Comparison of fluorescence probes for intracellular sodium imaging in prostate cancer cell lines

Abstract

Sodium (Na⁺) ions are known to regulate many signaling pathways involved in both physiological and pathological conditions. In particular, alterations in intracellular concentrations of Na⁺ and corresponding changes in membrane potential are known to be major actors of cancer progression to metastatic phenotype. Though the functionality of Na⁺ channels and the corresponding Na⁺ currents can be investigated using the patch-clamp technique, the latter is rather invasive and a technically difficult method to study intracellular Na⁺ transients compared to Na⁺ fluorescence imaging. Despite the fact that Na⁺ signaling is considered an important controller of cancer progression, only few data using Na⁺ imaging approaches are available so far, suggesting the persisting challenge within the scientific community. In this study, we describe in detail the approach for application of Na⁺ imaging technique to measure intracellular Na⁺ variations in human prostate cancer cells. Accordingly, we used three Na⁺-specific fluorescent dyes-Na⁺-binding benzofuran isophthalate (SBFI), CoroNa™ Green (Corona) and Asante NaTRIUM Green-2 (ANG-2). These dyes have been assessed for optimal loading conditions, dissociation constant and working range after different calibration methods, and intracellular Na⁺ sensitivity, in order to determine which probe can be considered as the most reliable to visualize Na⁺ fluctuations in vitro.

Keywords: ANG-2; CoroNa Green; Fluorescent dye; Prostate cancer cells; SBFI; Sodium imaging.

Fig. 1

Schematic representation of the molecular pathways used in this study to induce alterations [Na⁺]_i. Typical Na⁺ concentrations of the mammalian cells are 5–15 mM for cytosol ([Na⁺]_int) and 120–150 mM for extracellular ([Na⁺]_ext) medium. [Na⁺]_o was switched from 0 to 110 mM for investigating ion diffusion down the electrochemical gradient through Na⁺ leak channels. Ouabain inhibits the activity of Na⁺/K⁺ ATPase and hence restrains constant expulsion of the intracellular Na⁺. The application of voltage-gated Na⁺ channels (VGSCs) opener, veratridine, should also result in [Na⁺]_i increase. Ionomycin as an ionophore promotes Ca²⁺ entry through the plasma membrane and the depletion of endoplasmic reticulum (ER) Ca²⁺ stores and hence boosts the activity of Na⁺/Ca²⁺ exchanger (NCX)

Fig. 2

Chemical structures of the different Na⁺ indicator dyes used in this study in the form of acetoxymethyl esters (AM): Na⁺-binding benzofuran isophthalate (SBFI), CoroNa™ Green (CoroNa), and Asante NaTRIUM Green 2 (ANG-2)

Fig. 3

Calibration of SBFI in human prostate cancer PC-3 cells. The real-time [Na⁺]_o (0–120 mM) are indicated. Calibrations performed with choline solution are represented in a–c, whereas with K⁺ solution are shown in d–f. a, d Changes in emitted fluorescence intensities during excitation at 340 and 380 nm. b, e Fluorescence ratio 340 nm/380 nm derived from signals shown in a and d. In c and f the data shown is derived from b and e that have been plotted versus log₁₀ of the known Na⁺ concentrations and fitted using a logistic function. Data are represented as mean ± SD

Fig. 4

Calibration of CoroNa in human prostate cancer PC-3 cells. The real-time [Na⁺]_o (0–120 mM) are indicated. Calibrations performed with choline solution are represented in a, b, whereas with K⁺ solution in c, d. a, c Emitted fluorescence intensities at 488 nm. In b and e the data shown is derived from a and c that have been plotted versus log₁₀ of the known Na⁺ concentrations and fitted using a logistic function. Data are represented as mean ± SD

Fig. 5

Calibration of ANG-2 in human prostate cancer PC-3 cells. The real-time [Na⁺]_o (0–120 mM) are indicated. Calibrations performed with choline solution are represented in a, b, whereas with K⁺ solution in c, d. a, c Emitted fluorescence intensities at 540 nm.  In b and e the data shown is derived from a and c that have been plotted versus log₁₀ of the known Na⁺ concentrations and fitted using a logistic function. Data are represented as mean ± SD

Fig. 6

[Na⁺]_i levels during Na⁺ switch from 0 to 75 mM and then to 110 mM concentrations. Fluorescence intensities normalized to the initial values (F/F ₀). Data are represented as mean ± SD

Fig. 7

[Na⁺]_i levels after cytosolic Ca²⁺ increase induced by an application of 1 µM ionomycin. Fluorescence intensities normalized to the initial values (F/F ₀). Data are represented as mean ± SD

Fig. 8

[Na⁺]_i levels after Na⁺/K⁺-ATPase inhibition induced by an application of 250 µM ouabain. Fluorescence intensities normalized to the initial values (F/F ₀). Data are represented as mean ± SD

Fig. 9

[Na⁺]_i levels after voltage-gated Na⁺ channels opening induced by an application of 50 µM veratridine. Fluorescence intensities normalized to the initial values (F/F ₀). Data are represented as mean ± SD