- Binds directly to the DNA-binding domain of p53 (Kd = 60 ± 20 nM), near Ser240 and Ser241, disrupting the p53-MDM2 interaction and preventing p53 ubiquitination and degradation. [1]
- Upregulates Btf (Bcl-2-associated transcription factor), which represses MDM2 transcription and modulates p53, BAX, and Bcl-2 pathways. [2]

- C16-ceramide (water-soluble pyridinium derivative PC16, 1-5 μM) induced dose-dependent elevation of p53, nuclear translocation, and activation of downstream targets p21 and PUMA in A549, HCT116, and HepG2 cells. [1]
- Purified recombinant p53 bound biotinylated C16-ceramide in pull-down assays. Fluorescence titration showed high-affinity binding with Kd = 60 ± 20 nM. PC6 (C6-pyridinium ceramide) did not bind. [1]
- Membrane-binding assays showed p53 strongly preferred C16-ceramide over other acyl chain lengths (C12, C18, C20, C22, C24), C16-dihydroceramide, or PC6. [1]
- Photoactivatable ceramide (PAC) covalently modified p53 at Ser240 and Ser241 (AScore 1000). Mutations introducing bulky side chains at these residues (S240K, S241K, S241E) abolished ceramide binding; S240E or S241A did not. [1]
- Ceramide binding increased the thermal stability of the p53 DNA-binding domain (Tm increased from 41.6°C to 43.2°C). [1]
- PC16 (5 μM) disrupted the p53-MDM2 complex in live PC-3 cells (bimolecular fluorescence complementation assay), similar to Nutlin 3 (50 μM). ELISA showed concentration-dependent loss of p53-MDM2 interaction (0–100 nM PC16). [1]
- PC16 (60–100 nM) prevented p53 ubiquitination in in vitro ubiquitination assays, comparable to Nutlin 3 (80 nM). [1]
- In HCT116 cells, C16-ceramide (12 μM, 6 h) decreased cell viability in a time- and concentration-dependent manner (∼60% survival at 6 h). Apoptosis was confirmed by PARP cleavage, pro-caspase 3 decrease, and Annexin V/PI staining. [2]
- 2D-DIGE proteomics identified 51 differentially expressed proteins after C16-ceramide treatment (12 μM, 6 h), including upregulation of Btf (1.24- to 1.85-fold) and prohibitin (1.48-fold), and downregulation of stratifin (2.77-fold) and stathmin (1.11-fold). Western blot confirmed these changes. [2]
- C16-ceramide (12 μM) increased p53 expression (within 15–30 min), upregulated BAX, and decreased Mdm2 and phospho-Bcl-2 levels. [2]
- Btf overexpression (GFP-Btf transfection) decreased cell viability by ∼20%, increased p53 and BAX, and decreased phospho-Bcl-2. Btf knockdown (siRNA) increased resistance to ceramide-induced cell death and reduced caspases 3/7 activity by up to 60%. [2]
- C16-ceramide decreased MDM2 promoter activity (by ∼40% at 6 h) and Mdm2 protein levels (1.85-fold decrease). Btf overexpression also decreased MDM2 promoter activity (∼40%) and Mdm2 protein (1.30-fold). Btf silencing increased MDM2 promoter activity after ceramide treatment. [2]

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P53 and C16-ceramide interact within the core domain of the latter. In cells, p53 combines with natural C16-ceramide to form a complex [1]. HCT116 cell viability is significantly decreased by C16-ceramide (2.5–50 μM; 0-48 hours) in a concentration- and time-dependent manner [2]. Btf (Bcl-2-related transcription factor) is the mechanism by which C16-ceramide (12 μM; 48 hours) causes apoptosis in HCT116 cells [2]. p53 and BAX expression are upregulated by C16-ceramide (12 μM; 0–6 hours) and Btf expression. Through Btf, C16-ceramide inhibits the expression of Mdm2 [2].

