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Ascites Increases Expression/Function of Multidrug Resistance Proteins in Ovarian Cancer Cells.

Mo L, Pospichalova V, Huang Z, Murphy SK, Payne S, Wang F, Kennedy M, Cianciolo GJ, Bryja V, Pizzo SV, Bachelder RE - PLoS ONE (2015)

Bottom Line: One mechanism behind chemo-resistance involves the upregulation of multidrug resistance (MDR) genes (ABC transporters) that effectively transport (efflux) drugs out of the tumor cells.As a common symptom in stage III/IV ovarian cancer patients, ascites is associated with cancer progression.Functional studies show ascites-driven efflux is suppressible by specific inhibitors of either of two ABC transporters [Multidrug Related Protein (MRP1); Breast Cancer Related Protein (BCRP)].

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina, 27710, United States of America.

ABSTRACT
Chemotherapy resistance is the major reason for the failure of ovarian cancer treatment. One mechanism behind chemo-resistance involves the upregulation of multidrug resistance (MDR) genes (ABC transporters) that effectively transport (efflux) drugs out of the tumor cells. As a common symptom in stage III/IV ovarian cancer patients, ascites is associated with cancer progression. However, whether ascites drives multidrug resistance in ovarian cancer cells awaits elucidation. Here, we demonstrate that when cultured with ascites derived from ovarian cancer-bearing mice, a murine ovarian cancer cell line became less sensitive to paclitaxel, a first line chemotherapeutic agent for ovarian cancer patients. Moreover, incubation of murine ovarian cancer cells in vitro with ascites drives efflux function in these cells. Functional studies show ascites-driven efflux is suppressible by specific inhibitors of either of two ABC transporters [Multidrug Related Protein (MRP1); Breast Cancer Related Protein (BCRP)]. To demonstrate relevance of our findings to ovarian cancer patients, we studied relative efflux in human ovarian cancer cells obtained from either patient ascites or from primary tumor. Immortalized cell lines developed from human ascites show increased susceptibility to efflux inhibitors (MRP1, BCRP) compared to a cell line derived from a primary ovarian cancer, suggesting an association between ascites and efflux function in human ovarian cancer. Efflux in ascites-derived human ovarian cancer cells is associated with increased expression of ABC transporters compared to that in primary tumor-derived human ovarian cancer cells. Collectively, our findings identify a novel activity for ascites in promoting ovarian cancer multidrug resistance.

No MeSH data available.


Related in: MedlinePlus

Ascites increases efflux function in ID8 cells.(A). 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 (in vivo 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). Efflux function was measured by Rhodamine 123 assay. ID8 cells obtained from normal culture, from 7 day ascites pretreatment culture or from ascites in vivo 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 (C). 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.
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pone.0131579.g002: Ascites increases efflux function in ID8 cells.(A). 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 (in vivo 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). Efflux function was measured by Rhodamine 123 assay. ID8 cells obtained from normal culture, from 7 day ascites pretreatment culture or from ascites in vivo 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 (C). 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.

Mentions: We next investigated efflux function in ascites-treated ovarian cancer cells using two drug efflux assays (eFFlux ID green dye and rhodamine 123) [18]. The eFFlux ID green assay measures fluorescence intensity after incubating cells with fluorescent dye. Fig 2A shows reduced fluorescence intensity in both ascites pre-treated ID8 cells and ID8 cells isolated from ascites (syngeneic mouse model) compared to fluorescence intensity in untreated cells. These results suggest that ascites promotes efflux function in ovarian cancer cells. We next measured retention of rhodamine 123 dye in ID8 cells (+/- ascites treatment). As shown in Fig 2B and 2C, 2.5h after rhodamine removal, untreated ID8 cells had retained dye. In contrast, significantly reduced levels of dye were detected in ascites-preincubated ID8 cells, supporting the conclusion that ascites-treated tumor cells exhibit increased efflux. Together, these results suggest that ascites drives ovarian cancer efflux mechanisms.


