A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin

Abstract

A novel acetylenic tricyclic bis-(cyano enone), TBE-31, is a lead compound in a series of tricyclic compounds with enone functionalities in rings A and C. Nanomolar concentrations of this potent multifunctional molecule suppress the induction of the inflammatory protein, inducible nitric oxide synthase, activate phase 2 cytoprotective enzymes in vitro and in vivo, block cell proliferation, and induce differentiation and apoptosis of leukemia cells. Oral administration of TBE-31 also significantly reduces formation of aflatoxin-DNA adducts and decreases size and number of aflatoxin-induced preneoplastic hepatic lesions in rats by >90%. Because of the two cyano enones in rings A and C, TBE-31 may directly interact with DTT and protein targets such as Keap1 that contain reactive cysteine residues. The above findings suggest that TBE-31 should also be tested for chemoprevention and chemotherapy in relevant models of cancer and against other chronic, degenerative diseases in which inflammation and oxidative stress contribute to disease pathogenesis.

Fig. 1. Structures of tricylic bis-enones (TBE) and the oleanane triterpenoids CDDO, CDDO-Methyl ester and CDDO-Imidazolide

Fig. 2. TBEs block the induction of iNOS and induce phase 2 cytoprotective proteins

RAW264.7 mouse macrophage-like cells (A and B) or U937 cells (B) were treated with TBEs or CDDO-Imidazolide (Im) and IFN-γ (10 ng/ml) for 18 h (A) or with compounds alone for 6 h (B), and cell lysates were immunoblotted with iNOS, HO-1 or tubulin antibodies. Hepa1c1c7 cells were grown for 24 h and then treated with serial dilutions of compounds for 48 h. The concentration required to double (CD) the specific enzyme activity of NQO1 was used to quantify inducer potency (C). In (D), U937 cells were treated with TBEs or Im for 24 h. H₂DCFDA was added for 30 minutes, and then the cells were challenged with 250 µmol/L tert-butyl hydroperoxide for 20 minutes to induce the formation of reactive oxygen species. The mean fluorescence intensity of 10,000 cells per group was detected by flow cytometry, and results are expressed as percent of control.

Fig. 3. Increasing concentrations of TBEs induce differentiation, inhibit proliferation, and activate apoptosis in U937 leukemia cells

U937 cells were treated with various concentrations of TBE-31 and CDDO-Im for 4 days and CD11b expression was analyzed by flow cytometry (A) or were treated with 100 nM TBE-31 or 30 nM CDDO-Im (Im) for 8–24 h and Western blots of cell lysates were probed with Id1 and Id2 antibodies (B). To measure proliferation, cells were treated with TBEs or Im for 3 days and then evaluated using a [³H]thymidine incorporation assay (C). U937 cells also were treated for 24 h and Western blots were probed with PARP (cPARP, cleaved PARP) and tubulin antibodies to verify the induction of apoptosis (D). A = anisomycin (positive control, 10 µg/ml).

Fig 4. TBE-31 directly interacts with cysteine residues of Keap1 and blocks both the degradation of IκBα and constitutive STAT3 phosphorylation in HepG2 hepatocellular carcinoma cells

Spectrophotometric analysis (A) of 0.1 mM TBE-31 before (gray line) and after (black line) the addition of 1 mM DTT. (B) RAW cells and (C) HepG2 cells were pretreated with TBE-31 or CDDO-Im for 24 or 2 h, respectively, stimulated with TNFα for 15 min, and then immunoblotted with IκBα and tubulin antibodies. (D) HepG2 cells were also treated for 2 h and then immunoblotted with pSTAT3 and tubulin antibodies.

Fig. 5. Tissue levels (A) and stability of TBE-31

(B) TBE-31 and CDDO-Im were incubated in human plasma at 37°C, and samples were collected at 4 and 24 h and then analyzed by LCMS. (C) In an independent experiment, TBE-31 (1 µmol) was dissolved in propylene glycol (V = vehicle) and administered by i.v. or i.p. injection or by gavage to male CD-1 mice (3–4 per group). After 6 h, livers were harvested and homogenized, and Western blots of tissue lysates were probed with HO-1 and tubulin antibodies.

Fig. 6. TBE-31 reduces both the formation of aflatoxin-DNA adducts and pre-neoplasatic lesions in the livers of rats injected with aflatoxin

(A) Rats were gavaged with TBEs or CDDO-Im. Forty-eight h later, rats were gavaged with 25 µg/rat of AFB₁. Rats were sacrificed 2 h after treatment with AFB₁, and DNA from flash frozen livers was isolated and analyzed for levels of AFB₁-DNA adducts by liquid chromatography-mass spectrometry. * P < 0.05 compared to vehicle. Values are the mean ± SE (n=4). (B) Rats (n = 5) were gavaged with TBE-31 or CDDO-Im on Monday, Wednesday, and Friday mornings for 3 successive weeks. Beginning on the second week, AFB₁ (25µg/rat) was gavaged each Monday through Friday afternoon for 2 weeks. Five weeks after the final doses of drug and AFB₁, rats were sacrificed, and multiple sections of the left lateral lobe of the liver were fixed and embedded. Liver sections were stained for expression of GST-P positive foci and analyzed for preneoplastic tumor burden ("volume percent").