Ruxolitinib enhances cytotoxic and apoptotic effects of temozolomide on glioblastoma cells by regulating WNT signaling pathway-related genes.

Abstract

Although temozolomide is the primary chemotherapeutic agent in glioblastoma, current studies have focused on its combinational applications to overcome resistance by targeting multiple pathways. JAK/STAT and WNT are among the essential cancer-related signaling pathways. Ruxolitinib, the first approved JAK1/2 inhibitor, has promise in glioblastoma with its blood-brain barrier (BBB) crossing ability. The mentioned study aims to evaluate the anti-cancer potential of ruxolitinib individually and in combination with temozolomide on glioblastoma cells, brain cancer stem cells (BCSCs), and BBB-forming healthy cells. It also intends to determine the effects of JAK inhibitor treatment in combination with temozolomide on WNT signaling, which is known to cross-talk with the JAK/STAT pathway. The U87MG, BCSC, and HBMEC cell lines were the in vitro models. The cytotoxic and apoptotic effects of ruxolitinib and the combination were determined by the WST-1 test and Annexin V assay, respectively. The expression level changes of WNT signaling pathway genes caused by ruxolitinib and the combination treatments were defined by the qRT-PCR method. Network analysis of significantly upregulated and downregulated genes was performed via the GO KEGG pathway enrichment module of the String V11.5 database. The IC50 value of the ruxolitinib on U87MG glioblastoma cells was determined as 94.07µM at 24th h. The combination of temozolomide and ruxolitinib had a synergistic effect on U87MG cells at 24th h. The combination index (CI) was determined as 0.796, and ED60 values of ruxolitinib and temozolomide were determined as 89.75 and 391.48µM, respectively. Ruxolitinib improves the apoptotic effect of temozolomide on glioblastoma cells and brain cancer stem cells. Ruxolitinib regulates the WNT signaling pathway both individually and in combination with temozolomide. Our study indicates the potential of ruxolitinib to increase the cytotoxic and apoptotic activity of temozolomide in glioblastoma cells, also considering CSCs and healthy BBB-forming cells. As supported by gene expression and network analyses, the BBB-crossing agent ruxolitinib promises the potential to increase the efficacy of temozolomide in glioblastoma by affecting multiple signaling pathways in both cancer cells and CSCs.