Bioinformatics Group, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.
Pharmacogenomics Group, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.
BMC Cancer. 2023 Aug 29;23(1):806. doi: 10.1186/s12885-023-11330-2.
HeberFERON is a co-formulation of α2b and γ 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.
Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-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.
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.
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.
HeberFERON 是基于 α2b 和 γ 干扰素协同作用的联合制剂，已在基底细胞癌中显示出优于单独使用干扰素的临床优势。在胶质母细胞瘤（GBM）中，HeberFERON 显示出有前景的临床前和临床结果。这促使我们设计了一个微阵列实验，旨在鉴定 HeberFERON 与 U-87MG 模型中单独使用干扰素相比的独特作用所涉及的分子机制。
使用 Illumina HT-12 微阵列平台对包括对照（未处理）和接受 α2b-干扰素、γ-干扰素和 HeberFERON 治疗的三组的转录表达谱进行分析。应用无监督的基因和样本分组方法、差异表达基因的识别、功能富集和网络分析计算生物学方法，鉴定 HeberFERON 的独特转录模式。通过 qPCR 验证最具代表性的基因。对于用 HeberFERON 处理 24、48 和 72 小时的细胞的细胞周期分析，我们使用流式细胞术。
三种处理方法根据基因表达谱表现出不同的行为。富集分析鉴定了几个有丝分裂细胞周期相关事件，特别是从早前期到后期，这些事件仅由 HeberFERON 靶向。FOXM1 转录因子网络涉及多个细胞周期阶段，在 GBM 中高度表达，其表达显著下调。流式细胞术实验证实 HeberFERON 以剂量和时间依赖的方式作用于细胞周期，在治疗后 24 小时即可明显出现细胞阻滞。尽管 p53 未下调，但参与其调节活性的几个基因在功能上得到了富集。网络分析还揭示了 p53 与 HeberFERON 靶向的基因之间的强烈关系。我们提出了一种基于 PKR 和 ATF3 的同时激活、p53 磷酸化变化以及其 MDM2 介导的泛素化减少和从核输出到细胞质的机制模型，解释这种独特作用。PLK1、AURKB、BIRC5 和 CCNB1 等基因也受 FOXM1 调节，在该模型中也发挥核心作用。这些和其他相互作用可以解释 G2/M 期阻滞和 HeberFERON 对 U-87MG 增殖的影响。
我们提出了 HeberFERON 与 U-87MG 模型中单独使用干扰素相比的独特行为的分子机制，其中细胞周期相关事件具有重要意义。
