Colorectal cancer (CRC), is heterogeneous malignancy characterized by variations in molecular profiles and clinical manifestations. Therefore, treatments for CRC are based on histopathological type and clinical stage of disease. However, in patients with metastatic CRC, available therapeutic options have shown limited effect. In recent years, histone deacetylases (HDAC)s have been identified as relevant contributors to pathogenesis and metastatic invasion of CRC. As HDAC6 is often overexpressed in CRC and correlates with poor disease prognosis it represents a good potential therapeutic target. The aim of this study was to evaluate antitumor activity of four novel HDAC inhibitors with 1-benzhydryl piperazine as a surface recognition group, that differ in hydrocarbon linker in HT-29 (with BRAFV600 mutation) and HCT-116(BRAF wild type) human CRC lines.

HT-29 and HCT-116 human CRC cells cultured in RPMI1640 cell culture medium (CM) were treated with b2, b3, b4, and b7 HDAC inhibitors and b3 and b7 in combination with BRAF inhibitor vemurafenib for 72h with at 37°C and 5% CO2 in humid atmosphere. Cytotoxicity and IC50 values were estimated by MTT assay. Cell cycle analysis and PI/annexinV assay for apoptosis were performed by flow cytometry. Statistical differences between the control (CM) and treatment were estimated by one-way ANOVA and Student’s t-test.

b2 and b4 compounds with low potency for HDAC6 inhibition did not show any cytotoxic activity while b3 compound with higher potency for HDAC6 inhibition showed significant cytotoxic activity on HCT-116 and HT-29 cells with IC50 (µM) of 54.06 and 71.49, respectively. Furthermore, b7 panHDAC (HDAC1/3/6/8) inhibitor, which is potent in terms of HDAC6 inhibition, induced significant antiproliferative effect with IC50 ( µM) of 22.04 and 58.99 for HCT- 116 and HT-29, respectively. Antiproliferative effects of b3 and b7 were more profound on HCT-116 cells and in both cell lines accompanied with increased apoptosis and alterations in cell cycle distribution. Furthermore, it was evaluated on HT-29 cell line weather b3 and b7 compounds could improve the effect of BRAF inhibitor vemurafenib. Cytotoxic effect of combination treatment of b3 and b7 with vemurafenib was evaluated by Chou-Talalay model. Our results showed synergistic effect of b7 and slight synergistic effect for b3 for concentrations lower than IC50 (12.5µM and 15µM, respectively).

Our findings of synergistic antitumor effect of HDAC and BRAF inhibition may provide rationale for investigations of their joint use in therapy of patients with CRC harboring BRAF mutation and evaluation of the potential of this therapy to increase response rate and overcome acquired resistance to treatment with BRAF inhibitors.