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Vinodh Kakkassery, Sergej Skosyrski, Anja Lüth, Burkhard Kleuser, Markus van der Giet, Aline Riechardt, Norbert Kociok, Antonia M. Joussen; Weri Rb1 Etoposide-resistant Subclone Up-regulate Survival Favoring Sphingosine-1-phosphate After Etoposide Incubation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1294.
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Levels of sphingolipids influence programmed cell death or survive in different tumors. Objective of this study was to test, whether there are changes of sphingolipid levels in WERI RB1 and WERI RB1 etoposide-resistant subclone (WERI ETOR) after etoposide incubation.
Sphingolipid pathway enzymes were detected in WERI RB1, in WERI ETOR and in human retinoblastoma tissue by conventional PCR. WERI RB1 and WERI ETOR were incubated with 400 ng/ml etoposide for 24 h. Apoptosis and necrosis were measured by DAPI/propidium iodide assays. Levels of glucosyl-ceramides, ceramides, sphingosine, sphingosine-1-phosphate (S1P) were detected by Q-TOF mass spectrometry.
mRNA of ceramide synthase, sphingomyelin syntase 1 and 2, ceramide kinase, ceramide kinase like protein as well as acid, alkaline and neutral sphingomyelinase, sphingosine kinase 1 and 2, S1P receptor 1, 2 and 3 was detected in WERI RB1, in WERI ETOR and four human tissue samples. DAPI/propidium iodide assays confirmed the sensitivity of WERI-RB1, but resistance of WERI ETOR to etoposide. Significant up-regulation of sphingosine was seen in both cell lines (WERI RB1 vehicle control: 109 pmol sphingosine/mg protein versus WERI RB1 etoposide group: 481 pmol sphingosine/mg protein; WERI ETOR vehicle control: 128 pmol sphingosine/mg protein versus WERI ETOR etoposide group: 541 pmol sphingosine/mg protein). In contrast, S1P upregulation was only significantly increased in WERI ETOR, but not in WERI RB1 (WERI RB1 vehicle control: 82 pmol S1P/mg protein versus WERI RB1 etoposide group: 109 pmol S1P/mg protein; WERI ETOR vehicle control: 48 pmol sphingosine/mg protein versus WERI ETOR etoposide group: 102 pmol sphingosine/mg protein).
Both cell lines upregulate pro-apoptotic sphingosine after etoposide incubation. In contrast to etoposide sensitive WERI RB1, WERI ETOR produces additional survival favorable S1P. This phenomenon may be a key factor in retinoblastoma chemotherapy resistance and might offer S1P as a possible therapeutic target in retinoblastoma.
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