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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

Human ovarian cancer cells derived from ascites exhibit increased efflux compared to cells derived from the primary tumor.A and 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. 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). C. 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. D and E. 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 (D). E. 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 in vivo ascites cells (compared to untreated cells) are indicated. * indicates p<0.05. ** indicates p<.01, Student’s t-test.
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pone.0131579.g005: Human ovarian cancer cells derived from ascites exhibit increased efflux compared to cells derived from the primary tumor.A and 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. 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). C. 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. D and E. 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 (D). E. 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 in vivo ascites cells (compared to untreated cells) are indicated. * indicates p<0.05. ** indicates p<.01, Student’s t-test.

Mentions: Next, we sought to extend our findings to human ovarian cancer cells. We studied immortalized human ovarian cancer cells derived from patient ascites (HA1, HA2 cells) or from the primary tumor site (TD cells). Using the eFFlux ID green assay, we showed that HA1 and HA2 cells exhibited higher efflux than TD cells (Fig 5A and 5B), lending support to our finding that ascites drives the efflux function of ovarian cancer cells. Western blotting was performed to investigate efflux protein expression in these tumor-derived and ascites-derived human ovarian cancer cells. As shown in Fig 5C, expression of MDR1, MRP1 and BCRP was significantly higher in ascites-derived tumor cell lines (HA1, HA2) than in a primary tumor-derived cell line (TD). To determine which efflux proteins were functional in these cells, we next investigated effects of MDR inhibitors on efflux in tumor-derived and ascites-derived human ovarian cancer cells. MRP1 and BCRP inhibitors, but not an MDR1 inhibitor, suppressed dye efflux in HA1 and HA2 cells significantly. In contrast, inhibitors of MDR1, MRP1 and BCRP did not impact dye retention in TD cells (Fig 5D and 5E). We showed that combinations of these inhibitors did not increase fluorescence intensity of TD cells (data not shown), suggesting that activity of individual transporters was not compensated for by other transporters. These findings indicate ascites-derived human ovarian cancer cells, but not primary tumor-derived cells, support ABC transporter-dependent efflux.


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)

Human ovarian cancer cells derived from ascites exhibit increased efflux compared to cells derived from the primary tumor.A and 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. 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). C. 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. D and E. 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 (D). E. 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 in vivo ascites cells (compared to untreated cells) are indicated. * indicates p<0.05. ** indicates p<.01, Student’s t-test.
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pone.0131579.g005: Human ovarian cancer cells derived from ascites exhibit increased efflux compared to cells derived from the primary tumor.A and 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. 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). C. 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. D and E. 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 (D). E. 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 in vivo ascites cells (compared to untreated cells) are indicated. * indicates p<0.05. ** indicates p<.01, Student’s t-test.
Mentions: Next, we sought to extend our findings to human ovarian cancer cells. We studied immortalized human ovarian cancer cells derived from patient ascites (HA1, HA2 cells) or from the primary tumor site (TD cells). Using the eFFlux ID green assay, we showed that HA1 and HA2 cells exhibited higher efflux than TD cells (Fig 5A and 5B), lending support to our finding that ascites drives the efflux function of ovarian cancer cells. Western blotting was performed to investigate efflux protein expression in these tumor-derived and ascites-derived human ovarian cancer cells. As shown in Fig 5C, expression of MDR1, MRP1 and BCRP was significantly higher in ascites-derived tumor cell lines (HA1, HA2) than in a primary tumor-derived cell line (TD). To determine which efflux proteins were functional in these cells, we next investigated effects of MDR inhibitors on efflux in tumor-derived and ascites-derived human ovarian cancer cells. MRP1 and BCRP inhibitors, but not an MDR1 inhibitor, suppressed dye efflux in HA1 and HA2 cells significantly. In contrast, inhibitors of MDR1, MRP1 and BCRP did not impact dye retention in TD cells (Fig 5D and 5E). We showed that combinations of these inhibitors did not increase fluorescence intensity of TD cells (data not shown), suggesting that activity of individual transporters was not compensated for by other transporters. These findings indicate ascites-derived human ovarian cancer cells, but not primary tumor-derived cells, support ABC transporter-dependent efflux.

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