Cellular Pathophysiology of Leptospirosis: Role of Na/K-ATPase.

Inada and Ido identified Leptospira sp. as the pathogen responsible for Weil's Disease in 1915. Later, it was confirmed that Leptospira causes leptospirosis. The host microorganism's interaction at the cellular level remained misunderstood for many years. Although different bacterial components have been isolated and purified, the complexity of the molecular interactions between these components and the host and the molecular mechanisms responsible for the systemic dysfunctions still needs to be fully unveiled. Leptospirosis affects virtually all animal species. Its cellular pathophysiology must involve a ubiquitous cellular mechanism in all eukaryotes. Na/K-ATPase is the molecular target of the leptospiral endotoxin (glycolipoprotein-GLP). Na/K-ATPase dysfunctions on different types of cells give rise to the organ disorders manifested in leptospirosis. Concomitantly, the development of a peculiar metabolic disorder characterized by dyslipidemia, with increased levels of circulating free fatty acids and an imbalance in the fatty acid/albumin molar ratio, triggers events of cellular lipotoxicity. Synergistically, multiple molecular stimuli are prompted during the infection, activating inflammasomes and Na/K-ATPase signalosome, leading to pro-inflammatory and metabolic alterations during leptospirosis. Leptospirosis involves diverse molecular mechanisms and alteration in patient inflammatory and metabolic status. Nonetheless, Na/K-ATPase is critical in the disease, and it is targeted by GLP, its components, and other molecules, such as fatty acids, that inhibit or trigger intracellular signaling through this enzyme. Herein, we overview the role of Na/K-ATPase during leptospirosis infection as a potential therapeutic target or an indicator of disease severity.

Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography.

BACKGROUND: Leptospirosis is an emerging zoonosis that affects humans and animals. Immunochromatography rapid test is widely used for early diagnosis of leptospirosis, but with low sensitivity and specificity. OBJECTIVES: To evaluate Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography. METHODS: Insoluble fraction derived from the crude bacterial extract was obtained by serial centrifugation. The polypeptide profile was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immune reactivity of this fraction was assessed by Western Blotting and lateral flow immunochromatography (LFI). It was tested 160 microagglutination test (MAT)-positive sera from patients in the acute phase, 100 MAT-negative sera from patients with acute febrile illness, and 45 patients with other infectious diseases. FINDINGS: There was a predominance of low molecular mass-polypeptide bands, ranging from 2 to 37 kDa. The antibody reactivity of theses polypeptides was found to range from 13-50%, especially between 10 and 38 kDa. Among MAT-positive sera of patients with leptospirosis in the acute phase, 97% were also positive in LFI, indicating high sensitivity. Among MAT-negative sera, all were negative in LFI, indicating high specificity. Only 2% of cross-reactivity was detected. CONCLUSION: The insoluble fraction can be a valuable antigen source for development of point-of-care diagnosis test for leptospirosis.

Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography

BACKGROUND Leptospirosis is an emerging zoonosis that affects humans and animals. Immunochromatography rapid test is widely used for early diagnosis of leptospirosis, but with low sensitivity and specificity. OBJECTIVES To evaluate Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography. METHODS Insoluble fraction derived from the crude bacterial extract was obtained by serial centrifugation. The polypeptide profile was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immune reactivity of this fraction was assessed by Western Blotting and lateral flow immunochromatography (LFI). It was tested 160 microagglutination test (MAT)-positive sera from patients in the acute phase, 100 MAT-negative sera from patients with acute febrile illness, and 45 patients with other infectious diseases. FINDINGS There was a predominance of low molecular mass-polypeptide bands, ranging from 2 to 37 kDa. The antibody reactivity of theses polypeptides was found to range from 13-50%, especially between 10 and 38 kDa. Among MAT-positive sera of patients with leptospirosis in the acute phase, 97% were also positive in LFI, indicating high sensitivity. Among MAT-negative sera, all were negative in LFI, indicating high specificity. Only 2% of cross-reactivity was detected. CONCLUSION The insoluble fraction can be a valuable antigen source for development of point-of-care diagnosis test for leptospirosis.

The Na/K-ATPase role as a signal transducer in lung inflammation.

