Ablation of Acid Ceramidase Impairs Autophagy and Mitochondria Activity in Melanoma Cells

Abstract

Cutaneous melanoma is often resistant to therapy due to its high plasticity, as well as its ability to metabolise chemotherapeutic drugs. Sphingolipid signalling plays a pivotal role in its progression and metastasis. One of the ways melanoma alters sphingolipid rheostat is via over-expression of lysosomal acid ceramidase (AC), which catalyses the hydrolysis of pro-apoptotic long-chain ceramides into sphingosine and fatty acid. In this report, we examine the role of acid ceramidase in maintaining cellular homeostasis through the regulation of autophagy and mitochondrial activity in melanoma cell lines. We show that under baseline conditions, wild-type melanoma cells had 3-fold higher levels of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3 II), compared to AC-null cells. This difference was further magnified after cell starvation. Moreover, we noticed autophagy impairment in A375 AC-null cells, possibly due to local accumulation of non-metabolized ceramides. Nonetheless, we observed that AC-null cells exhibited a significant increase in mitochondrial membrane potential compared to control cells. Consistent with this observation, we found that, after total starvation, ~30% of AC-null cells undergo apoptosis compared to ~6% of wild-type cells. As expected, AC transfection restored viability in A375 AC-null cells. Together, these findings suggest that AC-null melanoma cells change and adapt their metabolism to survive in the absence of AC, although in a way that does not allow them to cope with the stress of nutrient deprivation.

Palmitoylethanolamide (PEA) Inhibits SARS-CoV-2 Entry by Interacting with S Protein and ACE-2 Receptor

“…Around 140 fields per well per experiment were analyzed using a 63× water objective. To quantify the number of LDs, we used a detailed protocol described previously [ 32 , 33 ].…”

N-acylethanolamine acid amide hydrolase is a novel target for drugs against SARS-CoV-2 and Zika virus

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 9, 2022. ; https://doi.org/10.1101/2022.02.08.479661 doi: bioRxiv preprint AGTGGGTGCACGTGTTAATC. To isolate single-cell clones, cells were seeded in 96-well performing a single-cell limiting dilution protocol as previously described (22,23). Edited clones were selected by sequencing analysis of PCR products, using the following primers: F 5'-GAGGCTGCAGATTGAGTGAC-3', R 5'-TGGAGGTTTCTAGGCAAGCA-3'.…”

“…Cells were tested for mycoplasma contamination as previously described (41) CRISPR/Cas9 design and transfection A549 and Huh-7 cells were transfected with CRISPR/Cas9 RNP (IDT, Coralville, Iowa) by nucleoporation (NEON Electroporation System, Thermo Fisher, Massachusetts, USA) using the following parameters: 1200 V, 30 ms, 2 pulses using the sgRNA guide: AGTGGGTGCACGTGTTAATC. To isolate single-cell clones, cells were seeded in 96-well performing a single-cell limiting dilution protocol as previously described (22,23). Edited clones were selected by sequencing analysis of PCR products, using the following primers: F 5'-GAGGCTGCAGATTGAGTGAC-3', R 5'-TGGAGGTTTCTAGGCAAGCA-3'.…”

“…Analyses performed 24h after infection revealed that ZIKV NS1 co-localizes with NAAA in ~30% of cytoplasmic vesicles (Figure 2b-d). To determine the nature of these vesicles, we took advantage of a fluorescence-based autophagy assay (LC3-GFP RFP assay), where the autophagic marker LC3, expressed as a fusion protein with two fluorochromes, allows to distinguish autophagosomes (GFP+/RFP+ vesicles) from autolysosomes (GFP-/RFP+ vesicles) in transfected cells (22,23). In this assay, LC3-GFP fluorescence is quenched when pH drops due to lysosome-autophagosome fusion.…”

Ceramide Metabolism Enzymes—Therapeutic Targets against Cancer

“…Acid ceramidase (ASAH1) has been detected to be overexpressed in melanomas, conferring resistance to chemotherapy. The involvement of ASAH1 in mitochondrial function and cellular autophagy in melanoma cells has been observed [ 137 ]. Furthermore, a recent study has shown that deletion of the ASAH1 gene blocks the induction of apoptosis mediated by doxorubicin [ 138 ].…”