- No direct in vivo data for C16-ceramide are reported in the provided texts. However, CerS6 (ceramide synthase 6) expression in cells elevated endogenous C16-ceramide and activated p53. [1]

- Fluorescence quenching: Steady-state fluorescence spectra of purified p53 core domain (1.0 μM) were recorded with excitation at 270 nm. Fluorescence quenching was measured with increasing PC16 or PC6 (10–2000 nM). Kd values were determined by non-linear fitting of emission at 304 nm. [1]
- Membrane-binding assay: Ceramides (0.5–4 μg) were spotted on PVDF membrane, blocked with 3% BSA, incubated with purified p53 or cell lysate overnight at 4°C, and bound p53 detected with p53-specific antibody. [1]
- Thermal shift assay: p53 DBD (100–300 aa) was incubated with or without PAC, and melting temperature (Tm) was measured. [1]
- In vitro ubiquitination assay: Reactions contained p53, E1, E2, MDM2, Mg²⁺-ATP, and ubiquitin, with or without PC16 (60, 80, 100 nM) or Nutlin 3 (80 nM), incubated at 37°C for 1 h, then analyzed by SDS-PAGE/WB. [1]
- Sandwich ELISA: 96-well plates coated with p53 polyclonal antibody were incubated with p53/MDM2 mixture with or without PC16 or Nutlin 3. Bound complex was detected with MDM2 monoclonal antibody and HRP-conjugated secondary antibody. [1]
- LC-MS/MS for sphingolipids: Lipid extraction with isopropyl alcohol/water/ethyl acetate (30:10:60). Samples were normalized to protein levels or lipid phosphate. [1]
- 2D-DIGE: Proteins (25 μg) from control and C16-ceramide-treated cells (12 μM, 6 h) were labeled with Cy3/Cy5, mixed with Cy2-labeled internal standard, separated on pH 3–10 NL IPG strips and 12% acrylamide gels, scanned, and analyzed with DeCyder software. [2]

Cell Viability Assay[2]
Cell Types: HCT116 Cell
Tested Concentrations: 2.5, 5, 10, 12, 20, 50 µM
Incubation Duration: 0-48 hrs (hours)
Experimental Results: Cell viability diminished dramatically in a time and concentration dependent manner.

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Western Blot Analysis[2]
Cell Types: HCT116 Cell
Tested Concentrations: 12 μM
Incubation Duration: 1, 3 and 6 hrs (hours)
Experimental Results: Increased PARP cleavage and diminished pro-caspase 3. diminished stratifin and stathmin levels, increased inhibin and btw. After treatment, RNAi-mediated Btf depletion also partially suppressed BAX expression. Luciferase activity and Mdm2 protein expression levels were Dramatically diminished.

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Cell viability (MTS assay): HCT116 cells were seeded in 96-well plates, treated with C16-ceramide (2.5–50 μM, 1.5–48 h), and MTS/PMS added for 2 h at 37°C. Absorbance measured at 492 nm. [2]
- Apoptosis detection (Annexin V/PI): Cells were stained with Annexin V-FLUOS and propidium iodide for 15 min and visualized by confocal microscopy. [2]
- Western blotting: Cells lysed in 1% SDS or RIPA buffer, separated by SDS-PAGE, transferred to PVDF, probed with antibodies against p53, PARP, caspase-3, Btf, Mdm2, BAX, phospho-Bcl-2, stratifin, stathmin, prohibitin, and α-tubulin. [1][2]
- Bimolecular fluorescence complementation (BiFC): PC-3 cells co-transfected with p53-V1 and MDM2-V2 were treated with PC16 (5 μM) or Nutlin 3 (50 μM) for 18 h, and fluorescence was imaged. [1]
- siRNA silencing: CerS6 or p53 were silenced using specific siRNAs; scrambled siRNA as control. Btf was silenced using ON-TARGETplus SMARTpool siRNA. Transfections performed with lipofectamine. [1][2]
- Transient transfection and luciferase assays: HCT116 cells transfected with MDM2-LUC or TP53-LUC reporter constructs and GFP-Btf expression vector. Luciferase activity measured 24 h post-transfection and normalized to protein concentration. [2]
- Caspase 3/7 assay: Cells transfected with GFP siRNA or Btf siRNA were treated with C16-ceramide, and caspase 3/7 activity measured using luminometric Caspase-Glo 3/7 assay. [2]

