Novel histone deacetylase inhibitor AR-42 exhibits antitumor activity in pancreatic cancer cells by affecting multiple biochemical pathways

<div><p>Objective</p><p>Pancreatic cancer is one of the most lethal types of cancer with a 5-year survival rate of ~5%. Histone deacetylases (HDACs) participate in many cellular processes, including carcinogenesis, and pharmacological inhibition of HDACs has emerged as a potential therapeutic strategy. In this study, we explored antitumor activity of the novel HDAC inhibitor AR-42 in pancreatic cancer.</p><p>Methods</p><p>Human pancreatic cancer cell lines BxPC-3 and PANC-1 were used in this study. Real-time PCR, RT-PCR, and western blotting were employed to investigate expression of specific genes and proteins, respectively. Translocation of apoptosis-inducing factor was investigated by immunofluorescence and subcellular fractionation. The number of apoptotic cells, cell cycle stages, and reactive oxygen species (ROS) generation levels were determined by flow cytometry. Cell invasiveness was examined by the Matrigel invasion assay. Efficacy of AR-42 <i>in vivo</i> was evaluated by utilizing BxPC-3 xenograft mouse model.</p><p>Results</p><p>AR-42 inhibited pancreatic cancer cell proliferation by causing G2/M cell cycle arrest via regulating expression levels of genes and proteins involved in cell cycle. AR-42 also induced ROS generation and DNA damage, triggering apoptosis of pancreatic cancer cells via both caspase-3-dependent and caspase-3-independent pathways. In addition, AR-42 increased expression levels of negative regulators of p53 (miR-125b, miR-30d, and miR33), which could contribute to lower expression level of mutant p53 in pancreatic cancer cells. Cell invasion assay showed that AR-42 reduced cancer cell aggressiveness and significantly diminished BxPC-3 xenograft tumor growth <i>in vivo</i>.</p><p>Conclusion</p><p>AR-42, a novel HDAC inhibitor, inhibited pancreatic cancer cells by regulating p53 expression, inducing cell cycle arrest, particularly at the G2/M stage, and activating multiple apoptosis pathways. Additionally, AR-42 inhibited cell invasiveness and potently suppressed pancreatic cancer tumors <i>in vivo</i>. We conclude that by virtue of its multiple mechanisms of action, AR-42 possesses a considerable potential as an antitumor agent in pancreatic cancer.</p></div

AR-42 induced ROS production and caused DNA damage in pancreatic cancer cells.

<p><b>(A</b>) BxPC-3 cells were treated by 1 μM AR-42 for 24 h. Concentrations of H₂O₂ and NAC comprised 1.467 mM and 25 mM, respectively. ROS generation was analyzed by flow cytometry using 2,7-dichlorodihydrofluorescein diacetate. All data are representative of three independent experiments with similar results. <b>(B)</b> Expression of γH₂AX was examined by western blot analysis.</p

AR-42 suppressed BxPC-3 cell invasiveness.

<p>(A) Cells invading Matrigel after 24 h were detected as stained cells on the opposite side of membrane. Top panel illustrates representative photomicrographs of cells treated with indicated concentrations of AR-42. Bottom panel illustrates quantitative analysis of invasion ability depending on AR-42 concentration. The ability of BxPC-3 cells to invade Matrigel was significantly attenuated by AR-42 in a concentration-dependent manner compared to that of DMSO-treated control cells. (B) The expression of E- and N-cadherin were determined by western blot analysis after BxPC-3 cells were treated with AR-42 for 24 h. Results were confirmed by repeated experiments. (C) The comparison of the inhibitory effects of AR-42, SAHA and TSA on invasiveness of BxPC-3 cells. Data of (A, C) are presented as the mean ± standard deviation of three independent experiments. Statistical significance of differences from the percentage of invasive cells in control experiments is indicated as follows: <i>*P <0</i>.<i>05</i>, <i>**P <0</i>.<i>01</i>, <i>***P <0</i>.<i>001</i>.</p

<i>In vivo</i> efficacy of AR-42 in BxPC-3 xenografted mice.

<p><b>(A)</b> BxPC-3 xenograft tumor volumes in mice treated via oral gavage with either vehicle or AR-42 (25, 50 mg/kg) every other day for 29 days. <b>(B)</b> Representative tumors from animals treated with vehicle or AR-42 (25, 50 mg/kg) <b>(C)</b> Body weight of xenografted mice in vehicle- and AR-42-treated groups. Data are presented as the mean ± standard deviation of measurements in 6 mice per group. Statistical significance of differences from the values in control group is indicated as follows: <i>*P <0</i>.<i>05</i>, <i>**P <0</i>.<i>01</i>, <i>***P <0</i>.<i>001</i>. (D) Western blot showing the expression of p53, survivin and cleaved caspase-3 proteins as analyzed from tumor protein extracts in each treatment group.</p

Schematic representation of multiple mechanisms affected by AR-42 during inhibition of pancreatic cancer cell growth.

<p>Schematic representation of multiple mechanisms affected by AR-42 during inhibition of pancreatic cancer cell growth.</p