Overfeeding in the early postnatal period aggravates inflammation and hepatic insulin sensitivity in the 5α-dihydrotestosterone-induced animal model of PCOS.

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age and is closely associated with chronic low-grade inflammation and insulin resistance. To clarify the contribution of prepubertal weight gain to the development of insulin resistance in PCOS, we investigated the effects of early postnatal overfeeding on inflammatory and energy-sensing pathways as well as on markers of insulin signaling in the liver of the PCOS rat model. Methods: Obesity induced by overfeeding was achieved by reducing litter size, while the PCOS-like condition was developed by treatment with 5α-dihydrotestosterone (DHT). Western blot and qPCR were used to analyze the expression of pro-inflammatory transcription factors and cytokines, as well as markers of the energy sensing and insulin signaling pathways. Results: The results showed that hepatic insulin sensitivity was impaired only in DHT-treated rats raised in small litters, as evidenced by increased phosphorylation of IRS1 on Ser307 and decreased expression of total IRS1. Postnatal overfeeding stimulated JNK1 activation independent of hyperandrogenemia; nevertheless, the synergistic effect of both factors triggered NLRP3 activation and increased IL1ß expression in the small litter DHT-treated group. This pro-inflammatory state was accompanied by decreased activatory phosphorylation of AMPK and reduced levels of its protein targets. Conclusions: Overfeeding in the early postnatal period leads to a decrease in hepatic insulin sensitivity in the rat model of PCOS, which is associated with decreased activation of AMPK and stimulation of the hepatic NLRP3-IL1ß signaling pathway. Accordingly, the inhibition of NLRP3 activation could provide a basis for the development of new therapeutic strategies for the treatment of insulin resistance in women with PCOS.

Crocus sativus tepals extract suppresses subcutaneous adipose tissue hypertrophy and improves systemic insulin sensitivity in mice on high-fat diet.

Obesity is a pressing problem worldwide for which standard therapeutic strategies have limited effectiveness. The use of natural products seems to be a promising approach to alleviate obesity and its associated complications. The tepals of Crocus sativus (Cr) plant, usually wasted in saffron production, are an unexplored source of bioactive compounds. Our aim was to elucidate the mechanisms of Cr tepals extract in obesity by investigating its effects on adipocyte differentiation, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) hypertrophy, and lipid metabolism in an animal model of diet-induced obesity. To this end, mouse 3T3-F442A preadipocytes were treated with Cr tepals extract and the expression of adipocyte differentiation genes was determined. Caloric intake, body mass, triglycerides, systemic insulin sensitivity, histology, insulin signaling, and lipid metabolism in VAT and SAT were analyzed in mice fed a 60% fat diet for 14 weeks and treated orally with Cr tepals extract during the last 5 weeks of the diet. We demonstrated for the first time that Cr tepals extract inhibits adipocyte differentiation in vitro. The animal model confirmed that oral treatment with Cr tepals extract results in weight loss, improved systemic insulin sensitivity, lower triglycerides, and improved lipid peroxidation. The suppressive effect of Cr tepals extract on adipocyte hypertrophy and inflammation was observed only in SAT, which, together with preserved SAT insulin signaling, most likely contributed to improved systemic insulin sensitivity. Our results suggest the functionality of SAT as a possible target for the treatment of obesity and its complications.

Coumarins-lipophilic cations conjugates: Efficient mitocans targeting carbonic anhydrases.

Being aware of the need to develop more efficient therapies against cancer, herein we disclose an innovative approach for the design of selective antiproliferative agents. We have accomplished the conjugation of a coumarin fragment with lipophilic cations (triphenylphosphonium salts, guanidinium) for providing mitochondriotropic agents that simultaneously target also carbonic anhydrases IX and XII, involved in the development and progression of cancer. The new compounds prepared herein turned out to be strong inhibitors of carbonic anhydrases IX and XII of human origin (low-to-mid nM range), also endowed with high selectivity, exhibiting negligible activity towards cytosolic CA isoforms. Key interactions with the enzyme were analysed using docking and molecular dynamics simulations. Regarding their in vitro antiproliferative activities, an increase of the tether length connecting both pharmacophores led to a clear improvement in potency, reaching the submicromolar range for the lead compounds, and an outstanding selectivity towards tumour cell lines (S.I. up to >357). Cytotoxic effects were also analysed on MDR cell lines under hypoxic and normoxic conditions. Chemoresistance exhibited by phosphonium salts, and not by guanidines, against MDR cells was based on the fact that the former were found to be substrates of P-glycoprotein (P-gp), the pump responsible for extruding foreign chemicals; this situation was reversed by administrating tariquidar, a third generation P-gp inhibitor. Moreover, phosphonium salts provoked a profound depolarization of mitochondria membranes from tumour cells, thus probably compromising their oxidative metabolism. To gain insight into the mode of action of title compounds, continuous live cell microscopy was employed; interestingly, this technique revealed two different antiproliferative mechanisms for both families of mitocans. Whereas phosphonium salts had a cytostatic effect, blocking cell division, guanidines led to cell death via apoptosis.

Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells.

Lung cancer remains the leading cause of cancer death globally, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Multidrug resistance (MDR), often caused by ATP-binding cassette (ABC) transporters, represents a significant obstacle in the treatment of NSCLC. While genetic profiling has an important role in personalized therapy, functional assays that measure cellular responses to drugs are gaining in importance. We developed an automated microplate-based immunofluorescence assay for the evaluation of MDR markers ABCB1, ABCC1, and ABCG2 in cells obtained from NSCLC patients through high-content imaging and image analysis, as part of a functional diagnostic approach. This assay effectively discriminated cancer from non-cancer cells within mixed cultures, which is vital for accurate assessment of changes in MDR marker expression in different cell populations in response to anticancer drugs. Validation was performed using established drug-sensitive (NCI-H460) and drug-resistant (NCI-H460/R) NSCLC cell lines, demonstrating the assay's capacity to distinguish and evaluate different MDR profiles. The obtained results revealed wide-ranging effects of various chemotherapeutic agents on MDR marker expression in different patient-derived NSCLC cultures, emphasizing the need for MDR diagnostics in NSCLC. In addition to being a valuable tool for assessing drug effects on MDR markers in different cell populations, the assay can complement genetic profiling to optimize treatment. Further assay adaptations may extend its application to other cancer types, improving treatment efficacy while minimizing the development of resistance.

Oxidative Stress Linking Obesity and Cancer: Is Obesity a 'Radical Trigger' to Cancer?

Obesity is on the rise worldwide, and consequently, obesity-related non-communicable diseases are as well. Nutritional overload induces metabolic adaptations in an attempt to restore the disturbed balance, and the byproducts of the mechanisms at hand include an increased generation of reactive species. Obesity-related oxidative stress causes damage to vulnerable systems and ultimately contributes to neoplastic transformation. Dysfunctional obese adipose tissue releases cytokines and induces changes in the cell microenvironment, promoting cell survival and progression of the transformed cancer cells. Other than the increased risk of cancer development, obese cancer patients experience higher mortality rates and reduced therapy efficiency as well. The fact that obesity is considered the second leading preventable cause of cancer prioritizes the research on the mechanisms connecting obesity to cancerogenesis and finding the solutions to break the link. Oxidative stress is integral at different stages of cancer development and advancement in obese patients. Hypocaloric, balanced nutrition, and structured physical activity are some tools for relieving this burden. However, the sensitivity of simultaneously treating cancer and obesity poses a challenge. Further research on the obesity-cancer liaison would offer new perspectives on prevention programs and treatment development.

Decreased TSPAN14 Expression Contributes to NSCLC Progression.

Tspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients' samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression.

The Role of the Thioredoxin Detoxification System in Cancer Progression and Resistance.

The intracellular redox homeostasis is a dynamic balancing system between the levels of free radical species and antioxidant enzymes and small molecules at the core of cellular defense mechanisms. The thioredoxin (Trx) system is an important detoxification system regulating the redox milieu. This system is one of the key regulators of cells' proliferative potential as well, through the reduction of key proteins. Increased oxidative stress characterizes highly proliferative, metabolically hyperactive cancer cells, which are forced to mobilize antioxidant enzymes to balance the increase in free radical concentration and prevent irreversible damage and cell death. Components of the Trx system are involved in high-rate proliferation and activation of pro-survival mechanisms in cancer cells, particularly those facing increased oxidative stress. This review addresses the importance of the targetable redox-regulating Trx system in tumor progression, as well as in detoxification and protection of cancer cells from oxidative stress and drug-induced cytotoxicity. It also discusses the cancer cells' counteracting mechanisms to the Trx system inhibition and presents several inhibitors of the Trx system as prospective candidates for cytostatics' adjuvants. This manuscript further emphasizes the importance of developing novel multitarget therapies encompassing the Trx system inhibition to overcome cancer treatment limitations.

