Targeting the proliferation of glioblastoma cells and enhancement of doxorubicin and temozolomide cytotoxicity through inhibition of PFKFB4 and HMOX1 genes with siRNAs

Glioblastoma multiforme continues to be one of the most aggressive brain cancers, posing a serious health challenge, as it offers a median survival of only 15–23 months and a 5-year survival rate of less than 6%. Current treatments often prove inadequate, underscoring the urgency for new therapeutic strategies. This study investigated the potential of silencing the PFKFB4 and HMOX1 genes in U87-MG glioblastoma cells using small interfering RNAs (siRNAs), both alone and alongside the chemotherapeutic agents temozolomide (TMZ) and doxorubicin (DOX). Through MTT assays, qPCR, and wound healing techniques, we assessed cell viability, gene expression, and cell migration. Notably, siPFKFB4 enhanced DOX’s cytotoxic effect, reducing its IC50 by six-fold, while having a milder impact with TMZ. When both siRNAs were combined with DOX, the IC50 decreased by two-fold without harming normal cells. Although siHMOX1 reduced cell migration, it only modestly affected cell proliferation with either DOX or TMZ. The gene expression analysis demonstrated that the siPFKFB4/DOX treatment led to an upregulation of pro-apoptotic genes such as DPYSL4, while simultaneously downregulating anti-apoptotic genes, including BCL-2 and PARP2. In contrast, the siHMOX1 combination influenced the expression of 14 genes, significantly enhancing the levels of CYLD, FAS, and CASP3, which are key promoters of apoptosis. In migration assays, siPFKFB4/DOX and siHMOX1/DOX reduced cell migration by 65 and 75%, respectively. These findings suggest that siPFKFB4 combined with DOX offers a promising pathway for GBM therapy, advocating further exploration into effective central nervous system drug delivery methods. © 2025 Elsevier B.V., All rights reserved.