Ceramide Synthase 6 Is a Novel Target of Methotrexate Mediating Its Antiproliferative Effect in a p53-Dependent Manner

We previously reported that ceramide synthase 6 (CerS6) is elevated in response to folate stress in cancer cells, leading to enhanced production of C16-ceramide and apoptosis. Antifolate methotrexate (MTX), a drug commonly used in chemotherapy of several types of cancer, is a strong inhibitor of folate metabolism. Here we investigated whether this drug targets CerS6. We observed that CerS6 protein was markedly elevated in several cancer cell lines treated with MTX. In agreement with the enzyme elevation, its product C16-ceramide was also strongly elevated, so as several other ceramide species. The increase in C16-ceramide, however, was eliminated in MTX-treated cells lacking CerS6 through siRNA silencing, while the increase in other ceramides sustained. Furthermore, the siRNA silencing of CerS6 robustly protected A549 lung adenocarcinoma cells from MTX toxicity, while the silencing of another ceramide synthase, CerS4, which was also responsive to folate stress in our previous study, did not interfere with the MTX effect. The rescue effect of CerS6 silencing upon MTX treatment was further confirmed in HCT116 and HepG2 cell lines. Interestingly, CerS6 itself, but not CerS4, induced strong antiproliferative effect in several cancer cell lines if elevated by transient transfection. The effect of MTX on CerS6 elevation was likely p53 dependent, which is in agreement with the hypothesis that the protein is a transcriptional target of p53. In line with this notion, lometrexol, the antifolate inducing cytotoxicity through the p53-independent mechanism, did not affect CerS6 levels. We have also found that MTX induces the formation of ER aggregates, enriched with CerS6 protein. We further demonstrated that such aggregation requires CerS6 and suggests that it is an indication of ER stress. Overall, our study identified CerS6 and ceramide pathways as a novel MTX target

CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress

Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response

Metabolic Reprogramming by Folate Restriction Leads to a Less Aggressive Cancer Phenotype

Folate coenzymes are involved in biochemical reactions of one-carbon transfer, and deficiency of this vitamin impairs cellular proliferation, migration and survival in many cell types. Here the effect of folate restriction on mammary cancer was evaluated using three distinct breast cancer subtypes differing in their aggressiveness and metastatic potential: non-invasive basal-like (E-Wnt), invasive but minimally metastatic claudin-low (M-Wnt), and highly metastatic claudin-low (metM-Wntliver) cell lines, each derived from the same pool of MMTV-Wnt-1 transgenic mouse mammary tumors. NMR-based metabolomics was used to quantitate 41 major metabolites in cells grown in folate-free medium versus standard medium. Each cell line demonstrated metabolic reprogramming when grown in folate-free medium. In E-Wnt, M-Wnt and metM-Wntliver cells 12, 29, and 25 metabolites, respectively, were significantly different (p<0.05 and at least 1.5-fold change). The levels of eight metabolites (aspartate, ATP, creatine, creatine phosphate, formate, serine, taurine and β-alanine) were changed in each folate-restricted cell line. Increased glucose, decreased lactate, and inhibition of glycolysis, cellular proliferation, migration and invasion occurred in M-Wnt and metM-Wntliver cells (but not E-Wnt cells) grown in folate-free versus standard medium. These effects were accompanied by altered levels of several folate-metabolizing enzymes, indicating that the observed metabolic reprogramming may result from both decreased folate availability and altered folate metabolism. These findings reveal that folate restriction results in metabolic and bioenergetic changes and a less aggressive cancer cell phenotype

Is ALDH1L1 Elevated in Lung Cancer? Comment on: Lee, S.-H.; et al. “The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer” Cancers 2020, 12, 1382

We read with interest the article by Lee et al [...

Epigenetic Silencing of ALDH1L1, a Metabolic Regulator of Cellular Proliferation, in Cancers

FDH (10-formyltetrahydrofolate dehydrogenase, the product of the ALDH1L1 gene), a major folate-metabolizing enzyme in the cytosol, is involved in the regulation of cellular proliferation. We have previously demonstrated that FDH is strongly and ubiquitously down-regulated in malignant human tumors and cancer cell lines. Here, we report that promoter methylation is a major mechanism controlling FDH levels in human cancers. A computational analysis has identified an extensive CpG island in the ALDH1L1 promoter region. It contains 96 CpG pairs and covers the region between −525 and +918 bp of the ALDH1L1 gene including the promoter, the entire exon 1, and a part of intron 1 immediately downstream of the exon. Bisulfite sequencing analysis revealed extensive methylation of the island (76%-95% of CpGs) in cancer cell lines. In agreement with these findings, treatment of FDH-deficient A549 cells with the methyltransferase inhibitor 5-aza-2′-deoxycytidine restored FDH expression. Analysis of the samples from patients with lung adenocarcinomas demonstrated methylation of the ALDH1L1 CpG island in tumor samples and a total lack of methylation in respective normal tissues. The same phenomenon was observed in liver tissues: the CpG island was methylation free in DNA extracted from normal hepatocytes but was extensively methylated in a hepatocellular carcinoma. Levels of ALDH1L1 mRNA and protein correlated with the methylation status of the island, with tumor samples demonstrating down-regulation of expression or even complete silencing of the gene. Our studies have also revealed that exon 1 significantly increases transcriptional activity of ALDH1L1 promoter in a luciferase reporter assay. Interestingly, the exon is extensively methylated in samples with a strongly down-regulated or silenced ALDH1L1 gene

