Gene expression profiles of HMEC-1 after exposure to the chemotherapeutic drugs bleomycin and cisplatin with untreated samples as control

Chemotherapy-related endothelial damage contributes to the early development of cardiovascular morbidity in testicular cancer patients. We aimed to identify relevant mechanisms of and search for candidate biomarkers for this endothelial damage. Human micro-vascular endothelial cells (HMEC-1) were exposed to bleomycin or cisplatin with untreated samples as control. 18k cDNA microarrays were used. Gene expression differences were analysed at single gene level and in gene sets clustered in biological pathways and validated by qRT-PCR. Protein levels of a candidate biomarker were measured in testicular cancer patient plasma before, during and after bleomycin-etoposide-cisplatin chemotherapy, and related to endothelial damage biomarkers (von Willebrand Factor (vWF), high-sensitivity C-Reactive Protein (hsCRP)). Microarray data identified several genes with highly differential expression; e.g. Growth Differentiation Factor 15 (GDF-15), Activating Transcription Factor 3 (ATF3) and Amphiregulin (AREG). Pathway analysis revealed strong associations with ‘p53’ and ‘Diabetes Mellitus’ gene sets. Based on known function, we measured GDF-15 protein levels in 41 testicular patients during clinical follow-up. Pre-chemotherapy GDF-15 levels equalled controls. Throughout chemotherapy GDF-15, vWF and hsCRP levels increased, and were correlated at different time-points. An unbiased approach in a preclinical model revealed genes related to chemotherapy-induced endothelial damage, like GDF-15. The increases in plasma GDF-15 levels in testicular cancer patients during chemotherapy and its association with vWF and hsCRP suggest that GDF-15 is a potentially useful biomarker related to endothelial damage. In an acute-exposure setting, HMEC-1 were left untreated as controls or were treated with 0.3 (IC50 (concentration inhibiting cell survival by 50%)) or 1.5 ug/mL (IC90) bleomycin and 2.6 (IC50) and 12.9 uM (IC90) cisplatin for 6, 24 and 48 hours. In a chronic-exposure setting, lower doses were administrated (IC10; bleomycin 0.06 ug/mL or cisplatin 0.52 uM) two times a week; cells were collected for analysis at day 30. Administration of cisplatin had to be withheld at the 7th administration because of considerable cell dead, but was continued at full dose thereafter. Bleomycin could be administrated without disruption. Total RNA was isolated from HMEC-1 and pooled for each time point and drug concentration from 2 independent experiments.