Search Results (16)

Endothelial–mesenchymal transition (EnMT) is the transversion of endothelial cells to mesenchymal cells under certain physiological or pathological conditions. When EnMT occurs in the corneal endothelium, corneal endothelial cells (CECs) lose their normal function and thus cannot maintain corneal clarity. Studies have shown that the mechanism of EnMT in CECs involves the transforming growth factor-β (TGF-β) signaling pathway, and one of the important inhibitors of the TGF-β/Smad2/3 pathway is sirtuin-1 (SIRT1). In this study, we used a rat model of corneal endothelium injury and TGF-β1-treated human CECs to induce EnMT, aiming to explore whether SIRT1 activation inhibits corneal EnMT in vivo and in vitro. SIRT1 was activated and suppressed using resveratrol (RSV) and EX527, respectively. The endothelial markers and mesenchymal markers were measured by immunofluorescence and Western blot assays. Co-immunoprecipitation was used to detect the interaction between SIRT1 and Smad2/3. The results showed that after mechanical injury, the group treated with RSV-activated SIRT1 regained corneal transparency and recovered from edema faster than the control group. Moreover, RSV-activated SIRT1 downregulated the expression levels of alpha smooth muscle actin (α-SMA), vimentin, and Snail and upregulated the expression levels of E-cadherin and Na⁺/K⁺-ATPase both in vivo and in vitro, but these effects were reversed when SIRT1 was inhibited by EX527. SIRT1 also upregulated the expression levels of TGF-β receptor 1 and phosphorylated Smad2/3. The interaction between SIRT1 and Smad2/3 in vitro was confirmed by co-immunoprecipitation. Overall, our results indicate that SIRT1 activation inhibits corneal EnMT via the TGF-β/Smad2/3 pathway, which may be a potential therapeutic target for corneal endothelium dysfunction. Full article

Since even low-level environmental exposure to cadmium (Cd) can lead to numerous unfavourable health outcomes, including damage to the nervous system, it is important to recognize the risk of health damage by this xenobiotic, the mechanisms of its toxic influence, and to find an effective protective strategy. This study aimed to evaluate, in a female Wistar rat model of current human environmental exposure to Cd (1 and 5 mg/kg of diet for 3–24 months), if the low-to-moderate treatment with this element can harm the brain and whether the supplementation with a 0.1% Aronia melanocarpa L. (Michx.) Elliott berries (chokeberries) extract (AE) can protect against this effect. The exposure to Cd modified the values of various biomarkers of neurotoxicity, including enzymes (acetylcholinesterase (AChE), sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase), phospholipase A2 (PLA2), and nitric oxide synthase 1 (NOS1)) and non-enzymatic proteins (calmodulin (CAM), nuclear factor erythroid 2-related factor 2 (Nrf2), and Kelch-like ECH-associated protein 1 (KEAP1)) crucial for the functioning of the nervous system, as well as the concentrations of calcium (Ca) and magnesium (Mg) and some metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the brain tissue. The co-administration of AE, partially or entirely, protected from most of the Cd-induced changes alleviating its neurotoxic influence. In conclusion, even low-level chronic exposure to Cd may adversely affect the nervous system, whereas the supplementation with A. melanocarpa berries products during the treatment seems a protective strategy. Full article

Currently, more than 55 million people around the world suffer from dementia, and Alzheimer’s Disease and Related Dementias (ADRD) accounts for nearly 60–70% of all those cases. The spread of Alzheimer’s Disease (AD) pathology and progressive neurodegeneration in the hippocampus and cerebral cortex is strongly correlated with cognitive decline in AD patients; however, the molecular underpinning of ADRD’s causality is still unclear. Studies of postmortem AD brains and animal models of AD suggest that elevated endoplasmic reticulum (ER) stress may have a role in ADRD pathology through altered neurocellular homeostasis in brain regions associated with learning and memory. To study the ER stress-associated neurocellular response and its effects on neurocellular homeostasis and neurogenesis, we modeled an ER stress challenge using thapsigargin (TG), a specific inhibitor of sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA), in the induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) of two individuals from our Mexican American Family Study (MAFS). High-content screening and transcriptomic analysis of the control and ER stress-challenged NSCs showed that the NSCs’ ER stress response resulted in a significant decline in NSC self-renewal and an increase in apoptosis and cellular oxidative stress. A total of 2300 genes were significantly (moderated t statistics FDR-corrected p-value ≤ 0.05 and fold change absolute ≥ 2.0) differentially expressed (DE). The pathway enrichment and gene network analysis of DE genes suggests that all three unfolded protein response (UPR) pathways, protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF-6), and inositol-requiring enzyme-1 (IRE1), were significantly activated and cooperatively regulated the NSCs’ transcriptional response to ER stress. Our results show that IRE1/X-box binding protein 1 (XBP1) mediated transcriptional regulation of the E2F transcription factor 1 (E2F1) gene, and its downstream targets have a dominant role in inducing G1/S-phase cell cycle arrest in ER stress-challenged NSCs. The ER stress-challenged NSCs also showed the activation of C/EBP homologous protein (CHOP)-mediated apoptosis and the dysregulation of synaptic plasticity and neurotransmitter homeostasis-associated genes. Overall, our results suggest that the ER stress-associated attenuation of NSC self-renewal, increased apoptosis, and dysregulated synaptic plasticity and neurotransmitter homeostasis plausibly play a role in the causation of ADRD. Full article

The relevant role played by the ATPase Inhibitory Factor 1 (IF1) as a physiological in vivo inhibitor of mitochondrial ATP synthase in cancer and non-cancer cells, and in the mitochondria of different mouse tissues, as assessed in different genetic loss- and gain-of-function models of IF1 has been extensively documented. In this review we summarize our findings and those of others that favor the implication of IF1 in metabolic reprogramming to an enhanced glycolytic phenotype, which is mediated by its binding and inhibition of the ATP synthase. Moreover, we emphasize that IF1 is phosphorylated in vivo in its S39 by the c-AMP-dependent PKA activity of mitochondria to render an inactive inhibitor that is unable to interact with the enzyme, thus triggering the activation of ATP synthase. Overall, we discuss and challenge the results that argue against the role of IF1 as in vivo inhibitor of mitochondrial ATP synthase and stress that IF1 cannot be regarded solely as a pro-oncogenic protein because in some prevalent carcinomas, it prevents metastatic disease. Full article

Inflamed and infected tissues can display increased local sodium (Na⁺) levels, which can have various effects on immune cells. In macrophages, high salt (HS) leads to a Na⁺/Ca²⁺-exchanger 1 (NCX1)-dependent increase in intracellular Na⁺ levels. This results in augmented osmoprotective signaling and enhanced proinflammatory activation, such as enhanced expression of type 2 nitric oxide synthase and antimicrobial function. In this study, the role of elevated intracellular Na⁺ levels in macrophages was investigated. Therefore, the Na⁺/K⁺-ATPase (NKA) was pharmacologically inhibited with two cardiac glycosides (CGs), ouabain (OUA) and digoxin (DIG), to raise intracellular Na⁺ without increasing extracellular Na⁺ levels. Exposure to HS conditions and treatment with both inhibitors resulted in intracellular Na⁺ accumulation and subsequent phosphorylation of p38/MAPK. The CGs had different effects on intracellular Ca²⁺ and K⁺ compared to HS stimulation. Moreover, the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) was not upregulated on RNA and protein levels upon OUA and DIG treatment. Accordingly, OUA and DIG did not boost nitric oxide (NO) production and showed heterogeneous effects toward eliminating intracellular bacteria. While HS environments cause hypertonic stress and ionic perturbations, cardiac glycosides only induce the latter. Cotreatment of macrophages with OUA and non-ionic osmolyte mannitol (MAN) partially mimicked the HS-boosted antimicrobial macrophage activity. These findings suggest that intracellular Na⁺ accumulation and hypertonic stress are required but not sufficient to mimic boosted macrophage function induced by increased extracellular sodium availability. Full article

Terpenes in Cannabis sativa exert analgesic effects, but the mechanisms are uncertain. We examined the effects of 10 terpenes on capsaicin responses in an established model of neuronal hypersensitivity. Adult rat DRG neurons cultured with neurotrophic factors NGF and GDNF were loaded with Fura2AM for calcium imaging, and treated with individual terpenes or vehicle for 5 min, followed by 1 µMol capsaicin. In vehicle treated control experiments, capsaicin elicited immediate and sustained calcium influx. Most neurons treated with terpenes responded to capsaicin after 6–8 min. Few neurons showed immediate capsaicin responses that were transient or normal. The delayed responses were found to be due to calcium released from the endoplasmic reticulum, as they were maintained in calcium/magnesium free media, but not after thapsigargin pre-treatment. Terpene inhibition of calcium influx was reversed after washout of medium, in the absence of terpenes, and in the presence of the Na⁺/K⁺ ATPase inhibitor ouabain, but not CB₁ or CB₂ receptor antagonists. Thus, terpenes inhibit capsaicin evoked calcium influx by Na⁺/K⁺ ATPase activation. Immunofluorescence showed TRPV1 co-expression with α1β1 Na⁺/K⁺ ATPase in most neurons while others were either TRPV1 or α1β1 Na⁺/K⁺ ATPase positive. Full article

Cancer poses a significant global health problem with profound personal and economic implications on National Health Care Systems. The reprograming of metabolism is a major trait of the cancer phenotype with a clear potential for developing effective therapeutic strategies to combat the disease. Herein, we summarize the relevant role that the mitochondrial ATP synthase and its physiological inhibitor, ATPase Inhibitory Factor 1 (IF1), play in metabolic reprogramming to an enhanced glycolytic phenotype. We stress that the interplay in the ATP synthase/IF1 axis has additional functional roles in signaling mitohormetic programs, pro-oncogenic or anti-metastatic phenotypes depending on the cell type. Moreover, the same axis also participates in cell death resistance of cancer cells by restrained mitochondrial permeability transition pore opening. We emphasize the relevance of the different post-transcriptional mechanisms that regulate the specific expression and activity of ATP synthase/IF1, to stimulate further investigations in the field because of their potential as future targets to treat cancer. In addition, we review recent findings stressing that mitochondria metabolism is the primary altered target in lung adenocarcinomas and that the ATP synthase/IF1 axis of OXPHOS is included in the most significant signature of metastatic disease. Finally, we stress that targeting mitochondrial OXPHOS in pre-clinical mouse models affords a most effective therapeutic strategy in cancer treatment. Full article

Cancer is a global health problem that 1 in 2–3 people can expect to experience during their lifetime. Several different modalities exist for cancer management, but all of these suffer from significant shortcomings in both diagnosis and therapy. Apart from developing completely new therapies, a viable way forward is to improve the efficacy of the existing modalities. One way is to combine these with each other or with other complementary approaches. An emerging latter approach is derived from ionic mechanisms, mainly ion channels and exchangers. We evaluate the evidence for this systematically for the main treatment methods: surgery, chemotherapy, radiotherapy and targeted therapies (including monoclonal antibodies, steroid hormones, tyrosine kinase inhibitors and immunotherapy). In surgery, the possible systemic use of local anesthetics to suppress subsequent relapse is still being discussed. For all the other methods, there is significant positive evidence for several cancers and a range of modulators of ionic mechanisms. This applies also to some of the undesirable side effects of the treatments. In chemotherapy, for example, there is evidence for co-treatment with modulators of the potassium channel (Kv11.1), pH regulation (sodium–hydrogen exchanger) and Na⁺-K⁺-ATPase (digoxin). Voltage-gated sodium channels, shown previously to promote metastasis, appear to be particularly useful for co-targeting with inhibitors of tyrosine kinases, especially epidermal growth factor. It is concluded that combining current orthodox treatment modalities with modulators of ionic mechanisms can produce beneficial effects including (i) making the treatment more effective, e.g., by lowering doses; (ii) avoiding the onset of resistance to therapy; (iii) reducing undesirable side effects. However, in many cases, prospective clinical trials are needed to put the findings firmly into clinical context. Full article

The mitochondrial synthesis of ATP makes a vital contribution to the growth and development of biological organisms, in which the enzyme mitochondrial F₁F₀-ATP synthase plays a pivotal role, in that it can either synthesize or hydrolyze cellular ATP. The finding of our previous study revealed that mitochondrial F₁F₀-ATPase inhibitor factor 1 (IF1) in Arabidopsis thaliana has a conserved function as an endogenous inhibitor affecting cellular energy status and plays an important role in plant growth and reproduction, particularly in fertility. In this study, to gain an insight into IF1-related traits, we performed isobaric tags for relative and absolute quantitation labeling analysis. In total, 67 of 4778 identified proteins were identified as differentially expressed proteins (DEPs; 59 up-regulated and 8 down-regulated) between wild-type and if1 mutant Arabidopsis thaliana seedlings. Gene ontology enrichment analysis revealed that these DEPs were the most significantly enriched in pathways such as “long-day photoperiodism, flowering,” “positive regulation of protein import into chloroplast stroma,” and “pollen sperm cell differentiation,” which are closely associated with reproductive development. Moreover, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that photosynthesis was the pathway most significantly enriched with DEPs. Collectively, our results revealed a global shift in protein abundance patterns corresponding to AtIF1 mutation, entailing changes in the abundance of multiple key proteins and metabolic processes, which will provide a valuable proteomic foundation for future studies. Full article

The most crucial function of corneal endothelial cells (CEnCs) is to maintain optical transparency by transporting excess fluid out of stroma. Unfortunately, CEnCs are not able to proliferate in vivo in the case of trauma or dystrophy. Visually impaired patients with corneal endothelial deficiencies that are waiting for transplantation due to massive global shortage of cadaveric corneal transplants are in a great need of help. In this study, our goal was to develop a defined, clinically applicable protocol for direct differentiation of CEnCs from human pluripotent stem cells (hPSCs). To produce feeder-free hPSC-CEnCs, we used small molecule induction with transforming growth factor (TGF) beta receptor inhibitor SB431542, GSK-3-specific inhibitor CHIR99021 and retinoic acid to guide differentiation through the neural crest and periocular mesenchyme (POM). Cells were characterized by the morphology and expression of human (h)CEnC markers with immunocytochemistry and RT-qPCR. After one week of induction, we observed the upregulation of POM markers paired-like homeodomain transcription factor 2 (PITX2) and Forkhead box C1 (FOXC1) and polygonal-shaped cells expressing CEnC-associated markers Zona Occludens-1 (ZO-1), sodium-potassium (Na⁺/K⁺)-ATPase, CD166, sodium bicarbonate cotransporter 1 (SLC4A4), aquaporin 1 (AQP1) and N-cadherin (NCAD). Furthermore, we showed that retinoic acid induced a dome formation in the cell culture, with a possible indication of fluid transport by the differentiated cells. Thus, we successfully generated CEnC-like cells from hPSCs with a defined, simple and fast differentiation method. Full article

Acute myeloid leukemia (AML) is among the top four malignancies in Saudi nationals, and it is the top leukemia subtype worldwide. Resistance to available AML drugs requires the identification of new targets and agents. Hsp90 is one of the emerging important targets in AML, which has a central role in the regulation of apoptosis and cell proliferation through client proteins including the growth factor receptors and cyclin dependent kinases. The objective of the first part of this study is to investigate the putative Hsp90 inhibition activity of three novel previously synthesized quinazolines, which showed HL60 cytotoxicity and VEGFR2 and EGFR kinases inhibition activities. Using surface plasmon resonance, compound 1 (HAA₂₀₂₀) showed better Hsp90 inhibition compared to 17-AAG, and a docking study revealed that it fits nicely into the ATPase site. The objective of the second part is to maximize the anti-leukemic activity of HAA₂₀₂₀, which was combined with each of the eleven standard inhibitors. The best resulting synergistic effect in HL60 cells was with the pan cyclin-dependent kinases (CDK) inhibitor dinaciclib, using an MTT assay. Furthermore, the inhibiting effect of the Hsp90α gene by the combination of HAA₂₀₂₀ and dinaciclib was associated with increased caspase-7 and TNF-α, leading to apoptosis in HL60 cells. In addition, the combination upregulated p27 simultaneously with the inhibition of cyclinD3 and CDK2, leading to abolished HL60 proliferation and survival. The actions of HAA₂₀₂₀ propagated the apoptotic and cell cycle control properties of dinaciclib, showing the importance of co-targeting Hsp90 and CDK, which could lead to the better management of leukemia. Full article

