J Korean Neurosurg Soc.  2020 Jul;63(4):444-454. 10.3340/jkns.2019.0252.

The Dose Dependent Effects of Ruxolitinib on the Invasion and Tumorigenesis in Gliomas Cells via Inhibition of Interferon Gamma-Depended JAK/STAT Signaling Pathway

• Delen E
• Doğanlar O

• KMID: 2504638
• DOI: http://doi.org/10.3340/jkns.2019.0252

Abstract

Objective
: Glioblastoma multiforme (GBM) is the most aggressive for of brain tumor and treatment often fails due to the invasion of tumor cells into neighboring healthy brain tissues. Activation of the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway is essential for normal cellular function including angiogenesis, and has been proposed to have a pivotal role in glioma invasion. This study aimed to determine the dose-dependent effects of ruxolitinib, an inhibitor of JAK, on the interferon (IFN)-I/IFN-α/IFN-β receptor/STAT and IFN-γ/IFN-γ receptor/STAT1 axes of the IFN-receptor-dependent JAK/STAT signaling pathway in glioblastoma invasion and tumorigenesis in U87 glioblastoma tumor spheroids.
Methods
: We administered three different doses of ruxolitinib (50, 100, and 200 nM) to human U87 glioblastoma spheroids and analyzed the gene expression profiles of IFNs receptors from the JAK/STAT pathway. To evaluate activation of this pathway, we quantified the phosphorylation of JAK and STAT proteins using Western blotting.
Results
: Quantitative real-time polymerase chain reaction analysis demonstrated that ruxolitinib led to upregulated of the IFN-α and IFN-γ while no change on the hypoxia-inducible factor-1α and vascular endothelial growth factor expression levels. Additionally, we showed that ruxolitinib inhibited phosphorylation of JAK/STAT proteins. The inhibition of IFNs dependent JAK/STAT signaling by ruxolitinib leads to decreases of the U87 cells invasiveness and tumorigenesis. We demonstrate that ruxolitinib may inhibit glioma invasion and tumorigenesis through inhibition of the IFN-induced JAK/STAT signaling pathway.
Conclusion
: Collectively, our results revealed that ruxolitinib may have therapeutic potential in glioblastomas, possibly by JAK/STAT signaling triggered by IFN-α and IFN-γ.

Keyword

Ruxolitinib; Glioblastoma; Carcinogenesis; Interferons; Signal transduction

Figure

• 

  Fig. 1. Tumour volume and invasion rate of 50, 100, and 200 nM ruxolitinib treated and untreated (control) U87 spheroids. B : Relative % of tumour inhibition and relative % of invasion area in control and experimental groups at 48 hours. C : Images belongs to sphere formation assay. Values expressed as mean±standard error (n=5). *Indicates significantly different values compared to control groups (one-way analysis of variance, Tukey honestly significant difference test : p≤0.05). †p≤0.01. ‡p≤0.001. §p<0.0001.

• 

  Fig. 2. The relative expression level of genes belongs to IFN-I and IFN-γ receptors depended JAK/STAT pathway and related downstream signalling in 50, 100, and 200 nM ruxolitinib treated and untreated (control) U87 spheroids. All data normalized with ribosomal RNA 18S levels. Values expressed as mean±standard error (n=5). *Indicates significantly different values compared to control groups (one-way analysis of variance, Tukey honestly significant difference test : p≤0.05). †p≤0.01. ‡p≤0.001. §p<0.0001. IFN : interferon, JAK/STAT : Janus kinase-signal transducer and activator of transcription.

• 

  Fig. 3. The relative expression level of genes belongs to hypoxia related angiogenesis (HIF1-α, HIF1-β, and VEGF) and specific JAK inhibitors SOCS family signalling in 50, 100, and 200 nM ruxolitinib treated and untreated (control) U87 spheroids. All data normalized with ribosomal RNA 18S levels. Values expressed as mean±standard error (n=5). *Indicates significantly different values compared to control groups (one-way analysis of variance, Tukey honestly significant difference test : p≤0.05). †p≤0.01. ‡p≤0.001. §p<0.0001. SOCS : the suppressor of cytokine signalling, HIF : hypoxia-inducible factor, VEGF : vascular endothelial growth factor, JAK : Janus kinase-signal transducer.

• 

  Fig. 4. A : JAK/STAT-IFNAR and JAK/STAT-IFN-γ receptor signaling pathway. B : Western blot analysis demonstrated the relative expressions of active JAK1, Phospho-JAK1 (p Y1022+Y1023), JAK2, Phospho-JAK2 (p Tyr1007, Tyr1008), Phospho-STAT1 [p Tyr701], STAT3, Phospho-STAT3 (p Tyr705), SOCS7 and β-actin in the 50, 100, and 200 nM ruxolitinib treated and untreated (control=1) U87 spheroids. CM : cell membrane, NM : nuclear membrane, JAK/STAT : Janus kinase-signal transducer and activator of transcription, IFNAR : interferon alpha receptor, IFN : interferon, SOCS : the suppressor of cytokine signalling.

Reference

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