Modulation of glioma-inflammation crosstalk profiles in human glioblastoma cells by indirubin-3'-(2,3 dihydroxypropyl)-oximether (E804) and 7-bromoindirubin-3'-oxime (7BIO)

Abstract

Indirubins E804 (indirubin-3'-(2,3 dihydroxypropyl)-oximether) and 7BIO (7-Bromoindirubin-3'-oxime) are synthetic derivatives of natural indirubin, the active compound in Danggui Longhui Wan, a traditional Chinese remedy for cancer and inflammation. Herein, we explore E804 and 7BIO for their potential to modulate key pro-inflammatory genes and cytokines in LN-18 and T98G glioblastoma cells. High grade gliomas typically secrete large amounts of inflammatory cytokines and growth factors that promote tumor growth in an autocrine fashion. Inflammation is emerging as a key concern in the success of new treatment modalities for glioblastomas. Studies indicate that select indirubin derivatives bind and activate signaling of the AHR pathway, as well as inhibit cyclin-dependent kinases and STAT3 signaling. AHR signaling is involved in hematopoiesis, immune function, cell cycling, and inflammation, and thus may be a possible target for glioma treatment. To determine the significance of the AHR pathway in LN-18 and T98G glioma inflammatory profiles, and on the effects of E804 and 7BIO on these profiles, we used 6,2',4'-trimethoxyflavone (TMF), a putative selective AHR antagonist. It was confirmed that E804 and 7BIO activates the AHR leading to cyp1b1 expression, and that TMF antagonizes expression. We then employed a commercial cancer inflammation and immunity crosstalk qRT-PCR array to screen for anti-inflammatory related properties. TMF alone inhibited expression of ifng, ptsg2, il12b, tnfa, il10, il13, the balance between pd1 and pdl1, and even expression of mhc1a/b. E804 was very potent in suppressing many pro-inflammatory genes, including il1a, il1b, il12a, ptgs2, tlr4, and others. E804 also affected expression of il6, vegfa, and stat3. Conversely, 7BIO induced cox2, but suppressed a different selection of pro-inflammatory genes including nos2, tnfa, and igf1. Secretion of IL-6 protein, an iconic inflammatory cytokine, was decreased by E804. VEGF (vascular endothelial growth factor) protein secretion was upregulated by 7BIO, yet downregulated by E804 and E804 plus TMF. Thus, E804 is both an AHR ligand and regulator of important pro-inflammatory cytokines such as IL-6 and oncogene STAT3, among others. Our results point to the use of E804 and TMF in combination as a promising new treatment for glioblastoma.

Keywords: 7BIO; Aryl hydrocarbon receptor; E804; Glioblastoma multiforme; Immunomodulation; Indirubin; Inflammation.