Intracellular safeguarding functions of metallothioneins (MTs) include sequestering transition and heavy metals, scavenging free radicals and protecting against electrophiles. We report that MT protection against Cu-induced cytotoxicity can be reversed and pro-oxidant and pro-apoptotic effects can be induced in HL-60 cells exposed to NO. We demonstrate that in ZnCl2-pretreated HL-60 cells loaded with copper nitrilotriacetate (Cu-NTA), exposure to an NO donor, S-nitroso-N-acetyl penicillamine, resulted in S-nitrosylation and oxidation of MT cysteines. This disruption of MT Cu-binding thiolate clusters caused loosening and release of redox-active Cu, enhanced redox-cycling activity of Cu and increased peroxidation of major classes of membrane phospholipids. We also found that Cu-induced oxidative stress in ZnCl2-pretreated/Cu-NTA-loaded HL-60 cells was accompanied by apoptosis documented by characteristic changes of nuclear morphology, internucleosomal DNA cleavage, externalization of phosphatidylserine, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. We conclude that in Cu-challenged cells, NO can reverse the protective role of MTs and convert them into pro-oxidant, pro-apoptotic implements.
In the report, we reflected our experience of minimally invasive percutaneous nephrolithotomy (Mini PCNL) in the treatment of staghorn kidney calculi. Our experience demonstrated that Mini PCNL by two-wavelength laser lithotripter with microsecond pulse duration and second harmonic generation is effective and safe procedure in treatment of staghorn nephrolithiasis.
The critical role of mitochondria in programmed cell death leads to the design of mitochondriotropic agents as a strategy in regulating apoptosis. For anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells and their suppression in surrounding normal cells represents a promising paradigm for new therapies. Different approaches targeting regulation of components of mitochondrial antioxidant system such as Mn-SOD demonstrated significant antitumor efficiency, particularly in combination therapy. This review is focused on a newly discovered early stage of mitochondria-dependent apoptosis - oxidative lipid signaling involving a mitochondria-specific phospholipid cardiolipin (CL). Cytochrome c (cyt c) acts as a CL-specific peroxidase very early in apoptosis. At this stage, the hostile events are still secluded within the mitochondria and do not reach the cytosolic targets. CL oxidation process is required for the release of pro-apoptotic factors into the cytosol. Manipulation of cyt c interactions with CL, inhibition of peroxidase activity, and prevention of CL peroxidation are prime targets for the discovery of anti-apoptotic drugs acting before the "point-of-no-return" in the fulfillment of the cell death program. Therefore, mitochondria-targeted disruptors and inhibitors of cyt c/CL peroxidase complexes and suppression of CL peroxidation represent new strategies in anti-apoptotic drug discovery.
Abstract Cyclo‐oxygenase‐2 (COX‐2) is believed to induce neuronal oxidative stress via production of radicals. While oxygen radicals are not directly involved in COX‐2‐catalytic cycle, superoxide anion radicals have been repeatedly reported to play a critical role in COX‐2‐associated oxidative stress. To resolve the controversy, we characterized production of free radicals in PC12 cells in which COX‐2 expression was manipulated either genetically or by direct protein transfection and compared them with those generated by a recombinant COX‐2 in a cell‐free system. Using spin‐traps α‐(4‐pyridyl‐1‐oxide)‐ N ‐t‐butylnitrone, 5,5‐dimethyl‐1‐pyrroline‐ N ‐oxide and 4‐((9‐acridinecarbonyl) amino)‐2,2,6,6‐ tetramethylpiperidine‐1‐oxyl (Ac‐Tempo), we observed arachidonic acid (AA)‐dependent production of carbon‐centered radicals by heme‐reconstituted recombinant COX‐2. No oxygen radicals or thiyl radicals have been detected. COX‐2 also catalyzed AA‐dependent one‐electron co‐oxidation of ascorbate to ascorbate radicals. Next, we used two different approaches of COX‐2 expression in cells, PCXII cells which express isopropyl‐1‐thio‐β‐ d ‐galactopyranoside inducible COX‐2, and PC12 cells transfected with COX‐2 using a protein delivery reagent, Chariot. In both models, COX‐2‐dependent AA‐induced generation of carbon‐centered radicals was documented using spin‐traps and Ac‐Tempo. No oxygen radical formation was detected in COX‐2‐transfected cells by either spin‐traps or fluorogenic probe, dihydroethidium. In the presence of ascorbate, AA‐induced COX‐2‐dependent ascorbate radicals were detected. AA caused a significant and selective oxidation of one of the major phospholipids, phosphatidylserine (PS). PS was not a direct substrate for COX‐2 but was co‐oxidized in the presence of AA. The radical generation and PS oxidation were inhibited by COX‐2 inhibitors, niflumic acid, nimesulide, or NS‐398. Thus, COX‐2 generated carbon‐centered radicals but not oxygen radicals or thiyl radicals are responsible for oxidative stress in AA‐challenged PC12 cells overexpressing COX‐2.
