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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

BCRP and MRP1 inhibitors suppress efflux function in ascites treated ID8 cells.(A). ID8 cells from normal culture, ascites pre-treatment culture, or ascites (in vivo 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 in vivo ascites cells (compared to untreated cells) are indicated. Error bars represent SDs from three independent experiments. * indicates p<0.05, Student’s t-test.
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pone.0131579.g004: BCRP and MRP1 inhibitors suppress efflux function in ascites treated ID8 cells.(A). ID8 cells from normal culture, ascites pre-treatment culture, or ascites (in vivo 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 in vivo ascites cells (compared to untreated cells) are indicated. Error bars represent SDs from three independent experiments. * indicates p<0.05, Student’s t-test.

Mentions: After demonstrating upregulated MDR-related ABC transporters in ascites treated ovarian cancer cells, we next sought to determine whether specific inhibitors of these transporters prevent efflux in these cells. We studied three MDR transporter inhibitors: verapamil (MDR1 inhibitor), MK-571 (MRP1/2 inhibitor), and Novobiocin (BCRP inhibitor). First, inhibitor effects on efflux function in ID8 cells cultured in normal medium was assessed. Inhibitors were added to cells for 10 minutes before the addition of eFFlux ID green dye. As shown in Fig 4A and 4B, only the MRP1 inhibitor increased fluorescence intensity of efflux ID green- labeled ID8 cells. These results indicate that untreated ID8 ovarian cancer cells support MRP1-dependent efflux, but not MDR1 or BCRP-dependent efflux. Next we studied effects of these inhibitors on efflux function in both ascites treated ID8 cells and in ID8 cells isolated directly from ascites (in vivo cells). MDR1 inhibitor did not increase fluorescence in ID8 cells pre-incubated with ascites for 7 days in vitro. However, MDR1 inhibitor did increase fluorescence in ID8 cells isolated from ascites in vivo (Fig 4A and 4B). Addition of either MRP1 inhibitor or BCRP inhibitor significantly increased fluorescence (MAF) in both ascites-treated ID8 cells and in ascites cells (in vivo) (Fig 4B). We also showed that combinations of these inhibitors did not increase fluorescence intensity of efflux ID green-labeled ID8 cells or ascites-treated cells over the fluorescence intensity obtained with single inhibitors, suggesting that activity of these individual transporters was not compensated for by other transporters (data not shown). Collectively, these data suggest that ascites promotes MRP1 and BCRP-dependent efflux in ID8 ovarian cancer cells.


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)

BCRP and MRP1 inhibitors suppress efflux function in ascites treated ID8 cells.(A). ID8 cells from normal culture, ascites pre-treatment culture, or ascites (in vivo 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 in vivo ascites cells (compared to untreated cells) are indicated. Error bars represent SDs from three independent experiments. * indicates p<0.05, Student’s t-test.
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Related In: Results  -  Collection

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pone.0131579.g004: BCRP and MRP1 inhibitors suppress efflux function in ascites treated ID8 cells.(A). ID8 cells from normal culture, ascites pre-treatment culture, or ascites (in vivo 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 in vivo ascites cells (compared to untreated cells) are indicated. Error bars represent SDs from three independent experiments. * indicates p<0.05, Student’s t-test.
Mentions: After demonstrating upregulated MDR-related ABC transporters in ascites treated ovarian cancer cells, we next sought to determine whether specific inhibitors of these transporters prevent efflux in these cells. We studied three MDR transporter inhibitors: verapamil (MDR1 inhibitor), MK-571 (MRP1/2 inhibitor), and Novobiocin (BCRP inhibitor). First, inhibitor effects on efflux function in ID8 cells cultured in normal medium was assessed. Inhibitors were added to cells for 10 minutes before the addition of eFFlux ID green dye. As shown in Fig 4A and 4B, only the MRP1 inhibitor increased fluorescence intensity of efflux ID green- labeled ID8 cells. These results indicate that untreated ID8 ovarian cancer cells support MRP1-dependent efflux, but not MDR1 or BCRP-dependent efflux. Next we studied effects of these inhibitors on efflux function in both ascites treated ID8 cells and in ID8 cells isolated directly from ascites (in vivo cells). MDR1 inhibitor did not increase fluorescence in ID8 cells pre-incubated with ascites for 7 days in vitro. However, MDR1 inhibitor did increase fluorescence in ID8 cells isolated from ascites in vivo (Fig 4A and 4B). Addition of either MRP1 inhibitor or BCRP inhibitor significantly increased fluorescence (MAF) in both ascites-treated ID8 cells and in ascites cells (in vivo) (Fig 4B). We also showed that combinations of these inhibitors did not increase fluorescence intensity of efflux ID green-labeled ID8 cells or ascites-treated cells over the fluorescence intensity obtained with single inhibitors, suggesting that activity of these individual transporters was not compensated for by other transporters (data not shown). Collectively, these data suggest that ascites promotes MRP1 and BCRP-dependent efflux in ID8 ovarian cancer cells.

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