Authenticated LNCaP cells (LNCaP-FGC) were purchased from American Type Culture Collection ATCC (cat. no. CRL-1740), and PC3, 22RV1, C4-2, DU145 and RWPE1 were provided by Dr Carlos Moreno and Dr Wei Zou at the Winship Cancer Institute of Emory University. Cells were previously authenticated by each investigator's laboratory. Cells were maintained in RPMI-1640 medium (ATCC) supplemented with 10% fetal bovine serum and 1% pen/strep (Thermo Fisher Scientific, Inc.) in a 5% CO₂-equilibrated 37°C incubator. For hypoxic exposure, cells were incubated in a humidified atmosphere of 1% O₂, 5% CO₂ at 37°C. N-cyanosulphonamide S0859 (cat. no. 1019331-10-2; Millipore Sigma; Merck KGaA) was added to media in experiments that required pharmacological inhibition of NBCs. Viable cells were counted using the trypan blue exclusion assay (34). Percent of viable cells was calculated by the ratio of live cell number to total cell number (live cells + trypan blue-stained cells).

Cells at the density of 1–2×10⁵ were plated in a 60-mm dish containing a poly-lysine-coated coverslip and incubated for 2 days. Cells on a coverslip were loaded with 6.5 µM of 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; cat. no. B1170; Thermo Fisher Sientific, Inc.) for 20 min and mounted in a closed perfusion chamber affixed to the stage of a Zeiss Axiovert inverted microscope. The dye was alternately excited with 440 and 490 nm lights using a Lambda LS/30 Xenon Arc lamp (Sutter Instruments), and 535 nm emission lights from both excitations were captured using a Nikon camera and analyzed using Nikon NIS Elements AR 3.0 imaging software (both from Nikon Corporation). The emission ratio 490/440 nm was calculated and converted to a pH value according to the nigericin method (35). The chamber was perfused with HEPES-buffered solution (mM: 140 NaCl, 1 KCl, 1.2 MgCl₂, 1 CaCl₂, 8.8 sucrose, 10 HEPES, pH 7.4) and then with a solution containing 5% CO₂, 28 mM HCO₃⁻ (NaHCO₃ replaced NaCl). Solutions contained 100 µM of amiloride to block endogenous NHEs. S0859 at 50 µM was added in experiments that required inhibition of NBCs. For Na⁺-free CO₂/HCO₃⁻ solution, LiCl replaced NaCl and choline bicarbonate replaced NaHCO₃. The rate of pH_i recovery (dpH/dt; pHi change per sec × 10⁻⁴) was calculated by drawing a slope in the first 4 min of recovery from a CO₂-induced acidification.

Total RNAs from the aforementioned cells were isolated using RNeasy Mini kit (Qiagen, Inc.) and transcribed using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using Applied Biosystems SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following primers purchased from OriGene Technologies, Inc.: SLC4A4 (cat. no. HP232301), SLC4A5 (cat. no. HP214119), SLC4A7 (cat. no. HP207103), SLC4A8 (cat. no. HP227521), SLC4A10 (cat. no. HP214322), β-actin ACTB (cat. no. HP204660), and 18S RNA (cat. no. HP220445). The sequences are listed Table SI. Reactions were performed using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Amplification was achieved at 50°C for 2 min and 95°C for 10 min for an initial denaturation, and then 40 cycles at 95°C for 15 sec and 60°C for 1 min. The quantification cycle (Cq) was determined using the software SDS 2.4 supplied with the instrument. Cq values of NBCs relative to a geometric mean from reference genes ACTB and 18S RNA were calculated, and fold changes relative to NBCe1 were determined using the 2^−ΔΔCq method (36).

Cells were homogenized in ice-cold buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100), supplemented with 1× protein inhibitor cocktail (Thermo Fisher Scientific, Inc.) and 1 mM phenylmethylsulfonyl fluoride. Cells were centrifuged at 13,200 × g for 10 min at 4°C to remove cell debris and supernatants were collected. Protein concentration was determined using Bradford reagents (Millipore Sigma; Merck KGaA). A total of 15 µg of proteins from samples were separated on a 4–15% SDS-polyacrylamide gel and blotted to a nitrocellulose membrane. The blot was incubated with mouse anti-human SLC4A4 monoclonal antibody (cat. no. sc-515543; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. The dilution was 1:500 with the blocking buffer (5% nonfat dry milk and 0.05% Tween-20 in TBS). The blot was washed and incubated with a goat horseradish peroxidase-conjugated antibody to mouse IgG (1:1,000 dilution; cat. no. 12-349; Millipore Sigma; Merck KGaA) for 2 h at room temperature. Immunoreactive bands were visualized using ECL chemiluminescence (Thermo Fisher Scientific, Inc.). The blot was striped and reprobed with rabbit anti-human β-actin polyclonal antibody (product code ab8227; 1:1,000 dilution; Abcam) for 1 h at room temperature. Densitometric analysis of immunoreactive bands was performed using ImageJ as previously described (37). NBCe1 pixel intensity was normalized to β-actin intensity after background subtraction.

