A co-formulation of interferons alpha2b and gamma distinctively targets cell cycle in the glioblastoma-derived cell line U-87MG

Background HeberFERON is a co-formulation of a2b and ? interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model.Methods Transcriptional expression profiling including a control (untreated) and three groups receiving a2b-interferon, ?-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry.Results The three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG.Conclusions We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.

Automated summary

HeberFERON displays a distinctive effect compared to individual interferons in the U-87MG model, particularly in cell cycle related events. Through transcriptional expression profiling, it was found that HeberFERON targets a series of genes related to the cell cycle, such as the transition from prometaphase to anaphase. Additionally, HeberFERON affects the cell cycle by regulating the FOXM1 transcription factor, p53 phosphorylation, and MDM2-mediated ubiquitination, and it plays a role in cell proliferation.