Anticancer effects of high-dose extracellular citrate treatment in pancreatic cancer cells under different glucose concentrations

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive solid tumor. Recently, the uptake of extracellular citrate by the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, has been demonstrated to exert profound effects on cancer cell metabolism. However, research on the function of extracellular citrate in PDAC pathogenesis and the relationship between NaCT expression and the tumor metabolic microenvironment is limited. Therefore, we aimed to evaluate the expression of citrate transporters across a spectrum of glucose concentrations in pancreatic cancer and systematically explore the effects of sodium citrate treatment on pancreatic cancer cells at different glucose concentrations. We observed a positive correlation between glucose concentration and NaCT expression in PDAC cell lines. Extracellular sodium citrate significantly reduced cell viability partially due to reduction in intracellular Ca²⁺ levels and decreased the migration of human PDAC cells. Furthermore, we observed a decrease in the levels of the stem cell marker prominin I (CD133) following sodium citrate treatment. Notably, the combination treatment of gemcitabine and extracellular sodium citrate exhibited a synergistic anticancer effect in both two-dimensional (2D) and three-dimensional (3D) culture systems. Additionally, we confirmed that pH slightly increased upon administration of sodium citrate, indicating that this could potentially augment the efficacy of gemcitabine. Altogether, these findings suggest that exogenous sodium citrate treatment, particularly in combination with gemcitabine, may represent a novel therapeutic strategy for treating PDAC. This approach holds promise for disrupting PDAC cell metabolism and inhibiting tumor progression.

Keywords: Extracellular citrate; Glucose; Pancreatic ductal adenocarcinoma; Sodium citrate; Synergistic anticancer effect.

Fig. 1

Expression of NaCT/SLC13A5 in patients with pancreatic cancer. (A) The expression of SLC13A5 in normal pancreases tissue (n = 171) and pancreatic cancer tissue (n = 179) in TCGA dataset of pancreatic cancer patients. *p < 0.05 (B) Formalin-fixed, paraffin-embedded-pancreatic cancer tissue arrays from patients with pancreatic cancer were used. Immunofluorescence was conducted using SLC13A5 (green) antibody. Counterstaining was conducted using DAPI. Scale bars: 50 μm. (B) The intensity of SLC13A5 in tumor regions from patients with pancreatic cancer (n = 8) and normal pancreatic tissue (n = 8). *p < 0.05. (C) The expression of NaCT/SLC13A5, SLC25A1, HK2, and β-actin in PANC-1 and MIA PaCa-2 cells. The quantification of band intensities from three independent experiments is shown in Supplementary Fig. 5. (D) The expression of NaCT/SLC13A5, SLC25A1, HK2, and β-actin in PANC-1 and MIA PaCa-2 cells after incubation with various concentrations of glucose (Glu) for 8 h. The quantification of band intensities from three independent experiments is shown in Supplementary Fig. 6. NaCT, sodium-dependent citrate transporter; SLC13A5, solute carrier family 13 member 5; DAPI, 4′,6-diamidino-2-phenylindole; TCGA, The Cancer Genome Atlas; SLC25A1, solute carrier family 25 member 1; HK2, hexokinase 2; PANC-1 and MIA PaCa-2, pancreatic cancer cell lines.

Fig. 2

Inhibition of cell proliferation after citrate treatment. (A) Cell viability in MIA CaPa-2 cells following 0, 1, or 10 mM sodium citrate treatment in media with varying glucose concentrations for 48 h. The experiment was performed in triplicate with multiple replicates. Results are presented as the mean ± SD; ****p < 0.0001 (B) The percentage of Ki67-positive cells after 0 or 10 mM sodium citrate treatment in a medium with various glucose concentrations for 48 h. Six random fields were quantified for each independent test (n = 3) at × 200 magnification, and the results are presented as the mean ± SD; **p < 0.01; ***p < 0.001 (C–E) Representative immunocytochemistry of Ki67 in MIA PaCa-2 cells after 0 or 10 mM citrate treatment in medium containing 1 mM (C), 5 mM (D), and 25 mM (E) glucose for 48 h. Scale bar: 50 μm. MIA CaPa-2, a pancreatic cancer cell line; SD, standard deviation.

Fig. 3

Inhibition of cell migration and CD133 expression after treatment with extracellular sodium citrate. (A) A scratch wound-healing assay was conducted on MIA PaCa-2 cells. Cell migration was monitored by inverted microscopy at 0 and 48 h after treatment with 0 or 10 mM sodium citrate in a medium with various concentrations of glucose. (B) The percentage of gap width of (A). (C) Migration assay using MIA PaCa-2 cells treated with various concentrations of sodium citrate and glucose for 24 h. The experiment was independently performed in triplicate. (D) Quantification of migrated cells of (C). Data are expressed as the mean ± SD. Five random fields of each test (n = 3) at × 200 magnification were counted. *p < 0.05; ****p < 0.0001. (E) CD133 and β-actin expression levels in MIA PaCa-2 cells after treatment under the indicated conditions for 24 h. The quantification of band intensities from three independent experiments is shown in Supplementary Fig. 7. MIA PaCa-2, a pancreatic cancer cell line; SD, standard deviation; CD133, prominin I, a cell-surface antigen.

Fig. 4

Reduction in MIA PaCa-2 cell-derived spheroids. (A) Spheroids after treatment with 0 or 10 mM sodium citrate in medium with the indicated glucose concentrations for 72 h, analyzed using an inverted microscope. Scale bar: 100 μm (B) The relative change in spheroid size (n = 3 in each of the three independent experiments) was estimated using Olympus CellSens software. The data are expressed as the mean ± SD. **p < 0.01. MIA PaCa-2, a pancreatic cancer cell line; SD, standard deviation.

Fig. 5

Synergic anticancer effect of sodium citrate and gemcitabine. (A) Cell viability in MIA CaPa-2 cells following treatment with 0 or 10 mM sodium citrate and 0, 10, or 100 nM gemcitabine. The experiment was conducted in triplicate with multiple replicates. Results are presented as the mean ± SD; ***p < 0.001, ****p < 0.0001. (B) Representative spheroids after treatment with 0 or 10 mM sodium citrate and 0, 10, or 100 nM gemcitabine for 72 h, analyzed using an inverted microscope. Scale bar: 100 μm. (C) The relative change in spheroid size (n = 3 in each of the three independent experiments) was estimated using Olympus CellSens software. Data are presented as the mean ± SD. ***p < 0.001, ****p < 0.0001. (D) Migration assay using MIA PaCa-2 cells treated with 0 or 10 mM sodium citrate and 0, 10, or 100 nM gemcitabine for 24 h. The experiment was independently performed in a triplicate. (E) Quantification of migrated cells of (D). The data are expressed as the mean ± SD. *p < 0.05, ***p < 0.001, ****p < 0.0001. MIA CaPa-2, a pancreatic cancer cell line; SD, standard deviation.