Alternatív sejtelhalási mechanizmusok, mint a tumor ellenes terápia célpontjai = Alternative mechanisms of the cell death as targets for anti-tumor therapy

A daganatellenes kemoterápiában alakalmazott vegyületek egyik fontos hatása az, hogy daganatsejtekben aktív sejtelhalást váltanak ki. Az alternatív sejtelhalási típusok közül legismeretebb az apoptotikus és a nekrotikus forma, amelyeknek eltérő immunológiai következménye is vélelmezhető. Az apoptotikus jelpálya centrális elemei a kaszpáz proteázok. Azonban kaszpázaktivitás hiányában is (génexpressziós eltérések, oxidatív környezet) indukálódhat aktív sejtelhalás és fordítva: a kaszpázaktivitás nem jár együtt mindig sejtelhalással. Kutatásaink célja az volt, hogy olyan, elsősorban proteolitikus aktivitásokat azonosítsunk daganatsejtekben, amelyek helyettesíthetik a kaszpázok hiányát, illetve kaszpáz aktiválódás mellett is befolyásolják a sejtelhalást. Távlati célunk az, hogy ezeket a proteolitikus aktivitásokat megcélozva hatékonyabb daganatellenes terápiák lehetőségét alapozzunk meg. Eredményeink azt mutatják, hogy a cisztein katepszinek nem helyettesítik a kaszpázok apoptótikus funkcióját az általunk kidolgozott leukémia modellekben, de befolyásolhatják a kialakuló sejtelhalás típusát (apoptózis-nekrózis váltás). A proteaszóma aktivitás pro- és antiapoptotikus hatású is lehet kaszpáz-mediálta sejtelhaláskor függően az indukáló szerektől és sejttípustól. Kidolgoztunk egy új eljárást nekrotikus sejtek detektálására áramlásos citométerrel. | Induction of active cell death is the main effect of therapeutic anticancer drugs. The most known alternative cell death forms are apoptosis and necrosis that have also alternative immunological consequences. Caspase proteases are the central elements of the apoptotic pathway. However, in the absence of caspase activity (change in gene expression, oxidative environment) cell death can proceed and vica versa: caspase activation is not always accompanied with cell death. The purpose of our project was to identify proteolytic activities that may replace caspases in apoptosis or modulate the outcome of apoptotic process in the presence of caspase activation in cancer cells. Taking a long view, our aim is to target these proteolytic activities to improve the anticancer activities of therapeutic drugs. Our resuls demonstrated that cystein cathepsins do not substitute caspase activity in our leukemic models, but influence the emerging mode of cell death (apoptosis-necrosis switch). Proteasome activity can promote both pro- and anti-apoptotic effects in caspase-mediated cell death depending on inducer drugs and cell types. We have developed a new method to detect necrotic cells by flow cytometry

Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells

For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme

A halálreceptor szignálutak működésének változásai a malignus transzformáció során. = Changes in death receptor signaling during malignant transformation

