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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

Na/H exchanger expression and activity in parental MDA-MB-231 (231-WT) and NHE1-knockout cells (231-KO)A, Western blot analysis of NHE1 expression in 231-WT, 231-KO, MDA-MB-468 and MCF7 cells probed with anti-NHE1 antibody. B, Changes in intracellular pH (pHi) in response to acid loading. Examples of traces illustrating the recovery from an acute acid load. Intracellular pH was examined in cells that were transiently acidified using ammonium chloride. Periods of NH4Cl, NaCl and Na-free solution are indicated. An entire example of the recovery is indicated for stimulated 231-WT cells (231s). An example of recovery is indicated for unstimulated 231-WT cells (231u) and MDA-MB-231 NHE1-knockout cells (231-KO). Fluorescence of BCECF, a pH-sensitive indicator dye, was used to record and quantify changes in pHi post-acute acid load induced by ammonium chloride. NHE1 activity was calculated from the slope of the first 20 sec of recovery from acidification and was expressed as ΔpH/sec. C, Relative rate of Na+/H+ exchange activity of 231-KO cells relative to 231-WT cells, and in MDA-MB-468 and MCF7 cells. Values of ΔpH/sec from 231-WT and 231-KO cells were normalized to the unstimulated control values for 231-WT cells. Data for MCF7 and MDA-MB-468 were normalized to their control unstimulated values respectively. No discernible exchanger activity was detected in the knockout cells. Background drift and buffering capacity were not significantly different between cell types. In stimulated conditions (0.2% serum), NHE1 activity is significantly increased in 231-WT, MDA-MB-468 and MCF7 cells [*P<0.001, + P<0.01, N=8].
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Figure 1: Na/H exchanger expression and activity in parental MDA-MB-231 (231-WT) and NHE1-knockout cells (231-KO)A, Western blot analysis of NHE1 expression in 231-WT, 231-KO, MDA-MB-468 and MCF7 cells probed with anti-NHE1 antibody. B, Changes in intracellular pH (pHi) in response to acid loading. Examples of traces illustrating the recovery from an acute acid load. Intracellular pH was examined in cells that were transiently acidified using ammonium chloride. Periods of NH4Cl, NaCl and Na-free solution are indicated. An entire example of the recovery is indicated for stimulated 231-WT cells (231s). An example of recovery is indicated for unstimulated 231-WT cells (231u) and MDA-MB-231 NHE1-knockout cells (231-KO). Fluorescence of BCECF, a pH-sensitive indicator dye, was used to record and quantify changes in pHi post-acute acid load induced by ammonium chloride. NHE1 activity was calculated from the slope of the first 20 sec of recovery from acidification and was expressed as ΔpH/sec. C, Relative rate of Na+/H+ exchange activity of 231-KO cells relative to 231-WT cells, and in MDA-MB-468 and MCF7 cells. Values of ΔpH/sec from 231-WT and 231-KO cells were normalized to the unstimulated control values for 231-WT cells. Data for MCF7 and MDA-MB-468 were normalized to their control unstimulated values respectively. No discernible exchanger activity was detected in the knockout cells. Background drift and buffering capacity were not significantly different between cell types. In stimulated conditions (0.2% serum), NHE1 activity is significantly increased in 231-WT, MDA-MB-468 and MCF7 cells [*P<0.001, + P<0.01, N=8].

Mentions: To examine the role of NHE1 in tumor cells we made a knockout of the protein in the parental MDA-MB-231 cells (231-WT). Western blot analysis was used to examine endogenous expression of NHE1 in the NHE1-knockout (231-KO), compared to the 231-WT cells, MCF7 and MDA-MB-468 cells. NHE1 was consistently observed as a band at approximately 100 kDa in multiple lysate preparations (Fig. 1A). A second smaller band was also observed and is typical of a partially or de-glycosylated NHE1 protein. As expected, the stable NHE1-knockout (231-KO) cells showed no NHE1 protein expression. We also compared the level of NHE1 protein in either stimulated (low-serum (0.2%) media) or unstimulated (10%-serum supplemented media) conditions as previously described [22]. When the level of NHE1 was quantitatively compared to that of actin, there was no up-regulation of NHE1 expression in any cell type under stimulated conditions (N=5, see Fig. 1 for example).


