Anticolon cancer activity of largazole, a marine-derived tunable histone deacetylase inhibitor

Abstract

Histone deacetylases (HDACs) are validated targets for anticancer therapy as attested by the approval of suberoylanilide hydroxamic acid (SAHA) and romidepsin (FK228) for treating cutaneous T cell lymphoma. We recently described the bioassay-guided isolation, structure determination, synthesis, and target identification of largazole, a marine-derived antiproliferative natural product that is a prodrug that releases a potent HDAC inhibitor, largazole thiol. Here, we characterize the anticancer activity of largazole by using in vitro and in vivo cancer models. Screening against the National Cancer Institute's 60 cell lines revealed that largazole is particularly active against several colon cancer cell types. Consequently, we tested largazole, along with several synthetic analogs, for HDAC inhibition in human HCT116 colon cancer cells. Enzyme inhibition strongly correlated with the growth inhibitory effects, and differential activity of largazole analogs was rationalized by molecular docking to an HDAC1 homology model. Comparative genomewide transcript profiling revealed a close overlap of genes that are regulated by largazole, FK228, and SAHA. Several of these genes can be related to largazole's ability to induce cell cycle arrest and apoptosis. Stability studies suggested reasonable bioavailability of the active species, largazole thiol. We established that largazole inhibits HDACs in tumor tissue in vivo by using a human HCT116 xenograft mouse model. Largazole strongly stimulated histone hyperacetylation in the tumor, showed efficacy in inhibiting tumor growth, and induced apoptosis in the tumor. This effect probably is mediated by the modulation of levels of cell cycle regulators, antagonism of the AKT pathway through insulin receptor substrate 1 down-regulation, and reduction of epidermal growth factor receptor levels.

Fig. 1.

Structures of largazole, FK228, and SAHA and modes of activation of largazole and FK228 to generate largazole thiol and redFK228, respectively.

Fig. 2.

Antiproliferative activity of largazole. A, performance of largazole across cell lines in the NCI-60 screen using three different measures of antiproliferative activity (growth-inhibitory effect, GI₅₀; cytostatic effect, TGI; cytotoxic effect, LC₅₀; concentration in M) (Paull et al., 1995). Color represents activity on the indicated continuous color scale based on log₁₀ transformed values. B, largazole induces G₁ arrest at lower concentration and G₂/M arrest at higher concentration in HCT116 cells (24-h treatment). C, largazole induces apoptosis in HCT116 cells. HCT116 cells were treated for 48 h with largazole, and apoptosis was measured as a function of caspase 3/7 activity by using Caspase-Glo 3/7. Error bars indicate S.D. from triplicate experiments.

Fig. 3.

SAR studies. A, structures of largazole and its analogs. B, antiproliferative activity in HCT116 cells, recombinant HDAC1, and cellular HDAC inhibition (8-h treatment) by largazole and its analogs. [Note: Thioesters were tested in the HDAC1 assay. The corresponding thiols are approximately 10 times more potent because the activity arises from the fraction that is hydrolyzed under assay conditions (equal hydrolysis rate assumed) (Ying et al., 2008a)]. C–H, molecular docking showing binding modes from different angles (C–E versus F–H). Largazole thiol (C and F), His analog of largazole thiol (D and G), and Tyr analog of largazole thiol (E and H) docked into a homology model of HDAC1 (Wang et al., 2005) using AutoDock Vina (Trott and Olson, 2010). In all cases the side chain bearing the thiol group interacts with the zinc ion (purple) resident at the active site of the enzyme. In all structures, the NH of the Gly-derived unit is able to form a hydrogen bond with Leu271 (solid red line). In addition, the His and Tyr residues of the analogs are able to form hydrogen bonds to the ω-carboxylic acid functionality (side chain) and amide carbonyl oxygen of Glu98, respectively (D and G versus E and H, solid red line).

Fig. 4.

Transcriptional analysis of HDAC inhibition in HCT116 cells and validation. A, dose optimization. Cells were treated with various concentrations of largazole, SAHA, or FK228 for 8 h. Protein lysates were collected and analyzed by immunoblot analysis for histone H3 acetylation as a measure for class I HDAC inhibition and for acetyl-α-tubulin as a measure of HDAC6 (class IIb) inhibition. Based on this analysis, it was estimated that largazole and FK228 are effective at 20 nM and SAHA is effective at 2 μM. B, hierarchical cluster analysis of probe sets with ≥2-fold mRNA changes for any of the three HDAC inhibitors. Transcriptome analysis (Affymetrix GeneChip Human Genome U133 plus 2.0 arrays) was carried out with duplicate biological samples. C, validation of transcriptional changes induced by largazole on the protein level (24-h treatment).

Fig. 5.

Effects of largazole on other colon cancer cell types with different susceptibility. A and B, effect of largazole on histone H3 acetylation in HT29 cells (A) and HCT15 cells (B) upon 8-h treatment, assessed by immunoblot analysis. C and D, DNA content analysis. As observed for HCT116 cells, largazole induces G₁ arrest at a lower concentration and G₂/M arrest at a higher concentration in HT29 cells (C) and HCT15 cells (D) (24-h treatment). Required largazole concentrations differ but correspond to the concentration range that induces histone H3 hyperacetylation (A and B).

Fig. 6.

Largazole stability and activation studies. Largazole and its metabolites were extracted with ethyl acetate, subjected to LC-MS, and monitored by using compound-specific MRM mode with harmine as internal standard. A, stability of largazole, liberated thiol, and thiol adduct in mouse serum. Largazole hydrolyzed in the presence of mouse serum to yield the largazole thiol adduct at earlier time points. Largazole thiol was generated from the largazole thiol adduct and peaked with 1-h incubation. B, LC-MS profile of largazole and metabolites. On the basis of specific MRM transitions and retention times, largazole and metabolites were identified. Largazole thiol and largazole thiol adduct showed the same MRM transition with different retention times. An authentic standard of largazole thiol eluted at t_R 4.7 min and also formed the adduct (t_R 2.2 min) when added directly to serum. C, cellular stability of largazole measured upon exposure to HCT116 protein lysate (0.7 mg/ml).

Fig. 7.

In vivo activity using a HCT116 xenograft mouse model. A, in vivo dose optimization. Largazole was dissolved in dimethyl sulfoxide and injected intraperitoneally into subcutaneous HCT116 tumor-bearing nu/nu mice. After 4 h, tumors were removed and analyzed for histone H3 acetylation by immunoblot analysis. B and C, efficacy studies. Tumor-bearing mice (n = 9) were injected with largazole (5 mg/kg i.p.) daily until the endpoint was reached in the control group (tumor length ≥15 mm or tumor volume ≥1000 mm³). Graphs depict time-dependent tumor volume (B) and overall growth rate (C) after 2 weeks of treatment. Error bars indicate S.E.M. D and E, immunohistochemistry. D, xenograft tumor sections were mounted on glass slides, stained for caspase-3, and counterstained with hematoxylin. Representative images are shown. E, Caspase-3 positive stained cells were quantified on 12 randomly selected fields from two independent tumors from each group. Error bars indicate S.E.M. F, biochemical analysis of largazole-induced protein changes in the tumor. Tumor tissue was harvested after 2 weeks of largazole or vehicle treatment and protein extracts were analyzed by immunoblot analysis.