In HT-29 colon cancer cells, 21 µM Droxinostat increased Annexin V/PI-defined apoptosis over 12–24 h. Pretreatment with the pan-caspase inhibitor Z-VAD-FMK attenuated the 24 h apoptotic and caspase-3 responses. [2]
In HT-29 cells, Droxinostat increased oxidant-sensitive fluorescence at 24 h. γ-Tocotrienol pretreatment reduced the oxidative and apoptotic responses. Caspase inhibition also partly reduced the oxidant-sensitive signal, indicating interaction between oxidative stress and apoptosis rather than establishing a single causal sequence. [2]
In cultured human amniotic epithelial cells, 5 µM Droxinostat for 48 h reduced the CCK-8 viability readout and increased histone H4K5 and H4K8 acetylation. Effects in this non-cancer epithelial model caution against assuming cancer-cell selectivity. [3]
Structural binding to S39E HDAC8
Biochemical assay · Recombinant human HDAC8 S39E phosphomimetic mutant, Zn2+-containing crystal complex [1]
Droxinostat occupies the HDAC8 active site and its hydroxamate chelates the catalytic zinc. This is a mutant complex, not a structure of phosphorylated wild-type enzyme.
Literature-Reported Treatment Conditions
- Treatment concentration
- 2 mM Droxinostat in the protein-containing crystallization solution before equal-volume mixing with precipitant
- Exposure duration
- Crystals appeared within 1 day; this is crystallization timing, not a treatment duration.
- Method
- Sitting-drop vapor-diffusion crystallization; structure refinement
- Reported limits
- No drug vehicle specified in the crystallization description; no affinity or cellular potency can be derived from the structure.
Methods and source locations
Compound reported in the paper: Droxinostat
Evidence concerns the reported research material; no Adooq lot validation or clinical efficacy is established.
Reagents; Crystallization and data collection; Phasing, model building, and structure refinement; Table 1; Results: Crystal structure of S39E HDAC8; Figure 2B
Resolution of the S39E HDAC8–Droxinostat crystal structure: X-ray crystallography; PDB accession 5BWZ — Table 1; Results: Crystal structure of S39E HDAC8
Active-site binding geometry: Crystallographic model and simulated-annealing omit map; Figure 2B viewed — Figure 2B and caption; Results: Crystal structure of S39E HDAC8
Apoptosis and caspase-inhibitor rescue in HT-29 cells
Cell-based study · Human HT-29 colon cancer cells [2]
Droxinostat increased Annexin V/PI-defined apoptosis and caspase-3 activity. Z-VAD-FMK pretreatment attenuated these responses, supporting a caspase-associated component rather than complete caspase dependence.
Literature-Reported Treatment Conditions
- Treatment concentration
- Droxinostat 21 µM; Z-VAD-FMK 10 µM in rescue groups
- Exposure duration
- Droxinostat 12 or 24 h for time course; rescue: 2 h inhibitor pretreatment followed by 24 h Droxinostat
- Method
- Cell exposure followed by flow cytometry and caspase substrate assay
- Vehicle
- DMSO vehicle control
- Co-treatment
- Z-VAD-FMK pretreatment in rescue groups; Droxinostat-alone and inhibitor-alone comparisons included.
- Reported limits
- Vehicle percentage not reported in the relevant methods. Time-course Figure 2A labels the baseline 0 h; no assumption of a separate time-matched vehicle sample for every plotted time.
Methods and source locations
Compound reported in the paper: Droxinostat
Evidence concerns the reported research material; no Adooq lot validation or clinical efficacy is established.
Reagents; Apoptotic assay and anti-apoptotic treatment; Caspase-3 activity measurement; Figure 2A–D and caption; corresponding Results
Annexin V/PI-defined early and late apoptosis: Annexin V-FITC/PI flow cytometry; mean ± SD of three independent experiments as reported in the caption. Vehicle and inhibitor-alone groups included; Figure 2 viewed. — Figure 2A–C and caption; Results: Droxinostat induced apoptosis in human colon cells
Caspase-3 activity: DEVD-p-NA substrate assay; absorbance at 405 nm. Qualitative direction from viewed Figure 2D; no values estimated from bars. — Figure 2D and caption; Caspase-3 activity measurement
Oxidative stress and antioxidant rescue in HT-29 cells
Cell-based study · Human HT-29 colon cancer cells [2]
Droxinostat increased oxidant-sensitive fluorescence at 24 h. γ-Tocotrienol attenuated the oxidative and apoptotic responses; Z-VAD-FMK also partially reduced the oxidant-sensitive signal, consistent with feedback between oxidative stress and apoptosis.
