Effects of a human amniotic membrane extract on ARPE-19 cells.

BACKGROUND: Human Amniotic Membrane (hAM) is endowed with several biological activities and might be considered an optimal tool in surgical treatment for different ophthalmic pathologies. We pioneered the surgical use of hAM to treat retinal pathologies such as macular holes, tears, and retinal detachments, and to overcome photoreceptor damage in age-related macular degeneration. Although hAM contributed to improved outcomes, the mechanisms of its effects are not yet fully understood. The characterization and explanation of the effects of hAM would allow the adoption of this new natural product in different retinal pathologies, operative contexts, and hAM formulations. At this end, we studied the properties of a hAM extract (hAME) on the ARPE-19 cells. METHODS AND RESULTS: A non-denaturing sonication-based technique was developed to obtain a suitable hAME. Viability, proliferation, apoptosis, oxidative stress, and epithelial-mesenchymal transition (EMT) were studied in hAME-treated ARPE-19 cells. The hAME was able to increase ARPE-19 cell viability even in the presence of oxidative stress (H2O2, TBHP). Moreover, hAME prevented the expression of EMT features, such as EMT-related proteins, fibrotic foci formation, and migration induced by different cytokines. CONCLUSIONS: Our results demonstrate that the hAME retains most of the properties observed in the whole tissue by others. The hAME, other than providing a manageable research tool, could represent a cost-effective and abundant drug to treat retinal pathologies in the future.

Zeta-crystallin: a moonlighting player in cancer.

Crystallins were firstly found as structural proteins of the eye lens. To this family belong proteins, such as ζ-crystallin, expressed ubiquitously, and endowed with enzyme activity. ζ-crystallin is a moonlighting protein endowed with two main different functions: (1) mRNA binding with stabilizing activity; (2) NADPH:quinone oxidoreductase. ζ-crystallin has been clearly demonstrated to stabilize mRNAs encoding proteins involved in renal glutamine catabolism during metabolic acidosis resulting in ammoniagenesis and bicarbonate ion production that concur to compensate such condition. ζ-crystallin binds also mRNAs encoding for antiapoptotic proteins, such as Bcl-2 in leukemia cells. On the other hand, the physiological role of its enzymatic activity is still elusive. Gathering research evidences and data mined from public databases, we provide a framework where all the known ζ-crystallin properties are called into question, making it a hypothetical pivotal player in cancer, allowing cells to hijack or subjugate the acidity response mechanism to increase their ability to resist oxidative stress and apoptosis, while fueling their glutamine addicted metabolism.

Enhanced Vasculogenic Capacity Induced by 5-Fluorouracil Chemoresistance in a Gastric Cancer Cell Line.

Chemotherapy is still widely used as a coadjutant in gastric cancer when surgery is not possible or in presence of metastasis. During tumor evolution, gatekeeper mutations provide a selective growth advantage to a subpopulation of cancer cells that become resistant to chemotherapy. When this phenomenon happens, patients experience tumor recurrence and treatment failure. Even if many chemoresistance mechanisms are known, such as expression of ATP-binding cassette (ABC) transporters, aldehyde dehydrogenase (ALDH1) activity and activation of peculiar intracellular signaling pathways, a common and universal marker for chemoresistant cancer cells has not been identified yet. In this study we subjected the gastric cancer cell line AGS to chronic exposure of 5-fluorouracil, cisplatin or paclitaxel, thus selecting cell subpopulations showing resistance to the different drugs. Such cells showed biological changes; among them, we observed that the acquired chemoresistance to 5-fluorouracil induced an endothelial-like phenotype and increased the capacity to form vessel-like structures. We identified the upregulation of thymidine phosphorylase (TYMP), which is one of the most commonly reported mutated genes leading to 5-fluorouracil resistance, as the cause of such enhanced vasculogenic ability.

Update on gastric cancer treatments and gene therapies.

