Search Results (104)

This study aimed to investigate the tyrosine kinase inhibitor Nintedanib and its potential role in reversing epithelial–mesenchymal transition (EMT) induced by transforming growth factor beta 2 (TGF-β2) in retinal pigment epithelial (RPE) cells, along with its therapeutic potential using a mouse model of subretinal fibrosis. We hypothesized that the blockade of angiogenesis promoting and fibrosis inducing signaling using the receptor tyrosine kinase inhibitor Nintedanib (OfevTM) can prevent or reverse EMT both in vitro and in our in vivo model of subretinal fibrosis. Primary human retinal pigment epithelial cells (phRPE) and adult retinal pigment epithelial cell line (ARPE-19) cells were treated with TGF-β210 ng/mL for two days followed by four days of Nintedanib (1 µM) incubation. Epithelial and mesenchymal phenotypes were assessed by morphological examination, quantitative real-time polymerase chain reaction(qPCR) (ZO-1, Acta2, FN, and Vim), and immunocytochemistry (ZO-1, vimentin, fibronectin, and αSMA). Metabolites were measured using luciferase-based assays. Extracellular acidification and oxygen consumption rates were measured using the Seahorse XF system. Metabolic-related genes (GLUT1, HK2, PFKFB3, CS, LDHA, LDHB) were evaluated by qPCR. A model of subretinal fibrosis using the two-stage laser-induced method in C57BL/6J mice assessed Nintedanib’s therapeutic potential. Fibro-vascular lesions were examined 10 days later via fluorescence angiography and immunohistochemistry. Both primary and ARPE-19 RPE stimulated with TGF-β2 upregulated expression of fibronectin, αSMA, and vimentin, and downregulation of ZO-1, consistent with morphological changes (i.e., elongation). Glucose consumption, lactate production, and glycolytic reserve were significantly increased in TGF-β2-treated cells, with upregulation of glycolysis-related genes (GLUT1, HK2, PFKFB3, CS). Nintedanib treatment reversed TGF-β2-induced EMT signatures, down-regulated glycolytic-related genes, and normalized glycolysis. Nintedanib intravitreal injection significantly reduced collagen-1+ fibrotic lesion size and Isolectin B4+ neovascularization and reduced vascular leakage in the two-stage laser-induced model of subretinal fibrosis. Nintedanib can induce Mesenchymal-to-Epithelial Transition (MET) in RPE cells and reduce subretinal fibrosis through metabolic reprogramming. Nintedanib can therefore potentially be repurposed to treat retinal fibrosis. Full article

Acute lung injury (ALI) is a fatal respiratory disease caused by excessive inflammation. Chelerythrine chloride (CH), an isoquinoline alkaloid, exhibits diverse biological activities. The research focused on assessing CH’s therapeutic effects against LPS-mediated ALI in mice and its underlying mechanisms. The anti-inflammatory effects of CH were evaluated both in LPS-induced RAW264.7 cells and ALI mouse model. An amount of 2.5 μM CH significantly inhibited the secretion of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in RAW264.7 cells. CH treatment notably mitigated the thickened alveolar septa and reduced edema in LPS-induced ALI in mice. Activity-based protein profiling (ABPP) technology was employed to identify the targets of CH. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was one of the direct targets of CH identified by ABPP. CH could downregulate the production of pyruvate. Furthermore, CH reduced the extracellular acidification rate (ECAR) while increasing the oxygen consumption rate (OCR) in LPS-stimulated RAW264.7 cells. All results suggest that CH mitigates LPS-induced ALI by targeting GAPDH and inhibiting glycolysis. This study reveals preliminary anti-inflammatory mechanisms of CH and its therapeutic potential for ALI. Full article

Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus and a leading cause of blindness in the working-age population. Current pharmacological treatments counteract DR’s later stages without targeting the earlier disease phases. Using computational approaches, our group previously identified the α1D and α2C adrenoceptors (α1DR and α2CR) as new putative drug targets for DR. Therefore, the aim of this work was to validate the role of these receptors in an in vitro model of DR, i.e., retinal pigmented epithelial cells (ARPE-19) challenged with high glucose (HG, 50 mM). We examined the effects of selective α1DR and α2CR agonists and antagonists on hyperglycemia-induced mitochondrial dysfunction and blood retinal barrier breakdown. Seahorse XFe was employed to assess the oxygen consumption rate and extracellular acidification rate. The integrity of the ARPE-19 barrier was evaluated through transepithelial electrical resistance measurements and a sodium fluorescein permeability test. α1DR pharmacological modulation through the α1DR antagonist BMY 7378 (0.1–1 µM, 24 h), but not α2CR, significantly attenuated HG-induced mitochondrial dysfunction. BMY 7378 (0.1–1 µM, 48 h) also prevented HG-mediated damage to retinal epithelial integrity. In contrast, the α1DR agonist phenylephrine (1–10 μM, 24 h) further reduced ARPE-19 mitochondrial activity compared to HG, indicating that α1D activation is directly implicated in DR-mediated mitochondrial dysfunction. In conclusion, the current in vitro study validated α1DR as a pharmacological target for DR. Full article

This study introduces novel CD44-targeted and redox-responsive nanoparticles (FNPs), proposed as doxorubicin (DOX) delivery devices for breast cancer. A cationized and redox-responsive Human Serum Albumin derivative was synthesized by conjugating Human Serum Albumin with cystamine moieties and then ionically complexing it with HA. The suitability of FNPs for cancer therapy was assessed through physicochemical measurements of size distribution (mean diameter of 240 nm), shape, and zeta potential (15.4 mV). Nanoparticles possessed high DOX loading efficiency (90%) and were able to trigger the drug release under redox conditions of the tumor environment (55% release after 2 h incubation). The use of the carrier increased the cytotoxic effect of DOX by targeting the CD44 protein. It was shown that, upon loading, the cytotoxic effect of DOX was enhanced in relation to CD44 protein expression in both 2D and 3D models. DOX@FNPs significantly decrease cellular metabolism by reducing both oxygen consumption and extracellular acidification rates. Moreover, they decrease the expression of proteins involved in the oxidative phosphorylation pathway, consequently reducing cellular viability and motility, as well as breast cancer stem cells and spheroid formation, compared to free DOX. This new formulation could become pioneering in reducing chemoresistance phenomena and increasing the specificity of DOX in breast cancer patients. Full article

α-glucosidase, a pharmacological target for type 2 diabetes mellitus (T2DM), is present in the intestinal brush border membrane and catalyzes the hydrolysis of sugar linkages during carbohydrate digestion. Since α-glucosidase inhibitors (AGIs) modulate intestinal metabolism, they may influence oxidative stress and glycolysis inhibition, potentially addressing intestinal dysfunction associated with T2DM. Herein, we report on a study of an ortho-carbonyl substituted hydroquinone series, whose members differ only in the number and position of methyl groups on a common scaffold, on radical-scavenging activities (ORAC assay) and correlate them with some parameters obtained by density functional theory (DFT) analysis. These compounds’ effect on enzymatic activity, their molecular modeling on α-glucosidase, and their impact on the mitochondrial respiration and glycolysis of the intestinal Caco-2 cell line were evaluated. Three groups of compounds, according their effects on the Caco-2 cells metabolism, were characterized: group A (compounds 2, 3, 5, 8, 9, and 10) reduces the glycolysis, group B (compounds 1 and 6) reduces the basal mitochondrial oxygen consumption rate (OCR) and increases the extracellular acidification rate (ECAR), suggesting that it induces a metabolic remodeling toward glycolysis, and group C (compounds 4 and 7) increases the glycolysis lacking effect on OCR. Compounds 5 and 10 were more potent as α-glucosidase inhibitors (AGIs) than acarbose, a well-known AGI with clinical use. Moreover, compound 5 was an OCR/ECAR inhibitor, and compound 10 was a dual agent, increasing the proton leak-driven OCR and inhibiting the maximal electron transport flux. Additionally, menadione-induced ROS production was prevented by compound 5 in Caco-2 cells. These results reveal that slight structural variations in a hydroquinone scaffold led to diverse antioxidant capability, α-glucosidase inhibition, and the regulation of mitochondrial bioenergetics in Caco-2 cells, which may be useful in the design of new drugs for T2DM and metabolic syndrome. Full article

Autism spectrum disorder (ASD) is associated with multiple physiological abnormalities. Current laboratory and clinical evidence most commonly report mitochondrial dysfunction, oxidative stress, and immunological imbalance in almost every cell type of the body. The present work aims to evaluate oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and inflammation-related molecules such as Cyclooxygenase-2 (COX-2), chitinase 3-like protein 1 (YKL-40), Interleukin-1 beta (IL-1β), Interleukin-9 (IL-9) in ASD children with and without regression compared to healthy controls. Children with ASD (n = 56) and typically developing children (TDC, n = 12) aged 1.11 to 11 years were studied. Mitochondrial activity was examined in peripheral blood mononuclear cells (PBMCs) isolated from children with ASD and from the control group, using a metabolic analyzer. Gene and protein levels of IL-1β, IL-9, COX-2, and YKL-40 were investigated in parallel. Our results showed that PBMCs of the ASD subgroup of regressed patients (ASD R(+), n = 21) had a specific pattern of mitochondrial activity with significantly increased maximal respiration, respiratory spare capacity, and proton leak compared to the non-regressed group (ASD R(-), n = 35) and TDC. Furthermore, we found an imbalance in the studied proinflammatory molecules and increased levels in ASD R(-) proving the involvement of inflammatory changes. The results of this study provide new evidence for specific bioenergetic profiles of immune cells and elevated inflammation-related molecules in ASD. For the first time, data on a unique metabolic profile in ASD R(+) and its comparison with a random group of children of similar age and sex are provided. Our data show that mitochondrial dysfunction is more significant in ASD R(+), while in ASD R(-) inflammation is more pronounced. Probably, in the group without regression, immune mechanisms (immune dysregulation, leading to inflammation) begin initially, and at a later stage mitochondrial activity is also affected under exogenous factors. On the other hand, in the regressed group, the initial damage is in the mitochondria, and perhaps at a later stage immune dysfunction is involved. Full article

The MAPK signaling pathway with BRAF mutations has been shown to drive the pathogenesis of 40–60% of melanomas. Inhibitors of this pathway’s BRAF and MEK components are currently used to treat these malignancies. However, responses to these treatments are not always successful. Therefore, identifying noninvasive biomarkers to predict treatment responses is essential for personalized medicine in melanoma. Using noninvasive ¹H magnetic resonance spectroscopy (¹H MRS), we previously showed that BRAF inhibition reduces lactate and alanine tumor levels in the early stages of effective therapy and could be considered as metabolic imaging biomarkers for drug response. The present work demonstrates that these metabolic changes observed by ¹H MRS and those assessed by ³¹P MRS are also found in preclinical human melanoma models treated with MEK inhibitors. Apart from ¹H and ³¹P MRS, additional supporting in vitro biochemical analyses are described. Our results indicate significant early metabolic correlations with response levels to MEK inhibition in the melanoma models and are consistent with our previous study of BRAF inhibition. Given these results, our study supports the potential clinical utility of noninvasive MRS to objectively image metabolic biomarkers for the early prediction of melanoma’s response to MEK inhibition. Full article

The mechanistic target of rapamycin complex (mTORC) regulates protein synthesis and can be activated by branched-chain amino acids (BCAAs). mTORC has also been implicated in the regulation of mitochondrial metabolism and BCAA catabolism. Some speculate that mTORC overactivation by BCAAs may contribute to insulin resistance. The present experiments assessed the effect of mTORC activation on myotube metabolism and insulin sensitivity using the mTORC agonist MHY1485, which does not share structural similarities with BCAAs. Methods: C2C12 myotubes were treated with MHY1485 or DMSO control both with and without rapamycin. Gene expression was assessed using qRT-PCR and insulin sensitivity and protein expression by western blot. Glycolytic and mitochondrial metabolism were measured by extracellular acidification rate and oxygen consumption. Mitochondrial and lipid content were analyzed by fluorescent staining. Liquid chromatography-mass spectrometry was used to assess extracellular BCAAs. Results: Rapamycin reduced p-mTORC expression, mitochondrial content, and mitochondrial function. Surprisingly, MHY1485 did not alter p-mTORC expression or cell metabolism. Neither treatment altered indicators of BCAA metabolism or extracellular BCAA content. Conclusion: Collectively, inhibition of mTORC via rapamycin reduces myotube metabolism and mitochondrial content but not BCAA metabolism. The lack of p-mTORC activation by MHY1485 is a limitation of these experiments and warrants additional investigation. Full article

