Abstract
Semi-synthetic triterpenoids are antioxidant inflammation modulator (AIM) compounds that inhibit tumor cell growth and metastasis. Compounds in the AIM class bind to Keap1 and attenuate Nrf2 degradation. In the nucleus, Nrf2 increases antioxidant gene expression and reduces pro-inflammatory gene expression. By increasing Nrf2 activity, AIMs reduce reactive oxygen species and inflammation in the tumor microenvironment, which reverses tumor-mediated immune evasion and inhibits tumor growth and metastasis. AIMs also directly inhibit tumor cell growth by modulating oncogenic signaling pathways, such as IKKβ/NF-κB. Here, we characterized the in vitro antioxidant, anti-inflammatory, and anticancer activities of RTA 408, a novel AIM that is currently being evaluated in patients with advanced malignancies. At low concentrations (≤ 25 nM), RTA 408 activated Nrf2 and suppressed nitric oxide and pro-inflammatory cytokine levels in interferon-γ-stimulated RAW 264.7 macrophage cells. At higher concentrations, RTA 408 inhibited tumor cell growth (GI50 = 260 ± 74 nM) and increased caspase activity in tumor cell lines, but not in normal primary human cells. Consistent with the direct effect of AIMs on IKKβ, RTA 408 inhibited NF-κB signaling and decreased cyclin D1 levels at the same concentrations that inhibited cell growth and induced apoptosis. RTA 408 also increased CDKN1A (p21) levels and JNK phosphorylation. The in vitro activity profile of RTA 408 is similar to that of bardoxolone methyl, which was well-tolerated by patients at doses that demonstrated target engagement. Taken together, these data support clinical evaluation of RTA 408 for cancer treatment.
Highlights
Antioxidant inflammation modulators (AIMs) include synthetic derivatives of oleanolic acid, a triterpenoid found in medicinal plants [1]
Consistent with this, we observed a decrease in nitric oxide synthase 2 (Nos2) protein levels in bardoxolone methyl-treated RAW 264.7 cells, which was attenuated when Nrf2 mRNA levels were reduced by siRNA (S1 Fig)
Phosphorylation of JNK in response to RTA 408 occurred at concentrations that increased cleavage of caspase-3 and caspase-9 (Fig 2C and S2 Fig). These results suggest that RTA 408 directly inhibits tumor cell growth and activates apoptosis in a manner similar to bardoxolone methyl and other AIMs
Summary
Antioxidant inflammation modulators (AIMs) include synthetic derivatives of oleanolic acid, a triterpenoid found in medicinal plants [1]. Oleanolic acid itself is a weak Nrf activator [2]; key alterations to the triterpenoid scaffold improved potency by more than 6 orders of magnitude [3]. The adaptor protein Kelch-like ECH-associated protein 1 (Keap1) targets Nrf for Cul3-Rbx1-mediated ubiquitination and constitutive proteasomal degradation, thereby maintaining low basal levels of Nrf2 [4]. AIMs increase Nrf levels by binding to Keap and blocking its ability to promote Nrf degradation [3,5]. Newly synthesized Nrf accumulates in the nucleus where it increases the expression of antioxidant genes and decreases the expression of pro-inflammatory genes [6,7]
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