human colon cancer cell lines hct116  (ATCC)

Journal: PLOS One

Article Title: Targeting ceramide metabolism to restore hypoxia-induced apoptosis in p53-deficient colon cancer cells

doi: 10.1371/journal.pone.0340295

Figure Lengend Snippet: A- Levels of ceramide species at 72 hours post-hypoxia. HCT116 p53 +/+ and p53 -/- cells were incubated in 21% and 1% O 2 and lipids were extracted. Ceramide species were quantified by LC/MS and normalized to protein content (pmol ceramide/mg protein). Values represent the average of two independent experiments ± S. D. *p < 0.05, **p < 0.01. B- mRNA expression levels of ceramide synthases ( CerS2 , CerS4 , and CerS6 ) were assessed by qRTPCR, normalized to β-actin expression and represented as log 2 fold change (hypoxia/normoxia). Values represent the average of two independent experiments.

Article Snippet: The human colon cancer cell line HCT116 p53 +/+ (p53 wild-type, or p53-proficient) was purchased from the American Tissue Culture Collection, ATCC, Manassas, VA.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: PLOS One

Article Title: Targeting ceramide metabolism to restore hypoxia-induced apoptosis in p53-deficient colon cancer cells

doi: 10.1371/journal.pone.0340295

Figure Lengend Snippet: A- Total ceramide levels at 72 hours post-hypoxia was measured by LC/MS and normalized to inorganic phosphate levels (pM of ceramide/ pM of inorganic phosphate). Values represent the average of two independent experiments ± S.D. *p < 0.05; **p < 0.01; *** p < 0.001. B- Cell viability was measured by MTT assay. Data are represented as fold change (treatment/DMSO control). HCT116 cells were treated with 1.5 mM of GW4869 (GW), 10 μM desipramine (Des), 10 μM 4-HPR (HPR), 10 μM SK1-I (SKI), 20 μM C6 DhCer or 20 μM sphingosine prior to their incubation in a hypoxic chamber for 72 hours. Values represent the average of three independent experiments ± S.D. **p < 0.01.

Article Snippet: The human colon cancer cell line HCT116 p53 +/+ (p53 wild-type, or p53-proficient) was purchased from the American Tissue Culture Collection, ATCC, Manassas, VA.

Techniques: Liquid Chromatography with Mass Spectroscopy, MTT Assay, Control, Incubation

Journal: PLOS One

Article Title: Targeting ceramide metabolism to restore hypoxia-induced apoptosis in p53-deficient colon cancer cells

doi: 10.1371/journal.pone.0340295

Figure Lengend Snippet: A- Protein levels of p53 were determined by western blot. GAPDH was used as a loading control. B- Cell morphology was observed in normoxia (21% O 2 ) or hypoxia (1% O 2 ) using phase-contrast microscopy. Images were captured at 100 × magnification and are representative of three independent experiments. C- Cell viability was measured by MTT assay on HCT116 p53 +/+ (white) and p53 -/- (grey) cells after 24, 48 and 72 hours of incubation in normoxia (plain) or hypoxia (patterned). The optical density (O.D.) was measured at 595 nm. Values represent the average of three independent experiments ± S. D. D- Percentage of cells in sub-G0 phase of the cell cycle was measured by flow cytometry. Each column represents the mean ± S. D. of two independent experiments. * p < 0.05, **p < 0.01, *** p < 0.001. E- Analysis of PARP cleavage by western blot. GAPDH was used as a loading control. Blots are representative of two independent experiments.

Article Snippet: The human colon cancer cell line HCT116 p53 +/+ (p53 wild-type, or p53-proficient) was purchased from the American Tissue Culture Collection, ATCC, Manassas, VA.

Techniques: Western Blot, Control, Microscopy, MTT Assay, Incubation, Flow Cytometry

Journal: PLOS One

Article Title: Targeting ceramide metabolism to restore hypoxia-induced apoptosis in p53-deficient colon cancer cells

doi: 10.1371/journal.pone.0340295

Figure Lengend Snippet: In p53 +/+ cells, hypoxia downregulates CerS2, 4, 6 and DEGS1. It also inhibits SK1 leading to apoptosis. However, in the absence of p53, CerS2 and 4 expression is sustained along with that of DEGS1. As a result, different ceramide species accumulate, particularly long and very long chain ceramides, of which C24:0 ceramide is the most abundant and promoting beside the low sphingosine levels to cell survival in response to oxygen deficit. Combined inhibition of DEGS1 and SK1 or treatment of HCT116 cells with sphingosine or C6 ceramide typically restored p53-like functions in p53-deficient cells exposed to hypoxia and sensitized these cells to hypoxia-induced apoptosis.

Article Snippet: The human colon cancer cell line HCT116 p53 +/+ (p53 wild-type, or p53-proficient) was purchased from the American Tissue Culture Collection, ATCC, Manassas, VA.

Techniques: Expressing, Inhibition