Kutató munkánk során a terveknek megfelelően vizsgáltuk a daganatsejtek aktív sejthalálát szabályozó jelátviteli utak működését. Vastagbélrák sejtekben kimutattuk, hogy a TRAIL halálligand által kiváltott apoptózis szabályozásában elsősorban az IAP (Inhibitor of Apoptózis) fehérjék, elsősorban a XIAP játszik szerepet. A XIAP gátlása közvetlenül siRNA-vel, vagy a SMAC/DIABLO mitokondriumból történő felszabadulását indukáló hatóanyagokkal illetve SMAC/DIABLO oligopeptidekkel fokozhatja a vastagbélráksejtek TRAIL érzékenységét. A rhabdomyoszarkómák esetében kimutattuk, hogy proteasoma gátlók a DR5 halálreceptor expressziójának és aggregációjának növelésével és a Bcl-2 expressziójának gátlásával fokozzák a TRAIL hatékonyságát. Tüdőrákokban kimutattuk, hogy az EGFR (Epidermális Növekedési Faktor Receptor) gátlókra rezisztens tüdődaganatok esetében a TRAIL kezelés hatékony lehet a jövőben. A mellékpajzsmirigy daganatok esetében a kutatási terv eredeti hipotézisével összhangban azt találtuk, hogy a transzformáció során a pro-apoptotikus gének (pl. TRAIL) expressziója emelkedik, azonban ezt az antiapoptotikus (pl. IAP) gének expressziója kompenzálhatja. Nemzetközi kollaborációban kimutattuk, hogy a FAS halálreceptor expressziójának szabályozásában szerepe lehet egy specifikus CpG sziget hypermetilációjának hólyagrákokban. Kutatásaink tehát számos lehetséges terápiás célpontot azonosítottak a halálreceptorok jelátviteli mechanizmusában. | During our research project we have studied several aspects of the signal transduction pathways regulating the active cell death in tumor cells. In colon cancer, we identified the IAP family of proteins (Inhibitor of Apoptosis Proteins), in particular XIAP as the main regulator of TRAIL sensitivity. Silencing of XIAP expression or indirect inhibition by the drug induced release of the SMAC/DIABLO peptide from the mitochondria or by SMAC/DIABLO mimetic oligopeptide restored TRAIL sensitivity. In rhabdomyosarcomas, we found the upregulation and increased aggregation of DR5 death receptor and downregulation of Bcl-2 as the main mechanism of synergism between TRAIL and proteasome inhibitors. In lung cancer cells we found that cells resistant to EGFR (Epidermal Growth Factor Receptor) inhibitors are often sensitive to TRAIL. In the malignancies of the parathyroid gland we found an upregulation of proapoptotic proteins (E.g. TRAIL) which is compensated by the upregulation of anti-apoptotic proteins (e.g. IAP). In international cooperation we found a single CpG site of which hypermethylation can regulate the expression of FAS death receptor in bladder cancer. This research project has identified several potential anti-cancer therapeutic targets in the signal transduction pathways of death receptors

Heparin can liberate high molecular weight DNA from secondary necrotic cells

The border-line between necrosis and apoptosis is not sharp, but the distinction between types of cell death is important, because necrosis may lead to local inflammation, while apoptosis usually does not. In certain autoimmune disorders, the inhibition of cell death is crucial, since macromolecules released from dead cells, may accelerate the autoimmune processes. In our study we used various cell death inhibitors to block N-(4-hydroxyphenil)-retinamide induced cell death in BL41 and U937 cell lines. VD-fmk, a general caspase inhibitor, inhibited DNA fragmentation induced by N-(4-hydroxyphenil)-retinamide, but not propidium-iodide uptake and necrosis. Interestingly heparin, a serine-protease inhibitor, lowered the propidium-iodide fluorescence of the dead cell population and increased the subG1 population measured by flow cytometry. We investigated the cause of these changes and found that heparin did not actually increase DNA fragmentation, it merely liberated high molecular weight DNA fragments from the dead cells. The exact mechanism is unclear, but we believe, that during secondary necrosis, heparin can enter the cells, bind ribonucleoproteins, and pull them out of the cells with the attached DNA where they are sensitive to enzymatic degradation. Our results suggest that heparin treatment may help the clearance of cell debris and decreases the immunogenity of secondary necrotic cells

CA inhibits either the TRAIL or STS-induced necroptosis in presence of caspase inhibitor.

<p>U937 cells were treated either with STS (1 µM) or with TRAIL (50 ng/mL) in the presence or absence of zVD (5 µM) for 20 hrs. Nec (10 µM) or CA (10 µM) were added 1 hr before cell death was induced. (A-B) CA (10 µM) considerably inhibited the (A) TRAIL (n = 4) or (B) STS-triggered necroptosis (n = 3). Percentage of PI positive cells was determined. (C) Time course analysis of cells with depolarized mitochondria is shown after DiOC₆(3) staining of, unfixed cells for STS treatment combined with the indicated inhibitors (n = 2). Values are mean±SD. *, P<0.05, **, P<0.01 and ***, P<0.001 calculated by Student’s t-probe. (D) PS distribution in the plasma membrane is shown in representative dot plots of Annexin V-FITC and PI stained, unfixed cells analyzed by flow cytometry. The values indicate the percentage of cells in the marked regions (n = 2). (E) Morphological signs of apoptosis and necrosis are shown in representative fluorescent microscopic images (400x) of Hoechst/PI double stained U937 cells (n = 2). Scale bar on the first subfigure applies to all the figures in the panel. (F) Distribution of cells with various volumes of acidic compartments (endo-lysosomes) is shown in representative overlay histograms of AO stained cells analyzed by flow cytometry. (G) Column diagram of percentage of cells with high AO red fluorescence intensity (n = 2). (H) Distribution of cells with various mitochondrial transmembrane potential is shown in representative overlay histograms of DiOC₆(3) stained cells analyzed by flow cytometry. (I) Column diagram of percentage of cells with high DiOC₆(3) fluorescence intensity (n = 3). Values are mean±SD. *, P<0.05, **, P<0.01 and ***, P<0.001 calculated by Student’s t-probe.</p

