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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) Western blotting. Expression levels of LC3B-II, phospho-mammalian target of rapamycin (p-mTOR) and phospho-extracelluar signal-regulated kinase 1/2 (p-ERK1/2) were analyzed after treatment with PD98059/C2-Cer. LC3B-II and p-ERK1/2 levels declined and p-mTOR levels were elevated compared with cells treated with C2-Cer only; (B) CCK8 assay. Treatment with 10 μM PD98059 significantly reduced the viability of cells exposed to C2-Cer; (C) CCK8 assay. CQ enhanced the growth-inhibition effect of C2-Cer compared with C2-Cer only. (*: p < 0.05).
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f6-ijms-15-03336: (A) Western blotting. Expression levels of LC3B-II, phospho-mammalian target of rapamycin (p-mTOR) and phospho-extracelluar signal-regulated kinase 1/2 (p-ERK1/2) were analyzed after treatment with PD98059/C2-Cer. LC3B-II and p-ERK1/2 levels declined and p-mTOR levels were elevated compared with cells treated with C2-Cer only; (B) CCK8 assay. Treatment with 10 μM PD98059 significantly reduced the viability of cells exposed to C2-Cer; (C) CCK8 assay. CQ enhanced the growth-inhibition effect of C2-Cer compared with C2-Cer only. (*: p < 0.05).

Mentions: Microtubule-associated protein 1 light chain 3 (LC3) -II transforms from LC3-I and acts as a marker for autophagic vesicles and autophagic activity, and LC3B-II (isoform of LC3-II) is correlated with elevated levels of autophagic vesicles. Endogenous LC3-II levels increased in HNSCC cells within 24 h after C2-Cer treatment in a concentration-dependent manner. LC3-II levels were higher in cells co-treated with the autophagy inhibitor chloroquine (CQ) compared with cells treated with C2-Cer alone. This result was confirmed by immunofluorescence. Fluoresence microscopic evaluation of LC3-II antibody-prestained cells revealed more stained particles in the cytoplasm of treated cells compared with control cells (Figure 5A). Specific autophagic vacuoles were observed in the cytoplasm via electron microscopy, 4 h after C2-Cer treatment (Figure 5B). LC3-II and phospho-extracelluar signal-regulated kinase 1/2 (p-ERK1/2) levels increased in concentration-dependently. However, the levels of phospho-AMP-activated protein kinase (p-AMPK), p-Akt and Beclin 1 showed no apparent differences, suggesting that autophagy may be induced through an ERK-related pathway (Figure 5C). Co-treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 reduced the expression levels of LC3B-II and p-ERK1/2, but increased p-mTOR (Figure 6A). In addition, cell viability was significantly reduced by PD98059, as shown via the CCK8 assay (Figure 6B; p < 0.05). These results suggest that PD98059 inhibited autophagy and increased the sensitivity of HNSCC cells to C2-Cer exposure.


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) Western blotting. Expression levels of LC3B-II, phospho-mammalian target of rapamycin (p-mTOR) and phospho-extracelluar signal-regulated kinase 1/2 (p-ERK1/2) were analyzed after treatment with PD98059/C2-Cer. LC3B-II and p-ERK1/2 levels declined and p-mTOR levels were elevated compared with cells treated with C2-Cer only; (B) CCK8 assay. Treatment with 10 μM PD98059 significantly reduced the viability of cells exposed to C2-Cer; (C) CCK8 assay. CQ enhanced the growth-inhibition effect of C2-Cer compared with C2-Cer only. (*: p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6-ijms-15-03336: (A) Western blotting. Expression levels of LC3B-II, phospho-mammalian target of rapamycin (p-mTOR) and phospho-extracelluar signal-regulated kinase 1/2 (p-ERK1/2) were analyzed after treatment with PD98059/C2-Cer. LC3B-II and p-ERK1/2 levels declined and p-mTOR levels were elevated compared with cells treated with C2-Cer only; (B) CCK8 assay. Treatment with 10 μM PD98059 significantly reduced the viability of cells exposed to C2-Cer; (C) CCK8 assay. CQ enhanced the growth-inhibition effect of C2-Cer compared with C2-Cer only. (*: p < 0.05).
Mentions: Microtubule-associated protein 1 light chain 3 (LC3) -II transforms from LC3-I and acts as a marker for autophagic vesicles and autophagic activity, and LC3B-II (isoform of LC3-II) is correlated with elevated levels of autophagic vesicles. Endogenous LC3-II levels increased in HNSCC cells within 24 h after C2-Cer treatment in a concentration-dependent manner. LC3-II levels were higher in cells co-treated with the autophagy inhibitor chloroquine (CQ) compared with cells treated with C2-Cer alone. This result was confirmed by immunofluorescence. Fluoresence microscopic evaluation of LC3-II antibody-prestained cells revealed more stained particles in the cytoplasm of treated cells compared with control cells (Figure 5A). Specific autophagic vacuoles were observed in the cytoplasm via electron microscopy, 4 h after C2-Cer treatment (Figure 5B). LC3-II and phospho-extracelluar signal-regulated kinase 1/2 (p-ERK1/2) levels increased in concentration-dependently. However, the levels of phospho-AMP-activated protein kinase (p-AMPK), p-Akt and Beclin 1 showed no apparent differences, suggesting that autophagy may be induced through an ERK-related pathway (Figure 5C). Co-treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 reduced the expression levels of LC3B-II and p-ERK1/2, but increased p-mTOR (Figure 6A). In addition, cell viability was significantly reduced by PD98059, as shown via the CCK8 assay (Figure 6B; p < 0.05). These results suggest that PD98059 inhibited autophagy and increased the sensitivity of HNSCC cells to C2-Cer exposure.

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