Substance abuse as compensation for stress factors involved in the performance of helping professions

THE ABSTRACT It has been shown recently that workload, stress, and burnout syndrome among the staff of the medical rescue service may be major risk factors in terms of triggering the use of psychoactive substances. Representing what is understandably a delicate issue, substance use among emergency medical staff has not been thoroughly studied in our country. Emergency medical workers' difficult working conditions and the chronic stress they are exposed to, in combination with a lack of support and care on the part of their employers, result in exhaustion and general distress, accompanied by the development of symptoms associated with both physical and mental disorders. This condition may lead to the use of psychoactive substances as a negative coping strategy. Consisting of both theoretical background and case studies, the paper points out the relationship between the chronic effect of stressors pertaining to the job of emergency medical workers and the use of psychoactive substances as a way of coping with and compensating for the implications of work-related stress and fatigue. Thorough case studies are presented to demonstrate the onset and development of addictive behaviour within a wider context, with special emphasis being placed on its association with coping with both acute and chronic occupational..

Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

The folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase, ALDH1L1), a metabolic regulator of proliferation, activates p53-dependent G1 arrest and apoptosis in A549 cells. In the present study, we have demonstrated that FDH-induced apoptosis is abrogated upon siRNA knockdown of the p53 downstream target PUMA. Conversely, siRNA knockdown of p21 eliminated FDH-dependent G1 arrest and resulted in an early apoptosis onset. The acceleration of FDH-dependent apoptosis was even more profound in another cell line, HCT116, in which the p21 gene was silenced through homologous recombination (p21−/− cells). In contrast to A549 cells, FDH caused G2 instead of G1 arrest in HCT116 p21+/+ cells; such an arrest was not seen in p21-deficient (HCT116 p21−/−) cells. In agreement with the cell cycle regulatory function of p21, its strong accumulation in nuclei was seen upon FDH expression. Interestingly, our study did not reveal DNA damage upon FDH elevation in either cell line, as judged by comet assay and the evaluation of histone H2AX phosphorylation. In both A549 and HCT116 cell lines, FDH induced a strong decrease in the intracellular ATP pool (2-fold and 30-fold, respectively), an indication of a decrease in de novo purine biosynthesis as we previously reported. The underlying mechanism for the drop in ATP was the strong decrease in intracellular 10-formyltetrahydrofolate, a substrate in two reactions of the de novo purine pathway. Overall, we have demonstrated that p21 can activate G1 or G2 arrest in the absence of DNA damage as a response to metabolite deprivation. In the case of FDH-related metabolic alterations, this response delays apoptosis but is not sufficient to prevent cell death

Methotrexate induces CerS6-dependent formation of ER membrane aggregates in A549 non-small cell lung carcinoma cells.

<p>(A) Treatment with MTX (10 nM) resulted in the increase of autophagosome number, but CerS6 was not associated with these organelles. (B) MTX (10 nM) induced aggregation of ER membranes and the accumulation of CerS6 in the aggregates. Arrows indicate ER membrane aggregates. Note the lack of such aggregates after lometrexol (1.0 μM) treatment. (C) MTX (10 nM) induced ER aggregation (detected by monitoring CALR-RFP) in the absence of exogenous CerS6. (D) A549 cells lacking CerS6 (siRNA silencing) did not form ER aggregates in response to MTX. Cells were co-transfected with GFP-CerS6 and LC3-RFP (panel A) or CALR-RFP (panel B), MTX was added 6 h after transfection and live cell images were captured 48 h later. In experiments with endogenous CerS6, cells were co-transfected with GFP and CALR-RFP. Six hours before co-transfection, cells were transfected with scrambled siRNA (<i>panel C</i>) or CerS6 siRNA (<i>panel D</i>). MTX was added 6 h after the second transfection and images were captured 48 h later. Pearson’s coefficient for co-localization (calculated using Fiji software) is indicated for each panel.</p