Novel TrxR1 Inhibitors Show Potential for Glioma Treatment by Suppressing the Invasion and Sensitizing Glioma Cells to Chemotherapy.

Currently, available glioblastoma (GBM) treatment remains ineffective, with relapse after initial response and low survival rate of GBM patients. The reasons behind limited capacities for GBM treatment are high tumor heterogeneity, invasiveness, and occurrence of drug resistance. Therefore, developing novel therapeutic strategies is of utmost importance. Thioredoxin reductase (TrxR) is a novel, promising target due to its overexpression in many cancer types and important role in cancer progression. Previous research on Ugi-type Michael acceptors-inhibitors of TrxR showed desirable anticancer properties, with significant selectivity toward cancer cells. Herein, two TrxR inhibitors, 5 and 6, underwent in-depth study on multidrug-resistant (MDR) glioma cell lines. Besides the antioxidative effects, 5 and 6 induced cell death, decreased cell proliferation, and suppressed invasion and migration of glioma cells. Both compounds showed a synergistic effect in combination with temozolomide (TMZ), a first-line chemotherapeutic for GBM treatment. Moreover, 5 and 6 affected activity of P-glycoprotein extrusion pump that could be found in cancer cells and in the blood-brain barrier (BBB), thus showing potential for suppressing MDR phenotype in cancer cells and evading BBB. In conclusion, investigated TrxR inhibitors are effective anticancer compounds, acting through inhibition of the thioredoxin system and perturbation of antioxidative defense systems of glioma cells. They are suitable for combining with other chemotherapeutics, able to surpass the BBB and overcome MDR. Thus, our findings suggest further exploration of Ugi-type Michael acceptors-TrxR inhibitors' potential as an adjuvant therapy for GBM treatment.

Further exploration of DVD-445 as a lead thioredoxin reductase (TrxR) inhibitor for cancer therapy: Optimization of potency and evaluation of anticancer potential.

A series of analogs of the earlier reported lead compound DVD-445 (thioredoxin reductase inhibitor with anticancer activity) has been synthesized via a modified Ugi reaction and investigated. Seven most potent compounds (with IC50 below 5.00 µM against recombinant rTrxR1 enzyme) were examined for their effect on cell growth and viability, oxidative stress induction and P-glycoprotein (P-gp) inhibition in human glioblastoma cells cell line U87 and its corresponding multidrug resistant (MDR) cell line U87-TxR. Several of these frontrunner compounds were shown to be superior over DVD-445. Besides providing promising candidates for anticancer therapy, our study further validates the small electrophilic Ugi Michael acceptor (UMA) chemotype as efficacious inhibitor of thioredoxin reductase.

Every-other-day feeding exacerbates inflammation and neuronal deficits in 5XFAD mouse model of Alzheimer's disease.

Food restriction has been widely associated with beneficial effects on brain aging and age-related neurodegenerative diseases such as Alzheimer's disease. However, previous studies on the effects of food restriction on aging- or pathology-related cognitive decline are controversial, emphasizing the importance of the type, onset and duration of food restriction. In the present study, we assessed the effects of preventive every-other-day (EOD) feeding regimen on neurodegenerative phenotype in 5XFAD transgenic mice, a commonly used mouse model of Alzheimer's disease. EOD feeding regimen was introduced to transgenic female mice at the age of 2 months and the effects on amyloid-ß (Aß) accumulation, gliosis, synaptic plasticity, and blood-brain barrier breakdown were analyzed in cortical tissue of 6-month-old animals. Surprisingly, significant increase of inflammation in the cortex of 5XFAD fed EOD mice was observed, reflected by the expression of microglial and astrocytic markers. This increase in reactivity and/or proliferation of glial cells was accompanied by an increase in proinflammatory cytokine TNF-α, p38 MAPK and EAAT2, and a decrease in GAD67. NMDA receptor subunit 2B, related to glutamate excitotoxicity, was increased in the cortex of 5XFAD-EOD mice indicating additional alterations in glutamatergic signaling. Furthermore, 4 months of EOD feeding regimen had led to synaptic plasticity proteins reduction and neuronal injury in 5XFAD mice. However, EOD feeding regimen did not affect Aß load and blood-brain barrier permeability in the cortex of 5XFAD mice. Our results demonstrate that EOD feeding regimen exacerbates Alzheimer's disease-like neurodegenerative and neuroinflammatory changes irrespective of Aß pathology in 5XFAD mice, suggesting that caution should be paid when using food restrictions in the prodromal phase of this neurodegenerative disease.

