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

Toxicity of oxidized phospholipids in cultured macrophages

Background: The interactions of oxidized low-density lipoprotein (LDL) and macrophages are hallmarks in the development of atherosclerosis. The biological activities of the modified particle in these cells are due to the content of lipid oxidation products and apolipoprotein modification by oxidized phospholipids. Results: It was the aim of this study to determine the role of short-chain oxidized phospholipids as components of modified LDL in cultured macrophages. For this purpose we investigated the effects of the following oxidized phospholipids on cell viability and apoptosis: 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and oxidized alkylacyl phospholipids including 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine (E-PGPC) and 1-O-hexadecyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (E-POVPC). We found that these compounds induced apoptosis in RAW264.7 and bone marrow-derived macrophages. The sn-2 carboxyacyl lipid PGPC was more toxic than POVPC which carries a reactive aldehyde function in position sn-2 of glycerol. The alkylacyl phospholipids (E-PGPC and E-POVPC) and the respective diacyl analogs show similar activities. Apoptosis induced by POVPC and its alkylether derivative could be causally linked to the fast activation of an acid sphingomyelinase, generating the apoptotic second messenger ceramide. In contrast, PGPC and its ether analog only negligibly affected this enzyme pointing to an entirely different mechanism of lipid toxicity. The higher toxicity of PGPC is underscored by more efficient membrane blebbing from apoptotic cells. In addition, the protein pattern of PGPC-induced microparticles is different from the vesicles generated by POPVC. Conclusions: In summary, our data reveal that oxidized phospholipids induce apoptosis in cultured macrophages. The mechanism of lipid toxicity, however, largely depends on the structural features of the oxidized sn-2 chain

A kisalföldi facéliavetésekben alkalmazott gazdálkodási módszerek felmérése

A kisalföldi régióban jelentős a facélia (Phacelia tanacetifolia Benth.) vetőmagtermesztése. Kutatásunk során a facéliavetésekben az utóbbi három évben alkalmazott gazdálkodási módszereket mértük fel online kérdőív segítségével, 50 gazdálkodó bevonásával. Tanulmányunk feltárta, hogy a megkérdezett gazdaságok jelentős részében a vetésforgó kedvelt eleme ez a kultúrnövény, melyet döntően kalászosok után vetettek, leggyakrabban március 10 és 20 között, 8-10 kg/ha vetőmag felhasználásával, gabona-sortávolságra. A legkedveltebb fajták a Lilla és az Angelia voltak. A növénytáplálásban az NPK műtrágyák mellett a bór-tartalmú lombtrágyák voltak népszerűek. A megkérdezett gazdaságok mindegyikében a kétmenetes betakarítást alkalmazták. A gazdaságok 60%-a alkalmazott vegyszeres gyomirtást, de csak 35%-uk volt megelégedve az engedélyezett gyomirtó szerek hatékonyságával. A termesztők többsége (~70%) szerint megnehezíti a növényvédelmet a linuron herbicid-hatóanyag kivonása, mindazonáltal hasonló arányban úgy vélik, hogy a gyomfésű kiválthatja a vegyszeres gyomirtást a facéliában. Eredményeink azt sugallják, hogy a gazdák nyitottak a teljesen vegyszermentes facélia termesztés-technológia bevezetése iránt, ami egy környezetkímélő megoldás lehet a kultúrnövény gyomszabályozásában

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

The guanine-quadruplex structure in the human c-myc gene's promoter is converted into B-DNA form by the human poly(ADP-ribose)polymerase-1.

The important regulatory role of the guanine-quadruplex (GQ) structure, present in the nuclease hypersensitive element (NHE) III(1) region of the human c-myc (h c-myc) gene's promoter, in the regulation of the transcription of that gene has been documented. Here we present evidences, that the human nuclear poly(ADP-ribose)polymerase-1 (h PARP-1) protein participates in the regulation of the h c-myc gene expression through its interaction with this GQ structure, characterized by binding assays, fluorescence energy transfer (FRET) experiments and by affinity pull-down experiments in vitro, and by chromatin immunoprecipitation (ChIP)-qPCR analysis and h c-myc-promoter-luciferase reporter determinations in vivo. We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form. The first Zn-finger structure present in h PARP-1 participates in this interaction. PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes

Complex regulation of autophagy in cancer - integrated approaches to discover the networks that hold a double-edged sword.

Autophagy, a highly regulated self-degradation process of eukaryotic cells, is a context-dependent tumor-suppressing mechanism that can also promote tumor cell survival upon stress and treatment resistance. Because of this ambiguity, autophagy is considered as a double-edged sword in oncology, making anti-cancer therapeutic approaches highly challenging. In this review, we present how systems-level knowledge on autophagy regulation can help to develop new strategies and efficiently select novel anti-cancer drug targets. We focus on the protein interactors and transcriptional/post-transcriptional regulators of autophagy as the protein and regulatory networks significantly influence the activity of core autophagy proteins during tumor progression. We list several network resources to identify interactors and regulators of autophagy proteins. As in silico analysis of such networks often necessitates experimental validation, we briefly summarize tractable model organisms to examine the role of autophagy in cancer. We also discuss fluorescence techniques for high-throughput monitoring of autophagy in humans. Finally, the challenges of pharmacological modulation of autophagy are reviewed. We suggest network-based concepts to overcome these difficulties. We point out that a context-dependent modulation of autophagy would be favored in anti-cancer therapy, where autophagy is stimulated in normal cells, while inhibited only in stressed cancer cells. To achieve this goal, we introduce the concept of regulo-network drugs targeting specific transcription factors or miRNA families identified with network analysis. The effect of regulo-network drugs propagates indirectly through transcriptional or post-transcriptional regulation of autophagy proteins, and, as a multi-directional intervention tool, they can both activate and inhibit specific proteins in the same time. The future identification and validation of such regulo-network drug targets may serve as novel intervention points, where autophagy can be effectively modulated in cancer therapy