancer treatment. We have previously reported that SK053, a peptidomimetic compound targeting the thioredoxin-thioredoxin reductase system, induces oxidative stress and demonstrates antitumor activity in mice. In this study, we investigated the mechanisms of SK053-mediated tumor cell death. Our results indicate that SK053 induces apoptosis of Raji cells accompanied by the activation of the endoplasmic reticulum (ER) stress and induction of unfolded protein response. Incubation of tumor cells with SK053 induces increase in BiP, CHOP, and spliced XBP-1 levels, which precede induction of apoptosis. CHOP-deficient (CHOP(-/-)) mouse embryonic fibroblasts are more resistant to SK053-induced apoptosis as compared with normal fibroblasts indicating that the apoptosis of tumor cells depends on the expression of this transcription factor. Additionally, the ER-stress-induced apoptosis, caused by SK053, is strongly related with Trx expression levels. Altogether, our results indicate that SK053 induces ER stress-associated apoptosis and reveal a link between thioredoxin inhibition and induction of UPR in tumor cells.

Adenanthin, a new inhibitor of thiol‐dependent antioxidant enzymes, impairs the effector functions of human natural killer cells

Natural killer (NK) cells are considered critical components of the innate and adaptive immune responses. Deficiencies in NK cell activity are common, such as those that occur in cancer patients, and they can be responsible for dysfunctional immune surveillance. Persistent oxidative stress is intrinsic to many malignant tumours, and numerous studies have focused on the effects of reactive oxygen species on the anti‐tumour activity of NK cells. Indeed, investigations in animal models have suggested that one of the most important thiol‐dependent antioxidant enzymes, peroxiredoxin 1 (PRDX1), is essential for NK cell function. In this work, our analysis of the transcriptomic expression pattern of antioxidant enzymes in human NK cells has identified PRDX1 as the most prominently induced transcript out of the 18 transcripts evaluated in activated NK cells. The change in PRDX1 expression was followed by increased expression of two other enzymes from the PRDX‐related antioxidant chain: thioredoxin and thioredoxin reductase. To study the role of thiol‐dependent antioxidants in more detail, we applied a novel compound, adenanthin, to induce an abrupt dysfunction of the PRDX‐related antioxidant chain in NK cells. In human primary NK cells, we observed profound alterations in spontaneous and antibody‐dependent NK cell cytotoxicity against cancer cells, impaired degranulation, and a decreased expression of activation markers under these conditions. Collectively, our study pinpoints the unique role for the antioxidant activity of the PRDX‐related enzymatic chain in human NK cell functions. Further understanding this phenomenon will prospectively lead to fine‐tuning of the novel NK‐targeted therapeutic approaches to human disease.

Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy

BackgroundAccumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT.MethodsIn vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method.ResultsPhotofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins.ConclusionsAltogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy.