Search Results (68)

A significant challenge in cancer treatment is the rising problem of drug resistance that reduces the effectiveness of therapeutic strategies. Current knowledge shows that multiple mechanisms play a role in cancer drug resistance. Another mechanism that has gained attention is the alteration in sphingolipid trafficking and the dysregulation of its metabolism, which was reported to cause cancer-associated drug resistance. Sphingolipids are lipids containing sphingosine and have multiple roles, ranging from lipid raft formation, apoptosis, and cell signaling to immune cell trafficking. Recent studies show that in developing cancer cells, altered or dysregulated sphingolipids are associated with drug efflux and promote the survival of cancer cells by bypassing apoptosis. Upregulated levels of the glucosylceramide synthase (GCS), an enzyme that functions in sphingolipid metabolism, lead to the upregulated ABCB1 gene that induces drug efflux from the cancer cells. These bypass mechanisms make drugs that induce apoptosis in tumor cells ineffective. By highlighting the current findings, this review aims to provide a mechanism of drug resistance caused by the dysregulation of glucosylceramide synthase, sphingosine kinase, and acid ceramidase enzymes as possible therapeutic targets to enhance the effectiveness of the currently used chemotherapeutic agents. Full article

Ceramide is a critical molecule in both the physiology and pathology of the central nervous system. The most studied aspect is its effect on embryonic/stem cells. A salient question is whether low doses of ceramide induce neuronal differentiation without interfering with sphingolipid metabolism and whether high doses can be used in glioblastoma for their cytotoxic effect. Here, we examined the effect of a high dose of ceramide (13 µM) on HN9.10e cells. Interestingly, 13 µM ceramide induced an immediate increase in cell viability, followed by an increase in the number of mitochondria. Microscopic and morphometric analysis revealed a decrease in the number of differentiated cells with 13 µM compared to 0.1 µM but with longer neurites. Furthermore, the lipidomic study demonstrated an increase in the formation of medium–long-chain ceramide and sphingomyelin species and sphingosine 1 phosphate. Sphingolipid modification correlated with SMPD3, ASAH2, and SPHK2 gene expression coding for neutral sphingomyenase 2, ceramidase 2, and sphingosine kinase 2, respectively. Overall, our data show that the variety of responses to ceramide of the same cell type is dependent on the concentration used. Low doses do not affect sphingolipid metabolism, and high doses do so with a different cellular response. Full article

The existing body of literature, in conjunction with our recent studies, shows that melatonin dysfunction can accelerate the aging process, with this effect depending on the specific age of the subject. The present study aims to ascertain the impact of pinealectomy on sphingolipid (SL) turnover in young adult (3-month-old), middle-aged (14-month-old), and old (18-month-old) rats. Ceramide (Cer) levels, neutral (NSMase) and acid sphingomyelinase (ASMase), acid ceramidase (ASAH1), and sphingosine-1-phosphate (S1P) levels in hippocampus and/or plasma, were evaluated by enzyme-linked immunosorbent assay. The accumulation of Cer and its metabolite second messenger S1P in the hippocampus and plasma was associated with increased levels and activity of hippocampal NSMase in the hippocampus and plasma. However, no such association was observed for hippocampal ASMase, whose levels and activity were reduced in middle-aged and old rats compared to young adult rats. Pinealectomy-induced melatonin deficiency in young adult rats showed an increase in hippocampal Cer content compared to the sham group. However, in contrast to young adult rats, pinealectomy had an inverse effect on age-related changes in hippocampal Cer, NSMase, and ASMase in middle-aged rats. Furthermore, pinealectomy exacerbated the age-related increase in S1P in the hippocampus of 18-month-old rats. Collectively, the results of the present study suggest that melatonin deficiency may influence the aging process by modulating SL turnover in an age-specific manner. Full article

Background: The aim of our study was to determine the role of sphingolipids, which control proliferation and apoptosis, in the placenta of pregnant women with pregnancy-associated breast cancer (PABC) after chemotherapy compared with healthy patients. Methods: We analyzed (by the PCR method) the gene expression of key sphingolipid metabolism enzymes (sphingomyelinases (SMPD1 and SMPD3), acid ceramidase (ASAH1), ceramide synthases (CERS 1–6), sphingosine kinase1 (SPHK1), sphingosine-1-phosphate lyase 1 (SGPL1), and sphingosine-1-phosphate receptors (S1PR1, S1PR2, and S1PR3)) and the content of subspecies of ceramides, sphingosine, and sphingosine-1-phosphate in seven patients with PABC after chemotherapy and eight healthy pregnant women as a control group. Results: We found a significant increase in the expression of genes of acid ceramidase (ASAH1), sphingosine-1-phosphate lyase 1 (SGPL1), sphingosine kinase (SPHK1), and ceramide synthases (CERS 1-3, 5, 6) in the samples of patients with PABC during their treatment with cytostatic chemotherapy. The increase in the expression of the enzymes’ genes was not accompanied by changes in the content of the studied sphingolipids. Such significant changes in the expression of genes controlling the level of CER, sphingosine, and S1P may indicate their ability to initiate the metabolism of pro-apoptotic and anti-apoptotic sphingolipids in the placenta of pregnant women with cancer undergoing chemotherapy in order to maintain levels typical of the placenta of healthy women. Conclusions: Our results may indicate the promising mechanism of placenta protection during chemotherapy for pregnant women with breast cancer and, consequently, of the newborn. This protective effect of the placenta and especially for the newborn has been discovered for the first time and requires more careful study. Full article

Sphingolipids play a major role in the regulation of hepatocellular apoptosis and proliferation. We have previously identified sphingolipid metabolites as biomarkers of chronic liver disease and hepatocellular carcinoma. Human hepatocellular carcinoma cell lines were transfected with a plasmid vector encoding for acid sphingomyelinase. Overexpressing cells were subsequently treated with mitomycin and cell proliferation, acid sphingomyelinase activity, sphingolipid concentrations, and generation of reactive oxygen species were assessed. The stimulation of acid sphingomyelinase-overexpressing cell lines with mitomycin showed a significant activation of the enzyme (p < 0.001) followed by an accumulation of various ceramide species (p < 0.001) and reactive oxygen radicals (p < 0.001) as compared to control transfected cells. Consequently, a significant reduction in cell proliferation was observed in acid sphingomyelinase-overexpressing cells (p < 0.05) which could be diminished by the simultaneous application of antioxidant agents. Moreover, the application of mitomycin induced significant alterations in mRNA expression levels of ceramidases and sphingosine kinases (p < 0.05). Our data suggest that the overexpression of the acid sphingomyelinase in human hepatoma cell lines enhances the in vitro antiproliferative potential of mitomycin via accumulation of ceramide and reactive oxygen species. The selective activation of acid sphingomyelinase might offer a novel therapeutic approach in the treatment of hepatocellular carcinoma. Full article

Following a High-Throughput Screening campaign to discover inhibitors of acid ceramidase, we report the novel and extremely potent covalent inhibitor, 1. Following resynthesis and stability monitoring, we discovered that 1 is chemically unstable and reacts with DMSO at room temperature. This mode of decomposition is likely general for this class of compound, and we urge caution for their use in drug discovery research. Full article

Glial cell dysfunction results in myelin loss and leads to subsequent motor and cognitive deficits throughout the demyelinating disease course.Therefore, in various therapeutic approaches, significant attention has been directed toward glial-restricted progenitor (GRP) transplantation for myelin repair and remyelination, and numerous studies using exogenous GRP injection in rodent models of hypomyelinating diseases have been performed. Previously, we proposed the transplantation of canine glial-restricted progenitors (cGRPs) into the double-mutant immunodeficient, demyelinated neonatal shiverer mice (shiverer/Rag2^−/−). The results of our previous study revealed the myelination of axons within the corpus callosum of transplanted animals; however, the extent of myelination and lifespan prolongation depended on the transplantation site (anterior vs. posterior). The goal of our present study was to optimize the therapeutic effect of cGRP transplantation by using a multisite injection protocol to achieve a broader dispersal of donor cells in the host and obtain better therapeutic results. Experimental analysis of cGRP graft recipients revealed a marked elevation in myelin basic protein (MBP) expression and prominent axonal myelination across the brains of shiverer mice. Interestingly, the proportion of galactosyl ceramidase (GalC) positive cells was similar between the brains of cGRP recipients and control mice, implying a natural propensity of exogenous cGRPs to generate mature, myelinating oligodendrocytes. Moreover, multisite injection of cGRPs improved mice survival as compared to non-transplanted animals. Full article

Aim: Sphingolipids are a class of complex and bioactive lipids that are involved in the pathological processes of cardiovascular disease. Fabry disease is an X-linked storage disorder that results in the pathological accumulation of glycosphingolipids in body fluids and the heart. Cardiac dyssynchrony is observed in patients with Fabry disease and left ventricular (LV) hypertrophy. However, little information is available on the relationship between plasma sphingolipid metabolites and LV remodelling after acute myocardial infarction (AMI). The purpose of this study was to assess whether the baseline plasma sphingomyelin/acid ceramidase (aCD) ratio predicts LV dyssynchrony at 6M after AMI. Methods: A total of 62 patients with AMI undergoing primary angioplasty were recruited. Plasma aCD and sphingomyelin were measured prior to primary angioplasty. Three-dimensional echocardiographic measurements of the systolic dyssynchrony index (SDI) were performed at baseline and 6 months of follow-up. The patients were divided into three groups according to the level of aCD and sphingomyelin above or below the median. Group 1 denotes lower aCD and lower sphingomyelin; Group 3 denotes higher aCD and higher sphingomyelin. Group 2 represents different categories of patients with aCD and sphingomyelin. Trend analysis showed a significant increase in the SDI from Group 1 to Group 3. Logistic regression analysis showed that the sphingomyelin/aCD ratio was a significant predictor of a worsening SDI at 6 months. Conclusions: AMI patients with high baseline plasma sphingomyelin/aCD ratios had a significantly increased SDI at six months. The sphingomyelin/aCD ratio can be considered as a surrogate marker of plasma ceramide load or inefficient ceramide metabolism. Plasma sphingolipid pathway metabolism may be a new biomarker for therapeutic intervention to prevent adverse remodelling after MI. Full article

We previously reported that a pathogenic abnormality in the barrier and water-holding functions of the stratum corneum (SC) in the skin of patients with atopic dermatitis (AD) is mainly attributable to significantly decreased levels of total ceramides in the SC. That decrease is mediated by the abnormal expression of a novel ceramide-reducing enzyme, sphingomyelin/glucosylceramide deacylase (SGDase), which is the β-subunit (ASAH1b) of acid ceramidase. In this study, we determined whether mice overexpressing ASAH1b in their epidermis develop AD-like skin symptoms. We generated transgenic (TG) mice overexpressing ASAH1b, regulated by the involucrin promoter, to localize its expression in the upper epidermis. After hair removal using a depilatory cream containing glycolic acid, the TG mice without any visible skin inflammation at 8 weeks of age had increased levels of ASAH1b and decreased levels of SC ceramide, with disrupted barrier functions measured by trans-epidermal water loss compared to the wild-type (WT) mice. Interestingly, enzymatic assays revealed that SGDase activity was not detectable in the skin of the TG mice compared to WT mice. Immunological staining revealed that there was an increased expression level of IL-33 in the epidermis and an accumulation of macrophages in the dermis of TG mice compared to WT mice, which are phenotypic characteristics of AD, that were exacerbated by tape-stripping of the skin. In the skin of the TG mice, the mRNA levels of IL-5, CCL11, IL-22, CXCL10, and IFNγ were significantly upregulated compared to the WT mice, and tape-stripping significantly increased the mRNA levels of IL-4, IL-33, CXCL1, CXCL12, TLR9, and CD163 compared to WT mice. These findings strongly indicate that the skin of the depilatory cream-treated TG mice exists in an atopic dry skin condition that is highly sensitive to various environmental stimuli. The sum of our results suggests that ASAH1b itself, even in the absence of its enzymatic activity, is a major etiologic factor for atopic dry skin symptoms via an unknown mechanism. Full article

The progressive myoclonus epilepsies (PME) are a diverse group of disorders that feature both myoclonus and seizures that worsen gradually over a variable timeframe. While each of the disorders is individually rare, they collectively make up a non-trivial portion of the complex epilepsy and myoclonus cases that are seen in tertiary care centers. The last decade has seen substantial progress in our understanding of the pathophysiology, diagnosis, prognosis, and, in select disorders, therapies of these diseases. In this scoping review, we examine English language publications from the past decade that address diagnostic, phenotypic, and therapeutic advances in all PMEs. We then highlight the major lessons that have been learned and point out avenues for future investigation that seem promising. Full article

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell-death-promoting signaling lipid that plays a central role in therapy-induced cell death. We previously determined that acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug LCL-805 across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 μM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 μM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide. Full article

The objective of this study was to investigate whether the activity of enzymes involved in sphingolipid catabolism could be biomarkers to predict early renal damage in streptozotocin (STZ)-induced diabetic rats and Angiotensin II (Ang II)-induced hypertension rats. Diabetic and hypertensive rats had no changes in plasma creatinine concentration. However, transmission electron microscopy (TEM) analysis showed slight ultrastructural changes in the glomeruli and tubular epithelial cells from diabetic and hypertensive rats. Our results show that the acid sphingomyelinase (aSMase) and neutral sphingomyelinase (nSMase) activity increased in the urine of diabetic rats and decreased in hypertensive rats. Only neutral ceramidase (nCDase) activity increased in the urine of diabetic rats. Furthermore, the immunofluorescence demonstrated positive staining for the nSMase, nCDase, and sphingosine kinase (SphK1) in glomerular mesangial cells, proximal tubule, ascending thin limb of the loop of Henle, thick ascending limb of Henle’s loop, and principal cells of the collecting duct in the kidney. In conclusion, our results suggest that aSMase and nCDase activity in urine could be a novel predictor of early slight ultrastructural changes in the nephron, aSMase and nCDase as glomerular injury biomarkers, and nSMase as a tubular injury biomarker in diabetic and hypertensive rats. Full article

