Fig 1.
Compound JA interferes with Wnt/β-catenin.
A) Structure of Jatrophone (JA). B) LiCl (25μM for 24 hours) and WNT3A activate Super 8xTOPFLASH Wnt reporter system in HEK293T cells. C) JA inhibits WNT3A activation of TOPFLASH. D) Constitutively active (ca) ΔLRP6 and ca β-catenin robustly active TOPFLASH Wnt reporter system in 293T cells. E) JA is capable of inhibiting Wnt reporter activity by ca ΔLRP6 in a concentration dependent manner but not ca β-catenin F). The firefly luciferase activity was normalized to Renilla luciferase activity. Following concentrations of JA were used: 0.1, 1, 2.5, 5, 10, 25 μM for both ca-LRP6 and ca-β-catenin. Concentration of JA for inhibiting WNT3A were 10 nM, 100 nM, 250 nM, 1 μM, 2 μM and 5 μM. The experiment in C and D was performed in four biological replicates, *p < 0.05, One-way Anova, Tukey´s post test.
Fig 2.
Determination of differential IC50 (DIC50) effects of JA on various TNBC cell lines and TNBC PDX-derived cell lines.
WST-1 proliferation assays for 48 hours utilizing JA at various dosages ranging from 100 nM-30 μM: (A) MSL-subtype TNBC cell lines MDA-MB-231 (EA) vs. MDA-MB-157 (AA). (B) BL1-subtype TNBC cell lines HCC-38 (EA) vs. MDA-MB-468 (AA). (C) TNBC PDX-derived “naïve” cell line HCl-2 vs. “chemoresistant” HCl-10. The IC50 of JA indicated for each cell lines determined by n = 3, in triplicate, using the two-tailed t-test p-values were calculated: *p < 0.05, **-p < 0.01 *** < 0.001 relative to vehicle control cells (DMSO).
Fig 3.
Compounds JA arrests cells at S-phase of cell cycle and induced apoptosis in MDA-MB-231 Cells.
A) Annexin V-FITC staining was used to detect apoptosis by flow analysis in control (DMSO), staurosporine (1μM), ICG-001 (10 μM) and JA (2.5 μM) treated cells for 48 hours. Cells were counter stain with propidium iodide (PI, 1 μg/ml). We show one representative FACS plot B) Quantification of alive, early and late apoptotic cells in cells from panel A. C) qPCR for Survivin (BIRC5) in control cells, ICG-001 and JA treated cells. (D) One representative FACS plot using PI/DNA content analyzed by flow cytometry (E) Quantification of cell cycle phases for in G0/G1, S and G2/M in both ICG-001 and JA treated cells. (F) qPCR for cell cycle markers CDK4, CCND1, CCNE1, CCNA1 and CCNB1 from the same cells as in panel E. Statistics on three biological independent experiments with duplicates for each of the above. T-test was used to determine p-values: ***-p < 0.001, **-p < 0.01 and *p < 0.05 vs. control.
Fig 4.
Immunofluorescence (IF) of non-phosphorylated β-catenin protein (amino acids Ser37; Thr41), which is commonly referred to as transcriptionally active β-catenin (ABC).
IF of ABC in vehicle control (1% DMSO) (A), JA (B) and ICG-001(C) treated MDA-MB-231 cells. Cells were counterstained with DAPI (Blue).
Fig 5.
JA inhibits expression on Wnt/β-catenin direct-target genes and degrades non-phosphorylated activated β-catenin protein levels.
(A) qPCR for BIRC5, AXIN2, HMGA2, CNND1, MYC and PCNA in ICG-001 and JA treated MDA-MB-231 cells. (B) Immunoblot analysis for AXIN2, HMGA2, CYCLIND1, PCNA and MYC. ACTIN serves as the loading control. (C) Immunoblot analysis for non-phosphorylated activated β-CATENIN (ABC), total pan-β-CATENIN and GS3KβSer9 from the same cells and extracts as in panel A and B. Quantification of triplicates using t-test p values are ***-p < 0.001, **-p < 0.01 and *p < 0.05 vs. control.
Fig 6.
JA inhibits EMT-markers and prevents cell migration in wound healing assays.
(A) qPCR for SLUG, FIBRONETIN, VIMENTIN and ZEB1 in ICG-001 and JA treated MDA-MB-231 cells. Results are expressed as mean ± SE, n = 3 *p<0.05. (B) The number of cells migrated into the scratched area was photographed (340) and calculated as a percentage of migration for 16 hours post treatment. Quantification of triplicates using t-test: ***-p < 0.001 vs. control.