Magdalena Gabrysiak

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.

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.

Investigation of cell death mechanisms in human lymphatic endothelial cells undergoing photodynamic therapy

BACKGROUND Photodynamic therapy (PDT) has been shown to induce ablation and functional occlusion of tumor-associated lymphatic vessels. However, direct effects of PDT on lymphatic endothelial cells (LECs) have not been studied so far. The aim of this study was to elucidate molecular mechanisms of cell death induced by PDT in human LECs. METHODS Verteporfin was used as a photosensitizer to investigate PDT-mediated damage of lymphatic vessels in mice using immunofluorescent staining and stereomicroscopy. In vitro dose-response studies were carried-out with crystal violet staining. Immunofluorescence, flow cytometry, immunoblotting and DNA electrophoresis were used to investigate the mechanisms of cell death in human LECs undergoing PDT. RESULTS PDT induced an increase in the number of propidium iodide positive lymphatic endothelial cells in the mouse dermis. In in vitro studies dose-dependent cytotoxic effects of PDT towards LECs were observed. Typical hallmarks of apoptotic cell death, including Annexin V binding, loss of mitochondrial membrane potential, caspase activation, cleavage of PARP as well as DNA fragmentation were observed in LECs when PDT was used at high irradiation conditions, causing >80% cell death. At lower light fluencies causing <50% cell death PDT induced autophagy rather than apoptosis, as revealed by conversion of LC3-I to the autophagosomal LC3-II and formation of LC3 puncta. Z-VAD-FMK, a caspase inhibitor, prevented cell death induced by high-dose PDT only, while 3-methyladenine, an autophagy suppressor, inhibited cell death induced by low-dose PDT. CONCLUSIONS Both apoptosis and autophagy are involved in cell death induced by verteporfin-PDT in LECs.

Epigenetic remodeling combined with photodynamic therapy elicits anticancer immune responses

Photodynamic therapy has been shown to induce strong immunity against tumor cells expressing exogenous tumor-associated antigens (TAAs), including P1A antigen. Cancer cells can evade the immune system by epigenetic silencing of TAAs, while DNA methyltransferase inhibitors, such as 5-aza-2’-deoxycytidine (5-aza-dC) can restore the expression of silenced or downregulated TAA. Thus, epigenetic remodeling with 5-aza-dC combined with PDT can elicit robust and durable antitumor immunity.

HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

Low dose of GRP78-targeting subtilase cytotoxin improves the efficacy of photodynamic therapy in vivo

Photodynamic therapy (PDT) exerts direct cytotoxic effects on tumor cells, destroys tumor blood and lymphatic vessels and induces local inflammation. Although PDT triggers the release of immunogenic antigens from tumor cells, the degree of immune stimulation is regimen-dependent. The highest immunogenicity is achieved at sub-lethal doses, which at the same time trigger cytoprotective responses, that include increased expression of glucose-regulated protein 78 (GRP78). To mitigate the cytoprotective effects of GRP78 and preserve the immunoregulatory activity of PDT, we investigated the in vivo efficacy of PDT in combination with EGF-SubA cytotoxin that was shown to potentiate in vitro PDT cytotoxicity by inactivating GRP78. Treatment of immunocompetent BALB/c mice with EGF-SubA improved the efficacy of PDT but only when mice were treated with a dose of EGF-SubA that exerted less pronounced effects on the number of T and B lymphocytes as well as dendritic cells in mouse spleens. The observed antitumor effects were critically dependent on CD8+ T cells and were completely abrogated in immunodeficient SCID mice. All these results suggest that GRP78 targeting improves in vivo PDT efficacy provided intact T-cell immune system.

Selection of an optimal promoter for gene transfer in normal B cells

Gene transfer into normal quiescent human B cells is a challenging procedure. The present study aimed to investigate whether it is possible to increase the levels of transgene expression by using various types of promoters to drive the expression of selected genes-of-interest. To produce lentiviral particles, the present study used the 2nd generation psPAX2 packaging vector and the vesicular stomatitis virus -expressing envelope vector pMD2.G. Subsequently, lentiviral vectors were generated containing various promoters, including cytomegalovirus (CMV), elongation factor-1 alpha (EF1α) and spleen focus-forming virus (SFFV). The present study was unable to induce satisfactory transduction efficiency in quiescent normal B cells; however, infection of normal B cells with Epstein-Barr virus resulted in increased susceptibility to lentiviral transduction. In addition, the SFFV promoter resulted in a higher level of transgene expression compared with CMV or EF1α promoters. As a proof-of concept that this approach allows for stable gene expression in normal B cells, the present study used bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins, as well as X-box binding protein-1 and binding immunoglobulin protein.

GRP78-targeting Sensitizes Cancer Cells to Cytotoxic Effects of Photodynamic Therapy

Photodynamic therapy (PDT) induces cytotoxic effects against tumor cells by triggering photochemical reactions leading to the production of singlet oxygen and reactive oxygen species. Intracellular proteins have been shown to undergo oxidation-related damage in response to PDT. A number of cytoprotective mechanisms have been demonstrated to relay on mechanisms associated with removal or re-folding of these proteins or leading to the induction of unfolded protein response. The latter is regulated by GRP78, a member of the heat shock protein family that undergoes up-regulation in tumor cells in response to PDT. The most selective GRP78-targeting compound is subtilase cytotoxin (SubAB) originally isolated from Shiga toxigenic Escherichia coli strains. We observed that a fusion protein consisting of the cytotoxin catalytic A subunit (SubA) with a human epidermal growth factor (EGF) designed to selectively target EGFR-positive tumor cells increases the cytotoxic effects of PDT. Although the combination treatment activated apoptotic pathways, tumor cell death occurred in cells resistant to apoptosis and was not inhibited by inhibitors of necrotic cell death or autophagy-associated death pathways. Instead, tumor cells undergo an atypical form of cell death, which is characterized by cellular vacuolization originating from the endoplasmic reticulum.