Novel Heat Shock Protein 90 Inhibitors Suppress P-Glycoprotein Activity and Overcome Multidrug Resistance in Cancer Cells.

Heat Shock Protein 90 (Hsp90) chaperone interacts with a broad range of client proteins involved in cancerogenesis and cancer progression. However, Hsp90 inhibitors were unsuccessful as anticancer agents due to their high toxicity, lack of selectivity against cancer cells and extrusion by membrane transporters responsible for multidrug resistance (MDR) such as P-glycoprotein (P-gp). Recognizing the potential of new compounds to inhibit P-gp function and/or expression is essential in the search for effective anticancer drugs. Eleven Hsp90 inhibitors containing an isoxazolonaphtoquinone core were synthesized and evaluated in two MDR models comprised of sensitive and corresponding resistant cancer cells with P-gp overexpression (human non-small cell lung carcinoma and colorectal adenocarcinoma). We investigated the effect of Hsp90 inhibitors on cell growth inhibition, P-gp activity and P-gp expression. Structure-activity relationship analysis was performed in respect to cell growth and P-gp inhibition. Compounds 5, 7, and 9 directly interacted with P-gp and inhibited its ATPase activity. Their potential P-gp binding site was identified by molecular docking studies. In addition, these compounds downregulated P-gp expression in MDR colorectal carcinoma cells, showed good relative selectivity towards cancer cells, while compound 5 reversed resistance to doxorubicin and paclitaxel in concentration-dependent manner. Therefore, compounds 5, 7 and 9 could be promising candidates for treating cancers with P-gp overexpression.

Novel electrophilic amides amenable by the Ugi reaction perturb thioredoxin system via thioredoxin reductase 1 (TrxR1) inhibition: Identification of DVD-445 as a new lead compound for anticancer therapy.

A series of peptidomimetic compounds incorporating an electrophilic moiety was synthesized using the Ugi reaction. These compounds (termed the Ugi Michael acceptors or UMAs) were designed to target the selenocysteine catalytic residue of thioredoxin reductase 1 (TrxR1), a promising cancer target. The compounds were assessed for their potential to inhibit TrxR1 using human neuroblastoma (SH-SY5Y) cell lysate. Based on this initial screening, six compounds were selected for testing against recombinant rat TrxR1 and in the insulin assay to reveal low-micromolar to submicromolar potency of these inhibitors. The same frontrunner compounds were evaluated for their ability to exert antiproliferative activity and induce cell death and this activity was compared to the UMA effects on the levels of reactive oxygen and nitrogen species (RONS). Collectively, the UMA compounds class presented itself as a rich source of leads for TrxR1 inhibitor discovery for anticancer application. Compound 7 (DVD-445) was nominated a lead for further optimization.

Sulfocoumarins, specific carbonic anhydrase IX and XII inhibitors, interact with cancer multidrug resistant phenotype through pH regulation and reverse P-glycoprotein mediated resistance.

New 6-triazolyl-substituted sulfocoumarins were described as potent inhibitors of the transmembrane human carbonic anhydrase isoforms, CAIX and CAXII. These membrane associated enzymes that maintain pH and CO2 homeostasis are involved in cancer progression, invasion, and resistance to therapy. Recently, it was shown that CAXII expression associates with the expression of P-glycoprotein in multidrug resistant cancer cells. CAXII regulates P-glycoprotein activity by maintaining high intracellular pHi. In this study, the activity of three new sulfocoumarins was evaluated in three sensitive and corresponding multidrug resistant cancer cell lines with increased P-glycoprotein expression (non-small cell lung carcinoma, colorectal carcinoma and glioblastoma). Compound 3 showed the highest potential for cancer cell growth inhibition in all tested cell lines. Flow cytometric analyses showed that compound 3 induced intracellular acidification, cell cycle arrest in G2/M phase and necrosis in non-small cell lung carcinoma cells. Compound 3 demonstrated irreversible, concentration- and time-dependent inhibition of P-glycoprotein activity in multidrug resistant non-small cell lung carcinoma cells. The suppression of P-glycoprotein activity was accompanied with increased P-glycoprotein expression suggesting a compensatory mechanism of multidrug resistant cancer cells. In addition, compound 3 was able to sensitize multidrug resistant non-small cell lung carcinoma cells to doxorubicin. Overall, results imply that compound 3 has multidrug resistance modulating effect through intracellular acidification and subsequent inhibition of P-glycoprotein activity.