Gastric cancer is an active topic of clinical and basic research due to high morbidity and mortality. To date, gastrectomy and chemotherapy are the only therapeutic options for gastric cancer patients, but drug resistance, either acquired or primary, is the main cause for treatment failure. Differences in development and response to cancer treatments have been observed among ethnically diverse GC patient populations. In spite of major incidence, GC Asian patients have a significantly better prognosis and response to treatments than Caucasian ones due to genetic discordances between the two populations. Gene therapy could be an alternative strategy to overcome such issues and especially CRISPR/Cas9 represents one of the most intriguing gene-editing system. Thus, in this review article, we want to provide an update on the currently used therapies for the treatment of advanced GC. Graphical abstract.

MAP Kinases Pathways in Gastric Cancer.

Gastric cancer (GC) is turning out today to be one of the most important welfare issues for both Asian and European countries. Indeed, while the vast majority of the disease burden is located in China and in Pacific and East Asia, GC in European countries still account for about 100,000 deaths per year. With this review article, we aim to focus the attention on one of the most complex cellular pathways involved in GC proliferation, invasion, migration, and metastasis: the MAP kinases. Such large kinases family is to date constantly studied, since their discovery more than 30 years ago, due to the important role that it plays in the regulation of physiological and pathological processes. Interactions with other cellular proteins as well as miRNAs and lncRNAs may modulate their expression influencing the cellular biological features. Here, we summarize the most important and recent studies involving MAPK in GC. At the same time, we need to underly that, differently from cancers arising from other tissues, where MAPK pathways seems to be a gold target for anticancer therapies, GC seems to be unique in any aspect. Our aim is to review the current knowledge in MAPK pathways alterations leading to GC, including H. pylori MAPK-triggering to derail from gastric normal epithelium to GC and to encourage researches involved in MAPK signal transduction, that seems to definitely sustain GC development.

Plasmatic carbonic anhydrase IX as a diagnostic marker for clear cell renal cell carcinoma.

Carbonic anhydrase (CA, EC 4.2.1.1) IX is regarded as a tumour hypoxia marker and CA inhibitors have been proposed as a new class of antitumor agents, with one such agent in Phase II clinical trials. The expression of some CAs, in particular the isoforms CA IX and CA XII, has been correlated with tumour aggressiveness and progression in several cancers. The aim of this study was to evaluate the possibility that CA IX could represent a marker related to clear cell Renal Cell Carcinoma (ccRCC). Bcl-2 and Bax, and the activity of caspase-3, evaluated in tissue biopsies from patients, were congruent with resistance to apoptosis in ccRCCs with respect to healthy controls, respectively. In the same samples, the CA IX and pro-angiogenic factor VEGF expressions revealed that both these hypoxia responsive proteins were strongly increased in ccRCC with respect to controls. CA IX plasma concentration and CA activity were assessed in healthy volunteers and patients with benign kidney tumours and ccRCCs. CA IX expression levels were found strongly increased only in plasma from ccRCC subjects, whereas, CA activity was found similarly increased both in plasma from ccRCC and benign tumour patients, compared to healthy volunteers. These results show that the plasmatic level of CA IX, but not the CA total activity, can be considered a diagnostic marker of ccRCCs. Furthermore, as many reports exist relating CA IX inhibition to a better outcome to anticancer therapy in ccRCC, plasma levels of CA IX could be also predictive for response to therapy.

Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.

Endothelial progenitor cell-dependent angiogenesis requires localization of the full-length form of uPAR in caveolae.

Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.

Gastric Cancer Vascularization and the Contribution of Reactive Oxygen Species.

Blood vessels are the most important way for cancer cells to survive and diffuse in the body, metastasizing distant organs. During the process of tumor expansion, the neoplastic mass progressively induces modifications in the microenvironment due to its uncontrolled growth, generating a hypoxic and low pH milieu with high fluid pressure and low nutrients concentration. In such a particular condition, reactive oxygen species play a fundamental role, enhancing tumor proliferation and migration, inducing a glycolytic phenotype and promoting angiogenesis. Indeed, to reach new sources of oxygen and metabolites, highly aggressive cancer cells might produce a new abnormal network of vessels independently from endothelial cells, a process called vasculogenic mimicry. Even though many molecular markers and mechanisms, especially in gastric cancer, are still unclear, the formation of such intricate, leaky and abnormal vessel networks is closely associated with patients' poor prognosis, and therefore finding new pharmaceutical solutions to be applied along with canonical chemotherapies in order to control and normalize the formation of such networks is urgent.

