Belinostat, at Its Clinically Relevant Concentrations, Inhibits Rifampicin-Induced CYP3A4 and MDR1 Gene Expression

Abstract

Activation of human pregnane X receptor (hPXR) has been associated with induction of chemoresistance. It has been proposed that such chemoresistance via cytochrome P450/drug transporters can be reversed with the use of antagonists that specifically abrogate agonist-mediated hPXR activation. Unfortunately, proposed antagonists lack the specificity and appropriate pharmacological characteristics that allow these features to be active in the clinic. We propose that, ideally, an hPXR antagonist would be a cancer drug itself that is part of a "cancer drug cocktail" and effective as an hPXR antagonist at therapeutic concentrations. Belinostat (BEL), a histone deacetylase inhibitor approved for the treatment of relapsed/refractory peripheral T-cell lymphoma, and often used in combination with chemotherapy, is an attractive candidate based on its hPXR ligand-like features. We sought to determine whether these features of BEL might allow it to behave as an antagonist in combination chemotherapy regimens that include hPXR activators. BEL represses agonist-activated hPXR target gene expression at its therapeutic concentrations in human primary hepatocytes and LS174T human colon cancer cells. BEL repressed rifampicin-induced gene expression of CYP3A4 and multidrug resistance protein 1, as well as their respective protein activities. BEL decreased rifampicin-induced resistance to SN-38, the active metabolite of irinotecan, in LS174T cells. This finding indicates that BEL could suppress hPXR agonist-induced chemoresistance. BEL attenuated the agonist-induced steroid receptor coactivator-1 interaction with hPXR, and, together with molecular docking studies, the study suggests that BEL directly interacts with multiple sites on hPXR. Taken together, our results suggest that BEL, at its clinically relevant therapeutic concentration, can antagonize hPXR agonist-induced gene expression and chemoresistance.

Fig. 1.

Effect of BEL on hPXR agonist–induced CYP3A4 and MDR1 gene expression. CYP3A4 and MDR1 mRNA expression was analyzed by quantitative reverse-transcription polymerase chain reaction in human primary hepatocytes (A and B) and LS174T cells (C and D) after treatment with vehicle DMSO, RIF, BEL ± RIF, SR, BEL + SR, KET, or KET ± RIF as indicated for 24 hours. Results are presented as the fold change over DMSO treatment. Data are expressed as the mean ± S.D. values. P < 0.05, compared with DMSO alone (#) or RIF or SR alone (*) by ANOVA with Dunnett’s multiple-comparisons test.

Fig. 2.

Effect of BEL on the viability of the hepatocytes and LS174T intestinal cells. Viability of the human primary hepatocytes (A) and LS174T cells (B) was determined under the same experimental conditions indicated in gene expression studies. Cell viability was measured by using CellTiter-Glo Reagent. Viability of DMSO-treated cells was expressed as 100%. Results are shown as the mean ± S.D. #P < 0.05, compared with DMSO alone by ANOVA and Dunnett’s multiple-comparisons test.

Fig. 3.

Effect of BEL on RIF-induced and basal CYP3A4 activity. (A) BEL inhibits RIF-induced CYP3A4 activity in the human primary hepatocytes. CYP3A4 activity was analyzed by the luminescent cytochrome P450-Glo CYP3A4 assays in the human primary hepatocytes after treatment with vehicle, DMSO, RIF, BEL ± RIF, or KET ± RIF as indicated for 24 hours. Results are presented as the fold change over DMSO treatment. Data represent the mean ± S.D. from four independent experiments performed on single-donor hepatocytes. P < 0.05; compared with DMSO alone (#) or RIF alone (*) by ANOVA with Dunnett’s multiple-comparisons test. (B) BEL does not inhibit the basal CYP3A4 activity in the human primary hepatocytes. DMSO, BEL, or KET was added to the hepatocytes only during the assay period to determine their direct effects on CYP3A4 activity. Results are presented as the fold change over DMSO treatment. Data represent the mean ± S.D. values from four independent experiments performed on single-donor hepatocytes. #P < 0.05; compared with DMSO alone by ANOVA with Dunnett’s multiple-comparisons test.

Fig. 4.

Effect of BEL on RIF-induced MDR1 activity and RIF-induced resistance to SN-38. (A) BEL inhibits RIF-induced MDR1 activity in LS174T cells. LS174T cells were treated with DMSO, RIF, or BEL ± RIF as indicated for 24 hours. R123 accumulation was then determined in the absence or presence of the MDR1-specific inhibitor PSC-833. The data are normalized to the DMSO treatment and are presented as the mean ± S.D. of at least four independent experiments. P < 0.05, compared with DMSO alone in the absence of PSC-833 (#) or RIF alone in the absence of PSC-833 (*) by ANOVA and Dunnett’s multiple comparisons test. (B) BEL attenuates RIF-induced resistance to SN-38 in LS174T cells. LS174T cells were treated with the indicated compounds for 24 hours, and viability was measured by using the CellTiter-Glo luminescent cell viability assay. The viability of DMSO-treated cells was set to 100%. Data represent the mean ± S.D. values from at least four experiments. Statistical significance (P < 0.05) was determined by ANOVA and Dunnett’s multiple-comparisons test.

Fig. 5.

hPXR molecular docking studies. (A) Ensemble-based molecular docking studies predict high affinity of BEL for multiple sites at hPXR. Score of top-ranked docked pose of ligands at different sites in hPXR, obtained from docking of ligands against an ensemble of hPXR conformations. (B) Mode of interaction of BEL at AF2 region (a) and α8 pocket (b). Dotted lines denote the H-bonding interaction, and the protein residues involved in hydrophobic interactions are shown by red spikes. H-bond distance is also shown alongside. The residues that are critical for SRC-1 interaction at AF2 region are highlighted in the rectangles. (C) Superposition of BEL and SRC-1 interaction at the AF2 region. The interacting residues common to both SRC-1 and BEL are highlighted.

Fig. 6.

Mechanisms of BEL interaction with hPXR. (A) BEL affects the recruitment of SRC-1 to the hPXR-LBD in in vitro SRC-1 coactivator recruitment assays. The TR-FRET ratio was calculated by dividing the emission signal at 520 nm from the acceptor fluorophore by the emission signal at 490 nm from the donor terbium. The TR-FRET ratios of the compounds were normalized to DMSO. An increase or a decrease of the normalized TR-FRET ratio in the presence of the compounds indicates increased or decreased binding of SRC-1 to hPXR-LBD, respectively. Data represent the mean ± S.D. values. P < 0.05, compared with DMSO alone (#) or SR12813 alone (*) by ANOVA and Dunnett’s multiple-comparisons test. (B) BEL binds to hPXR-LBD in an in vitro competitive ligand-binding assay. hPXR-LBD, Tb-anti-GST antibody, and a fluorescein-labeled hPXR ligand tracer were incubated together in the presence of DMSO (vehicle control), BEL (test compound), or SR (a known hPXR agonist). The TR-FRET ratio indicates the binding of the hPXR ligand tracer to hPXR-LBD, and a decrease of the TR-FRET ratio indicates the binding of agonists or antagonists to the hPXR-LBD by outcompeting the binding of the hPXR ligand tracer. Data represent the mean ± S.D. values from four independent determinations.

Fig. 7.

Proposed model for BEL repression of hPXR agonist-induced target gene expression and chemoresistance during combination chemotherapy.