Date of Award
Master of Science (MS)
Charles D Rice
Vincent S Gallicchio
Natural products containing derivatives of the basic indole backbone have gained significant interest in the use against cancer cells, inflammation, and a multitude of disorders in the human body. The indole backbone is present in several endogenous hormones such as serotonin and melatonin. Additionally, the indole-containing amino acid tryptophan is the starting structure for many endogenous metabolites controlling natural physiological homeostasis such as circadian rhythms, healthy gut microbiota, and gut health. Natural indirubin is a deep red bis-indole isomer of indigo blue, both of which are biologically active ingredients used to treat neoplasia, chronic inflammation, and enhance xenobiotics' detoxification. Naturally derived indirubins and other indole-containing compounds have been shown to have anti-proliferative effects, mainly attributed to the inhibition of the cell cycle-related kinases, such as cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3 ) with varying degrees of potency. Many indirubins are also aryl hydrocarbon receptor (AHR) agonists, with AHR-associated activities covering a wide range of potencies, depending on molecular structure. The AHR is a ligand-activated transcription factor that promotes drug-metabolizing enzymes leading to the degradation of these indirubins in an AHR-dependent manner. Our lab previously described the anti-inflammatory properties of indirubin-3'-(2,3 dihydroxypropyl)-oximether (E804), a novel indirubin derivative with potent STAT3 inhibitory properties, in murine RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). This study compared the effects of a novel STAT3 inhibitor, 6-nitrobenzo[b]thiophene 1,1-dioxide (STATTIC), which is structurally designed as an indole backbone to E804, on LPS-stimulated macrophage functions. I also determined if the effects of both STATTIC and E804 on these macrophage functions are modified by the AHR antagonist, 6,2’,4’-trimethoxyflavone (TMF). Initial studies using AHR reporter transactivation assays show that E804, but not STATTIC or TMF, is an AHR agonist, which further corroborates early studies showing that E804 induces the drug-metabolizing monooxygenases (CYP1A1 and CYP1B1). Additionally, I demonstrate that STATTIC is more potent than E804 in suppressing LPS-induced IL-6 secretion, iNOS protein expression, and nitric oxide production. Macrophage intracellular reactive oxygen species (ROS) naturally generated during LPS-stimulation was suppressed by low levels of STATTIC but not by higher levels. In contrast, similar concentrations of E804 suppress ROS production. STATTIC completely inhibited phagocytosis, and less so by E804. When examined alone, TMF has anti-inflammatory properties as well, and when combined with E804 and STATTIC, further enhanced these compounds’ effects. Collectively, these results indicate that E804 and STATTIC are potent modulators of pro-inflammatory profiles in LPS-treated macrophages. Additionally, these results suggest that AHR antagonism by TMF may antagonize the degradation of E804 and prolong its anti-inflammatory properties. Since STATTIC did not bind the AHR, any treatments using this novel small molecule may not require co-treatment such as TMF.
Tisch, Logan, "Comparing the Anti-Inflammatory Properties of STATTIC to Indirubin-E804 in RAW264.7 Macrophages" (2021). All Theses. 3504.