Article Figures & Data
Figures
- Fig. 1.
Sensitization of IGROV-1 cells to carboplatin by treatment with ABT-737. A, IGROV-1 cells were treated with either solvent (DMSO), 500 μmol/L of carboplatin, and/or 0.6 μmol/L of ABT-737. The cells were assessed by phase contrast microscopy after either 24 or 48 h. The results are representative of three experiments. B, IGROV-1 cells were treated with the indicated concentration of carboplatin in the absence (solid columns) or presence (striped columns) of 0.6 μmol/L of ABT-737. After either 24 or 48 h, the cells which remained attached were collected by trypsinization and counted. Columns, mean percentage of cell numbers measured in samples treated with DMSO alone; bars, SD (n = 4). C, IGROV-1 cells were treated with either 250 μmol/L of carboplatin or carboplatin and 0.6 μmol/L of ABT-737. After the indicated time, the cells were lysed and PARP cleavage assessed by Western blotting. D, IGROV-1 cells were treated with DMSO, ABT-737 (0.6 μmol/L), or carboplatin combined with either ABT-737 or ABT-737E. After 16 h, the cells were lysed and PARP cleavage was assessed. The results are representative of three experiments. E, OVCAR-3 or A2780 cells were treated with carboplatin (100 μmol/L) and either DMSO or ABT-737 (1 μmol/L). After the indicated times, the cells were lysed and PARP cleavage was assessed by Western blotting. The results are representative of three experiments. F, IGROV-1 cells were treated with the indicated concentrations of carboplatin and either DMSO (solid columns) or 0.6 μmol/L of ABT-737 (striped columns). After 16 h, the adherent and detached cells were combined and DNA fragmentation assessed using an ELISA to measure nucleosome formation. Columns, mean fraction of the results obtained from samples treated with 250 μmol/L of carboplatin alone; bars, SD (n = 3). *, P < 0.05, paired t test.
- Fig. 2.
Inhibition of Bcl-XL expression by siRNA increases the sensitivity to carboplatin. IGROV-1 (A), OVCAR-3 (B), or OVCAR-4 (C) cells were transfected with either two separate siRNA directed to Bcl-XL (○, Bcl-XL no. 2 or ▵, Bcl-XL no. 4), a nontargeting siRNA, an siRNA that does not enter the RISC complex (“RISC-free”), or two siRNA (SCNN1A no. 1 and no. 2) directed to a gene not previously implicated in drug resistance. Quantitative PCR studies indicated that in all three cell lines, the Bcl-XL siRNAs afforded >80% reduction in Bcl-XL mRNA (data not shown). The potency of carboplatin was subsequently measured in a sulforhodamine B assay (SRB). C, cells not treated with carboplatin (X-axis).
- Fig. 3.
Expression of Bcl-XL family members in ovarian cancer cells. Lysates were prepared from cultures of the indicated cell lines and protein expression assessed by Western blotting. The results are representative of three separate experiments.
- Fig. 4.
Drug sensitivity of primary cultures of epithelial cells derived from patients with ovarian cancer. Primary cultures were established from cells isolated from ascitic fluid collected from three patients diagnosed with ovarian cancer (Patient A: age 51, stage IV, poorly differentiated adenocarcinoma. Patient B: age 62, stage III papillary serous carcinoma. Patient C: age 49, stage III, serous carcinoma). The expression of Bcl-XL family members was measured in lysates prepared from cultures and protein expression assessed by Western blotting. Results representative of three separate experiments.
- Fig. 5.
Schedule-dependent sensitization with ABT-737. IGROV-1 cells were treated with a range of concentrations of carboplatin and either pretreated, cotreated, or subsequently treated with 0.6 μmol/L of ABT-737. After 72 h, the potency of carboplatin was determined using a sulforhodamine B assay. Rectangular boxes represent a new addition of drugs (and removal of any drug previously added). For example, in schedule no. 3, cells were treated for 24 h with carboplatin and ABT-737, then washed, and retreated with fresh ABT-737. In schedule no. 6, cells were treated with ABT-737 for 24 h, washed and the cells retreated with fresh ABT-737 together with carboplatin for a further 24 h. Combination indices (mean ± SD) are quoted at a fraction unaffected of 0.5, and where shown, are significantly different from unity (paired t test; **, P < 0.005; *, P < 0.05).
- Fig. 6.
Inhibition of IGROV-1 xenograft growth. Mice were s.c. inoculated with IGROV-1 cells. Once tumors became measurable, the animals were treated weekly with vehicle (▪), ABT-737 (▵, 100 mg/kg, i.p., q.d.), carboplatin (○, 30 mg/kg, i.p., q7d) or a combination of carboplatin and ABT-737(□). Tumor volume was monitored for 28 d after which drug dosing was ceased. None of the drug treatments reduced body weight by >5%.
Tables
- Table 1.
The potency of ABT-737 and carboplatin in cell proliferation/survival assays
Single agent Combination with carboplatin ABT-737, IC50 (μmol/L) ABT-737E, IC50 (μmol/L) ABT-737 (μmol/L) IC50 (μmol/L), no ABT-737 IC50 (μmol/L), with ABT-737 Combination index A2780 14 ± 1 26 ± 2 3.0 11 ± 1 10 ± 1 1.2 ± 0.1 cisA2780 11 ± 1 28 ± 1 3.0 110 ± 20 89 ± 14 1.2 ± 01 IGROV-1 8.2 ± 1.1 23 ± 1 0.6 21 ± 4 10 ± 3 0.54 ± 0.04* OVCAR-3 14 ± 3 27 ± 1 1.0 4.6 ± 0.9 2.7 ± 0.7 0.65 ± 0.05* OVCAR-4 10 ± 2 27 ± 2 1.0 25 ± 12 21 ± 9 0.93 ± 0.06 OVCAR-5 8.8 ± 0.9 25 ± 1 0.6 64 ± 10 47 ± 9 0.80 ± 0.04† OVCAR-8 13 ± 1 27 ± 1 1.0 70 ± 11 37 ± 7 0.61 ± 0.06* SK-OV-3 13 ± 3 28 ± 1 1.0 30 ± 4 25 ± 4 0.90 ± 0.04 NOTE: The potency of ABT-737 or ABT-737E as single agents was measured in cell proliferation/survival assays (columns 1 and 2; mean ± SD, n = 4-11). To measure the sensitization of ovarian cancer cells to carboplatin, cells were simultaneously treated with carboplatin and the indicated concentration of ABT-737 (column 3) and the potency of carboplatin determined (columns 4-5; mean ± SD, n = 4-7). Combination index values (mean ± SD) are quoted at a fraction unaffected of 0.5, and where shown, are significantly different from unity.
↵* P < 0.001, paired t test.
↵† P < 0.01, paired t test.
- Table 3.
The potency of ABT-737 and carboplatin in primary cultures of ovarian cancer
Patient ABT-737 IC50 (μmol/L) Combination index Carboplatin Carboplatin and ABT-737 A 26 ± 3 22 ± 3 11 ± 4 0.66 ± 0.12* B 19 ± 7 18 ± 7 15 ± 3 11 ± 0.3 C 12 ± 6 13 ± 6 10 ± 4 11 ± 0.3 NOTE: The IC50 (mean ± SD, n = 3-6) of ABT-737, carboplatin or the combination of ABT-737 (1 μmol/L) and carboplatin was measured using a sulforhodamine B assay.
↵* P < 0.005, paired t test.
Volume 13, Issue 23