The CAIX inhibitor SLC-0111 exerts anti-cancer activity on gastric cancer cell lines and resensitizes resistant cells to 5-Fluorouracil, taxane-derived, and platinum-based drugs.

Gastric cancer (GC) is the fifth most frequent malignancy and the fourth leading cause of worldwide cancer-related death. Despite the usage of multimodal perioperative chemotherapy (pCT), GC progressively gains chemoresistance, thereby, the identification of suitable targets to overcome drug resistance is fundamental. Amongst the potential biomarkers, carbonic anhydrase IX (CAIX) - associated with a poor prognosis of several solid cancers - has gained the most attention. In a cohort of GC patients who received perioperative FLOT (i.e., Leucovorin, 5-Fluouracil, Docetaxel, and Oxaliplatin) or FOLFOX (i.e., Leucovorin, 5-Fluouracil, and Oxaliplatin), non-responder patients showed an increased expression of tumor CAIX compared to responder group. Moreover, GC cell lines induced to be resistant to 5-Fluouracil, Paclitaxel, Cisplatin, or the combination of 5-Fluorouracil, Oxaliplatin, and Docetaxel, overexpressed CAIX compared to the control. Accordingly, CAIX-high-expressing GC cells showed increased therapy resistance compared to low-expressing cells. Notably, SLC0111 significantly improved the therapy response of both wild-type and resistant GC cells. Overall, these data suggest a correlation between CAIX and GC drug resistance highlighting the potential of SLC-0111 in re-sensitizing GC cells to pCT.

An Oleocanthal-Enriched EVO Oil Extract Induces the ROS Production in Gastric Cancer Cells and Potentiates the Effect of Chemotherapy.

Oleocanthal, a minor polar compound in extra-virgin olive (EVO) oil, contains anticancer properties, which should be encouraged in its use in oncology. Gastric Cancer (GC), a very aggressive human cancer, is often diagnosed at advanced stages, when surgery is substituted or supported by chemotherapy (CT). However, CT frequently fails due to the patient's resistance to the treatment. Thus, the aim of this study is to verify whether an OC-enriched EVO oil extract fraction (OCF) may be useful in order to overcome a resistance to GC. We evaluated the OCF effects on an AGS gastric adenocarcinoma cell line wild type (AGS wt) and on its subpopulations resistant to 5-fluorouracil (5FUr), Paclitaxel (TAXr) or cisplatin (CISr). We found that a 60 µM dose of the OCF acts on the AGS wt, 5FUr and TAXr, leading to the cell cycle inhibition and to a ROS production, but not on CISr cells. Resistance of CISr to the OCF seems to be due to higher levels of antioxidant-enzymes that can counteract the OCF-induced ROS production. Moreover, using the OCF plus 5-fluorouracil, Paclitaxel or cisplatin, we found a potentiating effect compared with a mono-treatment in all resistant GC cells, including CISr. In conclusion, the use of the OCF in the management of GC has shown very interesting advantages, opening-up the possibility to evaluate the efficacy of the OCF in vivo, as a valid adjuvant in the treatment of resistant GC.

zeta-Crystallin is a bcl-2 mRNA binding protein involved in bcl-2 overexpression in T-cell acute lymphocytic leukemia.

