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C2-ceramide induces cell death and protective autophagy in head and neck squamous cell carcinoma cells.

Zhu W, Wang X, Zhou Y, Wang H - Int J Mol Sci (2014)

Bottom Line: Ceramides are second messengers involved in several intracellular processes in cancer cells, amongst others.C2-Cer markedly increased the expression level of microtubule-associated protein 1 light chain 3B (LC3B) type II associated with protective autophagy.Furthermore, C2-Cer up-regulated the phosphorylation of extracellular signal-regulated kinase 1/2, but down-regulated its downstream substrate phospho-mammalian target of rapamycin (p-mTOR) during the autophagy process.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Surgery, the First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China. zwy555star@163.com.

ABSTRACT
Ceramides are second messengers involved in several intracellular processes in cancer cells, amongst others. The aim of this study was to evaluate the anti-tumor efficacy of C2-ceramide (C2-Cer; N-acetyl-D-sphingosine) by investigating cell death and autophagy in head and neck squamous cell carcinoma (HNSCC) cells. C2-Cer showed concentration-dependent cytotoxicity in HN4 and HN30 cell lines. It simultaneously induced caspase-3-independent apoptosis and programmed necrosis. C2-Cer markedly increased the expression level of microtubule-associated protein 1 light chain 3B (LC3B) type II associated with protective autophagy. An autophagy inhibitor enhanced C2-Cer-mediated cytotoxicity, while a programmed-necrosis inhibitor produced the opposite effect. Furthermore, C2-Cer up-regulated the phosphorylation of extracellular signal-regulated kinase 1/2, but down-regulated its downstream substrate phospho-mammalian target of rapamycin (p-mTOR) during the autophagy process. These results suggested that C2-Cer exerts anti-tumor effects by inducing programmed apoptosis and necrosis in HNSCC, and these cytotoxic effects are enhanced by an autophagy inhibitor.

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(A) Cell Counting Kit-8 (CCK8) assay. Cells were treated with different concentrations (0, 20, 40, 60 μM, respectively) of C2-Cer for 24 h. The cell proliferation inhibition rate increased after treatment with C2-Cer in a concentration-dependent manner; (B) Flow cytometry. Cells were treated with different concentrations (0, 20, 40, 60 μM, respectively) of C2-Cer for 24 h. Q2 + Q4 represent apoptotic cells. Apoptotic cells were significantly increased when exposed to C2-Cer in a concentration-dependent manner. (*: p < 0.05).
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f1-ijms-15-03336: (A) Cell Counting Kit-8 (CCK8) assay. Cells were treated with different concentrations (0, 20, 40, 60 μM, respectively) of C2-Cer for 24 h. The cell proliferation inhibition rate increased after treatment with C2-Cer in a concentration-dependent manner; (B) Flow cytometry. Cells were treated with different concentrations (0, 20, 40, 60 μM, respectively) of C2-Cer for 24 h. Q2 + Q4 represent apoptotic cells. Apoptotic cells were significantly increased when exposed to C2-Cer in a concentration-dependent manner. (*: p < 0.05).

Mentions: HN4 and HN30 cells were treated with various concentrations of C2-Cer for 24 h, and cell viability was assessed to determine the cytotoxic effects of C2-Cer. All cells showed morphological changes, including cell shrinkage, detachment from other cells, and formation of vesicles in the cytoplasm within 24 h of treatment, as observed via light microscopy. More C2-Cer-treated cells floated in the treated medium compared with control cells. C2-Cer significantly inhibited the proliferation of HNSCC cells in a concentration-dependent manner (Figure 1A; p < 0.05). The cytotoxic effect declined as the concentration increased. The LD50 in HN30 cells was 60 μM, though HN4 cells were less sensitive to C2-Cer than HN30 cells. Flow cytometric analysis showed that the ratios of propidium iodide (PI)+ cells were significantly higher in C2-Cer-treated cells than in controls in a concentration-dependent manner. C2-Cer induced apoptosis in both cell lines (Figure 1B). The expression level of cleaved caspase-3 was measured by Western blotting to determine the apoptosis mechanism of C2-Cer. There was no significant change in the level of caspase-3 cleavage after C2-Cer treatment (Figure 2A). To verify that apoptosis was induced by C2-Cer via a caspase-3-independent pathway, the pan-caspase inhibitor Z-VAD-FMK was added to C2-Cer-treated cells. Flow cytometric analysis showed that apoptosis was not inhibited by Z-VAD-FMK (Figure 2B).


