Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma.

A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments. Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis. In neuroblastic SH-SY5Y cells, we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation. Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment. Results were confirmed by mitochondrial mass analysis, electron microscopy and co-localization of mitochondria with GFP-LC3, suggesting the impaired clearance of damaged mitochondria. Thus, a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis. Inhibition of autophagy with small inhibitory RNAs against ATG5, ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis. In contrast, autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells: here reactive oxygen species (ROS) generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment. Moreover, autophagy inhibition determined sensitization of SK-N-AS to apoptosis. We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450, triggering rather necroptosis at higher doses. Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches.

Early downregulation of Mcl-1 regulates apoptosis triggered by cardiac glycoside UNBS1450.

Cardiac glycosides (CGs), prescribed to treat cardiovascular alterations, display potent anti-cancer activities. Despite their well-established target, the sodium/potassium (Na(+)/K(+))-ATPase, downstream mechanisms remain poorly elucidated. UNBS1450 is a hemi-synthetic cardenolide derived from 2″-oxovorusharin extracted from the plant Calotropis procera, which is effective against various cancer cell types with an excellent differential toxicity. By comparing adherent and non-adherent cancer cell types, we validated Mcl-1 as a general and early target of UNBS1450. A panel of CGs including cardenolides ouabain, digitoxin and digoxin as well as bufadienolides cinobufagin and proscillaridin A allowed us to generalize our findings. Our results show that Mcl-1, but not Bcl-xL nor Bcl-2, is rapidly downregulated prior to induction of apoptosis. From a mechanistic point of view, we exclude an effect on transcription and demonstrate involvement of a pathway affecting protein stability and requiring the proteasome in the early CG-induced Mcl-1 downregulation, without the involvement of caspases or the BH3-only protein NOXA. Strategies aiming at preventing UNBS1450-induced Mcl-1 downregulation by overexpression of a mutated, non-ubiquitinable form of the protein or the use of the proteasome inhibitor MG132 inhibited the compound's ability to induce apoptosis. Altogether our results point at Mcl-1 as a ubiquitous factor, downregulated by CGs, whose modulation is essential to achieve cell death.

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid.

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10(-9) vs. 10(-5)M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10(-5)M, but drops to 10(-9)M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

Cardiac glycosides in cancer therapy: from preclinical investigations towards clinical trials.

Cardiac glycosides have a long history in the treatment of cardiac disease. However, several preclinical studies as well as two phase I studies have shown that cardenolides may also possess anticancer effects. The mechanisms of these anticancer effects may include intracellular decrease of K(+) and increase of Na(+) and Ca(2+); intracellular acidification; inhibition of IL-8 production and of the TNF-α/NF-κB pathway; inhibition of DNA topoisomerase II and activation of the Src kinase pathway. To date three cardiac glycosides have been developed for treatment of cancer and were tested in a phase 1 clinical trial to determine dose limiting toxicities and maximum tolerated dose. Future studies of this novel class of anticancer drugs are warranted to determine their possible role in cancer treatment.

Epigenetically induced changes in nuclear textural patterns and gelatinase expression in human fibrosarcoma cells.

OBJECTIVE: Chromatin texture patterns of tumour cell nuclei can serve as cancer biomarkers, either to define diagnostic classifications or to obtain relevant prognostic information, in a large number of human tumours. Epigenetic mechanisms, mainly DNA methylation and histone post-translational modification, have been shown to influence chromatin packing states, and therefore nuclear texture. The aim of this study was to analyse effects of these two mechanisms on chromatin texture, and also on correlation with gelatinase expression, in human fibrosarcoma tumour cells. MATERIALS AND METHODS: We investigated effects of DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-azadC) and histone deacetylase inhibitor trichostatin A (TSA) on nuclear textural characteristics of human HT1080 fibrosarcoma cells, evaluated by image cytometry, and expression of gelatinases MMP-2 and MMP-9, two metalloproteinases implicated in cancer progression and metastasis. RESULTS: 5-azadC induced significant variation in chromatin higher order organization, particularly chromatin decondensation, associated with reduction in global DNA methylation, concomitantly with increase in MMP-9, and to a lesser extent, MMP-2 expression. TSA alone did not have any effect on HT1080 cells, but exhibited differential activity when added to cells treated with 5-azadC. When treated with both drugs, nuclei had higher texture abnormalities. In this setting, reduction in MMP-9 expression was observed, whereas MMP-2 expression remained unaffected. CONCLUSIONS: These data show that hypomethylating drug 5-azadC and histone deacetylase inhibitor TSA were able to induce modulation of higher order chromatin organization and gelatinase expression in human HT1080 fibrosarcoma cells.

