J Korean Med Sci.  2007 Apr;22(2):298-304. 10.3346/jkms.2007.22.2.298.

Sphingosine 1-Phosphate Triggers Apoptotic Signal for B16 Melanoma Cells via ERK and Caspase Activation

• Shin JH
• Choi GS
• Kang WH
• Myung KB

• KMID: 1713191
• DOI: http://doi.org/10.3346/jkms.2007.22.2.298

Abstract

The bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), recently was reported to induce apoptosis of some cancer cells and neurons, although it generally known to exert mitogenic and antiapoptotic effects. In this study, we investigated the effects of S1P on the cell growth, melanogenesis, and apoptosis of cultured B16 mouse melanoma cells. In results, S1P was found to induce apoptosis in B16 melanoma cells in a dose- and time-dependent manner, but exerted minimal effects on melanogenesis. Although receptors of sphingosine 1-phosphate (endothelial differentiation gene 1 [Edg]/S1P1, Edg5/S1P2, Edg3/S1P3) were expressed in B16 melanoma cells, they were shown not to be associated with S1P-induced apoptosis. In addition, pertussis toxin did not block the apoptotic effects of S1P on B16 melanoma cells. S1P induced caspase-3 activation and the extracellular signal-regulated kinase (ERK) activation. Interestingly, the ERK pathway inhibitor, UO126, reversed the apoptotic effects of S1P on B16 melanoma cells. These results suggest that S1P induced apoptosis of B16 melanoma cells via an Edg receptor-independent, pertussis toxin-insensitive pathway, and appears to be associated with the ERK and caspase-3 activation.

Keyword

Sphingosine 1-Phosphate; Melanoma; Apoptosis; Caspases; Extracellular Signal-Regulated Kinase

MeSH Terms

Sphingosine/administration & dosage/*analogs & derivatives
Signal Transduction/drug effects
Mice
Melanoma/*enzymology/*pathology
Lysophospholipids/*administration & dosage
Extracellular Signal-Regulated MAP Kinases/*metabolism
Enzyme Activation/drug effects
Cell Line
Caspase 3/*metabolism
Apoptosis/*drug effects
Animals

Figure

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  Fig. 1 Treatment with more than 5 µM inhibited the growth of B16 melanoma cells (*: p<0.05). The control was treated with vehicle instead of S1P for 24 hr. Optical density was determined at a wavelength of 540 nm. The experiment was repeated five times.

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  Fig. 2 Melanin content was not significantly affected by S1P (p>0.05).

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  Fig. 3 The number of apoptotic B16 melanoma cells increased in the S1P treatment group (10 µM for 24 hr) (B) as compared to the vehicle-treated control group (A). (TUNEL, ×200), (Inset: apoptotic cells, ×400).

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  Fig. 4 Western blot analysis of the cell extracts from B16 cells treated with 10 µM S1P using caspase-3 antibody showed decreased inactive casapse-3 (35 kDa) and increased active cleaved caspase-3 (17, 19 kDa).

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  Fig. 5 (A) Cell surface receptors of S1P are expressed abundantly in B16 melanoma cells (Immunocytochemistry, ×400). (B) Western blot analysis of cell extracts from B16 melanoma cells using specific anti-Edg antibodies shows 45 kDa proteins.

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  Fig. 6 MTT dye reduction assays were conducted as described in the methods section (Optical density at 540 nm). 24 hr of treatment with dihydro-S1P (10 µM), a structural analogue of S1P that binds and activates only Edg receptors, dose not induce cell death in contrast to the effect of S1P (*, p<0.05). This result suggested that the apoptotic effects of S1P were Edg-independent. This experiment was repeated three times.

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  Fig. 7 Serum-deprived cells were preincubated for 24 hr with 10 ng/mL of pertussis toxin (PTX) or vehicle, and incubated further in the presence of PTX or vehicle, together with S1P (10 µM). The MTT dye reduction assays were conducted as was described in the methods section. After time, cell viability was found to have been significantly decreased after treatment with S1P, with or without PTX (*: p<0.05). PTX failed to reverse the apoptotic effects of S1P. This experiment was repeated three times.

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  Fig. 8 Serum-deprived cells were preincubated for 1 hr with 10 µM of UO126 or vehicle, and incubated further in the presence of UO126 (the MAP kinase inhibitor) or vehicle, together with 10 µM of S1P or dihydro-S1P, for the indicated time periods. Western blot analysis was conducted as described in the methods section. This figure indicates that S1P activates ERK, and UO126 inhibits the activation of ERK.

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  Fig. 9 Serum-deprived cells were preincubated for 1 hr with 10 µM of UO126 or vehicle, and incubated further in the presence of UO126 or vehicle, together with 10 µM of S1P, for the indicated time periods. MTT dye reduction assays were conducted as described in the methods section. After 12 hr, S1P significantly induced cell death, and UO126 reversed S1P-induced cell death (*, p<0.05). This result indicated that S1P induced cell death via ERK activation in early stages. This experiment was repeated three times.

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