BCRP and MRP1 inhibitors suppress efflux function in ascites treated ID8 cells.

<p>(<b>A</b>). ID8 cells from normal culture, ascites pre-treatment culture, or ascites (<i>in vivo</i> cells) were treated with or without inhibitors targeting MDR1, MRP1 or BCRP for 10 min and then incubated with eFFlux ID Green dye for 30 min. fluorescence intensity of each condition was measured by flow cytometry. Multidrug resistance activity factor (MAF) was calculated for each sample, and mean MAF (+/- SD from triplicate samples) is shown for each condition in B. MAF values falling below background are indicated by gray border. Statistically significant increases in MAF between ascites-treated and <i>in vivo</i> ascites cells (compared to untreated cells) are indicated. Error bars represent SDs from three independent experiments. * indicates p<0.05, Student’s t-test.</p

Ascites derived from ovarian cancer-bearing mice increases chemo-resistance of ID8 cells.

<p>Murine ovarian cancer cells (ID8) from three different sources were studied: 1) ID8 cells from normal culture medium (Medium), 2) ID8 cells freshly isolated from ascites fluid in a syngeneic model (<i>In vivo</i> cells), and 3) ID8 cells treated for 7 days <i>in vitro</i> with ascites supernatant (Ascites treated 7 days). Each of these cell populations was exposed to paclitaxel<b>(A)</b> or docetaxel<b>(B)</b> at the indicated concentration for 24 h and [³H]-thymidine incorporation was determined. Three independent experiments were performed and a representative result is shown. Error bar represents SD of quadruplicates in each condition. * indicates p<0.05, *** p<0.001, Two way ANOVA. <b>A</b>. ID8 cells pre-treated with acellular ascites for 7 days (Ascites treated 7 days) or isolated from ascites (<i>in vivo</i> cells) have increased resistance to Paclitaxel compared to ID8 cells from normal medium. <b>B</b>. ID8 cells pre-treated with acellular ascites for 7 days (Ascites treated 7 days) have increased resistance to docetaxel at 2 and 4 nM compared to ID8 cells from normal culture. ID8 cells isolated from ascites (<i>in vivo</i> cells) have increased resistance to docetaxel compared to ID8 cells from normal medium (Medium). <b>C-D</b>. 7-AAD uptake was measured by flow cytometric analysis. The percent 7-AAD positive cells from three independent experiments is shown for paclitaxel (<b>C</b>) and docetaxel (<b>D</b>)-treated cells. Error bars represent SD. * indicates p<0.05, ** p<0.01, *** p<0.001, Two way ANOVA. ID8 cells pre-treated with acellular ascites for 7 days (Ascites treated 7 days) are more resistant to chemotherapy-induced cell death (<i>i</i>.<i>e</i>.,exhibit fewer 7-AAD+ cells) than ID8 cells from normal culture.</p

Ascites treatment increases expression of MDR1a/b and BCRP in ID8 cells.

<p>Total RNA was harvested from ID8 cells in normal culture as well as from ID8 cells pre-treated with ascites for 7 days. Expression levels of the indicated genes (relative to beta actin) were determined by real-time PCR(<b>A</b>). Fold increases in gene expression (ascites treated ID8 cells over normal ID8 cells) are shown in <b>B</b>. Three independent experiments were performed and error bar represents SD. * indicates p<0.05 and *** p<0.001, Student’s t-test.</p

Human ovarian cancer cells derived from ascites exhibit increased efflux compared to cells derived from the primary tumor.

<p><b>A and B.</b> Immortalized human ovarian cancer cells derived from patient ascites (HA1 and HA2 cells) or primary tumor site (TD cells) were incubated with eFFLux ID green dye for 40 min. Fluorescence intensity for each condition was measured by flow cytometry. Histogram of each cancer cell line derived from patient ascites (HA1 or HA2; blue) is overlaid with histogram of the primary tumor-derived line (TD; red)). <b>B.</b> Mean fluorescence intensity (MFI) was calculated for each line from three independent experiments. Fold decrease in geometric mean fluorescence intensity (GMFI) (relative to tumor-derived line) was calculated for each cell line in three independent experiments. Results are reported as the mean fold decrease from three independent trials treated (+/- SD). <b>C.</b> Total cellular extracts were obtained from a primary tumor-derived human ovarian cancer cell line (TD) and from each of two ascites-derived human ovarian cancer cell lines (HA1, HA2). Equivalent amounts of extracted proteins were subjected to SDS-PAGE and immunblotted with antibodies specific for MDR1, MRP1, BCRP, or beta actin, followed by the appropriate species IRdye-conjugated secondary antibody. Protein bands were detected by Odyssey infrared imaging. Protein bands were quantified using Image J software (NIH). Ratios of the indicated protein to beta actin are shown. <b>D and E.</b> Ascites-derived and tumor-derived human cell lines were incubated with or without inhibitors targeting MDR1, MRP or BCRP for 10 min and then incubated with eFFlux ID Green dye for 30 min. Samples were analyzed by flow cytometry. Histograms (+ inhibitor vs – inhibitor) were overlaid for each line (<b>D</b>). <b>E.</b> Multidrug resistance activity factor (MAF) was calculated for each sample, and mean MAF (+/- SD) from three independent experiments is shown for each condition in E. MAF values falling below background are indicated in gray. Statistically significant increases in MAF between ascites-treated and <i>in vivo</i> ascites cells (compared to untreated cells) are indicated. * indicates p<0.05. ** indicates p<.01, Student’s t-test.</p

Ascites increases efflux function in ID8 cells.

<p>(<b>A</b>). Efflux function was measured by eFFlux ID Green dye assay. ID8 cells isolated from normal culture, obtained after 7 day ascites treatment, or isolated from ascites (<i>in vivo</i> cells) were incubated with eFFlux ID Green dye for 40 min, allowing the cells to uptake and efflux this dye. ID8 cells pre-treated with ascites retained less dye compared to ID8 cells from normal culture, indicating an increased efflux function. (<b>B</b>). Efflux function was measured by Rhodamine 123 assay. ID8 cells obtained from normal culture, from 7 day ascites pretreatment culture or from ascites <i>in vivo</i> were incubated with Rhodamine 123 for 30 min. Then cells from each condition were washed with PBS and incubated with regular medium for 2.5 h. Fluorescent pictures were taken at 0 min (after dye removal and PBS wash) and at 2.5h hours after Rhodamine 123 removal. Quantification of the fluorescence intensity (minus background) in each group is shown in (<b>C</b>). Three independent experiments were performed and a representative result is shown. Error bar represents SD of fluorescent intensity measuring each cell in each image. * indicates p<0.05, Student’s t-test.</p