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Everolimus and sunitinib induce infantile hemangiomas cell apoptosis through P53 activation

ABSTRACT: Infantile hemangiomas (IH) are common vascular tumors in infants, yet their underlying mechanisms remain poorly understood. In this study, we isolated endothelial cells from IH and utilized hTERT and SV40 to establish an immortalized hemangioma-derived endothelial cell (iHemEC). We employed these iHemEC for in vitro drug screening and investigated the mechanisms of drug-induced inhibition of iHemEC via transcriptome sequencing, western blot analysis, and various cellular assays. We successfully established iHemEC that highly express VEGFR2, CD31, and GLUT-1, markers characteristic of endothelial cells. Screening of 18 putative working drugs were performed on iHemEC. Sunitinib, Elimusertib, HIF-1 inhibitor-4, Rebastinib, and Everolimus can inhibit iHemEC with lower IC50 than Propranolol and Rapamycin. We further performed transcriptome profiling in iHemEC upon Everolimus and Sunitinib intervention. GSEA showed that both drugs can significantly downregulate Hallmark of MYC target V1&V2, E2F Target and G2M Checkpoint which are all cell cycle related Hallmark. PI3K/AKT/mTOR pathway is only downregulated in Everolimus treated cells which is validated by western blot. Chromosome instability was found specifically in Sunitinib treated cells which was reported to cause DNA damage. DHR123 was used to detect DNA damage induced ROS and released extracellular ROS can only be observed in Sunitinib treated cells. Finally, P53 activation was observed in western blot, we found that Sunitinib can trigger P53 activation and BCL2 downregulation from 0.2 µM which is fifty times lower than Everolimus at 10 µM. Our study successfully developed an iHemEC line suitable for in vitro drug screening and mechanistic study. Everolimus and Sunitinib are two of working drugs and both drugs can induce P53 dependent apoptosis on iHemEC might through PI3K/AKT/mTOR inhibition and chromosome instability respectively. These findings provide a valuable basis for further research and potential clinical application in the treatment of IH.

ORGANISM(S): Homo sapiens

PROVIDER: GSE279596 | GEO | 2025/10/16

REPOSITORIES: GEO

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Project description:Glomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte cytoskeleton. To elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury. Upon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involving cytoskeletal-associated pathways, adhesion, migration and extracellular matrix composition to be affected. Everolimus is capable of protecting podocytes from injury, both on the transcriptome and protein level. Rescued genes included TUBB2B and DCDC2, both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Confirming gene expression data, Western-blot analysis in cultured podocytes showed an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Microtubule-inhibitor experiments led to a maldistribution of TUBB2B and DCDC2 as well as an aberrant reorganization of the actin cytoskeleton. Tubb2bbrdp/brdp mice showed a delay in glomerular podocyte and capillary development. Taken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton might involve regulation of microtubules, revealing a potential novel role of TUBB2B and DCDC2 in glomerular podocyte development We analyzed the effect of everolimus on gene expression in a puromycin aminonucleoside experimental in vitro model of podocyte injury using microarrays

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Everolimus and sunitinib induce infantile hemangiomas cell apoptosis through P53 activation

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