Cells at the density of 1–2×10⁴ were plated in 24-well plates and transfected with siRNA oligonucleotides the following day. The siRNA 27-mer duplexes targeting human SLC4A4 (cat. no. SR305704) and the scrambled negative control duplex (cat. no. SR30004) were purchased from OriGene Technologies, Inc. (Table SII). Three SLC4A4 siRNA duplexes were pooled at equal concentrations for transfection (total 10 and 20 nM). Transfection was performed with Lipofectamine RNAiMax (Thermo Fisher) according to the manufacturer's instructions. After transfection, cells were incubated in hypoxia for 72–96 h in a 37°C incubator, and the efficacy of knockdown was determined by immunoblotting.

Cell death was measured using the LDH release assay as previously described (38) with slight modification. Briefly, cells in 24-well plates were incubated with 1% Triton X-100 or water for 45 min and LDH released from cells was quantitated using CyQuant LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, Inc.). The amount of formazan produced by LDH-mediated NADH oxidation was determined by absorbances at 490 and 680 nm (background). Background absorbance at 680 nm was subtracted and absorbance in media only (no cells) was also subtracted. Cell death was calculated as a percentage of spontaneous LDH release to total LDH release.

The formalin-fixed, paraffin-embedded human prostate carcinoma tissue microarrays containing 41 cases of prostate cancer and 9 cases of normal prostate tissue were purchased from US Biolab Corporation, Inc. (cat. no. PRO501). Information on pathology grade, Gleason grade, Gleason score, TNM classification and clinical stages are available on the company website. The purpose of using human tissue samples in this study was to examine the characteristics of cancerous tissue, not to develop treatments; thus, a respective Intuitional Review Board was not required for the use of the tissue microarrays in our study.

A sphere formation assay was performed as previously described by Zhang et al (39) with slight modification. Falcon 8-well chamber slides (product no. 354118; Corning, Inc.) were precoated with 50 µl of LDEV-free growth factor-reduced Geltrex (cat. no. A1413201; Thermo Fisher Scientific, Inc.). Cells were plated at 3,000 cells/well in the aforementioned culture medium supplemented with 1% Geltrex. One day later, the cells were treated with 0 or 100 µM of S0859 and incubated at 37°C for 6 days to form spheres. Images of spheres at a magnification of ×10 were captured using a Keyence BZ-X700 fluorescence microscope (Keyence Corporation). To quantify sphere growth, the number of spheres was counted and Feret diameters were measured using the FIJI version of ImageJ.

The tissue microarrays were heated and subjected to deparaffinization in xylene, rehydration in graded series of ethanol, and rinsing with distilled water. The slides were then heat treated with the target retrieval solution DIVA Decloaker (Biocare Medical) using an electric pressure cooker for 20 min. After washing, the slides were blocked with Background Sniper (Biocare Medical) at room temperature for 10 min, washed and incubated with anti-NBCe1/SLC4A4 antibody (product no. HPA035628; Millipore Sigma; Merck KGaA) diluted at 1:200 at 4°C overnight. The slides were washed and then incubated with MACH 2 Rabbit AP-Polymer (cat. no. RALP525; Biocare Medical) for 30 min at room temperature. The slides were stained with the chromogen solution Warp Red (BioCare Medical) at room temperature for 7 min. Nuclei were counterstained with hematoxylin at room temperature for 45 sec. Digital images of stained slides were captured using a Biotek Lionheart FX microscope. The images were then evaluated by a histopathologist.