Tumor heterogeneity leads to drug resistance in cancer treatment with the crucial role of sphingolipids in cell fate and stress signaling. We analyzed sphingolipid metabolism and autophagic flux to study chemotherapeutic interactions on the A549 lung cancer model. Loaded cells with fluorescent sphingomyelin analog (BODIPY) and mCherry-EGFP-LC3B were used to track autophagic flux and assess cytotoxicity when cells are exposed to chemotherapy (epirubicin, cisplatin, and paclitaxel) together with sphingolipid pathway inhibitors and autophagy modulators. Our cell model approach employed fluorescent sphingolipid biosensors and a Gaussian Mixture Model of cell heterogeneity profiles to map the influence of chemotherapy on the sphingolipid pathway and infer potential synergistic interactions. Results showed significant synergy, especially when combining epirubicin with autophagy inducers (rapamycin and Torin), reducing cell viability. Cisplatin also synergized with a ceramidase inhibitor. However, paclitaxel often led to antagonistic effects. Our mapping model suggests that combining chemotherapies with autophagy inducers increases vesicle formation, possibly linked to ceramide accumulation, triggering cell death. However, the in silico model proposed ceramide accumulation in autophagosomes, and kinetic analysis provided evidence of sphingolipid colocalization in autophagosomes. Further research is needed to identify specific sphingolipids accumulating in autophagosomes. These findings offer insights into potential strategies for overcoming chemotherapy resistance by targeting the sphingolipid pathway. Full article

In Gaucher disease (GD), a relatively common sphingolipidosis, the mutant lysosomal enzyme acid β-glucocerebrosidase (GCase), encoded by the GBA1 gene, fails to properly hydrolyze the sphingolipid glucosylceramide (GlcCer) in lysosomes, particularly of tissue macrophages. As a result, GlcCer accumulates, which, to a certain extent, is converted to its deacylated form, glucosylsphingosine (GlcSph), by lysosomal acid ceramidase. The inability of mutant GCase to degrade GlcSph further promotes its accumulation. The amount of mutant GCase in lysosomes depends on the amount of mutant ER enzyme that shuttles to them. In the case of many mutant GCase forms, the enzyme is largely misfolded in the ER. Only a fraction correctly folds and is subsequently trafficked to the lysosomes, while the rest of the misfolded mutant GCase protein undergoes ER-associated degradation (ERAD). The retention of misfolded mutant GCase in the ER induces ER stress, which evokes a stress response known as the unfolded protein response (UPR). GD is remarkably heterogeneous in clinical manifestation, including the variant without CNS involvement (type 1), and acute and subacute neuronopathic variants (types 2 and 3). The present review discusses animal models developed to study the molecular and cellular mechanisms underlying GD. Full article

Acid ceramidase (AC) is a lysosomal enzyme required to hydrolyze ceramide to sphingosine by the removal of the fatty acid moiety. An inherited deficiency in this activity results in two disorders, Farber Lipogranulomatosis and spinal muscular atrophy with myoclonic epilepsy, leading to the accumulation of ceramides and other sphingolipids in various cells and tissues. In addition to ceramide hydrolysis, several other activities have been attributed to AC, including a reverse reaction that synthesizes ceramide from free fatty acids and sphingosine, and a deacylase activity that removes fatty acids from complex lipids such as sphingomyelin and glycosphingolipids. A close association of AC with another important enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM), has also been observed. Herein, we used a highly purified recombinant human AC (rhAC) and novel UPLC-based assay methods to investigate the recently described deacylase activity of rhAC against three sphingolipid substrates, sphingomyelin, galactosyl- and glucosylceramide. No deacylase activities were detected using this method, although we did unexpectedly identify a significant ASM activity using natural (C-18) and artificial (Bodipy-C12) sphingomyelin substrates as well as the ASM-specific fluorogenic substrate, hexadecanoylamino-4-methylumbelliferyl phosphorylcholine (HMU-PC). We showed that this ASM activity was not due to contaminating, hamster-derived ASM in the rhAC preparation, and that the treatment of ASM-knockout mice with rhAC significantly reduced sphingomyelin storage in the liver. However, unlike the treatment with rhASM, this did not lead to elevated ceramide or sphingosine levels. Full article