The human antiapoptotic bcl-2 gene has been discovered in t(14;18) B-cell leukemias/lymphomas because of its overexpression caused at a transcriptional control level by the bcl-2/IgH fusion gene. We were the first to disclose the post-transcriptional control of bcl-2 expression mediated by interactions of an adenine + uracil (AU)-rich element (ARE) in the 3'-UTR of bcl-2 mRNA with AU-binding proteins (AUBPs). Here, we identify and characterize zeta-crystallin as a new bcl-2 AUBP, whose silencing or overexpression has impact on bcl-2 mRNA stability. An increased Bcl-2 level observed in normal phytohemagglutinin (PHA)-activated T lymphocytes, acute lymphatic leukemia (ALL) T-cell lines, and T cells of patients with leukemia in comparison with normal non-PHA-activated T lymphocytes was concomitant with an increase in zeta-crystallin level. The specific association of zeta-crystallin with the bcl-2 ARE was significantly enhanced in T cells of patients with ALL, which accounts for the higher stability of bcl-2 mRNA and suggests a possible contribution of zeta-crystallin to bcl-2 overexpression occurring in this leukemia.

Streptomycin sulphate loaded solid lipid nanoparticles show enhanced uptake in macrophage, lower MIC in Mycobacterium and improved oral bioavailability.

Present study addresses the challenge of incorporating hydrophilic streptomycin sulphate (STRS; log P -6.4) with high dose (1 g/day) into a lipid matrix of SLNs. Cold high-pressure homogenization technique used for SLN preparation achieved 30% drug loading and 51.17 ± 0.95% entrapment efficiency. Polyethylene glycol 600 as a supporting-surfactant assigned small size (218.1 ± 15.46 nm) and mucus-penetrating property. It was conceived to administer STRS-SLNs orally rather than intramuscularly. STRS-SLNs remained stable on incubation for varying times in SGF or SIF. STRS-SLNs were extensively characterised for microscopic (TEM and AFM), thermal (DSC), diffraction (XRD) and spectroscopic (NMR and FTIR) properties and showed zero-order controlled release. Enhanced (60 times) intracellular uptake was observed in THP-1 and Pgp expressing LoVo and DLD-1 cell lines, using fluorescein-SLNs. Presence of SLNs in LoVo cells was also revealed by TEM studies. STRS-SLNs showed 3 times reduction in MIC against Mycobacterium tuberculosis H37RV (256182) in comparison to free STRS. It also showed better activity against both M. bovis BCG and Mycobacterium tuberculosis H37RV (272994) in comparison to free STRS. Cytotoxicity and acute toxicity studies (OECD 425 guidelines) confirmed in vitro and in vivo safety of STRS-SLNs. Single-dose oral pharmacokinetic studies in rat plasma using validated LCMS/MS technique or the microbioassay showed significant oral absorption and bioavailability (160% - 710% increase than free drug).

Lipo-PEG nano-ocular formulation successfully encapsulates hydrophilic fluconazole and traverses corneal and non-corneal path to reach posterior eye segment.

The present study describes a special lipid-polyethylene glycol matrix solid lipid nanoparticles (SLNs; 138 nm; -2.07 mV) for ocular delivery. Success of this matrix to encapsulate (entrapment efficiency - 62.09%) a hydrophilic drug, fluconazole (FCZ-SLNs), with no burst release (67% release in 24 h) usually observed with most water-soluble drugs, is described presently. The system showed 164.64% higher flux than the marketed drops (Zocon®) through porcine cornea. Encapsulation within SLNs and slow release did not compromise efficacy of FCZ-SLNs. Latter showed in vitro and in vivo antifungal effects, including antibiofilm effects comparable to free FCZ solution. Developed system was safe and stable (even to sterilisation by autoclaving); and showed optimal viscosity, refractive index and osmotic pressure. These SLNs could reach up to retina following application as drops. The mechanism of transport via corneal and non-corneal transcellular pathways is described by fluorescent and TEM images of mice eye cross sections. Particles streamed through the vitreous, crossed inner limiting membrane and reached the outer retinal layers.

Selective inhibition of carbonic anhydrase IX decreases cell proliferation and induces ceramide-mediated apoptosis in human cancer cells.