Literature-Reported Treatment Conditions
- Treatment concentration
- Droxinostat 21 µM; GT3 (γ-tocotrienol) 10 µM or Z-VAD-FMK 10 µM in their respective rescue designs
- Exposure duration
- Droxinostat 12 or 24 h for ROS time course; rescue experiments: 2 h pretreatment then 24 h Droxinostat
- Method
- Cell exposure with antioxidant or caspase inhibitor comparisons; flow cytometry and caspase substrate assay
- Vehicle
- DMSO vehicle control
- Co-treatment
- GT3 and Z-VAD-FMK were separate pretreatments, not a joint triple combination.
- Reported limits
- Vehicle percentage not reported. DCFDA is an oxidant-sensitive readout and does not identify one ROS species; rescue supports involvement but not a uniquely ordered causal pathway.
Methods and source locations
Compound reported in the paper: Droxinostat
Evidence concerns the reported research material; no Adooq lot validation or clinical efficacy is established.
ROS assay and antioxidant treatment; Apoptotic assay and anti-apoptotic treatment; Caspase-3 activity measurement; Figures 3A–B, 4A, 5A–B,D and captions; corresponding Results
Intracellular oxidant-sensitive fluorescence: DCFDA flow cytometry, mean fluorescence intensity; mean ± SD of three independent experiments. H2O2 positive control in Figure 3; vehicle and GT3-alone controls in Figure 4. No exact fold change inferred from images. — Figure 3A–B; Figure 4A; ROS assay and antioxidant treatment; corresponding Results
Apoptosis and caspase-3 response after antioxidant pretreatment: Annexin V/PI flow cytometry and DEVD-p-NA assay at 405 nm; apoptosis caption reports mean ± SD of three independent experiments. Figure 5 viewed. — Figure 5A–B and caption; corresponding Results
Oxidant-sensitive fluorescence after caspase inhibition: DCFDA flow cytometry; mean ± SD of three independent experiments as reported. Vehicle, inhibitor-alone and Droxinostat-alone groups present. — Figure 5D and caption; Results: Oxidative stress induced by droxinostat contributes to cellular apoptosis
Viability and histone H4 acetylation in amniotic epithelial cells
Cell-based study · Cultured human amniotic epithelial (HAEpi) cells from ScienCell Research Laboratories [3]
Droxinostat reduced the CCK-8 viability readout and increased H4K5/H4K8 acetylation in a non-cancer epithelial model. These findings limit extrapolation to cancer-selective cytotoxicity.
Literature-Reported Treatment Conditions
- Treatment concentration
- Droxinostat 0, 1, 5, 10, 20 µM in viability screen; 5 µM for histone acetylation
- Exposure duration
- 24, 48, 72 h in viability screen; 48 h for histone acetylation
- Method
- Cell exposure followed by CCK-8 or Western blot
- Vehicle
- DMSO control
- Co-treatment
- Droxinostat tested as a single agent; chidamide experiments are separate and unadopted.
- Reported limits
- DMSO percentage not specified. CCK-8 is a viability/metabolic readout and does not itself establish cell-death mechanism.
Methods and source locations
Compound reported in the paper: Droxinostat
Evidence concerns the reported research material; no Adooq lot validation or clinical efficacy is established.
Treating cells with HDAC inhibitors; CCK-8 assay; Western blot analysis; Statistical analysis; Figure 3A–B and caption; corresponding Results
CCK-8 viability readout: CCK-8 absorbance at 450 nm; concentration/time screen: 0, 1, 5, 10, 20 µM at 24, 48, 72 h. Caption: mean ± SD from three independent experiments. Figure 3A viewed. — Figure 3A and caption; Results: Effects of droxinostat on cell viability and the levels of MMPs, H4K5ac and H4K8ac in HAEpi cells
Histone H4K5ac and H4K8ac abundance: Western blot, total histone H4 internal/loading control; caption: mean ± SD of three independent experiments. Figure 3B viewed; no fold changes estimated from the graph. — Figure 3B and caption; Western blot analysis; corresponding Results
