Article Figures & Data
Figures
- Fig. 1.
Effects of paclitaxel on the growth of two human ovarian cancer cell lines exhibiting different levels of HER-2/neu and E1A expression. Seventy-two h after paclitaxel treatment, cells were analyzed by MTT assay. The percentage of cell growth was calculated by defining the absorption of cells not treated with paclitaxel as 100%. Error bars, SDs. All data points have error bars within the SD range of 1–3%. Cell lysates of each transfectant and parental cell lines were subjected to 12% SDS-PAGE and blotted with anti-p185 and anti-E1A antibodies. Equal loading was confirmed by probing the same SDS-PAGE gel with anti-actin antibody. HER-2/neu expression signal intensity was adjusted based on the original strength of the signal. The SKOV3.ip1 and 2774-c-10 nitrocellulose membranes were exposed at different time points. A, the effects of E1A on the expression of HER-2/neu in a human ovarian cancer cell line. B, enhanced growth inhibition by paclitaxel was seen on the viable fractions of the E1A-transfected HER-2/neu-overexpressing SKOV3.ip1 cell lines, but not on those of the control cell lines. C, no enhanced growth inhibition by paclitaxel was seen on the viable fractions of either the E1A-transfected low-HER-2/neu-expressing 2774-c-10 cell line or the control cell lines.
- Fig. 2.
Effects of paclitaxel on cell cycle progression in human ovarian cancer cells. After treatment with paclitaxel, cells exhibiting different levels of HER-2/neu and E1A expression were stained with propidium iodide and analyzed for DNA content by flow cytometry. The brackets indicate sub-G0 fractions. A, cells were treated with paclitaxel (0.1 or 1μ m) for 33 h. Increased sub-G0 fractions were found in SKOV3.ip1 cells with HER-2/neu overexpression down-regulated by E1A, SKOV3.ip1-E1A2 cells, and SKOV3.ip1-E1A16 cells. B, cells were treated with paclitaxel (0.1 or 1 μm) for 24–33 h. The series of 2774-c-10 cell lines all underwent apoptosis, regardless of the level of E1A expression.
- Fig. 3.
Effects of paclitaxel on internucleosomal DNA fragmentation in human ovarian cancer cells. After treatment with paclitaxel (0.1 μm) for 48 h, cells were collected and analyzed for internucleosomal DNA fragmentation by agarose gel electrophoresis. Internucleosomal DNA fragmentation was observed in both cells with a low basal HER-2/neu expression level (series of 2774-c-10 cell lines) and cells in which HER-2/neu overexpression was down-regulated by E1A (SKOV3.ip1-E1A2 and SKOV3.ip1-E1A16 cells).
- Fig. 4.
Effects of E1A on proapoptotic and antiapoptotic molecules. Cell lysates were subjected to 12% SDS-PAGE and hybridized with anti-p185, anti-E1A, proapoptotic (Bax and Bad), or antiapoptotic (Bcl-2 and Bcl-XL) molecules in human HER-2/neu-overexpressing ovarian cancer SKOV3.ip1 cell lines. Down-regulation of HER-2/neu or E1A did not affect the expression level of the apoptosis-related molecules in human HER-2/neu-overexpressing ovarian cancer cells. Equal loading was confirmed by probing the same SDS-PAGE gel with anti-actin antibody. Shifting of the original bands of Bcl-2 and Bcl-XL as a result of phosphorylation is indicated by the two arrowheads. There was an overall decrease in the Bad expression level after paclitaxel treatment.
- Fig. 5.
Activation of caspase-3 by paclitaxel-induced apoptosis in HER-2/neu-overexpressing human ovarian cancer cells. After treatment with paclitaxel (0.1 μm), cell lysate samples were collected every 6 h, subjected to 12% SDS-PAGE, and then hybridized with anti-PARP, anti-caspase-3, and anti-caspase-7 antibodies. A, 24 h after treatment the Mr 116,000 PARP was observed to be cleaved into an Mr 85,000 form in E1A-transfected cells but not in either the parental cells or the control SKOV3.ip1-Efs cells. B, cleaved caspase-3 (Mr 17,000) products were observed only in SKOV3.ip1-E1A cells. No cleaved caspase-7 (Mr 20,000) products were detected.
- Fig. 6.
Partial inhibition of paclitaxel-induced apoptosis by a cell-permeable caspase-3 inhibitor in HER-2/neu-overexpressing ovarian cancer cells. Cells were pretreated for 1 h with either the pan-caspase inhibitor (Z-VAD-FMK) or the caspase-3 inhibitor (Z-DEVD-FMK). Paclitaxel (0.1μ m) was then applied, and after Hoechst staining, the cells were examined for signs of apoptosis, that is, changes in cell morphology and chromatin condensation. The number of total cells and the number of apoptotic cells were counted in four areas under the high-power field of a fluorescent microscope. Three independent experiments were conducted. The percentage of apoptotic cells was calculated in relation to the total number of untreated control cells, which was set at 100%. Error bars, SDs.
- Fig. 7.
Prolonged survival of nude mice treated with paclitaxel and E1A/DC-Chol complex. Each female nude mouse received an injection of 2 × 106 viable HER-2/neu-ovexpressing human cancer SKOV3.ip1 cells. The survival curves are based on the total number of survival days for each mouse in the different groups and were analyzed by the method of Kaplan and Meier (38) . Treated mice survived significantly longer than mice left untreated (P = 0.00217), mice treated with paclitaxel alone, or mice treated with E1A alone.
Volume 6, Issue 1