Recently, carbonic anhydrase (CA) inhibitors have been proposed as a potential new class of antitumor agents. The aim of this study was to evaluate the antitumor activity of three CA inhibitors, namely acetazolamide (AZ) and two newly synthesized aromatic sulfonamides with high affinity for CA IX, 2-(4-sulfamoylphenyl-amino)-4,6-dichloro-1,3,5-triazine (TR1) and 4-[3-(N,N-dimethylaminopropyl)thioreidophenylsulfonylaminoethyl]benzenesulfonamide (GA15), against human tumor cells. The effects of AZ, TR1, and GA15 on cell proliferation and apoptosis were evaluated in CA IX-positive HeLa and 786-O cells and CA IX-negative 786-O/von Hippel-Lindau (VHL) cells. We also investigated whether the potential antitumor activity of these molecules might be mediated by an increase in ceramide production. AZ, TR1, and GA15 could significantly reduce cell proliferation and induce apoptosis in HeLa and 786-O cells. Moreover, all three inhibitors could decrease intracellular pH (pH(i)) and increase ceramide production in the same cells. Treatment with the ceramide synthase inhibitor fumonisin B1 prevented the apoptotic effects of the three CA inhibitors. In all experiments, the effects of aromatic sulfonamides were more pronounced than those of AZ. The three inhibitors did not show any antitumor activity in CA IX-negative 786-O/VHL cells and failed to lower pH(i) or increase intracellular ceramide levels in the same cells. In conclusion, CA inhibition can decrease cell proliferation and induce apoptosis in human tumor cells. The ability of CA inhibitors to decrease pH(i) might trigger cell apoptosis through mediation of ceramide synthesis. Activation of this apoptotic cascade probably is mediated by inhibition of the CA IX isoform.

The acidic tumor microenvironment drives a stem-like phenotype in melanoma cells.

Acidosis characterizes the microenvironment of most solid tumors and is considered a new hallmark of cancer. It is mainly caused by both "aerobic" and "anaerobic" glycolysis of differently adapted cancer cells, with the final product lactic acid being responsible of the extracellular acidification. Many evidences underline the role of extracellular acidosis in tumor progression. Among the different findings, we demonstrated that acidosis-exposed cancer cells are characterized by an epithelial-to-mesenchymal transition phenotype with high invasive ability, high resistance to apoptosis, anchorage-independent growth, and drug therapy. Acidic melanoma cells over-express SOX2, which is crucial for the maintenance of their oxidative metabolism, and carbonic anhydrase IX, that correlates with poor prognosis of cancer patients. Considering these evidences, we realized that the profile outlined for acid cancer cells inevitably remind us the stemness profile. Therefore, we wondered whether extracellular acidosis might induce in cancer cells the acquisition of stem-like properties and contribute to the expansion of the cancer stem cell sub-population. We found that a chronic adaptation to acidosis stimulates in cancer cells the expression of stem-related markers, also providing a high in vitro/in vivo clonogenic and trans-differentiating ability. Moreover, we observed that the acidosis-induced stem-like phenotype of melanoma cells was reversible and related to the EMT induction. These findings help to characterize a further aspect of stem cell niche, contributing to the sustainment and expansion of cancer stem cell subpopulation. Thus, the usage of agents controlling tumor extracellular acidosis might acquire great importance in the clinic for the treatment of aggressive solid tumor. KEY MESSAGES: • Extracellular acidosis up-regulates EMT and stem-related markers in melanoma cells • Acidic medium up-regulates in vitro self-renewal capacity of melanoma cells • Chronic acidosis adaptation induces trans-differentiation ability in melanoma cells • Melanoma cells adapted to acidosis show higher tumor-initiating potential than control cells • Extracellular acidosis promotes a stem-like phenotype in prostate and colorectal carcinoma cells.

Correction to: Atorvastatin-loaded solid lipid nanoparticles as eye drops: proposed treatment option for age-related macular degeneration (AMD).

In the original version of this article Ana Guzman-Aranguez's name appeared incorrect.

5-Fluorouracil Conversion Pathway Mutations in Gastric Cancer.