C2-ceramide induces cell death and protective autophagy in head and neck squamous cell carcinoma cells.

Zhu W, Wang X, Zhou Y, Wang H - Int J Mol Sci (2014)

(A) Cell Counting Kit-8 (CCK8) assay. Cells were treated with different concentrations (0, 20, 40, 60 μM, respectively) of C2-Cer for 24 h. The cell proliferation inhibition rate increased after treatment with C2-Cer in a concentration-dependent manner; (B) Flow cytometry. Cells were treated with different concentrations (0, 20, 40, 60 μM, respectively) of C2-Cer for 24 h. Q2 + Q4 represent apoptotic cells. Apoptotic cells were significantly increased when exposed to C2-Cer in a concentration-dependent manner. (*: p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3958915&req=5

f1-ijms-15-03336: (A) Cell Counting Kit-8 (CCK8) assay. Cells were treated with different concentrations (0, 20, 40, 60 μM, respectively) of C2-Cer for 24 h. The cell proliferation inhibition rate increased after treatment with C2-Cer in a concentration-dependent manner; (B) Flow cytometry. Cells were treated with different concentrations (0, 20, 40, 60 μM, respectively) of C2-Cer for 24 h. Q2 + Q4 represent apoptotic cells. Apoptotic cells were significantly increased when exposed to C2-Cer in a concentration-dependent manner. (*: p < 0.05).
Mentions: HN4 and HN30 cells were treated with various concentrations of C2-Cer for 24 h, and cell viability was assessed to determine the cytotoxic effects of C2-Cer. All cells showed morphological changes, including cell shrinkage, detachment from other cells, and formation of vesicles in the cytoplasm within 24 h of treatment, as observed via light microscopy. More C2-Cer-treated cells floated in the treated medium compared with control cells. C2-Cer significantly inhibited the proliferation of HNSCC cells in a concentration-dependent manner (Figure 1A; p < 0.05). The cytotoxic effect declined as the concentration increased. The LD50 in HN30 cells was 60 μM, though HN4 cells were less sensitive to C2-Cer than HN30 cells. Flow cytometric analysis showed that the ratios of propidium iodide (PI)+ cells were significantly higher in C2-Cer-treated cells than in controls in a concentration-dependent manner. C2-Cer induced apoptosis in both cell lines (Figure 1B). The expression level of cleaved caspase-3 was measured by Western blotting to determine the apoptosis mechanism of C2-Cer. There was no significant change in the level of caspase-3 cleavage after C2-Cer treatment (Figure 2A). To verify that apoptosis was induced by C2-Cer via a caspase-3-independent pathway, the pan-caspase inhibitor Z-VAD-FMK was added to C2-Cer-treated cells. Flow cytometric analysis showed that apoptosis was not inhibited by Z-VAD-FMK (Figure 2B).

Bottom Line: Ceramides are second messengers involved in several intracellular processes in cancer cells, amongst others.C2-Cer markedly increased the expression level of microtubule-associated protein 1 light chain 3B (LC3B) type II associated with protective autophagy.Furthermore, C2-Cer up-regulated the phosphorylation of extracellular signal-regulated kinase 1/2, but down-regulated its downstream substrate phospho-mammalian target of rapamycin (p-mTOR) during the autophagy process.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Surgery, the First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China. zwy555star@163.com.

ABSTRACT
Ceramides are second messengers involved in several intracellular processes in cancer cells, amongst others. The aim of this study was to evaluate the anti-tumor efficacy of C2-ceramide (C2-Cer; N-acetyl-D-sphingosine) by investigating cell death and autophagy in head and neck squamous cell carcinoma (HNSCC) cells. C2-Cer showed concentration-dependent cytotoxicity in HN4 and HN30 cell lines. It simultaneously induced caspase-3-independent apoptosis and programmed necrosis. C2-Cer markedly increased the expression level of microtubule-associated protein 1 light chain 3B (LC3B) type II associated with protective autophagy. An autophagy inhibitor enhanced C2-Cer-mediated cytotoxicity, while a programmed-necrosis inhibitor produced the opposite effect. Furthermore, C2-Cer up-regulated the phosphorylation of extracellular signal-regulated kinase 1/2, but down-regulated its downstream substrate phospho-mammalian target of rapamycin (p-mTOR) during the autophagy process. These results suggested that C2-Cer exerts anti-tumor effects by inducing programmed apoptosis and necrosis in HNSCC, and these cytotoxic effects are enhanced by an autophagy inhibitor.

Show MeSH
Related in: MedlinePlus