RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity

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.

Fig 1. RTA 408 Increases Expression of Nrf2 Target Genes and Decreases Expression of Inflammatory Mediators in RAW 264.7 Cells.

RAW 264.7 macrophages were treated with the indicated concentrations of RTA 408 or bardoxolone methyl for two hours, and then treated with 20 ng/mL IFNγ for an additional 24 hours. A, B, Nitrite (NO₂-) concentrations in the media were measured by Griess reaction and cell viability was assessed using WST-1 reagent. Viability is presented as percent survival relative to survival in cells treated with IFNγ alone. Data are representative of three experiments. C, mRNA levels of Nrf2 target genes Nqo1, Gclc, and Txnrd1 were measured by qRT-PCR. Data are presented relative to expression in cells treated with IFNγ alone. Data points are the mean and SD of three experiments. D, mRNA levels of pro-inflammatory genes Nos2, Ptgs2, Ccl2, and Ccl5 were measured by qRT-PCR. Values are presented relative to expression in cells treated with IFNγ alone. Data points are the mean and SD of three experiments. E, protein levels of Nos2 and Ptgs2 were evaluated by Western blot. Actin served as a loading control. Data are representative of two experiments. F, protein levels of Ccl2 and Ccl5 in the culture media were measured by ELISA. Data are representative of two experiments. For C and D, statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test. *, P < 0.05; **, P <0.01 compared to cells treated with IFNγ alone.

Fig 2. RTA 408 Inhibits Growth and Induces Apoptosis in Human Tumor Cell Lines.

A, Cells were treated with RTA 408 for 72 hours and viability was assessed using the SRB assay. Values are presented as percent growth in RTA 408-treated cells relative to growth in vehicle-treated cells. B, Human tumor cell lines were treated with RTA 408 for 24 hours and cleavage of the DEVD-AFC peptide was measured as a surrogate for caspase activity. Data are presented as caspase activity in RTA 408-treated cells relative to activity in vehicle-treated cells. Data points are the mean and SD of three experiments. Statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test. *, P < 0.05; **, P < 0.01 compared to vehicle-treated cells. C, Cells were treated with RTA 408 for 24 hours and protein levels of caspase-3 and caspase-9 were evaluated by Western blot. Actin served as a loading control. Data are representative of two experiments. D, Human tumor cell lines and normal primary human cells were treated with RTA 408 for 24 hours and cleavage of the DEVD-AFC peptide was measured as a surrogate for caspase activity. Data are presented as caspase activity in RTA 408-treated cells relative to activity in vehicle-treated cells. Data points are the mean and SD of three experiments.

Fig 3. RTA 408 Inhibits Proliferation and Colony Formation in Wild-Type and Keap1-/- MEFs.

A, Growth of WT and Keap1 ^-/- MEFs treated with RTA 408. MEFs were treated with RTA 408 and cells were counted at 24-hour intervals. Statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test. *, P < 0.05; **, P < 0.01 compared to vehicle-treated cells at the same time point. B, Colony formation by WT and Keap1 ^-/- MEFs treated with RTA 408. Statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test. *, P < 0.05; **, P < 0.01 compared to vehicle-treated cells. C, Nrf2 target gene expression in WT and Keap1 ^-/- MEFs treated with RTA 408 for 18 hours. mRNA levels of Nqo1 and Gclm were measured by qRT-PCR. Values are presented as fold-induction relative to vehicle-treated WT MEFs. D, RTA 408 increases expression of Nrf2 target genes in human tumor cell lines. MDA-MB-231, HCT 116, and G361 cells were treated with the indicated concentrations of RTA 408 for 18 hours. mRNA levels of NQO1, HMOX1, GCLM, and GCLC, were measured by qRT-PCR. Data are presented as fold-induction relative to vehicle-treated cells for each cell line. Statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test.*, P < 0.05; **, P <0.01; ***, P < 0.001 compared to vehicle-treated cells. E, MDA-MB-231, HCT 116, and G361 cells were treated with RTA 408 for 48 hours and cell viability was determined using the SRB assay. Data are presented as percent survival relative to survival in vehicle-treated cells. In all panels, data points are the mean of three independent experiments and error bars are SD.

Fig 4. RTA 408 Inhibits NF-κB and Activates JNK.

A, HeLa/NF-κB-Luc or A549/NF-κB-Luc cells were treated with RTA 408 for one hour and then treated with 10 ng/mL TNFα. Five hours later, luminescence was measured to assess NF-κB activity. Data are presented as percent activity relative to cells treated with TNFα alone. Data points for HeLa and A549 are the mean and SD of three and four experiments, respectively. B, HeLa cells were pre-treated with RTA 408 or bardoxolone methyl for 6 hours at the indicated concentrations followed by a five-minute treatment with TNFα. Protein levels of phospho-IκBα and total IκBα were evaluated by western blot. Actin was used as loading control. Data are representative of four experiments. C, Cells were treated with RTA 408 for 24 hours and protein levels of cyclin D1, CDKN1A (p21), and total and phospho-JNK were evaluated by western blot. Actin served as a loading control. Data are representative of two experiments.

Fig 5. RTA 408 Mechanism of Action.

The coordinated anticancer activities of RTA 408 affect both tumor cells and cells within the tumor microenvironment. By activating Nrf2 and reducing ROS and inflammation in the tumor microenvironment, RTA 408 reverses tumor-mediated immune suppression and prevents tumor growth and metastasis. Within the tumor cells, transient Nrf2 activation by RTA 408 does not promote growth or survival. RTA 408 also modulates the activity of oncogenic signaling pathways (such as NF-κB, cyclin D1, JNK, and CDKN1A (p21)) and promotes growth arrest and apoptosis of tumor cells.