To date, 5-Fluorouracil (5FU) is a major component of several chemotherapy regimens, thus its study is of fundamental importance to better understand all the causes that may lead to chemoresistance and treatment failure. Given the evident differences between prognosis in Asian and Caucasian populations, triggered by clear genetic discordances and given the extreme genetic heterogeneity of gastric cancer (GC), the evaluation of the most frequent mutations in every single member of the 5FU conversion and activation pathway might reveal several important results. Here, we exploited the cBioPortal analysis software to query a large databank of clinical and wide-genome studies to evaluate the components of the three major 5FU transformation pathways. We demonstrated that mutations in such ways were associated with a poor prognosis and reduced overall survival, often caused by a deletion in the TYMP gene and amplification in TYMS. The use of prodrugs and dihydropyrimidine dehydrogenase (DPD) inhibitors, which normally catabolizes 5FU into inactive metabolites, improved such chemotherapies, but several steps forward still need to be taken to select better therapies to target the chemoresistant pools of cells with high anaplastic features and genomic instability.

Atorvastatin-loaded solid lipid nanoparticles as eye drops: proposed treatment option for age-related macular degeneration (AMD).

Statins, widely prescribed for cardiovascular diseases, are also being eyed for management of age-related macular degeneration (AMD). Poor bioavailability and blood-aqueous barrier may however limit significant ocular concentration of statins following oral administration. We for the first time propose and investigate local application of atorvastatin (ATS; representative statin) loaded into solid lipid nanoparticles (SLNs), as self-administrable eye drops. Insolubility, instability, and high molecular weight > 500 of ATS, and ensuring that SLNs reach posterior eye were the challenges to be met. ATS-SLNs, developed (2339/DEL/2014) using suitable components, quality-by-design (QBD) approach, and scalable hot high-pressure homogenization, were characterized and evaluated comprehensively for ocular suitability. ATS-SLNs were 8 and 12 times more bioavailable (AUC) in aqueous and vitreous humor, respectively, than free ATS. Three-tier (in vitro, ex vivo, and in vivo) ocular safety, higher corneal flux (2.5-fold), and improved stability (13.62 times) including photostability of ATS on incorporation in ATS-SLNs were established. Autoclavability and aqueous nature are the other highlights of ATS-SLNs. Presence of intact fluorescein-labeled SLNs (F-SLNs) in internal eye tissues post-in vivo application as eye drops provides direct evidence of successful delivery. Perinuclear fluorescence in ARPE-19 cells confirms the effective uptake of F-SLNs. Prolonged residence, up to 7 h, was attributed to the mucus-penetrating nature of ATS-SLNs. Graphical abstract.

Role of NADPH oxidase in H9c2 cardiac muscle cells exposed to simulated ischaemia-reperfusion.

Oxidative stress is associated with several cardiovascular pathologies, including hypertension, cardiac hypertrophy and heart failure. Although oxidative stress is also increased after ischaemia-reperfusion (I/R), little is known about the role and the activation mechanisms, in cardiac myocytes under these conditions, of NADPH oxidase, a superoxide-producing enzyme. We found that rat cardiac muscle cells (H9c2) subjected to an in vitro simulated ischaemia (substrate-free medium plus hypoxia) followed by 'reperfusion', displayed increased reactive oxygen species (ROS) production attributable to a parallel increase of NADPH oxidase activity. Our investigation on mechanisms responsible for NADPH oxidase activation showed a contribution of both the increase of NOX2 expression and p47(phox) translocation to the membrane. We also found that the increase of NADPH oxidase activity was associated with higher levels of lipid peroxidation, the activation of redox-sensitive kinases, in particular ERK and JNK, and with cell death. Diphenyleneiodonium (DPI), a flavoprotein inhibitor used as NADPH oxidase inhibitor, prevented I/R-induced ROS formation in treated cells, together with the related lipoperoxidative damage, and JNK phosphorylation without affecting ERK activation, resulting in protection against cell death. Our results provide evidence that NADPH oxidase is a key enzyme involved in I/R-induced oxidant generation and suggest it can be a possible target in cardioprotective strategies against I/R injury, a condition of great importance in human pathology.