Article Figures & Data
Figures
- Fig. 1.
BCI-215 is nontoxic to rat hepatocytes and developing zebrafish embryos. (A–C) Rat hepatocytes were treated in 96-well plates with 10-point concentration gradients of DUSP inhibitors and menadione as a positive control for hepatotoxicity. Sanguinarine, NSC95397, BCI, and menadione, but not BCI-215 produced dose-dependent cell death in rat hepatocytes as measured by (A) PI uptake and (B) loss of mitochondrial membrane integrity. (C) Hepatocyte toxicity correlated with production of ROS. (D and E) In contrast to other DUSP inhibitors, BCI-215 did not generate ROS in developing zebrafish embryos. Data and images are from a single experiment that has been repeated once. Scale bar, 500 µm.
- Fig. 2.
BCI and BCI-215 cause apoptotic cell death at concentrations that induce ERK phosphorylation. MDA-MB-231 cells were treated with vehicle (DMSO), BCI, or BCI-215 and stained with Hoechst 33342 and anti-phospho-ERK and anti-cleaved caspase-3 antibodies, respectively. (A) Fluorescence micrographs show pyknotic nuclei indicative of early apoptosis. Images are maximum projections of a 10-plane, 0.25 µm each, z-series acquired using a 60X objective on a Molecular Devices ImageXpress Ultra High Content Reader (Sunnyvale, CA). BCI and BCI-215 were at 22 µM. Scale bar, 30 µm. (B) Multiparametric analysis of chromatin condensation, caspase-3 cleavage, and ERK phosphorylation by HCA. Each box plot is the aggregate of four (caspase) or five (nuclear condensation and ERK phosphorylation) independent experiments. Boxes show upper and lower quartiles; whiskers, range; dot, mean. *P < 0.05; **P < 0.01; ****P < 0.001 versus DMSO by one-way analysis of variance with Dunnett’s multiple comparisons test. The last data point for cleaved caspase is n = 3 for 50 µM BCI-215 with two of the three values being identical. (C and D) Confirmation of apoptosis with secondary cell lysis by flow cytometry. Data in (D) are the average ± S.E.M. values of three independent flow cytometry experiments. Early apoptosis, Q3, annexin V positive and PI negative; late apoptosis, Q2, annexin V and PI positive; necrosis, Q1, PI positive and annexin V negative.
- Fig. 3.
BCI-215 sensitizes breast cancer cells to immune cell kill. (A) MDA-MB-231 cells were treated overnight in 384-well plates with vehicle or 3 µM BCI-215 followed by washout. Cells were subsequently exposed to various ratios of PBMC-derived LAK. After 24 hours, cells were fixed and stained with Hoechst 33342. Cells were imaged on the ArrayScan II, cancer cell nuclei identified and gated by their larger size compared with PBMC, and enumerated. Cell densities were normalized to vehicle or BCI-215 in the absence of activated immune cells, respectively. Data are the average ± S.E.M. values from four independent experiments, each performed in triplicate. (B) Comparison of BCI-215 versus clinically used antineoplastic agents, doxorubicin (DOX) and cisplatin (CDDP). MDA-MB-231 cells were either stained with CellTracker green or transduced with a mitochondrial-targeted, GFP-labeled cytochrome c biosensor, and processed and analyzed as in (A) except that cancer cells were specifically identified by green fluorescence instead of nucleus size gating. Each data point represents the mean ± S.E.M. value of three independent experiments, each performed in triplicate.
- Fig. 4.
BCI-215 activates mitogen-activated and SAPK cascades in the absence of oxidative stress. (A) Activation kinetics. MDA-MB-231 human breast cancer cells were treated with BCI or BCI-215 (20 µM) for the indicated time points and analyzed for phosphorylation of the DUSP1/MKP-1 and DUSP6/MKP-3 substrates, ERK, JNK/SAPK, and p38, as well as their upstream activators MEK1 and MKK4/SEK1 by Western blot. (B) Activation of kinase cascades in three different cell lines. Cells were treated for 1 hour with vehicle (DMSO), 20 µM BCI-215 (215), or 5 µM doxorubicin (DOX). Data in (A) and (B) are from a single experiment that was repeated once. (C and D) ROS generation. MDA-MB-231 cells were prelabeled with Hoechst 33342 and chloromethyl-fluorescein diacetate, acetyl ester (CM-H2-DCFDA) for 30 minutes followed by treatment with test agents for up to 5 hours. (C) At the indicated time points, cells were imaged and the percentage of ROS positive cells enumerated. (D) Concentration response at the 2-hour time point. Each data point is the mean of four wells ± S.E.M. from a single experiment that was repeated twice.
- Fig. 5.
Effect of MAPK inhibition of BCI-215 toxicity. MDA-MB-231 cells were pretreated with concentration gradients of MAPK inhibitors followed by vehicle or a proapoptotic concentration of BCI-215 (25 µM). After 24 hours, cells were stained with Hoechst 33342 and an antibody against cleaved caspase-3, and analyzed for (A) cell density, (B and C) nuclear morphology, and (D) caspase cleavage. Data on graphs depict percent rescue from BCI, calculated as 1 − [(data point + DMSO)/(DMSO + BCI-215)] × 100. Images in (E) illustrate cell loss and nuclear morphology with vehicle (DMSO) and BCI-215 alone, or of BCI-215 in the presence of SCH771984 (375 nM), SB203580 (18 µM), SP600125 (18 µM), or JNK-IN8 (1.8 µM). Data are the averages of 4–7 independent experiments ± S.E.M., each performed in quadruplicate. Images are from an ArrayScan VTI using a 20X objective. Scale bar, 30 µm.
- Fig. 6.
Phospho-kinase profiling of BCI-215 in MDA-MB-231 cells. Lysates from cells treated with vehicle (0.1% DMSO) or BCI-215 (20 µM) for 30 minutes were analyzed for phosphorylation levels of 43 human kinases. Bar graph shows mean ± range of two independent repeats; inset shows correlation of two independent replicate runs.
Additional Files
Data Supplement
Files in this Data Supplement:
- Supplemental Data - Supplemental methods, 3 figures, 1 table.