Ascites Increases Expression/Function of Multidrug Resistance Proteins in Ovarian Cancer Cells.

Mo L, Pospichalova V, Huang Z, Murphy SK, Payne S, Wang F, Kennedy M, Cianciolo GJ, Bryja V, Pizzo SV, Bachelder RE - PLoS ONE (2015)

Ascites increases efflux function in ID8 cells.(A). 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 (in vivo 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). Efflux function was measured by Rhodamine 123 assay. ID8 cells obtained from normal culture, from 7 day ascites pretreatment culture or from ascites in vivo 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 (C). 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493087&req=5

pone.0131579.g002: Ascites increases efflux function in ID8 cells.(A). 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 (in vivo 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). Efflux function was measured by Rhodamine 123 assay. ID8 cells obtained from normal culture, from 7 day ascites pretreatment culture or from ascites in vivo 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 (C). 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.
Mentions: We next investigated efflux function in ascites-treated ovarian cancer cells using two drug efflux assays (eFFlux ID green dye and rhodamine 123) [18]. The eFFlux ID green assay measures fluorescence intensity after incubating cells with fluorescent dye. Fig 2A shows reduced fluorescence intensity in both ascites pre-treated ID8 cells and ID8 cells isolated from ascites (syngeneic mouse model) compared to fluorescence intensity in untreated cells. These results suggest that ascites promotes efflux function in ovarian cancer cells. We next measured retention of rhodamine 123 dye in ID8 cells (+/- ascites treatment). As shown in Fig 2B and 2C, 2.5h after rhodamine removal, untreated ID8 cells had retained dye. In contrast, significantly reduced levels of dye were detected in ascites-preincubated ID8 cells, supporting the conclusion that ascites-treated tumor cells exhibit increased efflux. Together, these results suggest that ascites drives ovarian cancer efflux mechanisms.

Bottom Line: One mechanism behind chemo-resistance involves the upregulation of multidrug resistance (MDR) genes (ABC transporters) that effectively transport (efflux) drugs out of the tumor cells.As a common symptom in stage III/IV ovarian cancer patients, ascites is associated with cancer progression.Functional studies show ascites-driven efflux is suppressible by specific inhibitors of either of two ABC transporters [Multidrug Related Protein (MRP1); Breast Cancer Related Protein (BCRP)].

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina, 27710, United States of America.

ABSTRACT
Chemotherapy resistance is the major reason for the failure of ovarian cancer treatment. One mechanism behind chemo-resistance involves the upregulation of multidrug resistance (MDR) genes (ABC transporters) that effectively transport (efflux) drugs out of the tumor cells. As a common symptom in stage III/IV ovarian cancer patients, ascites is associated with cancer progression. However, whether ascites drives multidrug resistance in ovarian cancer cells awaits elucidation. Here, we demonstrate that when cultured with ascites derived from ovarian cancer-bearing mice, a murine ovarian cancer cell line became less sensitive to paclitaxel, a first line chemotherapeutic agent for ovarian cancer patients. Moreover, incubation of murine ovarian cancer cells in vitro with ascites drives efflux function in these cells. Functional studies show ascites-driven efflux is suppressible by specific inhibitors of either of two ABC transporters [Multidrug Related Protein (MRP1); Breast Cancer Related Protein (BCRP)]. To demonstrate relevance of our findings to ovarian cancer patients, we studied relative efflux in human ovarian cancer cells obtained from either patient ascites or from primary tumor. Immortalized cell lines developed from human ascites show increased susceptibility to efflux inhibitors (MRP1, BCRP) compared to a cell line derived from a primary ovarian cancer, suggesting an association between ascites and efflux function in human ovarian cancer. Efflux in ascites-derived human ovarian cancer cells is associated with increased expression of ABC transporters compared to that in primary tumor-derived human ovarian cancer cells. Collectively, our findings identify a novel activity for ascites in promoting ovarian cancer multidrug resistance.

No MeSH data available.


Related in: MedlinePlus