Acute respiratory distress syndrome (ARDS) is marked by damage to the capillary endothelium and alveolar epithelium following edema formation and cell infiltration. Currently, there are no effective treatments for severe ARDS. Pathologies such as sepsis, pneumonia, fat embolism, and severe trauma may cause ARDS with respiratory failure. The primary mechanism of edema clearance is the epithelial cells' Na/K-ATPase (NKA) activity. NKA is an enzyme that maintains the electrochemical gradient and cell homeostasis by transporting Na+ and K+ ions across the cell membrane. Direct injury on alveolar cells or changes in ion transport caused by infections decreases the NKA activity, loosening tight junctions in epithelial cells and causing edema formation. In addition, NKA acts as a receptor triggering signal transduction in response to the binding of cardiac glycosides. The ouabain (a cardiac glycoside) and oleic acid induce lung injury by targeting NKA. Besides enzymatic inhibition, the NKA triggers intracellular signal transduction, fostering proinflammatory cytokines production and contributing to lung injury. Herein, we reviewed and discussed the crucial role of NKA in edema clearance, lung injury, and intracellular signaling pathway activation leading to lung inflammation, thus putting the NKA as a protagonist in lung injury pathology.

Irisin, Exercise, and COVID-19.

Muscle and adipose tissue produce irisin during exercise. Irisin is thermogenic adipomyokine, improves glucose and lipid metabolism, and ameliorates the effects of obesity-driven inflammation, metabolic syndrome, and diabetes. In addition, exercise-induced irisin activates anti-inflammatory pathways and may play an essential role in improving the outcomes of inflammatory conditions, such as coronavirus disease (COVID-19). COVID-19 infection can activate different intracellular receptors and modulate various pathways during the course of the disease. The cytokine release storm (CRS) produced is significant because it promotes the context for systemic inflammation, which increases the risk of mortality in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). In addition, viral infection and the resulting organ damage may stimulate the mitogen-activated protein kinase(MAPK) and toll-like receptor 4 (TLR4)/toll interleukin receptor (TIR)-domain-containing adaptor (MyD88) pathways while negatively modulating the AMP-activated protein kinase (AMPK) pathway, leading to increased inflammatory cytokine production. Exercise-induced irisin may counteract this inflammatory modulation by decreasing cytokine production. Consequently, increased irisin levels, as found in healthy patients, may favor a better prognosis in patients with SARS-CoV2. This review aims to explore the molecular mechanisms underlying the anti-inflammatory properties of irisin in mitigating CRS and preventing severe outcomes due to infection with SARS-CoV2.

Na+/K+-ATPase as a Target of Cardiac Glycosides for the Treatment of SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), identified for the first time in Wuhan, China, causes coronavirus disease 2019 (COVID-19), which moved from epidemic status to becoming a pandemic. Since its discovery in December 2019, there have been countless cases of mortality and morbidity due to this virus. Several compounds such as chloroquine, hydroxychloroquine, lopinavir-ritonavir, and remdesivir have been tested as potential therapies; however, no effective treatment is currently recommended by regulatory agencies. Some studies on respiratory non-enveloped viruses such as adenoviruses and rhinovirus and some respiratory enveloped viruses including human respiratory syncytial viruses, influenza A, parainfluenza, SARS-CoV, and SARS-CoV-2 have shown the antiviral activity of cardiac glycosides, correlating their effect with Na+/K+-ATPase (NKA) modulation. Cardiac glycosides are secondary metabolites used to treat patients with cardiac insufficiency because they are the most potent inotropic agents. The effects of cardiac glycosides on NKA are dependent on cell type, exposure time, and drug concentration. They may also cause blockage of Na+ and K+ ionic transport or trigger signaling pathways. The antiviral activity of cardiac glycosides is related to cell signaling activation through NKA inhibition. Nuclear factor kappa B (NFκB) seems to be an essential transcription factor for SARS-CoV-2 infection. NFκB inhibition by cardiac glycosides interferes directly with SARS-CoV-2 yield and inflammatory cytokine production. Interestingly, the antiviral effect of cardiac glycosides is associated with tyrosine kinase (Src) activation, and NFκB appears to be regulated by Src. Src is one of the main signaling targets of the NKA α-subunit, modulating other signaling factors that may also impair viral infection. These data suggest that Src-NFκB signaling modulated by NKA plays a crucial role in the inhibition of SARS-CoV-2 infection. Herein, we discuss the antiviral effects of cardiac glycosides on different respiratory viruses, SARS-CoV-2 pathology, cell signaling pathways, and NKA as a possible molecular target for the treatment of COVID-19.

