Article Figures & Data
Figures
- Fig. 1.
New chemical entities: E3330 analogs. (A) Schematic of E3330 and the new compounds. Groups that were investigated include the quinone series, 3-position series, alkyl sidechain series, and carboxylic acid/amine series (denoted as A–D, respectively). (B) Current new molecules with more potent Ref-1 redox inhibition. The drawing, displaying, and characterizing of chemical structures, substructures, and reactions were performed as described in the Materials and Methods.
- Fig. 2.
Pretreatment with E3330 and APX2009, but not APX2007 or APX2032, attenuates cisplatin-induced cell death in sensory neuronal cultures. (A) Each column represents the mean ± S.E.M. of the percent survival of cells from cultures treated with various concentrations of drugs as indicated for 24 hours. Cell viability as measured by trypan blue exclusion was determined on day 14 in culture from three independent harvests. An asterisk indicates a significant difference in survival after drug treatment compared with no drug treatment, using analysis of variance and the Tukey post hoc test. (B) Neuronal cultures were exposed to vehicle (DMSO) or to 20 µM E3330, APX2007, APX2009, or APX2032 drugs (as indicated) for 72 hours and to various concentrations of cisplatin for 24 hours. Each column represents the mean ± S.E.M. of the percent survival of cells as measured by trypan blue exclusion. An asterisk indicates a significant difference in cultures not treated with cisplatin compared with cultures treated with the drug, using analysis of variance and the Tukey post hoc test DMSO, dimethylsulfoxide. *P < 0.05.
- Fig. 3.
E3330 and APX2009 do not alter CGRP release from sensory neurons in culture, but they attenuate the cisplatin-induced reduction in capsaicin-evoked release of CGRP. Each column represents the mean ± S.E.M. of basal release (light-gray columns) or capsaicin-stimulated release (dark-gray columns) of CGRP in femtomoles per well per minute. (A) Cultures were exposed to medium or to 10 or 20 µM of the various drugs (as indicated) for 72 hours prior to release experiments. (B) Cultures were exposed to medium or to 10 or 20 µM of the various drugs (as indicated) for 72 hours and to cisplatin for 24 hours prior to release experiments. An asterisk indicates a significant difference in capsaicin-stimulated release compared with untreated cells, using analysis of variance and the Tukey post hoc test.
- Fig. 4.
APX2009, but not APX2007 or APX2032, attenuates the cisplatin-induced phosphorylation of H2AX in sensory neuronal cultures. The top panel shows representative Western blots of pH2AX and vinculin from cultures prior to and after 24 and 48 hours of exposure to 10 μM cisplatin. Cultures were exposed to DMSO as a vehicle control or to 20 µM APX2007, APX2009, or APX2032 for 72 hours before and during cisplatin treatment as indicated. The bottom panel represents the mean ± S.E.M. of the densitometry of pH2AX expression normalized to vinculin from three independent experiments. Treatment with APX2009 resulted in a decrease in pH2AX at all time points as determined using analysis of variance and the Tukey post hoc test.
- Fig. 5.
APX2009 attenuates the oxaliplatin-induced toxicity of sensory neurons in culture. (A) Each column represents the mean ± S.E.M. of the percentage of surviving cells as measured by trypan blue exclusion after 24-hour exposure to various concentrations of oxaliplatin as indicated. Cultures are treated for 72 hours with DMSO as a vehicle control (left), 10 µM APX2009 (center), or 20 µM APX2009 (right). (B) Columns represent the mean ± S.E.M. of the basal release of CGRP (light-gray columns) or release stimulated by 30 nM capsaicin (dark-gray columns) in femtomoles per well per minute. The horizontal bar indicates cultures exposed to 30 µM oxaliplatin for 24 hours and 10 or 20 µM APX2009 for 72 hours prior to release experiments. (C) The top panel shows representative Western blots of pH2AX and vinculin from cultures prior to and after 24 and 48 hours of exposure to 30 μM oxaliplatin and DMSO or 20 µM APX2009 for 72 hours before and during cisplatin treatment as indicated. The bottom panel represents the mean ± S.E.M. of the densitometry of pH2AX expression normalized to vinculin from three independent experiments. An asterisk indicates a statistically significant difference on oxaliplatin treated cultures compared with controls, using analysis of variance and the Tukey post hoc test. DMSO, dimethylsulfoxide. *P < 0.05.
- Fig. 6.
Tumor, but not CAF, cell killing by APX2009 in a PDAC three-dimensional model. Pa03C tumor cells (transduced with TdTomato) were grown in three-dimensional cultures in the presence and absence of CAFs (transduced with enhanced green fluorescent protein). Spheroids were treated with APX2009, and the area and intensity were quantified after 12 days in culture. A) The intensity of the tumor spheroids alone (black bars), tumor spheroids with CAFs (red bars), and intensity of the CAFs (green bars) is shown. B) The area of the tumor spheroids alone (black bars), tumor spheroids with CAFs (red bars), and intensity of the CAFs (green bars) is shown. C) Representative images of the spheroids are shown. The overlay indicates the coculture with both tumor (red) and CAFs (green). Differences were determined using both the t test (vehicle control versus drug treatment at each dose) and one-way analysis of variance, and statistical differences were observed for the tumor alone or tumor cocultured with CAFs. *P, 0.05; ** P, 0.01; ***P, 0.001. No differences from the control were observed in CAFs treated with APX2009, similar to what has been observed for E3330 (Logsdon et al., 2015).
Additional Files
Data Supplement
Files in this Data Supplement:
- Supplemental Data - 4 supplemental figures.
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