Development of a matrix-assisted laser desorption/ionization-mass spectrometry screening test to evidence reversible and irreversible inhibitors of CDC25 phosphatases.

The cell division cycle 25 phosphatases (CDC25s) are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers, and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25A and -C inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS) to detect reversible inhibitors or submitted to peptide mass fingerprint (PMF) analysis to reveal irreversible inhibitors covalently bound to the protein active site. After its validation, the protocol is applied to the detection of a novel candidate inhibitor of CDC25s named SV37. The screening procedure, as well as the preliminary biological results, demonstrates that this compound behaves as a reversible inhibitor.

From the deepest sea shelf to the uppermost kitchen cabinet shelf: the quest for novel TNF-α inhibitors.

TNF-α was discovered more then 20 years ago as a cytokine implicated in a wide range of cell signaling pathways, many of which are known to lead to the activation of genes involved in inflammation and carcinogenesis. TNF-α is involved in the pathogenesis of many diseases, including Crohn's disease, diabetes, septic shock, tumorigenesis, rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease. Multidisciplinary research endeavoring to understand the biomolecular and biomedicinal properties of TNF-α has never faded away, and the search for natural products able to inhibit TNF-α remains, to date, a hot topic of investigation. Over the last 10 years, many TNF-α-inhibiting natural compounds have been discovered, and their anti-TNF-α activities have been described. The present review describes the major cell signaling pathways activated by TNF-α and discusses the chemical and biological properties of TNF-α-inhibiting natural products, focusing on compounds that are able to inhibit TNF-α-related signal transduction pathways or TNF-α gene expression.

Natural chalcones as dual inhibitors of HDACs and NF-κB.

Histone deacetylase enzymes (HDACs) are emerging as a promising biological target for cancer and inflammation. Using a fluorescence assay, we tested the in vitro HDAC inhibitory activity of twenty-one natural chalcones, a widespread group of natural products with well-known anti-inflammatory and antitumor effects. Since HDACs regulate the expression of the transcription factor NF-κB, we also evaluated the inhibitory potential of the compounds on NF-κB activation. Only four chalcones, isoliquiritigenin (no. 10), butein (no. 12), homobutein (no. 15) and the glycoside marein (no. 21) showed HDAC inhibitory activity with IC50 values of 60-190 µM, whereas a number of compounds inhibited TNFα-induced NF-κB activation with IC50 values in the range of 8-41 µM. Interestingly, three chalcones (nos. 10, 12 and 15) inhibited both TNFα-induced NF-κB activity and total HDAC activity of classes I, II and IV. Molecular modeling and docking studies were performed to shed light into dual activity and to draw structure-activity relationships among chalcones (nos. 1-21). To the best of our knowledge this is the first study that provides evidence for HDACs as potential drug targets for natural chalcones. The dual inhibitory potential of the selected chalcones on NF-κB and HDACs was investigated for the first time. This study demonstrates that chalcones can serve as lead compounds in the development of dual inhibitors against both targets in the treatment of inflammation and cancer.

Power from the garden: plant compounds as inhibitors of the hallmarks of cancer.

On December 23rd, 1971, President Richard Nixon signed the National Cancer Act and invested more than $ 100 million "to launch an intensive campaign to find a cure for cancer". Today, despite these considerable efforts, cancer still remains a very aggressive silent killer all over the world. Moreover, over the last decade, novel synthetic chemotherapeutic agents currently in use in the clinics did not succeed in fulfilling their expectations even though they are very cost-intensive. In parallel, there is increasing evidence for the potential of plant-derived compounds on the inhibition of different steps of tumor genesis and associated inflammatory processes, underlining the importance of these products in cancer prevention and therapy. This review summarizes the impact of selected natural compounds on the eight major alterations, known as the cancer hallmarks, and also on their two enabling characteristics that were coined by Hanahan and Weinberg earlier. Altogether these ten alterations are responsible for the progressive transition of healthy cells into neoplastic ones and their further dissemination in the body. With this review, we try to highlight molecular mechanisms by which plant extracts and their purified active components fight and overcome these pathological variations of the cell signaling pathways for the improvement of prevention and therapy. We truly believe that all diseases can be found in Nature and that Nature also provides the efficient cures.

Pure NOTES sigmoid resection in an animal survival model.

