Dipeptidyl peptidase-IV (DPP-IV), calcium-sensing receptor (CaSR), telomerase, cell cycle regulators (Rb, Cdk2). Ceramide (endogenous long chain species such as C₁₆:₀ and C₂₄:₁) mediates growth inhibition, G₀/G₁ cell cycle arrest, and telomerase inhibition in A549 lung adenocarcinoma cells. It also acts as a competitive inhibitor of DPP-IV and activates CaSR to impart kokumi taste. No specific IC₅₀ or Kᵢ values were reported for ceramide in these studies. [1,2]
Endogenous metabolite

Endogenous ceramide generation in A549 cells: Treatment of A549 human lung adenocarcinoma cells with exogenous D-erythro-C₆-ceramide (20 μM, 24 h) resulted in a 5- to 6-fold increase in endogenous long chain ceramides (mainly C₁₆:₀ and C₂₄:₁). This increase was inhibited by fumonisin B1 (FB1, 50 μM), an inhibitor of CoA-dependent ceramide synthase, but not by myriocin (MYR, 50 nM), an inhibitor of serine palmitoyltransferase (de novo pathway). [1]
Mechanism of endogenous ceramide generation: Using dual-labeled C₆-ceramide ([sphingosine-3-³H]D-erythro-C₆-ceramide and N-[N-hexanoyl-1-¹⁴C]D-erythro-C₆-ceramide), it was demonstrated that the sphingosine backbone, but not the fatty acid chain, was incorporated into newly synthesized long chain ceramides. This incorporation was blocked by FB1, indicating a salvage pathway involving deacylation (by ceramidase) and reacylation (by ceramide synthase). [1]
Stereospecificity and Golgi requirement: The generation of endogenous long chain ceramide was stereospecific, occurring only with D-erythro-C₆-ceramide and not with L-erythro-C₆-ceramide or D-erythro-C₆-dihydroceramide. This process was completely blocked by brefeldin A (10 μg/mL), which causes Golgi disassembly, suggesting a requirement for intact Golgi apparatus. [1]
Biological activities mediated by endogenous ceramide: D-erythro-C₆-ceramide (20 μM, 24 h) induced G₀/G₁ cell cycle arrest, growth inhibition (~50%), and telomerase inhibition in A549 cells. These effects were blocked by FB1, demonstrating that endogenous long chain ceramide mediates these responses. L-erythro-C₆-ceramide and D-erythro-C₆-dihydroceramide, which did not generate endogenous ceramide, had no effect. [1]
Apoptosis in HL-60 cells: Both D-erythro- and L-erythro-C₆-ceramides were equally effective in inducing apoptosis in HL-60 human myeloid leukemia cells, with IC₅₀ values of 11 μM and 13 μM, respectively, at 24 h. This effect was not dependent on the generation of endogenous long chain ceramide. [1]
Skin barrier lipids in atopic dermatitis: In human epidermis, protein-bound ω-hydroxyceramides (ω-OH-Cer) accounted for 46-53 wt% of total protein-bound lipids in healthy skin, but decreased to 23-28 wt% in nonlesional and 10-25 wt% in lesional atopic skin. Free very long chain fatty acids (VLCFA, >C24) were reduced to 25% of control in lesional areas. Metabolic labeling with [¹⁴C]-serine showed that de novo synthesis of free ceramides and glucosylceramides was decreased by 46% and 77%, respectively, in lesional atopic skin compared to healthy controls. [2]
In the A549 human lung cancer cell line, endogenous ceramide (generated by bacterial sphingomyelinase overexpression or daunorubicin therapy) suppresses telomerase reverse transcriptase mRNA production via deactivating the c-Myc transcription factor and telomerase activity[1]. The biochemical recycling of the sphingosine backbone of C6-ceramide is necessary for the continuous production of long-chain endogenous ceramides. This process entails the deacylation and reacylation of ceramides to produce endogenous long-chain ceramides, primarily C16:0- and C24:1-ceramide. The most likely source of these ceramides is CoA-dependent ceramide synthase, which fumonisin B1 inhibits. Long-chain endogenous ceramide synthesis in A549 cells mediates the effects of exogenous C6-ceramide on telomerase activity modulation, cell cycle arrest, and growth suppression [1].