Terpinen-4-ol (T-4-O) is an important component of tea tree oil and has anti-inflammatory effects. Currently, there are very few studies on the mechanisms by which T-4-O improves lipopolysaccharide (LPS)-induced macrophage inflammation. In this study, LPS-stimulated mouse RAW264.7 macrophages were used as a model to analyze the effects of T-4-O on macrophage inflammatory factors and related metabolic pathways in an inflammatory environment. The results showed that T-4-O significantly decreased the expression levels of inflammatory cytokines induced by LPS. Cellular metabolism results showed that T-4-O significantly decreased the ratio of the extracellular acidification rate and oxygen consumption rate. Non-targeted metabolomics results showed that T-4-O mainly affected glutamine and glutamate metabolism and glycine, serine, and threonine metabolic pathways. qPCR results showed that T-4-O increased the transcript levels of GLS and GDH and promoted glutamine catabolism. Western blotting results showed that T-4-O inhibited the mTOR and IκB, thereby decreasing NF-κB activity. The overall results showed that T-4-O inhibited mTOR phosphorylation to promote glutamine metabolism and increased cell oxidative phosphorylation levels, thereby inhibiting the expression of LPS-induced inflammatory cytokines. Full article

Fructose is a commonly consumed monosaccharide implicated in developing several metabolic diseases. Previously, elevated branched-chain amino acids (BCAA) have been correlated with the severity of insulin resistance. Most recently, the effect of fructose consumption on the downregulation of BCAA catabolic enzymes was observed. Thus, this mechanistic study investigated the effects of physiologically attainable levels of fructose, both with and without concurrent insulin resistance, in a myotube model of skeletal muscle. Methods: C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values. Results: Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells. Discussion: Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance. Full article

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism. Full article

Advanced urothelial bladder cancer (UBC) patients are tagged by a dismal prognosis and high mortality rates, mostly due to their poor response to standard-of-care platinum-based therapy. Mediators of chemoresistance are not fully elucidated. This work aimed to study the metabolic profile of advanced UBC, in the context of cisplatin resistance. Three isogenic pairs of parental cell lines (T24, HT1376 and KU1919) and the matching cisplatin-resistant (R) sublines were used. A set of functional assays was used to perform a metabolic screening on the cells. In comparison to the parental sublines, a tendency was observed towards an exacerbated glycolytic metabolism in the cisplatin-resistant T24 and HT1376 cells; this glycolytic phenotype was particularly evident for the HT1376/HT1376R pair, for which the cisplatin resistance ratio was higher. HT1376R cells showed decreased basal respiration and oxygen consumption associated with ATP production; in accordance, the extracellular acidification rate was also higher in the resistant subline. Glycolytic rate assay confirmed that these cells presented higher basal glycolysis, with an increase in proton efflux. While the results of real-time metabolomics seem to substantiate the manifestation of the Warburg phenotype in HT1376R cells, a shift towards distinct metabolic pathways involving lactate uptake, lipid biosynthesis and glutamate metabolism occurred with time. On the other hand, KU1919R cells seem to engage in a metabolic rewiring, recovering their preference for oxidative phosphorylation. In conclusion, cisplatin-resistant UBC cells seem to display deep metabolic alterations surpassing the Warburg effect, which likely depend on the molecular signature of each cell line. Full article