Data were reported as the mean ± standard error of the mean (SEM). The significance of the difference between means was determined using: i) Unpaired, two-tailed Student's t-test for comparison of dpH/dt and immunoblotting in normoxia vs. hypoxia, control vs. knockdown, and control vs. S0859 treatment; ii) paired, two-tailed Student's t-test for comparison of dpH/dt before and after Na⁺ addition, as well as S0859 sensitivity; iii) one-way ANOVA with Turkey post hoc test for comparison of NBC qPCR; and iv) two-way ANOVA with Fisher's LSD or Sidak post hoc test for comparison of viable cell number and cell death over time after S0859 treatment. P<0.05 was considered to indicate a statistically significant difference. Analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc.) and Microsoft Office Excel add-in Analysis ToolPak (Microsoft Corporation).

qPCR with primers specific to each member of NBCs was performed to determine their expression levels in human prostate cancer cell lines LNCaP, C4-2, PC3, 22RV1, DU145, and the prostate cell line RWPE-1. The expression ratios of each isoform relative to NBCe1 after being normalized to a geometric mean of reference genes ACTB and 18S RNA is presented in Fig. 1A-F. Notably, NBCe1 expression levels were predominantly high in LNCaP cells and its subline C4-2 cells (P<0.01 for both; n=4/group). NBCe1 expression was also observed in other cells, but its level was not predominant and detected together with NBCe2, NDCBE, NCBE and weakly with NBCn1. The expression profiles in PC3, 22RV1, and DU145 were to a certain extent similar to that in RWPE1. The comparison of NBCe1 expression levels among all six different cell lines is presented in Fig. 2. The level was significantly higher in LNCaP cells.

Because NBCe1 is highly abundant in LNCaP cells, this cell line was focused on for analysis of Na/HCO₃-dependent acid extrusion and its response to hypoxia. Immunoblotting data from cells incubated in normoxia vs. hypoxia (1% O₂, 5% CO₂) for 4 days are presented in Fig. 3A. NBCe1 was markedly upregulated in hypoxia. Densitometric quantitation of immunoreactive NBCe1 normalized to β-actin resulted in a 5.1-fold increase (P<0.01, Student's t-test; n=3; Fig. 3B). In parallel experiments, pH_i measurement with the fluorescence dye BCECF was performed to assess whether acid extrusion is enhanced in hypoxia. The average pH_i traces (n=11 cells/group) when cells were perfused with 5% CO₂, 28 mM HCO3^– (plus 100 µM of amiloride to block endogenous NHEs) are presented in Fig. 3C and D. Comparison of pH_i recovery rates resulted in a 3-fold increase in hypoxia (0.90±0.48 ×10⁻⁴ dpH_i/sec in normoxia vs. 2.78±0.71 ×10⁻⁴ dpH_i/sec in hypoxia; P<0.05, Student's t-test; Fig. 3E). The properties of the pH_i recovery from a CO₂-induced acidification were further evaluated by assessing its Na⁺ dependence and S0859 sensitivity. An average pH_i recovery in the absence and presence of Na⁺ (n=11) is presented in Fig. 3F. The recovery was minimal in Na⁺-free CO₂/HCO₃⁻ solution, indicating that the major acid extrusion in LNCaP cells is dependent upon Na⁺. The recovery was increased when Na⁺ was applied. The dpH/dt in this Na⁺-containing solution was 5-fold higher than the value in Na⁺-free solution (P<0.05, paired Student's t-test; Fig. 3G), indicating that Na/HCO₃ transport largely governs acid extrusion in hypoxia. In other experiments, the sensitivity to the Na/HCO₃ inhibitor S0859 (50 µM) was examined (Fig. 3H). This concentration was selected based on a previous study (40) where S0859 at >30 µM fully inhibited NBCs in cardiomyocytes. Comparison of pH_i recoveries in the absence vs. presence of S0859 revealed a significant inhibition by the drug. The average inhibition was 77% (P<0.01, Student's t-test; n=16 cells/group; Fig. 3I). Collectively with the predominant expression of NBCe1, these pH_i data demonstrated that NBCe1 plays a major role in acid extrusion in LNCaP cells.

To examine whether NBCe1 affects growth and survival of LNCaP cells, NBCe1 gene expression was disrupted using siRNA oligonucleotides. The knockdown efficacy determined by immunoblotting 96 h after transfection is presented in Fig. 4A. Compared to the control siRNA/random 27-mers, the siRNA/NBCe1 decreased NBCe1 protein levels by over 90% (P<0.01, Student's t-test; n=3; Fig. 4B). In parallel experiments, the number of viable cells in the trypan blue exclusion assay was counted. As shown in Fig. 4C, the knockdown decreased the number of viable cells by 54% (from 2.4×10⁵ cells/ml to 1.1×10⁵ cells/ml; P<0.01; n=6/group) when determined at 4 days after treatment. The cell viability (i.e., percentage of viable cell to total cells) was also decreased, but the magnitude of the change was relatively small (13%; P<0.01; Fig. 4D), implying that the decrease in cell number is not tightly related to the decrease in viability. Consistent with this implication, the knockdown caused 10–12% cell death, determined by the LDH release assay (n=5 at 10 nM and n=6 at 20 nM of siRNA/NBCe1), markedly smaller than the percent change in cell number (Fig. 4E). Doubling the amounts of siRNA/NBCe1 oligonucleotides for transfection did not further increase the cell death (P>0.05), indicating that the knockdown has reached a maximum level of cell death.

