Article Figures & Data
Figures
- Figure 1.
A, structural formula of RITA [2,5-bis(5-hydroxymethyl-2-thienyl)furan] and nutlin [4-(4,5-bis-(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl)-piperazin-2-one]. B, C, and D, RITA induces growth arrest and apoptosis of MM cells harboring wild-type p53. MM cells were plated in 96-well plates and cultured either with or without different concentrations of RITA (0.25–20 μmol/L). After 48 h, the cells were treated with MTT for 4 h at 37°C, and MTT activity was measured. Survival of the cells were expressed as percentage of the DMSO-treated control in cells harboring wild-type p53 (B) or mutant p53 (U266) or null p53 (8266R5) (C). Results represent the mean ± SD of three independent experiments done in triplicate. D, MM cells were treated with various concentrations of RITA (0.25–12.5 μmol/L) for 48 h. The cells were subsequently stained with FITC-Annexin V and propidium iodide and analyzed by flow cytometry. Each column represents the mean of apoptotic cells (Annexin V and propidium iodide positive) of three independent experiments after normalizing the data with vehicle treatment, and each bar represents SD.
- Figure 2.
RITA induces activation of the p53 pathway in MM cells in a time-dependent manner. MM.1S (A) and H929 (B) cells were treated with 0.5 and 2.5 μmol/L RITA, respectively, at different time periods (4–24 h). Total cell lysates were prepared at the indicated hours after treatment of the cells with RITA and analyzed by Western blot for the indicated proteins. Data shown are representative of three independent experiments.
- Figure 3.
RITA induces p53-dependent activation of the p53 pathway in MM cells in a dose-dependent manner. MM.1S (A), H929 (B), 8226R5 (C), and U266 (D) cells were treated with RITA for 6 hours at various concentrations (0.25–10 μmol/L). Total cell lysates were prepared and analyzed by Western blot for the expression of p53 and its transcriptional targets as well as apoptosis-associated proteins. Data shown are representative of three independent experiments.
- Figure 4.
RITA induces caspase-dependent apoptosis in MM cells harboring wild-type p53. MM.1S or H929 cells were treated with 20 μmol/L pan-caspase inhibitor, Z-VAD-FMK, or 100 μmol/L caspase-8 inhibitor, Z-IETD-FMK, before treating the cells with RITA. A, after 6 h, cells were lysed and analyzed by Western blot for the expression of caspase-3 and/or caspase-8 and PARP. B, apoptosis was measured by flow cytometry in MM.1S and H929 cells treated with RITA in the presence or absence of Z-VAD-FMK or Z-IETD-FMK. Each column represents the mean of apoptotic cells (Annexin V–positive cells) of three independent experiments after normalizing the data with vehicle treatment, and each bar represents SD. *, significance at P < 0.02 by two-tailed t test. C, primary MM samples from four MM patients or BMMNC from two healthy volunteers were grown in normal growth medium. PBMNCs from three healthy volunteers were grown in the presence of 1% phytohemagglutinin. Cells were treated with escalating doses of RITA (0–20 μmol/L), and after 48 h, cell viability was measured by MTT assay.
- Figure 5.
RITA and nutlin display synergistic cytotoxic effect in MM cell lines (A) and in primary MM samples (B). MM cells were seeded at 20 × 104/mL and were treated simultaneously with escalating doses of RITA and nutlin. After 48 h, cell viability was measured by MTT assay. CI value was calculated as described in Materials and Methods. C, a combination of RITA plus nutlin enhances killing of MM cells at 48 h as measured by flow cytometry. Graph represents data from three independent experiments. *, significance at P < 0.05 by two-tailed t test. Filled, empty, and hatched columns represent the percentage of apoptosis in nultin-, RITA-, or nutlin plus RITA–treated cells, respectively.
- Figure 6.
RITA induces transcriptional regulation of a set of p53 target genes. A and B, microarray analysis of gene expression in MM.1S cells after 6 h treatment with RITA. The heat map of genes differentially expressed in MM cells. Vertical rows indicate separate arrays, and horizontal rows indicate genes. Values are normalized to untreated control. Green indicates low expression; red indicates high expression. C, mRNA levels of oncogenes were detected by qRT-PCR in MM.1S cells 6 h after treatment with 1 μmol/L RITA. Values were normalized to those obtained for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as log2-fold induction over DMSO-treated cells (mean ± SD).
Additional Files
Supplementary Daa, Saha, et al.
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Volume 9, Issue 11