Diacylglycerol kinase (DGK) assay for ceramide quantitation: Total endogenous ceramide levels were measured using the DGK method. Total cellular lipids were extracted, dried, and resuspended. The DGK assay utilized E. coli diacylglycerol kinase to phosphorylate ceramide and diacylglycerol in the presence of radiolabeled ATP. The phosphorylated products (ceramide phosphate and phosphatidic acid) were separated by TLC, identified by comparison with standards, and quantified by scintillation counting. Results were normalized to internal phosphate levels. [1]
HPLC/MS analysis of ceramide subspecies: Endogenous ceramide species (C₁₂–C₂₆) were analyzed by normal phase HPLC coupled to atmospheric pressure chemical ionization mass spectrometry. Separations were performed using a ThermoFinnigan LCQ ion trap mass spectrometer. [1]
DPP-IV inhibition assay: DPP-IV inhibitory activity was measured using a fluorometric screening kit. Assays were performed in black-walled 96-well plates with DPP-IV assay buffer (20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl and 1 mM EDTA). Fluorescence change at 360/460 nm was monitored over 30 minutes. IC₅₀ values were calculated by logarithmic regression analysis. [1]
Ceramide biosynthesis in epidermis: Freshly prepared human epidermis was floated on MCDB 153 medium containing [³-¹⁴C]-serine (1 μCi/mL) and incubated for 24 h at 37°C in 5% CO₂. After incubation, lipids were extracted, separated by TLC, and incorporated radioactivity was quantified by liquid scintillation counting and visualized by phosphoimager analysis. [2]
Fatty acid analysis by gas chromatography: Free extractable fatty acids were methylated with diazomethane and analyzed by capillary gas chromatography using an HP-1 column (polydimethylsiloxane). The temperature program was: 4 min at 150°C, then increased to 250°C at 4°C/min, maintained at 250°C for 10 min. Fatty acid methyl esters were detected by flame ionization detection and identified by comparison with reference standards. [2]

Cell viability (MTT and trypan blue): A549 cells (5 × 10³ cells/well in 96-well plates) were treated with increasing concentrations of C₆-ceramide for 72 h, then MTT (25 μL) was added for 4-5 h. After lysis, absorbance was read at 570 nm. For trypan blue exclusion, cells (1 × 10⁵) were treated in 6-well plates for 24 h, then counted using a hematocytometer with trypan blue solution. [1]
Cell cycle analysis by flow cytometry: A549 cells treated with C₆-ceramide (20 μM, 24 h) were stained with propidium iodide (50 μg/mL) after RNase treatment, and DNA content was analyzed by flow cytometry (15,000 events per sample). [1]
Telomerase activity (TRAP assay): Telomerase activity was measured using the TRAPeze kit. Cell extracts (50-100 ng protein) were added to TRAP reaction mixture containing dNTPs, TS primer, reverse primer mix, and Taq polymerase. Extended products were amplified by PCR (94°C for 30 s, 60°C for 30 s, 27 cycles). Products were separated by 12.5% acrylamide gel electrophoresis and visualized by autoradiography. [1]
Western blot analysis (PARP cleavage): Total protein (50 μg/lane) was separated by 4-15% SDS-PAGE, transferred to Immobilon membrane, and probed with rabbit polyclonal anti-PARP antibody (1 μg/mL) and peroxidase-conjugated secondary antibody (1:2500). [1]
Metabolic labeling with [³H]palmitate: A549 cells (1 × 10⁵) were grown in 6-well plates with [³H]palmitate (1 μCi/mL) with or without 20 μM C₆-ceramide for various time points. Lipids were extracted by the Bligh and Dyer method and separated by TLC using chloroform/methanol/2× NH₄OH (40:10:1). [1]

[1]. Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line. Role for endogenous ceramide in mediating the action of exogenous ceramide. J Biol Chem. 2002 Apr 12;277(15):12960-9.

[2]. Deficiency of epidermal protein-bound omega-hydroxyceramides in atopic dermatitis. J Invest Dermatol. 2002 Jul;119(1):166-73.

N-Oleoyl Phytosphingosine is a phytoceramide with an N-acyl group of oleoyl (9Z-octadecenoyl). Functionally, it is related to oleic acid. Ceramide NP is a lipid molecule belonging to the ceramide class of lipid molecules. Ceramides are the main lipid components of the stratum corneum of human skin. Ceramide 3 is formed by the N-acylation of a phytosphingosine backbone with a saturated fatty acid (stearic acid). It is widely used as a moisturizer in various cosmetics and personal care products. Ceramide 3 works synergistically with ceramide 1 to enhance the skin barrier function. See also: Ceramide 3 (note moved here).

C46H82N2O23

1031.14288

581.538

100403-19-8

57378373

Off-white to light yellow solid powder

12.4

4

4

32

41

568

3

CCCCCCCCCCCCC/C=C/[C@H]([C@H](COC1O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O[C@@H]3O[C@H](CO)[C@@H](O[C@@H]4O[C@H](CO)[C@H](O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3NC(=O)C)[C@H]2O)[C@H](O)[C@H]1O)NC(=O)C)O

ATGQXSBKTQANOH-UWVGARPKSA-N

InChI=1S/C36H71NO4/c1-3-5-7-9-11-13-15-17-18-19-21-23-25-27-29-31-35(40)37-33(32-38)36(41)34(39)30-28-26-24-22-20-16-14-12-10-8-6-4-2/h17-18,33-34,36,38-39,41H,3-16,19-32H2,1-2H3,(H,37,40)/b18-17-/t33-,34+,36-/m0/s1

(Z)-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]octadec-9-enamide

100403-19-8; N-(1,3,4-TRIHYDROXYOCTADECAN-2-YL)OCTADEC-9-ENAMIDE; RefChem:124459; Ceramide VI (AP); SCHEMBL29009460;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL
H2O : ~33.33 mg/mL
MEthanol : ~25 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (Infinity mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)