The Effects of Indirubin Derivative E804 on Inflammatory Profiles in Glioblastoma Multiforme Cell Lines and Subsequent Crosstalk Effect on Tumor Associated THP-1 Macrophages

Glioblastoma Multiforme (GBM) is a highly invasive brain tumor that affects approximately 18,000 people annually in the USA alone yet remains without curative treatment. Median survival with treatment is 14 months, with most treatments only prolonging life for several months while causing severe adverse side effects. There is a need for new therapeutic modalities. Herein I explore two small molecules that show promise in modulating inflammatory signaling in an in vitro GBM model. Indirubins E804 (indirubin-3’-(2,3 dihydroxypropyl)-oximether) and 7BIO (7-Bromoindirubin-3’-oxime) are synthetic derivatives of natural indirubin. Natural indirubin is a bisindole alkaloid derived from tryptophan precursors and is an agonist for the ligand-activated transcription factor aryl hydrocarbon receptor (AHR). Indirubin has been shown to disrupt important cancer-driving signaling such as JAK/STAT3, GSK3, and CDKs. To determine AHR pathway significance in my results, I added a selective AHR antagonist, 6,2’,4’-trimethoxyflavone (TMF), in addition to indirubins. Herein I show that E804 modulates a large array of inflammatory genes, including down-regulation of important autocrine GBM signaling molecules like IL-6 and VEGF, which help drive tumor development. 7BIO, but not E804, increased the expression of STAT3, suggesting that this compound is not suitable for glioma therapy. Additional studies showed that E804 does not fully activate the unfolded protein response (UPR). The UPR is an ER stress pathway that can induce indiscriminate apoptosis - an undesirable side effect in an area as sensitive as the brain. Finally, using transcriptomics combined with pathway analysis, I examined the effects that E804- or E804+TMF-treated glioblastoma cells have on differentiated THP-1 macrophages to model the effects that gliomas may have on glioma-associated macrophages. Glioma-associated macrophages can comprise up to 30% of GBM mass, and, if activated by certain glioma-derived signals, have been implicated in tumor promotion. I found that treated-GBM supernatants inhibited gene profiles in THP-1 macrophages that are linked to tumor progression, such as the Wnt, TLR, and Cell Cycle pathways; enriched gene ontology (GO) terms in up-regulated gene lists involving vascularization and hormone metabolism; and modulated expression of genes associated with macrophage polarization. Taken together, the results of this study demonstrate that E804 and E804 + TMF modulate signaling in GBM cells, with the ability to influence macrophages. These treatments hold promise as an alternative to current modalities using very toxic nitrosourea compounds, and future work should continue to investigate crosstalk between macrophages and GBM, both in vitro and in vivo