Article Figures & Data
Figures
- Fig. 1.
CDDO-Imidazolide (CDDO-Im) rapidly induces apoptosis in myeloma cells. Human myeloma cells (A and B, RPMI-8226; C and D, JJN3) were treated with 500 nmol/L CDDO-Imidazolide for 4 hours followed by washout of the drug. At 24 hours, cells were analyzed by cytospin (left) or fluorescence-activated cell sorting (right) for Annexin V and propidium iodine staining. Red arrows, membrane blebbing; black arrowheads, nuclear fragmentation.
- Fig. 2.
CDDO-Imidazolide suppresses STAT phosphorylation (A-C) and its nuclear action (D-E) in myeloma cells. JJN3 human myeloma cells (A, D, and E), in which IL-6 is an autocrine growth factor, or murine 1254 plasmacytoma cells (B and C), which were stimulated with IL-6 (10 ng/mL), were treated with CDDO-Imidazolide (250 nmol/L unless indicated otherwise) for various times. Cell lysates were immunoprecipitated with phosphotyrosine antibodies (A and B), whereas whole-cell extracts (C) or nuclear lysates (D) were immunoblotted with specific antibodies to p-STAT5. Nuclear lysates of JJN3 cells were used to measure STAT3 binding to consensus DNA sequences (E); cells were treated with 300 nmol/L CDDO-Imidazolide, and an ELISA that used immobilized oligonucleotides was done on these lysates. Negative (−) and positive (+) controls included in the kit (Active Motif).
- Fig. 3.
CDDO-Imidazolide decreases constitutive STAT phosphorylation, inhibits proliferation, and induces apoptosis in lung cancer cells. A549 (A) and H358 (B) human lung cancer cells were treated with 0.3 to 1 μmol/L (A) or 1 μmol/L CDDO-Imidazolide (B) for 1 to 4 hours, and whole-cell extracts were immunoblotted with p-STAT3 and tubulin antibodies. To measure cell proliferation, A549 cells were treated with CDDO-Imidazolide for 48 hours, and evaluated by a [3H]thymidine incorporation assay. A549 cells also were treated with CDDO-Imidazolide for 24 hours and then analyzed for apoptosis by flow cytometry for Annexin V and propidium iodide staining (D).
- Fig 4.
CDDO-Imidazolide regulates STAT target genes and induces SOCS-1 and SHP-1. A, SOCS-1 mRNA expression from A549 cells treated with 1 μmol/L CDDO-Imidazolide for 0 to 24 hours was analyzed by real-time PCR. Lysates of JJN3 cells treated with 300 nmol/L CDDO-Imidazolide for 0 to 2 hours were immunoprecipitated with SHP antibodies and then immunoblotted for SHP-1 (B) or p-STAT5 (C). p-inhibitor, calyculin A (1 nmol/L).
Tables
- Table 1.
Genes in the STAT pathway either up-regulated or down-regulated by the treatment with CDDO-Imidazolide
Gene Fold induction CEBPB 2.1 IL22RA1 0.5 IL2RG 0.6 ISGF3G 5.7 JUNB 2.2 NMI 0.2 PIM1 3.2 SMAD1 2.4 SMAD7 3.0 SOCS1 5.9 NOTE: A549 cells were treated with 1 μmol/L CDDO-Imidazolide for 4 hours, and total RNA was isolated and analyzed using a human JAK/STAT Oligo GEArray blot. The expression of the listed genes changed following treatment with CDDO-Imidazolide compared with untreated control cells.
Volume 12, Issue 14
