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Identification of dysregulation of sphingolipids in retinoblastoma using liquid chromatography-mass spectrometry
Omkar Surendra Khade1, Sruthy Sasidharan1, Ankit Jain2
1Institute of Bioinformatics, Bangalore, Karnataka, India; Manipal Academy of Higher Education, Mangalore, Karnataka, India.
Abstract:
Retinoblastoma (RB) is a rare ocular cancer seen in children that counts for approximately 3% of all childhood cancers. It is found that mutation in RB1, a tumour Suppressor Gene on chromosome 13 as the cause of malignancy. Retinoblastoma protein is the target for ceramide to cause apoptosis. We studied lipidomics of two RB cell lines, one aggressive cell line (NCC-RbC-51) derived from a metastatic site and one non aggressive cell line (WERI-Rb1) in comparison with a control cell line (MIO-M1). Lipid profiles of all the cell lines were studied using high resolution mass spectrometer coupled to high performance liquid chromatography. Data acquired from all the three cell lines in positive mode were analyzed to identify differentially expressed metabolites. Several phospholipids and lysophospholipids were found to be dysregulated. We observed upregulation of hexosyl ceramides, and down regulation of dihydroceramides and higher order sphingoglycolipids hinting at a hindered sphingolipid biosynthesis. The results obtained from liquid chromatography-mass spectrometry are validated by using qPCR and it was observed that genes involved in ceramide biosynthesis pathway are getting down regulated.
Insights
This study reveals altered lipid profiles in retinoblastoma (RB) cells, indicating impaired sphingolipid biosynthesis is linked to this childhood eye cancer. These findings offer new insights into RB development.
Area of Science:
- Oncology
- Biochemistry
- Genetics
Background:
- Retinoblastoma (RB) is a rare pediatric eye cancer, accounting for 3% of childhood cancers, often caused by RB1 tumor suppressor gene mutations.
- The retinoblastoma protein is known to be a target for ceramide-induced apoptosis, suggesting a role for sphingolipids in RB.
- Understanding the lipidomic landscape of RB is crucial for identifying potential therapeutic targets.
Purpose of the Study:
- To investigate and compare the lipid profiles of aggressive and non-aggressive retinoblastoma cell lines with a control cell line.
- To identify specific lipid metabolites and pathways dysregulated in retinoblastoma.
- To explore the relationship between lipid dysregulation and sphingolipid biosynthesis in RB.
Main Methods:
- Lipidomic analysis of three cell lines (NCC-RbC-51, WERI-Rb1, MIO-M1) using high-resolution mass spectrometry coupled with high-performance liquid chromatography (LC-MS).
- Differential metabolite analysis of lipid profiles in positive mode.
- Validation of LC-MS findings using quantitative polymerase chain reaction (qPCR) to assess gene expression related to ceramide biosynthesis.
Main Results:
- Significant dysregulation of phospholipids and lysophospholipids was observed in RB cell lines compared to controls.
- Upregulation of hexosyl ceramides and downregulation of dihydroceramides and higher-order sphingoglycolipids were identified, suggesting impaired sphingolipid biosynthesis.
- qPCR analysis confirmed the downregulation of genes involved in the ceramide biosynthesis pathway in RB cells.
Conclusions:
- The study demonstrates a distinct lipidomic signature in retinoblastoma, characterized by hindered sphingolipid biosynthesis.
- Dysregulated sphingolipid metabolism, particularly ceramide pathway alterations, is implicated in the pathogenesis of retinoblastoma.
- These findings highlight potential therapeutic avenues targeting lipid metabolism in retinoblastoma treatment.