The relationship of oleic acid/albumin molar ratio and clinical outcomes in leptospirosis.

Human leptospirosis is an acute infectious zoonosis presenting specific lipid disorders. Previous in vitro studies showed both leptospira glycolipoprotein endotoxin, and high oleic acid levels were associated with Na/K-ATPase inhibition that is amplified by the reduction of circulating albumin levels. In this study, we aimed to investigate the relationship of oleic acid/albumin (OA/A) molar ratio and clinical outcomes in Leptospirosis. Through a prospective observational cohort study employing high-performance liquid chromatography (HPLC) we sequentially determined serum concentrations of nonesterified fatty acids (NEFA) and albumin in twenty-eight patients with severe leptospirosis since their hospital admission. Twenty patients recovered, and eight died. Data was distributed in two groups according to clinical outcomes. Oleic acid/albumin molar ratios (OA/A), initial samples, were higher than those in healthy donors. The ratio OA/A, however, persisted high in dying patients, whereas patients who survived had a reduction matching to healthy donors. Biochemical alterations suggest that cure is correlated to the reestablishment of the OA/A molar ratio, while fatal outcomes related to persisting OA/A imbalances. Analysis by receiver operating characteristic (ROC) showed the area under the curve of 0.864 and the cutoff value of 0.715 being associated with a high odds ratio. Lipid analysis from patients with leptospirosis had an acute high serum OA/A molar ratio, and sustained imbalance has a high odds ratio and strong correlation with mortality.

Na/K-ATPase: Their role in cell adhesion and migration in cancer.

Na/K-ATPase (NKA) is a p-type transmembrane enzyme formed by three different subunits (α, ß, and γ gamma). Primarily responsible for transporting sodium and potassium through the cell membrane, it also plays a critical role in intracellular signaling. The activation of diverse intracellular pathways may trigger cell death, survival, or even cell proliferation. Changes in the NKA functions or expression in isoforms subunits impact pathological conditions, such as cancer. The NKA function affects cell adhesion, motility, and migration, which are different in the physiological and pathological states. All enzyme subunits take part in the cell adhesion process, with the ß subunit being the most studied. Thus, herein we aim to highlight NKA' central role in cell adhesion, motility, and migration in cancer cells.

Alphavirus Replication: The Role of Cardiac Glycosides and Ion Concentration in Host Cells.

Alphaviruses are arthropod-borne viruses that can cause fever, rash, arthralgias, and encephalitis. The mosquito species Aedes aegypti and Aedes albopictus are the most frequent transmitters of alphaviruses. There are no effective vaccines or specific antivirals available for the treatment of alphavirus-related infections. Interestingly, changes in ion concentration in host cells have been characterized as critical regulators of the alphavirus life cycle, including fusion with the host cell, glycoprotein trafficking, genome translation, and viral budding. Cardiac glycosides, which are classical inhibitors of the Na+ K+ ATPase (NKA), can inhibit alphavirus replication although their mechanisms of action are poorly understood. Nonetheless, results from multiple studies suggest that inhibition of NKA may be a suitable strategy for the development of alphavirus-specific antiviral treatments. This review is aimed at exploring the role of changes in ion concentration during alphavirus replication and at considering the possibility of NKA as a potential therapeutic target for antiviral drugs.

Serum albumin-fatty acid saturation test.

Circulating non-esterified fatty acids (NEFA) are toxic to mammalian cells. They increase in diseases such as diabetes and sepsis. Herein we propose a serum albumin-fatty acid saturation test. •We based our test on three methodologies: isoelectric focusing (IF) of human plasma albumin, staining proteins after isoelectric focusing in gels with Coomassie Brilliant Blue, and serum albumin measurement with bromocresol green.•The test consists in the determination of albumin IF and staining with bromocresol green. If albumin is saturated with NEFA, it focuses on lower pH, meaning it is the threshold to bind to them. Excessive NEFA is free and toxic. Many other tests are available for NEFA quantification as NEFA kit assay. All colorimetric assays are used for quantification of NEFA and other tests need expensive equipment to read out the results, and they do not measure albumin levels.•Our method focused on albumin-NEFA saturation instead of just NEFA quantification. Critically ill patients have an alteration in both albumin and NEFA. Therefore, our test undergoes less daytime variation compared to assays that measure absolute NEFA values, allowing a more reliable use as an indicator of albumin-fatty acid saturation and NEFA toxicity.

