In vitro activity: Chidamide (formerly known as CS055, Tucidinostat, and HBI-8000, is a potent and orally available benzamide-type histone deacetylase (HDAC) inhibitor with IC50s of 95, 160, 67 and 78 nM for HDAC1, HDAC2, HDAC3 and HDAC10, respectively. Chidamide is approved by the Chinese FDA for relapsed or refractory peripheral T-cell lymphoma (PTCL), and has orphan drug status in Japan. As of April 2015 it is only approved in China. Chidamide selectively binds to and inhibits HDAC leading to an increase of acetylation levels of histone protein H3. It also inhibits the expression of signaling kinases in the PI3K/Akt and MAPK/Ras signaling pathways and may result in cell cycle arrest and the induction of tumor cell apoptosis, which may inhibit tumor cell proliferation in susceptible tumor cells. Chidamide is being investigated as a treatment for pancreatic cancer. However, it is not US FDA approved for the treatment of pancreatic cancer.

Kinase Assay: HDAC activity is detected as described in the Colorimetric HDAC Activity Assay kit. Each reaction (100 μL) contains nuclear protein (50 μg) extract from leukaemia cells and HDAC substrate. To test the effect of HDACis, Chidamide (CS055) and MS-275 are added to the mixtures and incubated at 37°C for 1 h. The HDAC activities are measured by a microplate readers at 405 nm. The positive control (only nuclear extract and vehicle) is set as 100% and double-distilled water containing 10 μM Trichostatin A, a known strong HDACi, is used as a negative control and set as 0%.

Cell Assay: Proliferation of the PaTu8988 cells is evaluated using CCK-8 assay. PaTu8988 cells are randomly into 4 groups and incubated in the absence or presence of concentrations of 0, 1.25, 2.5 and 5 μM) of Chidamide for 48 h. Subsequently, 10 μL CCK-8 is added in each well and incubated for 2 h. The optical density of each well is then measured with a microplate reader at 450 nm. The cell survival rate is calculated using the formula: Cell survival rate (%)=1-(ODctrl-ODsample)/ODctrl×100%.