Schematic diagram about the action of inhibitors on TRAIL and STS- induced cell death pathways.

<p>Both TRAIL and STS triggered necroptosis in caspase depleted U937 cells upon prolonged incubation time. RIPK1 inhibitors Nec and GA could completely inhibit the TRAIL-evoked necroptosis, but had partial inhibitory potential to the STS-provoked process. Contrarily CA and NSA abolished both the TRAIL and STS-induced necroptosis. In absence of caspase inhibitor TRAIL and STS induced apoptosis which is followed by secondary necrosis. PJ-34 delayed the necrotic plasma membrane disruption during secondary necrosis, but failed to inhibit necroptosis.</p

STS induces primary necrosis in the presence of caspase inhibitor.

<p>(A-B) Nec (10 µM) and (C) GA (1 µM) significantly inhibited the STS-triggered necroptosis. Cells were exposed to STS (1 µM) in the presence or absence of zVD (5 µM) for 12 hrs. Percentage of PI positive cells was determined by flow cytometry (A, C). (n = 4) and by Hoechst/PI double staining technique (B) (representative of n = 2), (400x). Scale bar on the first subfigure applies to all the figures in the panel. (D) Nec (10 µM) arrested the STS-induced (1 µM) necroptosis in the presence of zVD (5 µM) after 20 hrs incubation. The mitochondrial transmembrane potential and plasma membrane integrity is shown in representative dot plots of DiOC₆(3) and PI stained, unfixed cells. The values indicate the percentage of cells in the marked regions (n = 13). (E-G) Nec (10 µM) (E, F) and GA (1 µM) (G) partially inhibited the STS-triggered necroptosis. Cells were exposed to STS (1 µM) in the presence or absence of zVD (5 µM) for 20 hrs. Percentage of PI positive cells was determined by flow cytometric analysis (E, G) (n = 13 for Nec and n = 4 for GA), and by Hoechst/PI double staining technique (F) (n = 2). Scale bar on the first subfigure applies to all the figures in the panel. Values are mean±SD. *, P<0.05, **, P<0.01 and ***, P<0.001 calculated by Student’s t-probe.</p

PJ3-4 does not arrest either the TRAIL or STS-induced necroptosis in presence of caspase inhibitor.

<p>U937 cells were treated either with STS (1 µM) or with TRAIL (50 ng/mL) in the presence or absence of zVD (5 µM) for 20 hrs. PJ-34 (1 µM) was added 1 hr before cell death was induced. (A) Column diagram of percentage of cells with high DiOC₆(3) fluorescence intensity (n = 3). (B-C) PJ-34 (1 µM) considerably inhibited the (B) STS (n = 8) or (C) TRAIL-triggered (n = 8) secondary necrosis but not the necroptosis. Percentage of PI positive cells was determined. Values are mean±SD. *, P<0.05, **, P<0.01 and ***, P<0.001 calculated by Student’s t-probe. (D) Column diagram of percentage of cells with high AO fluorescence intensity (n = 2). (E) Dot plot distribution of cells stained with Annexin V-FITC and PI analyzed by flow cytometry. The values indicate the percentage of cells in the marked regions (representative of n = 2). (F) Representative histograms of distribution of PI stained, ethanol-fixed (sub-G1) cells were analyzed by flow cytometry (n = 8). The numbers indicate the percentage of cells in the marked regions. (G) PJ-34 concentration dependently reduced the proportion of PI positive cells for STS treatment for 20 hrs – representative experiment.</p