Serum albumin saturation test based on non-esterified fatty acids imbalance for clinical employment.

Fatty acids are fundamental as energy and structural source to the human cells. They are not usually found free in human circulation. Alteration in fatty acids metabolism is linked to diseases such as diabetes, preeclampsia, heart disease, and some infectious diseases. Increased levels of non-esterified fatty acids (NEFA) may cause cell dysfunction and lipotoxicity. Since physiologically fatty acids are transported bound to albumin, we propose here a simple and cheap test that consists of albumin isoelectric focusing determination to measure the potential systemic NEFA cytotoxicity. For validation of this method, albumin isoelectric focusing in 51 serum samples from 40 critically ill patients and 11 controls was compared with NEFA/albumin ratios measured by HPLC. We called this approach an albumin saturation test. This test may indicate to physicians the potential NEFA lipotoxicity guiding them throughout better patient management. The albumin saturation test can point out serum albumin-NEFA saturation through a cheap assay that could be performed by any care facility.

Effect of polygodial and its direct derivatives on the mammalian Na+/K+-ATPase activity.

The sesquiterpene polygodial is an agonist of the transient receptor potential vanilloid 1 (TRPV1). Our group recently reported the synthesis and anticancer effects of polygodial and its derivatives, and showed that these compounds retain activity against apoptosis- and multidrug-resistant cancer cells. Herein, we tested the inhibitory effect of these compounds on the activity of the enzyme Na+/K+-ATPase (NKA) from kidney (α1 isoform) and brain (α2 and α3 isoforms) guinea pig extracts. Polygodial (1) displayed a dose-dependent inhibition of both kidney and brain purified NKA preparations, with higher sensitivity for the cerebral isoforms. Polygo-11,12-diol (2) and C11,C12-pyridazine derivative (3) proved to be poor inhibitors. Unsaturated ester (4) and 9-epipolygodial (5) inhibited NKA preparations from brain and kidney, with the same inhibitory potency. Nevertheless, they did not achieve maximum inhibition even at higher concentration. Comparing the inhibitory potency in crude homogenates and purified preparations of NKA, compounds 4 and 5 revealed a degree of selectivity toward the renal enzyme. Kinetic studies showed a non-competitive inhibition for Na+ and K+ by compounds 1, 4 and 5 and for ATP by 1 and 4. However, compound 5 presented a competitive inhibition type. Furthermore, K+-activated p-nitrophenylphosphatase activity of these purified preparations was not inhibited by 1, 4 and 5, suggesting that these compounds acted in the initial phase of the enzyme's catalytic cycle. These findings suggest that the antitumor action of polygodial and its analogues may be linked to their NKA inhibitory properties and reinforce that NKA may be an important target for cancer therapy.

Antileukemic Properties of Sesquiterpene Lactones: A Systematic Review.

This review summarizes the reported molecular mechanisms underlying the antileukemic property of Sesquiterpene Lactones (SLs). This systematic review was registered in the PROSPERO database and conducted following the PRISMA Statements. The MeSH terms, Sesquiterpenes, Lactones and Leukemia were used in four databases (Pubmed, Web of Science, Scopus and Bireme). There were 281 studies selected, but after exclusions, due to replication (n = 172) or not following PECOS criteria (n = 24), 148 studies remained. Of the 148 articles, only 22 were submitted to quality assessment and were scored in high level if more than two techniques, to elucidate antileukemic properties, were described, and then data were extracted. The studies mostly used human leukemia cell lines including primary and established cells, with or without chemotherapy resistance. The SLs used were obtained principally from plants. The antileukemic properties of SLs were extracted from 22 high level studies. They included cell death induction, mainly by apoptosis, as well as cell differentiation, cell cycle disruption, leukemia cancer stem cell growth inhibition and NF-κB pathway inhibition; the latter is a promising therapeutic target for lymphoid malignancies. We concluded that, in spite of the necessity of better toxicological profile characterization of SLs, the antileukemic properties of these compounds support the proposal that the SLs are promising candidates for the treatment of leukemia.