Modulation of Antioxidant Potential with Coenzyme Q10 Suppressed Invasion of Temozolomide-Resistant Rat Glioma In Vitro and In Vivo.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity.

Design, synthesis, and biological evaluation of novel derivatives of dithiodiglycolic acid prepared via oxidative coupling of thiols.

Human thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing enzyme which plays a crucial role in regulating numerous redox signalling pathways within the cell. While its functioning is important in all cells, levels of TrxR1 expression are higher in cancer cells, possibly as an adaptation to much higher levels of reactive oxygen species and the need for more extensive DNA synthesis. This makes TrxR1 an attractive target for cancer therapy development. Inspired by the structure of disulphide compounds which have advanced through various stages of clinical development, we designed a series of dithiodiglycolic acid derivatives. These were prepared from respective thiol synthons using an iodine- or benzotriazolyl chloride-promoted oxidative disulphide bond formation. Inhibition of TrxR present in cell lysates from human neuroblastoma cells (SH-SY5Y) and rat liver cells indicated several compounds with a potential for TrxR inhibition. Some of these compounds were also tested for growth inhibition against two human cancer cell lines and normal human keratinocytes.

Early Impairments of Hippocampal Neurogenesis in 5xFAD Mouse Model of Alzheimer's Disease Are Associated with Altered Expression of SOXB Transcription Factors.

Dysregulation of neurogenesis in the subgranular zone (SGZ) of the hippocampal dentate gyrus has been related to cognitive deficits and memory loss in neurodegenerative diseases, such as Alzheimer's disease (AD). Members of the B group of SOX transcription factors play critical roles in regulating neurogenesis in the embryonic and adult nervous system, including maintaining the multipotency, renewal, and cell fate decision of neural stem/progenitor cells. The aim of the present study was to evaluate the expression patterns of selected SOXB proteins in the SGZ, of 8-week-old male and female 5xFAD mice, which represent a transgenic model of AD with a severe and very early development of amyloid pathology. Immunohistochemical analysis showed a significant decrease in the number of cells expressing SOX1, SOX2, and SOX21 transcription factors within the SGZ of 5xFAD mice in comparison to their non-transgenic counterparts which coincidences with reduced number of doublecortin immunoreactive immature neurons found in Tg males. Despite observed changes in expressional pattern of examined SOXB proteins, the proliferative capacity evaluated by the number of Ki-67 immunoreactive cells remained unaffected in transgenic mice of both genders. Based on our results, we suggest that SOXB proteins might be considered as new biomarkers for the detection of early impairments in adult neurogenesis in different animal models or/and new targets in human regenerative medicine.

A single high dose of dexamethasone increases GAP-43 and synaptophysin in the hippocampus of aged rats.

The administration of dexamethasone, a synthetic glucocorticoid receptor agonist, has been reported to modulate cognitive performance in both animals and humans. In the present study, we demonstrate the effects of a single high dose of dexamethasone on the expression and distribution of synaptic plasticity-related proteins, growth-associated protein-43 (GAP-43) and synaptophysin, in the hippocampus of 6-, 12-, 18- and 24-month-old rats. Acute dexamethasone treatment significantly altered the expression of GAP-43 at the posttranslational level by modulating the levels of phosphorylated GAP-43 and proteolytic GAP-43-3 fragment. The effect was the most pronounced in the hippocampi of the aged animals. The total GAP-43 protein was increased only in 24-month-old dexamethasone-treated animals, and was concomitant with a decrease in calpain-mediated proteolysis. Moreover, by introducing the gray level co-occurrence matrix method, a form of texture analysis, we were able to reveal the subtle differences in the expression pattern of both GAP-43 and synaptophysin in the hippocampal subfields that were not detected by Western blot analysis alone. Therefore, the current study demonstrates, through a novel combined approach, that dexamethasone treatment significantly affects both GAP-43 and synaptophysin protein expression in the hippocampus of aged rats.

Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites.

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 µM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3 % of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.