Characterization of inorganic phosphate transport in the triple-negative breast cancer cell line, MDA-MB-231

Abstract

Background: Recent studies demonstrate that interstitial inorganic phosphate is significantly elevated in the breast cancer microenvironment as compared to normal tissue. In addition it has been shown that breast cancer cells express high levels of the NaPi-IIb carrier (SLC34A2), suggesting that this carrier may play a role in breast cancer progression. However, the biochemical behavior of inorganic phosphate (Pi) transporter in this cancer type remains elusive.

Methods: In this work, we characterize the kinetic parameters of Pi transport in the aggressive human breast cancer cell line, MDA-MB-231, and correlated Pi transport with cell migration and adhesion.

Results: We determined the influence of sodium concentration, pH, metabolic inhibitors, as well as the affinity for inorganic phosphate in Pi transport. We observed that the inorganic phosphate is dependent on sodium transport (K0,5 value = 21.98 mM for NaCl). Furthermore, the transport is modulated by different pH values and increasing concentrations of Pi, following the Michaelis-Menten kinetics (K0,5 = 0.08 mM Pi). PFA, monensin, furosemide and ouabain inhibited Pi transport, cell migration and adhesion.

Conclusions: Taken together, these results showed that the uptake of Pi in MDA-MB-231 cells is modulated by sodium and by regulatory mechanisms of intracellular sodium gradient. General Significance: Pi transport might be regarded as a potential target for therapy against tumor progression.

Fig 1. Comparative indices of ³²Pi influx in breast cancer cell lines.

Intact MCF-7, T47-D or MDA-MB-231 cells (5 x 10⁴ cells/mL = 1.45 mg protein/mL) (A) and 67NR and 4T1 (B) were incubated for 1 h at 37°C in a reaction mixture containing 116 mM NaCl or 116 mM choline cloride, 5.5 mM Glucose, 5.4 KCl, 10 mM HEPES, 0.8 mM MgCl₂, 0.1 mM KH₂PO₄ and 2.5 μCi / nmol ³²Pi. The results are the means ± SE of at least 3 experiments, with different cell suspensions. Asterisks mark significant differences (p≤ 0.05) from MDA-MB-231, as determined by One-Way analysis of variance (ANOVA), using Turkey’s multiple comparisons test.

Fig 2. Gene expression analysis of Pi transporters in MDA-MB-231 cells.

Total RNA was purified from MDA-MB-231 and, after treatment of RNA with DNase I, a full-length cDNA was generated from RNA. qPCR was carried. Gene expression data were normalized to an endogenous reference β-actin (ACTB). The results are the means ± SE of 7 experiments, with different cell suspensions. Asterisks mark significant differences (p≤ 0.05) from NaPi1, as determined by One-Way analysis of variance (ANOVA), using Turkey’s multiple comparisons test.

Fig 3. Time course, NaCl dependence and Pi dependence of MDA-MB-231 ³²Pi influx.

Intact cells (5 x 10⁴ cells/mL = 1.45 mg protein/mL) were incubated at 37°C in a reaction mixture containing 116 mM NaCl or 116 mM choline cloride, 5.5 mM Glucose, 5.4 KCl, 10 mM HEPES, 0.8 mM MgCl₂, 0.1 mM KH₂PO₄ and 2.5 μCi/nmol ³²Pi at various times (A), several NaCl concentrations (0–150 mM) (B) or various concentrations of KH₂PO₄ (0–0.5 mM) (C). The results are the means ± SE of at least 3 experiments, with different cell suspensions.

Fig 4. Effect of pH on NaCl-dependent ³²Pi transport in MDA-MB-231.

Intact cells (5 x 10⁴ cells/mL = 1.45 mg protein/mL) were incubated for 1 h at 37°C in a reaction mixture containing 116 mM NaCl or 116 mM choline cloride, 5.5 mM Glucose, 5.4 KCl, 0.8 mM MgCl₂, 0.1 mM KH₂PO₄, 2.5 μCi/nmol ³²Pi and 10 mM HEPES, 15 mM Tris, 15 mM MES with pH ranging from 6.4 to 9.2 (A). In these pH ranges, the cells remained viable throughout the experiment according CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (B). These results are the means ± SE of at least 3 experiments, with different cell suspensions.

Fig 5. Effect of PFA on NaCl-dependent ³²Pi transport in MDA-MB-231.

Intact cells (5 x 10⁴ cells/mL = 1.45 mg protein/mL) were incubated for 1 h at 37°C in a reaction mixture containing 116 mM NaCl or 116 mM choline cloride, 5.5 mM Glucose, 5.4 KCl, 0.8 mM MgCl₂, 0.1 mM KH₂PO₄, 2.5 μCi/nmol ³²Pi and 10 mM HEPES, in the presence of increasing concentrations of PFA. In these PFA concentrations, the cells remained viable throughout the experiment. The results are the means ± SE of at least 3 experiments, with different cell suspensions. Asterisks mark significant differences (p≤ 0.05) from control, as determined by One-Way analysis of variance (ANOVA), using Turkey’s multiple comparisons test.

Fig 6

Effect of inhibitors on cell migration (A) and cell adhesion (B) in MDA-MB-231. Intact cells (5 x 10⁵ cells/mL) were incubated for 1 h at 37°C in a Boyden Chamber Assay™ migration (A) or in a adhesion chamber (B) in the presence or absence (control) of inhibitors indicated in the abscissa: ouabain (1 mM), furosemide (1 mM), Monensin (100 μM) and PFA (5 mM). In the presence of these inhibitors at their respective concentrations, the cells remained viable throughout the experiment. The results are the means ± SE of at least 3 experiments, with different cell suspensions. Asterisks mark significant differences (p≤ 0.05) from control, as determined by One-Way analysis of variance (ANOVA), using Turkey’s multiple comparisons test.

Fig 7. Proposed mechanism for Pi transport mechanism in MDA-MB-231 cells: Na⁺, K⁺-ATPase ouabain-sensitive in the plasma membrane and Na⁺-ATPase furosemide-sensitive plasma membrane coupled to Pi transport.

Arrows indicate the direction of ion flow.