Na/K Pump and Beyond: Na/K-ATPase as a Modulator of Apoptosis and Autophagy.

Lung cancer is a leading cause of global cancer deaths. Na/K-ATPase has been studied as a target for cancer treatment. Cardiotonic steroids (CS) trigger intracellular signalling upon binding to Na/K-ATPase. Normal lung and tumour cells frequently express different pump isoforms. Thus, Na/K-ATPase is a powerful target for lung cancer treatment. Drugs targeting Na/K-ATPase may induce apoptosis and autophagy in transformed cells. We argue that Na/K-ATPase has a role as a potential target in chemotherapy in lung cancer treatment. We discuss the effects of Na/K-ATPase ligands and molecular pathways inducing deleterious effects on lung cancer cells, especially those leading to apoptosis and autophagy.

Omega-9 Oleic Acid Induces Fatty Acid Oxidation and Decreases Organ Dysfunction and Mortality in Experimental Sepsis.

Sepsis is characterized by inflammatory and metabolic alterations, which lead to massive cytokine production, oxidative stress and organ dysfunction. In severe systemic inflammatory response syndrome, plasma non-esterified fatty acids (NEFA) are increased. Several NEFA are deleterious to cells, activate Toll-like receptors and inhibit Na+/K+-ATPase, causing lung injury. A Mediterranean diet rich in olive oil is beneficial. The main component of olive oil is omega-9 oleic acid (OA), a monounsaturated fatty acid (MUFA). We analyzed the effect of OA supplementation on sepsis. OA ameliorated clinical symptoms, increased the survival rate, prevented liver and kidney injury and decreased NEFA plasma levels in mice subjected to cecal ligation and puncture (CLP). OA did not alter food intake and weight gain but diminished reactive oxygen species (ROS) production and NEFA plasma levels. Carnitine palmitoyltransferase IA (CPT1A) mRNA levels were increased, while uncoupling protein 2 (UCP2) liver expression was enhanced in mice treated with OA. OA also inhibited the decrease in 5' AMP-activated protein kinase (AMPK) expression and increased the enzyme expression in the liver of OA-treated mice compared to septic animals. We showed that OA pretreatment decreased NEFA concentration and increased CPT1A and UCP2 and AMPK levels, decreasing ROS production. We suggest that OA has a beneficial role in sepsis by decreasing metabolic dysfunction, supporting the benefits of diets high in monounsaturated fatty acids (MUFA).

Possible mechanisms of Pseudomonas aeruginosa-associated lung disease.

Pseudomonas aeruginosa is an opportunistic bacterium causing lung injury in immunocompromised patients correlated with high morbidity and mortality. Many bacteria, including P. aeruginosa, use extracellular signals to synchronize group behaviors, a process known as quorum sensing (QS). In the P. aeruginosa complex QS system controls expression of over 300 genes, including many involved in host colonization and disease. P. aeruginosa infection elicits a complex immune response due to a large number of immunogenic factors present in the bacteria or released during infection. Here, we focused on the mechanisms by which P. aeruginosa triggers lung injury and inflammation, debating the possible ways that P. aeruginosa evades the host immune system, which leads to immune suppression and resistance.

Acute Respiratory Distress Syndrome: Role of Oleic Acid-Triggered Lung Injury and Inflammation.

Lung injury especially acute respiratory distress syndrome (ARDS) can be triggered by diverse stimuli, including fatty acids and microbes. ARDS affects thousands of people worldwide each year, presenting high mortality rate and having an economic impact. One of the hallmarks of lung injury is edema formation with alveoli flooding. Animal models are used to study lung injury. Oleic acid-induced lung injury is a widely used model resembling the human disease. The oleic acid has been linked to metabolic and inflammatory diseases; here we focus on lung injury. Firstly, we briefly discuss ARDS and secondly we address the mechanisms by which oleic acid triggers lung injury and inflammation.

Synthetic and Biological Studies of Sesquiterpene Polygodial: Activity of 9-Epipolygodial against Drug-Resistant Cancer Cells.