Between 2009 and 2012, we examined and treated 38 patients with bilateral kidney stones. In all 38 patients, stones were removed by percutaneous nephrolithotripsy (PCNL). Out of 38 patients with bilateral staghorn kidney stones, 26 patients (68.5%) had separate surgeries on either kidney, 9 patients (23.7%) had one-step surgery, and 3 patients (7.8%) had PCNL on one kidney and percutaneous nephrostomy (PCN) on the other. Out of 9 patients who had one-step surgery, PCNL allowed for a complete removal of stones from both kidneys in 4 patients and a removal of stones from one kidney in 3 patients. The other 5 patients had bilateral residual kidney stones after PCNL. For these 5 patients, the second step of treatment included stone extraction, which was done through a fistula and allowed for a complete removal of calculi in 1 patient. For the other 4 patients, stone extraction through a fistula was repeated, allowing for a complete removal of stones in 2 patients. Out of 26 patients with bilateral staghorn kidney stones who had a separate stone-removal surgery on either kidney, 9 patients had all stones removed from one kidney after PCNL, in seven patients the kidney was stone-free when stones were extracted through a fistula. As for surgery for the second kidney, PCNL allowed for a complete removal of stones in 10 patients, and extraction of stones through a fistula in 12 patients. When indicated, one-step bilateral nephrolithotripsy is an effective treatment option for patients with bilateral staghorn kidney stones.
Objective. To test the hypothesis that enhanced oxidative stress during pregnancies complicated by preeclampsia is associated with improper copper (Cu) binding by plasma albumin, resulting in enhanced Cu redox-cycling activity and that altered Cu binding, in turn, is caused by interactions of excessive amounts of free fatty acids with albumin.Study Design. We studied binding and redox-cycling activity of Cu in 17 normal pregnancy and 17 preeclampsia plasma samples. Binding of exogenous Cu in plasma samples was quantified indirectly using spectrophotometric measurements of its complex with a specific chelator of Cu(I), bathocuproine disulfonate. Redox-cycling activity of Cu in plasma samples was estimated by electron paramagnetic resonance (EPR) spectroscopy of ascorbate radicals formed during one-electron oxidation of ascorbate by redox-active catalytic Cu. Formation of Cu/albumin complexes in model systems in the presence and absence of fatty acids was studied using EPR spectroscopy of Cu(II)/albumin.Results. We found that preeclampsia plasma (as compared to normal pregnancy plasma) (1) displays elevated endogenous ascorbate redox-cycling that is normalized by a Cu(II) chelator, cuprizone I, (2) has lowered capacity to bind and redox-regulate exogenously added Cu, and (3) responds to treatment with fatty-acid-free albumin by diminished ascorbate oxidizing activity. Conversely, addition of free fatty acid (oleic acid) to normal pregnancy plasma sample yields increased ascorbate redox-cycling activity. Our model experiments showed that Cu-dependent redox-cycling activity of purified human serum albumin is significantly increased by excess free fatty acids.Conclusion. Mishandling of Cu by albumin contributes to oxidative stress in preeclampsia. Cu chelators may represent promising mechanism-based antioxidants to attenuate oxidative stress in preeclampsia.
Abstract In recent years, chimeric antigen receptor (CAR) T cell therapy has emerged as a promising approach for treating various malignancies, most notably in hematologic cancers. However, its efficacy in solid tumors is often hindered by an immunosuppressive tumor microenvironment (TME), which, in particular, is frequently characterized by the presence of tumor-associated cell-free DNA (cfDNA), especially in the form of neutrophil extracellular traps (NETs). In adoptive immunotherapies NETs, composed primarily of neutrophil-derived DNA, histones and neutrophil granule enzymes (e.g., MPO, NE), act as physical barriers and induce the secretion of immunosuppressive factors that impair CAR T cell function. This study investigates the potential of co-administration of deoxyribonuclease I (DNase I) with CAR T cells in a syngeneic B16 melanoma murine model of lung metastasis. Bioluminescent imaging of melanoma metastatic processes has shown that a single injection of DNase I (10 mg/kg) suppresses B16-EGFR lung metastasis at early stages in comparison to the vehicle control group but does not improve survival. However, co-administration of DNase I with murine EGFR-CAR T cells significantly suppresses tumor burden, decreases the number of metastatic foci, and substantially prolongs survival compared to the CAR T cell monotherapy group. Degrading of NETs [RB1] by DNase I increases the amount of tumor-infiltrating T and CAR T cells and reduces the immunosuppressive effects of the TME. This is reflected in the CD8 population of tumor-infiltrating CAR T cells from the DNase I treated group, which have lower expression of PD-1 and TIM-3 exhaustion markers. This research highlights the critical role of the NETs in modulating CAR T cell efficacy and provides a compelling rationale for incorporating DNase I as an adjunctive treatment to improve therapeutic responses in patients undergoing CAR T cell therapy. However, further clinical studies are warranted to validate these findings and explore the translational potential of this combinatorial approach in enhancing cancer treatment. [RB1]Again - this takes the emphasis and the reader away from NETs and into the cfDNA universe. Citation Format: Alexey Stepanov, Wenjian Wang, Yingqin Hou, Ivan Chernikov Ivan Chernikov, Reid Bissonnette, George Tetz, Grigory Borisenko, Dmitry Genkin. Neutrophil extracellular traps-targeting DNase I enhances the therapeutic efficacy of CAR-T cell adoptive immunotherapy in a syngeneic murine metastasis model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B048.