Lysosomal sequestration of anticancer therapeutics lowers their cytotoxic potential, reduces drug availability at target sites, and contributes to cancer resistance. Only recently has it been shown that lysosomal sequestration of weak base drugs induces lysosomal biogenesis mediated by activation of transcription factor EB (TFEB) which, in turn, enhances their accumulation capacity, thereby increasing resistance to these drugs. Here, we addressed the question of whether lysosomal biogenesis is the only mechanism that increases lysosomal sequestration capacity. We found that lysosomal sequestration of some tyrosine kinase inhibitors (TKIs), gefitinib (GF) and imatinib (IM), induced expansion of the lysosomal compartment. However, an expression analysis of lysosomal genes, including lysosome-associated membrane proteins 1, 2 (LAMP1, LAMP2), vacuolar ATPase subunit B2 (ATP6V1B2), acid phosphatase (ACP), and galactosidase beta (GLB) controlled by TFEB, did not reveal increased expression. Instead, we found that both studied TKIs, GF and IM, induced lysosomal fusion which was dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) mediated Ca²⁺signaling. A theoretical analysis revealed that lysosomal fusion is sufficient to explain the enlargement of lysosomal sequestration capacity. In conclusion, we demonstrated that extracellular TKIs, GF and IM, induced NAADP/Ca²⁺ mediated lysosomal fusion, leading to enlargement of the lysosomal compartment with significantly increased sequestration capacity for these drugs without apparent lysosomal biogenesis. Full article

Increasing evidences show that the ATPase Inhibitory Factor 1 (IF1), the physiological inhibitor of the ATP synthase, is overexpressed in a large number of carcinomas contributing to metabolic reprogramming and cancer progression. Herein, we show that in contrast to the findings in other carcinomas, the overexpression of IF1 in a cohort of colorectal carcinomas (CRC) predicts less chances of disease recurrence, IF1 being an independent predictor of survival. Bioinformatic and gene expression analyses of the transcriptome of colon cancer cells with differential expression of IF1 indicate that cells overexpressing IF1 display a less aggressive behavior than IF1 silenced (shIF1) cells. Proteomic and functional in vitro migration and invasion assays confirmed the higher tumorigenic potential of shIF1 cells. Moreover, shIF1 cells have increased in vivo metastatic potential. The higher metastatic potential of shIF1 cells relies on increased cFLIP-mediated resistance to undergo anoikis after cell detachment. Furthermore, tumor spheroids of shIF1 cells have an increased ability to escape from immune surveillance by NK cells. Altogether, the results reveal that the overexpression of IF1 acts as a tumor suppressor in CRC with an important anti-metastatic role, thus supporting IF1 as a potential therapeutic target in CRC. Full article

Background: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. Objectives and Methods: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study. Results: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. Conclusion: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE. Full article

Pathological osteolysis is commonly associated with osteoporosis, bone tumors, osteonecrosis, and chronic inflammation. It involves excessive resorption of bone matrix by activated osteoclasts. Suppressing receptor activator of NF-κB ligand (RANKL) signaling pathways has been proposed to be a good target for inhibiting osteoclast differentiation and bone resorption. Bajijiasu—a natural compound derived from Morinda officinalis F. C. How—has previously been shown to have anti-oxidative stress property; however, its effect and molecular mechanism of action on osteoclastogenesis and bone resorption remains unclear. In the present study, we found that Bajijiasu dose-dependently inhibited RANKL-induced osteoclast formation and bone resorption from 0.1 mM, and reached half maximal inhibitory effects (IC₅₀) at 0.4 mM without toxicity. Expression of RANKL-induced osteoclast specific marker genes including cathepsin K (Ctsk), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP), vacuolar-type H⁺-ATPase V0 subunit D2 (V-ATPase d2), and (matrix metalloproteinase-2 (MMP2) was inhibited by Bajijiasu treatment. Luciferase reporter gene studies showed that Bajijiasu could significantly reduce the expression and transcriptional activity of NFAT as well as RANKL-induced NF-κB activation in a dose-dependent manner. Further, Bajijiasu was found to decrease the RANKL-induced phosphorylation of extracellular signal-regulated kinases (ERK), inhibitor of κB-α (IκB-α), NFAT, and V-ATPase d2. Taken together, this study revealed Bajijiasu could attenuate osteoclast formation and bone resorption by mediating RANKL signaling pathways, indicative of a potential effect of Bajijiasu on osteolytic bone diseases. Full article