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)

Na/H exchanger expression and activity in parental MDA-MB-231 (231-WT) and NHE1-knockout cells (231-KO)A, Western blot analysis of NHE1 expression in 231-WT, 231-KO, MDA-MB-468 and MCF7 cells probed with anti-NHE1 antibody. B, Changes in intracellular pH (pHi) in response to acid loading. Examples of traces illustrating the recovery from an acute acid load. Intracellular pH was examined in cells that were transiently acidified using ammonium chloride. Periods of NH4Cl, NaCl and Na-free solution are indicated. An entire example of the recovery is indicated for stimulated 231-WT cells (231s). An example of recovery is indicated for unstimulated 231-WT cells (231u) and MDA-MB-231 NHE1-knockout cells (231-KO). Fluorescence of BCECF, a pH-sensitive indicator dye, was used to record and quantify changes in pHi post-acute acid load induced by ammonium chloride. NHE1 activity was calculated from the slope of the first 20 sec of recovery from acidification and was expressed as ΔpH/sec. C, Relative rate of Na+/H+ exchange activity of 231-KO cells relative to 231-WT cells, and in MDA-MB-468 and MCF7 cells. Values of ΔpH/sec from 231-WT and 231-KO cells were normalized to the unstimulated control values for 231-WT cells. Data for MCF7 and MDA-MB-468 were normalized to their control unstimulated values respectively. No discernible exchanger activity was detected in the knockout cells. Background drift and buffering capacity were not significantly different between cell types. In stimulated conditions (0.2% serum), NHE1 activity is significantly increased in 231-WT, MDA-MB-468 and MCF7 cells [*P<0.001, + P<0.01, N=8].
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Figure 1: Na/H exchanger expression and activity in parental MDA-MB-231 (231-WT) and NHE1-knockout cells (231-KO)A, Western blot analysis of NHE1 expression in 231-WT, 231-KO, MDA-MB-468 and MCF7 cells probed with anti-NHE1 antibody. B, Changes in intracellular pH (pHi) in response to acid loading. Examples of traces illustrating the recovery from an acute acid load. Intracellular pH was examined in cells that were transiently acidified using ammonium chloride. Periods of NH4Cl, NaCl and Na-free solution are indicated. An entire example of the recovery is indicated for stimulated 231-WT cells (231s). An example of recovery is indicated for unstimulated 231-WT cells (231u) and MDA-MB-231 NHE1-knockout cells (231-KO). Fluorescence of BCECF, a pH-sensitive indicator dye, was used to record and quantify changes in pHi post-acute acid load induced by ammonium chloride. NHE1 activity was calculated from the slope of the first 20 sec of recovery from acidification and was expressed as ΔpH/sec. C, Relative rate of Na+/H+ exchange activity of 231-KO cells relative to 231-WT cells, and in MDA-MB-468 and MCF7 cells. Values of ΔpH/sec from 231-WT and 231-KO cells were normalized to the unstimulated control values for 231-WT cells. Data for MCF7 and MDA-MB-468 were normalized to their control unstimulated values respectively. No discernible exchanger activity was detected in the knockout cells. Background drift and buffering capacity were not significantly different between cell types. In stimulated conditions (0.2% serum), NHE1 activity is significantly increased in 231-WT, MDA-MB-468 and MCF7 cells [*P<0.001, + P<0.01, N=8].
Mentions: To examine the role of NHE1 in tumor cells we made a knockout of the protein in the parental MDA-MB-231 cells (231-WT). Western blot analysis was used to examine endogenous expression of NHE1 in the NHE1-knockout (231-KO), compared to the 231-WT cells, MCF7 and MDA-MB-468 cells. NHE1 was consistently observed as a band at approximately 100 kDa in multiple lysate preparations (Fig. 1A). A second smaller band was also observed and is typical of a partially or de-glycosylated NHE1 protein. As expected, the stable NHE1-knockout (231-KO) cells showed no NHE1 protein expression. We also compared the level of NHE1 protein in either stimulated (low-serum (0.2%) media) or unstimulated (10%-serum supplemented media) conditions as previously described [22]. When the level of NHE1 was quantitatively compared to that of actin, there was no up-regulation of NHE1 expression in any cell type under stimulated conditions (N=5, see Fig. 1 for example).

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