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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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Characterization of NHE1-knockout (231-KO) MDA-MB-231 cellsA, B, Cell proliferation and viability in response to increasing paclitaxel doses was assessed by spectrophotometric analysis of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) absorbance at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of mean OD values at indicated time points for each cell type relative to 0 hr. A, Proliferation of 231-WT and 231-KO cells. In stimulated (0.2% serum) conditions, proliferation of knockout cells is not significantly different from parental MDA-MB-231 cells. However, both 231-WT and 231-KO cells are more proliferative over 48 hr. compared to MDA-MB-468 (468) and MCF7 cells. B, Effect of paclitaxel on cell viability of 231-WT and 231-KO cells. Paclitaxel was significantly more cytotoxic to NHE1-knockout cells compared to parental MDA-MB-231 cells at concentrations of 0.1 nM and higher in stimulated conditions over 24 hours [*P<0.001, + P<0.01, # P<0.05, N=3]. In contrast, stimulated MDA-MB-468 and MCF7 cells did not show any changes in viability dependent on paclitaxel concentration.
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Figure 2: Characterization of NHE1-knockout (231-KO) MDA-MB-231 cellsA, B, Cell proliferation and viability in response to increasing paclitaxel doses was assessed by spectrophotometric analysis of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) absorbance at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of mean OD values at indicated time points for each cell type relative to 0 hr. A, Proliferation of 231-WT and 231-KO cells. In stimulated (0.2% serum) conditions, proliferation of knockout cells is not significantly different from parental MDA-MB-231 cells. However, both 231-WT and 231-KO cells are more proliferative over 48 hr. compared to MDA-MB-468 (468) and MCF7 cells. B, Effect of paclitaxel on cell viability of 231-WT and 231-KO cells. Paclitaxel was significantly more cytotoxic to NHE1-knockout cells compared to parental MDA-MB-231 cells at concentrations of 0.1 nM and higher in stimulated conditions over 24 hours [*P<0.001, + P<0.01, # P<0.05, N=3]. In contrast, stimulated MDA-MB-468 and MCF7 cells did not show any changes in viability dependent on paclitaxel concentration.

Mentions: To further characterize differences between parental and NHE1-knockout MDA-MB-231 cells, we studied cell proliferation in unstimulated and stimulated conditions over 48 hr. In serum-deprived conditions typical of the tumor microenvironment, both 231-WT and 231-KO cells were more proliferative compared to MCF7 and MDA-MB-468 cells over 48 hr. (Fig. 2A; P<0.001, N=5). There was no difference in proliferation rates between stimulated 231-WT and 231-KO cells. All cell types had similar rates of proliferation in unstimulated conditions (data not shown). We also examined the effect of increasing concentrations of paclitaxel on cell viability. Interestingly, stimulated 231-KO cells appeared to be more susceptible to paclitaxel-mediated cytotoxicity at 0.1 nM and higher concentrations (P<0.05 to P<0.001, N=3) compared to 231-WT cells (Fig. 2B). In addition, there were no significant changes in the viability of MCF7 and MDA-MB-468 cells in response to paclitaxel at the tested concentrations. In other experiments, we examined the effect of NHE1 inhibitors on cell viability. EMD87580 was not cytotoxic to cells at up to 100 μM, but high concentrations of HMA (100 μM) resulted in less than 10% viability in all cell types (data not shown).


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)

Characterization of NHE1-knockout (231-KO) MDA-MB-231 cellsA, B, Cell proliferation and viability in response to increasing paclitaxel doses was assessed by spectrophotometric analysis of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) absorbance at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of mean OD values at indicated time points for each cell type relative to 0 hr. A, Proliferation of 231-WT and 231-KO cells. In stimulated (0.2% serum) conditions, proliferation of knockout cells is not significantly different from parental MDA-MB-231 cells. However, both 231-WT and 231-KO cells are more proliferative over 48 hr. compared to MDA-MB-468 (468) and MCF7 cells. B, Effect of paclitaxel on cell viability of 231-WT and 231-KO cells. Paclitaxel was significantly more cytotoxic to NHE1-knockout cells compared to parental MDA-MB-231 cells at concentrations of 0.1 nM and higher in stimulated conditions over 24 hours [*P<0.001, + P<0.01, # P<0.05, N=3]. In contrast, stimulated MDA-MB-468 and MCF7 cells did not show any changes in viability dependent on paclitaxel concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359231&req=5

Figure 2: Characterization of NHE1-knockout (231-KO) MDA-MB-231 cellsA, B, Cell proliferation and viability in response to increasing paclitaxel doses was assessed by spectrophotometric analysis of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) absorbance at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of mean OD values at indicated time points for each cell type relative to 0 hr. A, Proliferation of 231-WT and 231-KO cells. In stimulated (0.2% serum) conditions, proliferation of knockout cells is not significantly different from parental MDA-MB-231 cells. However, both 231-WT and 231-KO cells are more proliferative over 48 hr. compared to MDA-MB-468 (468) and MCF7 cells. B, Effect of paclitaxel on cell viability of 231-WT and 231-KO cells. Paclitaxel was significantly more cytotoxic to NHE1-knockout cells compared to parental MDA-MB-231 cells at concentrations of 0.1 nM and higher in stimulated conditions over 24 hours [*P<0.001, + P<0.01, # P<0.05, N=3]. In contrast, stimulated MDA-MB-468 and MCF7 cells did not show any changes in viability dependent on paclitaxel concentration.
Mentions: To further characterize differences between parental and NHE1-knockout MDA-MB-231 cells, we studied cell proliferation in unstimulated and stimulated conditions over 48 hr. In serum-deprived conditions typical of the tumor microenvironment, both 231-WT and 231-KO cells were more proliferative compared to MCF7 and MDA-MB-468 cells over 48 hr. (Fig. 2A; P<0.001, N=5). There was no difference in proliferation rates between stimulated 231-WT and 231-KO cells. All cell types had similar rates of proliferation in unstimulated conditions (data not shown). We also examined the effect of increasing concentrations of paclitaxel on cell viability. Interestingly, stimulated 231-KO cells appeared to be more susceptible to paclitaxel-mediated cytotoxicity at 0.1 nM and higher concentrations (P<0.05 to P<0.001, N=3) compared to 231-WT cells (Fig. 2B). In addition, there were no significant changes in the viability of MCF7 and MDA-MB-468 cells in response to paclitaxel at the tested concentrations. In other experiments, we examined the effect of NHE1 inhibitors on cell viability. EMD87580 was not cytotoxic to cells at up to 100 μM, but high concentrations of HMA (100 μM) resulted in less than 10% viability in all cell types (data not shown).

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