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The Na⁺/H⁺ exchanger (NHE1) as a novel co-adjuvant target in paclitaxel therapy of triple-negative breast cancer cells.

Amith SR, Wilkinson JM, Baksh S, Fliegel L - Oncotarget (2015)

Bottom Line: In vivo xenograft tumor growth in athymic nude mice was also dramatically decreased compared to parental MDA-MB-231 cells.Loss of NHE1 expression also increased the susceptibility of knockout cells to paclitaxel-mediated cell death.NHE1 inhibition, in combination with paclitaxel, resulted in a dramatic decrease in viability, and migratory and invasive potential of triple-negative breast cancer cells, but not in hormone receptor-positive, luminal MCF7 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Dysregulation of Na⁺/H⁺ exchanger isoform one (NHE1) activity is a hallmark of cells undergoing tumorigenesis and metastasis, the leading cause of patient mortality. The acidic tumor microenvironment is thought to facilitate the development of resistance to chemotherapy drugs and to promote extracellular matrix remodeling leading to metastasis. Here, we investigated NHE1 as a co-adjuvant target in paclitaxel chemotherapy of metastatic breast cancer. We generated a stable NHE1-knockout of the highly invasive, triple-negative, MDA-MB-231 breast cancer cells. The NHE1-knockout cells proliferated comparably to parental cells, but had markedly lower rates of migration and invasion in vitro. In vivo xenograft tumor growth in athymic nude mice was also dramatically decreased compared to parental MDA-MB-231 cells. Loss of NHE1 expression also increased the susceptibility of knockout cells to paclitaxel-mediated cell death. NHE1 inhibition, in combination with paclitaxel, resulted in a dramatic decrease in viability, and migratory and invasive potential of triple-negative breast cancer cells, but not in hormone receptor-positive, luminal MCF7 cells. Our data suggest that NHE1 is critical in triple-negative breast cancer metastasis, and its chemical inhibition boosts the efficacy of paclitaxel in vitro, highlighting NHE1 as a novel, potential co-adjuvant target in breast cancer chemotherapy.

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Related in: MedlinePlus

Effect of paclitaxel in combination with NHE1 inhibitors on cell viability of wild type (231-WT) and NHE1-knockout (231-KO) MDA-MB-231 cells, and MDA-MB-468 (468) and MCF7 cellsCells were treated for 24 hours in reduced (0.2%) serum media (stimulated conditions), with 1 nM paclitaxel (TAX), 10 μM EMD87580 (EMD) (A), or 10 nM HMA (HMA) (B), or with both paclitaxel and EMD87580 (ET, A), or paclitaxel and HMA (HT, B) [*P<0.001, + P<0.01, N=5]. Media only control cells (CTRL) were left untreated. Viability was assessed by the catalytic conversion of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan in live cells. Spectrophotometric quantitation of this colorimetric change was recorded as the optical density of the sample at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of sample means over mean control values for each treatment for each cell type. In 231-KO cells, a significant loss of viability is observed when cells are treated with paclitaxel alone [+ P<0.01, N=5]. In stimulated conditions, both triple-negative MDA-MB-231 and MDA-MB-468 cells showed significantly reduced viability when treated with paclitaxel in the presence of NHE1 inhibitors EMD87580 or HMA [*P<0.001, N=5]. MCF7 cell viability, however, was unaffected by drug treatments.
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Figure 4: Effect of paclitaxel in combination with NHE1 inhibitors on cell viability of wild type (231-WT) and NHE1-knockout (231-KO) MDA-MB-231 cells, and MDA-MB-468 (468) and MCF7 cellsCells were treated for 24 hours in reduced (0.2%) serum media (stimulated conditions), with 1 nM paclitaxel (TAX), 10 μM EMD87580 (EMD) (A), or 10 nM HMA (HMA) (B), or with both paclitaxel and EMD87580 (ET, A), or paclitaxel and HMA (HT, B) [*P<0.001, + P<0.01, N=5]. Media only control cells (CTRL) were left untreated. Viability was assessed by the catalytic conversion of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan in live cells. Spectrophotometric quantitation of this colorimetric change was recorded as the optical density of the sample at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of sample means over mean control values for each treatment for each cell type. In 231-KO cells, a significant loss of viability is observed when cells are treated with paclitaxel alone [+ P<0.01, N=5]. In stimulated conditions, both triple-negative MDA-MB-231 and MDA-MB-468 cells showed significantly reduced viability when treated with paclitaxel in the presence of NHE1 inhibitors EMD87580 or HMA [*P<0.001, N=5]. MCF7 cell viability, however, was unaffected by drug treatments.

Mentions: As noted above, we found that stimulated 231-WT cells have elevated Na+ /H+ exchanger activity independent of changes in protein expression. We hypothesized that with NHE1 inhibition they would be more susceptible to paclitaxel-induced cell death because of a dependence on this elevated rate of Na+ /H+ exchange. We indeed found this to be the case. The viability of stimulated 231-WT cells was significantly decreased (P<0.001, N=5) when cells were treated with 1 nM paclitaxel in combination with either 10 μM EMD87580 (Fig. 4A), or 10 nM HMA (Fig 4B). This effect was significant when compared to cells treated with paclitaxel, EMD87580, or HMA alone at the same concentrations. Similar results were observed in MDA-MB-468 cells (P<0.001, N=5), but not in MCF7 cells. In contrast, in unstimulated 231-WT cells, viability was not further reduced by the combination of paclitaxel and NHE inhibitors (not shown). Likewise, in 231-KO cells, no significant differences were observed between treatments; however, treatment with 1 nM paclitaxel alone slightly but significantly reduced viability (P<0.05, N=5).