Next, LNCaP cells were treated with 50 µM of S0859 to assess whether pharmacological inhibition of NBCe1 produces similar effects. As shown in Fig. 5A, S0859 treatment decreased the number of viable cells (23% at 4 days after treatment, P<0.01, n=15/group; and 19% at 6 days after treatment, P<0.05, n=6/group). These decreases were smaller than the decrease by the aforementioned NBCe1 knockdown. Furthermore, cell death was not observed at 4 days after treatment but increased at 6 days after treatment (Fig. 5B). Consistent with this lack of cell death at 4 days after treatment, the viability was unchanged during the same treatment days (Fig. 5C) and NBCe1 protein levels were also unaltered (Fig. 5D and E). Thus, the pharmacological inhibition of NBCe1 decreases cell proliferation, similar to the knockdown, but the two methods appear to have different mechanisms affecting cell death.

The qPCR results revealed the most exclusively abundant expression of NBCe1 in LNCaP and C4-2 cells, but moderate co-expression with other NBCs in cells such as PC3. This leads to the possibility that NBCe1 contribution to cell proliferation and viability may vary depending upon cell types. To address this possibility, NBCe1 knockdown was performed in PC3 cells and cell numbers were counted. As shown in Fig. 6A, the knockdown decreased the number of viable cells by 48% (from 1.71×10⁵ to 0.89×10⁵ cells/ml at 4 days after transfection; P<0.05, n=9/group). The knockdown also caused a small increase in cell death (3%; Fig. 6B). Thus, NBCe1 affected the growth and viability of PC3 cells, similar to those in LNCaP cells. Next, cells were treated with S0859, which should inhibit all NBCs, and viable cell number and cell death at 4 days after treatment were assessed. Interestingly, S0859 had no effect at 50 µM but decreased viable cell numbers at 100 µM (38% decrease; P<0.05, n=4/group; Fig. 6C). The decrease was more severe at 6 days after treatment (31% decrease at 50 µM and 75% decrease at 100 µM; P<0.01 for both, n=3-8/group). As anticipated, S0859 caused a small increase cell death (6% at both concentrations; Fig. 6D). A higher amount of S0859 was required to decrease the proliferation of PC3 cells, in comparison to LNCaP cells. Conclusively, NBCe1 knockdown in PC3 cells decreased cell proliferation to the level similar to that by the same knockdown in LNCaP cells, indicating that NBCe1 significantly affects PC3 cell growth.

The effects of NBC inhibition on LNCaP and PC3 cell growth were further examined in 3D cultures. The images of cell spheres formed 6 days after treatment with 100 µM of S0859 or none are presented in Fig. 7A. Compared to the control, S0859 decreased sphere formation in both cell lines. The number of LNCaP cell spheres was decreased by 64% (P<0.01, n=4/group; Fig. 7B) and the Feret diameter was decreased by 23% (P<0.01, n=13–34 spheres/group; Fig. 7C). Similarly, the number of PC3 cell spheres was decreased by 31% (P<0.01, n=4/group; Fig. 7D) and the Feret diameter was decreased by 27% (P<0.01, n=39–56 spheres/group; Fig. 7E). Thus, similar as in 2D cultures, pharmacological inhibition of NBCe1 reduces LNCaP and PC3 cell growth in 3D cultures.

The robust expression of NBCe1 in LNCaP cells led us to a localization study of this transporter in human prostate tissue and prostatic cancer. For this experiment, NBCe1 immunohistochemistry was performed on human prostate cancer tissue microarrays containing 9 cases of normal prostate tissue and 41 cases of prostate cancer (aforementioned in the Materials and methods). NBCe1 was localized to the basolateral side of the glandular epithelial cells in normal prostate (Fig. 8A), consistent with its basolateral localization in a variety of secretory glands (11). In prostatic cancer, NBCe1 was highly abundant in adenocarcinoma in Gleason grades 3–5 (Fig. 8B-F). The plasma membrane staining progressively disappeared in higher Gleason grades, consistent with the lost ability of cancer cells to form glands in more advanced tumor stages.