INTRODUCTION: The potential to use single-site transluminal access to perform major surgical procedures is limited. In the current study, a pure natural orifice transluminal endoscopic surgery (NOTES) technique was developed for sigmoid resection, with combined transgastric and transvaginal access to the abdominal cavity and assisted by colonoscopy. METHODS: This experimental study was conducted on a porcine model. Transgastric access was achieved by needle-knife incision and balloon dilation. Colonoscopy was used to maneuver the colon and expose the colic mesentery. Mesocolic dissection close to the bowel was carried out gastroscopically using a coagulating forceps. To prepare the anastomosis, a circular stapler anvil was introduced endoluminally. Subsequently, sigmoid resection was performed using a roticulating linear stapler inserted transvaginally. Bowel extraction was performed by invagination transrectally. After extracorporeal distal linear stapling of the sigmoid, colorectal anastomosis was completed by application of a circular stapling device transrectally. Gastric access closure was achieved using the over-the-scope clipping system (OTSC). RESULTS: The procedure was successful in all animals, with the operation time ranging from 150 to 270 minutes. The first animal died at postoperative day 5 from peritonitis due to an infected hematoma following spleen injury by an observation trocar. All other animals gained weight postoperatively. Animals were sacrificed after postoperative day 35. The work-up showed complete anastomotic healing and healed gastric closure. The OTSC clip was still in situ in all animals. CONCLUSION: The presented study shows that pure NOTES resection and anastomosis of the large bowel are feasible. Intraluminal organ manipulation provided excellent organ exposition and rendered one additional access site unnecessary. Transgastric preparation was shown to be safe and effective.

Quercetin downregulates Mcl-1 by acting on mRNA stability and protein degradation.

BACKGROUND: We recently demonstrated that quercetin, a flavonoid naturally present in food and beverages belonging to the large class of phytochemicals, was able to sensitise leukaemic cells isolated from patients with chronic lymphocytic leukaemia (CLL) when associated with recombinant tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) or anti-CD95. We also showed that quercetin potentiated the effect of fludarabine on resistant B cells from CLL patients. Resistance to therapy in CLL depends on the expression and activity of anti-apoptotic proteins of the Bcl-2 family. Among these, myeloid cell leukaemia-1 (Mcl-1) has been associated with apoptotic resistance in CLL. Therefore, we investigate here whether the sensitising activity of this flavonoid, which leads to increased apoptosis in both cell lines and CLL, could be related to Mcl-1 expression and stability. RESULTS: B cells isolated from CLL patients showed different levels of Mcl-1 protein expression, resulting, in several cases, in increased sensitivity to fludarabine. Quercetin significantly enhanced the downregulation of Mcl-1 in B cells isolated from selected patients expressing detectable levels of Mcl-1. In U-937 cells, quercetin increased Mcl-1 mRNA instability in the presence of actinomycin D. When cells were treated with MG-132, a proteasome inhibitor, Mcl-1 protein level increased. However, quercetin, in the presence of Z-Vad-FMK, continued to lower Mcl-1 protein expression, indicating its independence from caspase-mediated degradation. In contrast, co-treatment of quercetin and MG-132 did not revert the effect of MG-132 mono-treatment, thus suggesting a possible interference of quercetin in regulating the proteasome-dependent degradation of Mcl-1. Gossypol, a small-molecule inhibitor of Bcl-2 family members, mimics the activity of quercetin by lowering Mcl-1 expression and sensitising U-937 cells to apoptosis induced by recombinant TRAIL and the Fas-ligand. CONCLUSION: This study demonstrates that in U-937 cells, quercetin downregulates Mcl-1 acting directly or indirectly on its mRNA stability and protein degradation, suggesting that the same mechanism may bypass resistance to apoptosis in leukaemic cells isolated from CLL patients and sensitise B cells to apoptosis induced by drugs and death receptor inducers.

Erythropoietin, erythropoiesis and beyond.

Erythropoietin (EPO) is a glycoprotein that is mainly produced in the adult kidney, and it was initially highlighted for its action on the hematopoietic system. Moreover, EPO is also expressed in several non-hematopoietic tissues, where it plays a role in the protection from apoptosis and inflammation due to hypoxia, toxicity or injury. These protective effects are mainly known and studied in cardioprotection and neuroprotection but are also reported in retina degeneration, auditory injury and pancreatic-related diseases. The tissue protective effect of EPO is mainly mediated through the interaction with the heterodimeric receptor EPOR/ßcR. Human recombinant EPO (HuREPO), which has been developed to treat anemia, is not adequate for tissue protection. The low affinity of the alternative receptor for EPO involves the injection of excessive concentration of erythropoiesis-stimulating agents (ESAs), implicating side effects due to the cross-talk with hematopoietic activity. For these reasons, EPO derivatives with less affinity for the EPO homodimeric receptor are under development. In this review, we provide an overview of the erythroid and non-erythroid functions of EPO by detailing the molecular mechanisms activated by the binding of EPO to its receptors in different tissues.

