Anticancer activity of MPT0G157, a derivative of indolylbenzenesulfonamide, inhibits tumor growth and angiogenesis

Abstract

Histone deacetylases (HDACs) display multifaceted functions by coordinating the interaction of signal pathways with chromatin structure remodeling and the activation of non-histone proteins; these epigenetic regulations play an important role during malignancy progression. HDAC inhibition shows promise as a new strategy for cancer therapy; three HDAC inhibitors have been approved. We previously reported that N-hydroxy-3-{4-[2-(2-methyl-1H-indol-3-yl)-ethylsulfamoyl]-phenyl}-acrylamide (MPT0G157), a novel indole-3-ethylsulfamoylphenylacrylamide compound, demonstrated potent HDAC inhibition and anti-inflammatory effects. In this study, we evaluated its anti-cancer activity in vitro and in vivo. MPT0G157 treatment significantly inhibited different tumor growth at submicromolar concentration and was particularly potent in human colorectal cancer (HCT116) cells. Apoptosis and inhibited HDACs activity induced by MPT0G157 was more potent than that by the marketed drugs PXD101 (Belinostat) and SAHA (Vorinostat). In an in vivo model, MPT0G157 markedly inhibited HCT116 xenograft tumor volume and reduced matrigel-induced angiogenesis. The anti-angiogenetic effect of MPT0G157 was found to increase the hyperacetylation of heat shock protein 90 (Hsp90) and promote hypoxia-inducible factor-1α (HIF-1α) degradation followed by down-regulation of vascular endothelial growth factor (VEGF) expression. Our results demonstrate that MPT0G157 has potential as a new drug candidate for cancer therapy.

Keywords: HDAC; HIF-1α; Hsp90; angiogenesis; tumor microenvironment.

Figure 1. The synthesis of MPT0G157

Reagents and conditions: A. 4-bromobenzenesulfonyl chlorides, pyridine, ACN, r.t.; B. t-Butyl acrylate, Pd₂(dba)₃, [(t-Bu)₃P]BF₄, K₂CO₃, TEA, DMF, 100–105°C; C. (i) TFA, 0°C to r.t.; (ii) EDC·HCl, HOBt, N-methylmorphine, NH₂OTHP, DMF, r.t.; (iii) 10% TFA, methanol, r.t. Abbreviation: CAN, acetonitrile; DMF, dimethylformamide; K₂CO₃, potassium carbonate; Pd₂(dba)₃, tris(dibenzylideneacetone) dipalladium; [(t-Bu)₃P]BF₄, tris-t-tubylphosphonium tetrafluoroborate; TFA, trifluoroacetic acid; EDC, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzotriazole; NH₂OTHP, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine.

Figure 2. MPT0G157 inhibited tumor growth but less sensitive against normal bone marrow cells

A. human colorectal cancer (HCT116), breast cancer cell line (MDA-MB-231), lung cancer cell line (A549), pancreatic cancer cell line (AsPC-1) or B. human normal bone marrow stromal cells (HS-5) (1 × 10⁴) were incubated with or without indicated concentrations of MPT0G157, PXD101, and SAHA for 48 h. Cell proliferation was evaluated by sulforhodamine B (SRB) assay. Results are shown as mean ± SEM from three independent experiments.

Figure 3. MPT0G157 exhibited potent HDACs inhibitory effect

A. HCT116 cells (1 × 10⁶) were incubated with indicated concentrations of MPT0G157, PXD101 or SAHA for 24 h, the nuclear proteins were isolated to determine the inhibition of total HDAC enzyme activity. B. HCT116 cells (1 × 10⁶) treated as explained in (A), whole-cell extracts were subjected to western blotting for the indicated proteins. Quantitative analysis of acetyl-histone H3 C. and acetyl-α-tubulin D. expression in western blot were determined by ImageQuant (Molecular Dynamics, USA). E. Immunohistochemical analysis of the HDAC1, 2 and 6 expression of a tissue microarray containing normal colon tissues and tumor tissue derived from patients with colon adenocarcinoma. Scale bars are indicated. Results in (A–D) represent mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group.

Figure 4. MPT0G157 treatment induced apoptosis in HCT116 cells

A, B. Cells (1 × 10⁶) were incubated for 48 h with or without MPT0G157, PXD101, and SAHA, fixed and then stained with propidium iodide to analyze (A) the DNA contents by flow cytometry and (B) cell cycle distributions. C. Percentages of subG1 phase in response to drug treated as explained in (A) D. Time-dependent effects of MPT0G157, PXD101, and SAHA (0.1, 1 μM) on subG1 population. E. Cells were incubated as explained in (A), then total cell lysates were prepared for western blot analysis of the indicated proteins; arrowhead indicated the cleavage form of indicated proteins. Results in (C–E) are mean ± SEM from three independent experiments. **p < 0.01, ***p < 0.001 compared with the control group.

Figure 5. The effect of MPT0G157 in HCT116 xenograft model

Mice bearing established HCT116 tumors (~100 mm³) were divided into three group (n = 5) and dosed as Materials and Methods. The tumor volumes A. and body weight B. of mice were measured. Results are mean ± SEM. **p < 0.01 versus control group.

Figure 6. MPT0G157 treatment inhibited HIF-1α expression in HCT116 cells

A. HCT116 cells (1 × 10⁶) treated with Cobalt(II) chloride (300 μM) for different period as indicated, whole-cell extracts were subjected to western blotting. B. Cell viability was determined after 4 or 12 h of treatment with 100–300 μM of Cobalt(II) chloride using the MTT assay (compared with the control group). C. Cells were incubated with MPT0G157, PXD101 or SAHA (0.1 μM) for 24 h prior to Cobalt(II) chloride (300 μM) treatment for another 4 h, and total cell lysates were subjected to western blotting for the HIF-1α, Hsp90, β-actin or immunoprecipitated with 1 μg of an anti-acetyl-lysine antibody and immunoblotted for Hsp90 antibody. D. Cells were incubated as explained in (C), the levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) mRNA were measured by RT-PCR. Results are shown as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the relevant control group; #p < 0.05 compared with Colbalt(II) chloride-only group.

Figure 7. MPT0G157 treatment inhibited the in vivo angiogenesis

Nude mice were subcutaneously injected with matrigel mixed with or without MPT0G157 (0.1 and 1 μM) (n = 3). A. After seven days, the animals were sacrificed and the plugs were excised from the mice and photographed. Scale bar represents 0.5 cm. B. Sections of H&E, Masson's trichrome stained and CD31 stained matrigel plugs were examined by light microscopy under 200 × magnification. Scale bar = 100 μm. C. Quantification of the hemoglobin contents of matrigel plugs by spectrophotometer measured at 540 nm. Data represent the mean ± SEM. *p < 0.05 and ***p < 0.001 versus control group.