The Na⁺/H⁺ exchanger (NHE1) as a novel co-adjuvant target in paclitaxel therapy of triple-negative breast cancer cells.

Amith SR, Wilkinson JM, Baksh S, Fliegel L - Oncotarget (2015)

Effect of paclitaxel in combination with NHE1 inhibitors on cell viability of wild type (231-WT) and NHE1-knockout (231-KO) MDA-MB-231 cells, and MDA-MB-468 (468) and MCF7 cellsCells were treated for 24 hours in reduced (0.2%) serum media (stimulated conditions), with 1 nM paclitaxel (TAX), 10 μM EMD87580 (EMD) (A), or 10 nM HMA (HMA) (B), or with both paclitaxel and EMD87580 (ET, A), or paclitaxel and HMA (HT, B) [*P<0.001, + P<0.01, N=5]. Media only control cells (CTRL) were left untreated. Viability was assessed by the catalytic conversion of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan in live cells. Spectrophotometric quantitation of this colorimetric change was recorded as the optical density of the sample at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of sample means over mean control values for each treatment for each cell type. In 231-KO cells, a significant loss of viability is observed when cells are treated with paclitaxel alone [+ P<0.01, N=5]. In stimulated conditions, both triple-negative MDA-MB-231 and MDA-MB-468 cells showed significantly reduced viability when treated with paclitaxel in the presence of NHE1 inhibitors EMD87580 or HMA [*P<0.001, N=5]. MCF7 cell viability, however, was unaffected by drug treatments.
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Figure 4: Effect of paclitaxel in combination with NHE1 inhibitors on cell viability of wild type (231-WT) and NHE1-knockout (231-KO) MDA-MB-231 cells, and MDA-MB-468 (468) and MCF7 cellsCells were treated for 24 hours in reduced (0.2%) serum media (stimulated conditions), with 1 nM paclitaxel (TAX), 10 μM EMD87580 (EMD) (A), or 10 nM HMA (HMA) (B), or with both paclitaxel and EMD87580 (ET, A), or paclitaxel and HMA (HT, B) [*P<0.001, + P<0.01, N=5]. Media only control cells (CTRL) were left untreated. Viability was assessed by the catalytic conversion of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan in live cells. Spectrophotometric quantitation of this colorimetric change was recorded as the optical density of the sample at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of sample means over mean control values for each treatment for each cell type. In 231-KO cells, a significant loss of viability is observed when cells are treated with paclitaxel alone [+ P<0.01, N=5]. In stimulated conditions, both triple-negative MDA-MB-231 and MDA-MB-468 cells showed significantly reduced viability when treated with paclitaxel in the presence of NHE1 inhibitors EMD87580 or HMA [*P<0.001, N=5]. MCF7 cell viability, however, was unaffected by drug treatments.
Mentions: As noted above, we found that stimulated 231-WT cells have elevated Na+ /H+ exchanger activity independent of changes in protein expression. We hypothesized that with NHE1 inhibition they would be more susceptible to paclitaxel-induced cell death because of a dependence on this elevated rate of Na+ /H+ exchange. We indeed found this to be the case. The viability of stimulated 231-WT cells was significantly decreased (P<0.001, N=5) when cells were treated with 1 nM paclitaxel in combination with either 10 μM EMD87580 (Fig. 4A), or 10 nM HMA (Fig 4B). This effect was significant when compared to cells treated with paclitaxel, EMD87580, or HMA alone at the same concentrations. Similar results were observed in MDA-MB-468 cells (P<0.001, N=5), but not in MCF7 cells. In contrast, in unstimulated 231-WT cells, viability was not further reduced by the combination of paclitaxel and NHE inhibitors (not shown). Likewise, in 231-KO cells, no significant differences were observed between treatments; however, treatment with 1 nM paclitaxel alone slightly but significantly reduced viability (P<0.05, N=5).

Bottom Line: In vivo xenograft tumor growth in athymic nude mice was also dramatically decreased compared to parental MDA-MB-231 cells.Loss of NHE1 expression also increased the susceptibility of knockout cells to paclitaxel-mediated cell death.NHE1 inhibition, in combination with paclitaxel, resulted in a dramatic decrease in viability, and migratory and invasive potential of triple-negative breast cancer cells, but not in hormone receptor-positive, luminal MCF7 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Dysregulation of Na⁺/H⁺ exchanger isoform one (NHE1) activity is a hallmark of cells undergoing tumorigenesis and metastasis, the leading cause of patient mortality. The acidic tumor microenvironment is thought to facilitate the development of resistance to chemotherapy drugs and to promote extracellular matrix remodeling leading to metastasis. Here, we investigated NHE1 as a co-adjuvant target in paclitaxel chemotherapy of metastatic breast cancer. We generated a stable NHE1-knockout of the highly invasive, triple-negative, MDA-MB-231 breast cancer cells. The NHE1-knockout cells proliferated comparably to parental cells, but had markedly lower rates of migration and invasion in vitro. In vivo xenograft tumor growth in athymic nude mice was also dramatically decreased compared to parental MDA-MB-231 cells. Loss of NHE1 expression also increased the susceptibility of knockout cells to paclitaxel-mediated cell death. NHE1 inhibition, in combination with paclitaxel, resulted in a dramatic decrease in viability, and migratory and invasive potential of triple-negative breast cancer cells, but not in hormone receptor-positive, luminal MCF7 cells. Our data suggest that NHE1 is critical in triple-negative breast cancer metastasis, and its chemical inhibition boosts the efficacy of paclitaxel in vitro, highlighting NHE1 as a novel, potential co-adjuvant target in breast cancer chemotherapy.

Show MeSH
Related in: MedlinePlus