Anemia in cancer.

Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), among others, play a major role in the pathophysiology of anemia in the cancer patient not only through complex mechanisms of the purely inflammatory situation but also through genetic regulatory aspects of erythropoiesis via GATA-1 and GATA-2, and other factors. In terms of therapy, iron is used more and more; the late side effects of transfusions are not really understood and the recent controversy regarding erythropoietin usage has resulted in regulatory authorities and scientific societies providing several recommendations and guidelines. These various aspects are addressed herein.

Pro-inflammatory cytokine-mediated anemia: regarding molecular mechanisms of erythropoiesis.

Anemia of cancer and chronic inflammatory diseases is a frequent complication affecting quality of life. For cancer patients it represents a particularly bad prognostic. Low level of erythropoietin is considered as one of the causes of anemia in these pathologies. The deficiency in erythropoietin production results from pro-inflammatory cytokines effect. However, few data is available concerning molecular mechanisms involved in cytokine-mediated anemia. Some recent publications have demonstrated the direct effect of pro-inflammatory cytokines on cell differentiation towards erythroid pathway, without erythropoietin defect. This suggested that pro-inflammatory cytokine-mediated signaling pathways affect erythropoietin activity. They could interfere with erythropoietin-mediated signaling pathways, inducing early apoptosis and perturbing the expression and regulation of specific transcription factors involved in the control of erythroid differentiation. In this review we summarize the effect of tumor necrosis factor (TNF)alpha, TNF-related apoptosis-inducing ligand (TRAIL), and interferon (IFN)-gamma on erythropoiesis with a particular interest for molecular feature.

Oxidation-dependent maturation and survival of explanted blood monocytes via Bcl-2 up-regulation.

Monocytes isolated and cultured according to standard procedures from the blood of 22 healthy donors display an activation process, monitored as adhesion and increased exposure of CD11. Starting from very early time points, monocytes undergo a deep redox modulation, i.e., they increase reactive oxygen species (ROS) formation and decrease glutathione content; at the same time, the anti-apoptotic protein Bcl-2 is substantially up-regulated. The cause-effect relationship between these parameters was investigated. On the one side, pharmacological glutathione depletion with BSO further increases ROS formation and Bcl-2 levels. On the other side, scavenging of ROS by Trolox prevents Bcl-2 up-regulation. Two lipoxygenase (LOX) inhibitors (CAPE or AA861) prevent ROS increase and, accordingly, also prevent Bcl-2 up-regulation. All this evidence supports the redox-sensitivity of Bcl-2 regulation. Trolox, CAPE and AA861, i.e., all treatments that abolish ROS increase and prevent Bcl-2 up-regulation, increase the rate of cell loss, whereas BSO, increasing Bcl-2, reduces cell loss and induces chemo-resistance. Thus, explanted healthy monocytes seem to undergo an oxidation-dependent maturation implying increased survival via Bcl-2 up-regulation, perhaps mimicking physiological activation.

Antimicrobial efficacy and potential application of a newly developed plasma-based ultraviolet irradiation facility.

A low-pressure mercury vapour discharge tube generating high-intensity ultraviolet (UV) resonance radiation at 254 nm was designed to achieve a nearly simultaneous all-round UV irradiation of products. Testing this 'universal homogeneous ultraviolet (UHUV) irradiation facility' with suspended Bacillus subtilis spores, resulted in a 10(6)-fold reduction in viable count within 30 s applying irradiation energy of 0.3 mW/cm(2). Moreover, this irradiation dose reduced the number of immobilized B. subtilis spores on several material surfaces (wood-free paper, aluminium foil, polystyrol, polypropylene, and polyethylene foil) 10(2)-10(4)-fold. To evaluate potential applications of this UHUV irradiation technique under more realistic conditions, dental hand pieces and orthodontic forceps were contaminated by a blood-saliva mix containing Staphylococcus aureus. Under these conditions, a reduction in viable count of 10(5)-10(6)-fold was achieved within 5-15 min, but higher irradiation energy levels up to 13 mW/cm(2) were necessary. Because of its construction, the shape of the newly developed UHUV irradiation device can be adapted to various shapes to achieve a fast and effective antimicrobial treatment.