Hepatocellular carcinoma (HCC), the main pathological type of liver cancer, is related to risk factors such as viral hepatitis, alcohol intake, and non-alcoholic fatty liver disease (NAFLD). The constitutive activation of the PI3K/AKT signaling pathway is common in HCC and has essential involvement in tumor progression. The serine/threonine kinase AKT has several downstream substrates, which have been implicated in the regulation of cellular metabolism. However, the contribution of each of the three AKT isoforms, i.e., AKT1, AKT2 and AKT3, to HCC metabolism has not been comprehensively investigated. In this study, we analyzed the functional role of AKT1, AKT2 and AKT3 in HCC metabolism. The overexpression of activated AKT1, AKT2 and AKT3 isoforms in the human HCC cell lines Hep3B and Huh7 resulted in higher oxygen consumption rate (OCR), ATP production, maximal respiration and spare respiratory capacity in comparison to vector-transduced cells. Vice versa, lentiviral vector-mediated knockdowns of each AKT isoform reduced OCR in both cell lines. Reduced OCR rates observed in the three AKT isoform knockdowns were associated with reduced extracellular acidification rates (ECAR) and reduced lactate production in both analyzed cell lines. Mechanistically, the downregulation of OCR by AKT isoform knockdowns correlated with an increased phosphorylation of the pyruvate dehydrogenase on Ser232, which negatively regulates the activity of this crucial gatekeeper of mitochondrial respiration. In summary, our data indicate that each of the three AKT isoforms is able to upregulate OCR, ECAR and lactate production independently of each other in human HCC cells through the regulation of the pyruvate dehydrogenase. Full article

Background: Defects in apoptosis regulation are one of the classical features of cancer cells, often associated with more aggressiveness and failure to therapeutic options. We investigated the combinatorial antitumor effects of a natural product, physachenolide C (PCC) and bortezomib, in KRAS^mut/P53^mut lung cancer cells and xenograft mice models. Methods: The in vitro anticancer effects of the bortezomib and PCC combination were investigated using cell viability, migration, and invasion assays in 344SQ, H23, and H358 cell lines. Furthermore, the effects of combination treatment on the critical parameters of cellular metabolism, including extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation based on the oxygen consumption rate of cancer cells were assessed using Seahorse assay. Finally, the antitumor effect of the bortezomib (1 mg/kg) and PCC (10 mg/kg) combination was evaluated using xenograft mice models. Results: Our data showed that the bortezomib–PCC combination was more effective in reducing the viability of lung cancer cells in comparison with the individual treatments. Similarly, the combination treatment showed a significant inhibition of cell migration and invasion of cancer cells. Additionally, the key anti-apoptotic protein c-FLIP was significantly inhibited along with a substantial reduction in the key parameters of cellular metabolism in cancer cells. Notably, the bortezomib or PCC inhibited the tumor growth compared to the control group, the tumor growth inhibition was much more effective when bortezomib was combined with PCC in tumor xenograft mice models. Conclusion: These findings demonstrate that PCC sensitizes cancer cells to bortezomib, potentially improving the antitumor effects against KRAS^mut/P53^mut lung cancer cells, with an enhanced efficacy of combination treatments without causing significant side effects. Full article

The application of doxorubicin (DOX) is hampered by cardiotoxicity, with diastolic dysfunction as the earliest manifestation. Fibrosis leads to impaired relaxation, but the mechanisms that operate shortly after DOX exposure are not clear. We asked whether the activation of cardiac fibroblasts (CFs) anticipates myocardial dysfunction and evaluated the effects of DOX on CF metabolism. CFs were isolated from the hearts of rats after the first injection of DOX. In another experiment, CFs were exposed to DOX in vitro. Cell phenotype and metabolism were determined. Early effects of DOX consisted of diastolic dysfunction and unchanged ejection fraction. Markers of pro-fibrotic remodeling and evidence of CF transformation were present immediately after treatment completion. Oxygen consumption rate and extracellular acidification revealed an increased metabolic activity of CFs and a switch to glycolytic energy production. These effects were consistent in CFs isolated from the hearts of DOX-treated animals and in naïve CFs exposed to DOX in vitro. The metabolic switch was paralleled with the phenotype change of CFs that upregulated markers of myofibroblast differentiation and the activation of pro-fibrotic signaling. In conclusion, the metabolic switch and activation of CFs anticipate DOX-induced damage and represent a novel target in the early phase of anthracycline cardiomyopathy. Full article