- In HCT116 cells, C16-ceramide (12 μM, 6 h) decreased cell viability by ∼40%. Higher concentrations (20–50 μM) caused greater toxicity. [2]
- p53-deficient cells (PC-3) and p53-silenced cells were insensitive to C16-ceramide toxicity. [1]

[1]. C16-ceramide is a natural regulatory ligand of p53 in cellular stress response. Nat Commun. 2018 Oct 8;9(1):4149.

[2]. The proapoptotic C16-ceramide-dependent pathway requires the death-promoting factor Btf in colon adenocarcinoma cells. J Proteome Res. 2009 Oct;8(10):4810-22.

- C16-ceramide is a natural sphingolipid generated by ceramide synthase 6 (CerS6). It binds directly to the p53 DNA-binding domain (Kd ∼60 nM), a previously unknown mechanism for p53 activation by a physiological metabolite. This interaction disrupts the p53-MDM2 complex, preventing p53 ubiquitination and degradation, leading to p53 accumulation and activation of downstream targets. [1]
- Ceramide binding site is near the Box V motif of p53, a secondary MDM2 contact region. The C10 atom of the ceramide acyl chain is proximal to Ser240 and Ser241. [1]
- Endogenous C16-ceramide bound to p53 was detected in cells expressing CerS6, but not in control cells. Only C16-ceramide, not other ceramide species (C18, C20, C22, C24, dhC16), was found in complex with p53. [1]
- Serum starvation or folate stress elevated CerS6 and C16-ceramide, leading to p53 activation. CerS6 silencing prevented this response. [1]
- Btf (BCLAF1) is a nuclear protein that interacts with Bcl-2 and Bcl-xL. It acts as a transcriptional repressor and pro-apoptotic factor. In the ceramide pathway, Btf is upregulated and represses MDM2 transcription, contributing to p53 stabilization. [2]

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N-Hexadecanoylsphingosine is an N-acylsphingosine in which the N-acyl group of the ceramide is designated as hexadecanoyl (palmitoyl). It functions as a metabolite of Mycoplasma genitalium and human serum. It is an N-acylsphingosine, Cer(d34:1), and N-palmitoylsphingosine base. It is functionally related to hexadecanoic acid. N-palmitoylsphingosine has been reported in Trypanosoma japonicum and Trypanosoma bournei, and relevant data are available.

C34H67NO3

537.900691270828

537.512

24696-26-2

C16-Ceramide-d31;852043-41-5;C16-Ceramide-13C16;C16-Ceramide-d9;2260669-51-8

5283564

White to off-white solid powder

0.919g/cm3

675.396ºC at 760 mmHg

94-95ºC

362.267ºC

0mmHg at 25°C

1.48

9.953

3

3

30

38

508

2

CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@@H](/C=C/CCCCCCCCCCCCC)O

YDNKGFDKKRUKPY-TURZORIXSA-N

InChI=1S/C34H67NO3/c1-3-5-7-9-11-13-15-17-19-21-23-25-27-29-33(37)32(31-36)35-34(38)30-28-26-24-22-20-18-16-14-12-10-8-6-4-2/h27,29,32-33,36-37H,3-26,28,30-31H2,1-2H3,(H,35,38)/b29-27+/t32-,33+/m0/s1

N-[(E,2S,3R)-1,3-dihydroxyoctadec-4-en-2-yl]hexadecanamide

C16 Ceramide; C16-ceramide; DTXSID301317974; RefChem:573083; ...; 24696-26-2;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Ethanol : ~20 mg/mL (~37.18 mM; warming and heat to 60°C)
DMSO: < 1 mg/mL
DMF : 20 mg/mL (~37.18 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (3.72 mM) (saturation unknown) in 10% EtOH + 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 20.0 mg/mL clear EtOH + stock solution to 900 μL of corn oil and mix evenly.

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