Polygodial, a terpenoid dialdehyde isolated from Polygonum hydropiper L., is a known agonist of the transient receptor potential vanilloid 1 (TRPV1). In this investigation a series of polygodial analogues were prepared and investigated for TRPV1-agonist and anticancer activities. These experiments led to the identification of 9-epipolygodial, which has antiproliferative potency significantly exceeding that of polygodial. 9-Epipolygodial was found to maintain potency against apoptosis-resistant cancer cells as well as those displaying the multidrug-resistant (MDR) phenotype. In addition, the chemical feasibility for the previously proposed mechanism of action of polygodial, involving the formation of a Paal-Knorr pyrrole with a lysine residue on the target protein, was demonstrated by the synthesis of a stable polygodial pyrrole derivative. These studies reveal rich chemical and biological properties associated with polygodial and its direct derivatives. These compounds should inspire further work in this area aimed at the development of new pharmacological agents, or the exploration of novel mechanisms of covalent modification of biological molecules with natural products.

Wittig derivatization of sesquiterpenoid polygodial leads to cytostatic agents with activity against drug resistant cancer cells and capable of pyrrolylation of primary amines.

Many types of cancer, including glioma, melanoma, non-small cell lung cancer (NSCLC), among others, are resistant to proapoptotic stimuli and thus poorly responsive to current therapies based on the induction of apoptosis in cancer cells. The current investigation describes the synthesis and anticancer evaluation of unique C12-Wittig derivatives of polygodial, a sesquiterpenoid dialdehyde isolated from Persicaria hydropiper (L.) Delabre. These compounds were found to undergo an unprecedented pyrrole formation with primary amines in a chemical model system, a reaction that could be relevant in the biological environment and lead to the pyrrolation of lysine residues in the target proteins. The anticancer evaluation of these compounds revealed their promising activity against cancer cells displaying various forms of drug resistance, including resistance to proapoptotic agents. Mechanistic studies indicated that compared to the parent polygodial, which displays fixative general cytotoxic action against human cells, the C12-Wittig derivatives exerted their antiproliferative action mainly through cytostatic effects explaining their activity against apoptosis-resistant cancer cells. The possibility for an intriguing covalent modification of proteins through a novel pyrrole formation reaction, as well as useful activities against drug resistant cancer cells, make the described polygodial-derived chemical scaffold an interesting new chemotype warranting thorough investigation.

Na/K-ATPase as a target for anticancer drugs: studies with perillyl alcohol.

BACKGROUND: Na/K-ATPase (NKA) is inhibited by perillyl alcohol (POH), a monoterpene used in the treatment of tumors, including brain tumors. The NKA α1 subunit is known to be superexpressed in glioblastoma cells (GBM). This isoform is embedded in caveolar structures and is probably responsible for the signaling properties of NKA during apoptosis. In this work, we showed that POH acts in signaling cascades associated with NKA that control cell proliferation and/or cellular death. METHODS: NKA activity was measured by the amount of non-radioactive Rb(+) incorporation into cultured GBM cell lines (U87 and U251) and non-tumor cells (mouse astrocytes and VERO cells). Cell viability was measured by lactate dehydrogenase levels in the supernatants of POH-treated cells. Activated c-Jun N-terminal Kinase (JNK) and p38 were assessed by western blotting. Apoptosis was detected by flow cytometry and immunocytochemistry, and the release of interleukins was measured by ELISA. RESULTS: All four cell types tested showed a similar sensitivity for POH. Perillic acid (PA), the main metabolite of POH, did not show any effect on these cells. Though the cell viability decreased in a dose-dependent manner when cells were treated with POH, the maximum cytotoxic effect of PA obtained was 30% at 4 mM. 1.5 mM POH activated p38 in U87 cells and JNK in both U87 and U251 cells as well as mouse astrocytes. Dasatinib (an inhibitor of the Src kinase family) and methyl ß-cyclodextrin (which promotes cholesterol depletion in cell membranes) reduced the POH-induced activation of JNK1/2 in U87 cells, indicating that the NKA-Src complex participates in this mechanism. Inhibition of JNK1/2 by the JNK inhibitor V reduced the apoptosis of GBM cells that resulted from POH administration, indicating the involvement of JNK1/2 in programmed cell death. 1.5 mM POH increased the production of interleukin IL-8 in the U251 cell supernatant, which may indicate a possible strategy by which cells avoid the cytotoxic effects of POH. CONCLUSIONS: A signaling mechanism mediated by NKA may have an important role in the anti-tumor action of POH in GBM cells.