Extracellular Adenosine in Gastric Cancer: The Role of GCSCs

Abstract

Gastric cancer (GC) is among the most common and deadliest types of cancer, with a poor prognosis primarily due to late-stage detection and the presence of cancer stem cells (CSCs). This study investigates the mechanisms regulating extracellular adenosine levels in gastric cancer stem-like cells (GCSCs) derived from the MKN-74 cell line. Our results show that GCSCs release more ATP into the extracellular medium and exhibit higher levels of CD39 expression, which enables them to hydrolyze a greater amount of ATP. Furthermore, we also found that GCSCs possess a greater capacity to hydrolyze AMP, primarily due to the activity of the CD73 protein, with no significant changes in CD73 transcripts and protein levels between GCSCs and differentiated cells. Additionally, adenosine transport is primarily mediated by members of the equilibrative nucleoside transporter (ENT) family in GCSCs, where a significant increase in the expression level of the ENT2 protein is observed compared to non-GCSCs MKN-74 cells. These findings suggest that targeting the adenosine metabolism pathway in GCSCs could be a potential therapeutic strategy for gastric cancer.

Keywords: adenosine; gastric cancer; gastric cancer stem-like cells (GCSCs); stomach cancer.

Figure 1

The gastric cancer stem cells (GCSCs) exhibit a high level of extracellular adenosine. MKN-74 cells were subjected to various culture conditions to cultivate GCSCs and non-GCSCs, as delineated in the Methods section. (A) Spheres at 3 days and (B) 7 days; light microscopy images of spheres (GCSCs) derived from the MKN-74 cell line. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of (C) ICAM1, (D) CD44, (E) CD24, and (F) EPCAM in GCSCs and non-GCSCs. Values were normalized to ACTB mRNA levels. (G) The levels of extracellular adenosine were quantified in the culture medium, as described in Section 4.5. The plots represent the means ± SD. * p < 0.05. n = 5.

Figure 2

The gastric cancer stem cells (GCSCs) exhibit a high capacity to hydrolyze ATP. MKN-74 cells were subjected to different culture conditions to generate GCSCs and non-GCSCs, as detailed in the Materials and Methods (Section 4). (A) Extracellular ATP levels were quantified in the culture medium as described in Section 4.4. (B) Extracellular ATP levels were quantified in the culture medium exposed to 100 µM ATP and/or 100 µM POM-1 (to inhibit CD39). (C) qRT-PCR analysis of ENTPD1 in GCSCs and non-GCSCs. Values were normalized to ACTB mRNA. (D) Representative Western blot of CD39 and β-actin in GCSCs and non-GCSCs. (E) The graph represents the quantification of CD39 signals in Western blot normalized against β-actin signals. The plots represent the means ± SD. * p < 0.05. n = 4.

Figure 3

Immunofluorescence staining for CD39 and CD73. (A) Representative images of spheroids derived from MKN-74 cells, compared to adherent MKN-74 cells. The expression of CD39 and (C) CD73 was visualized using a green fluorescent dye, while cell nuclei were counterstained with DAPI. Scale bar: 50 μm. (B,D) show the quantification of mean fluorescence intensity (MFI) for CD39 and CD73, respectively, in both cell types. Data represent the analysis of six representative images from distinct fields per condition, obtained from three independent experiments. Graphs show means ± SD. * p < 0.05; n = 6.

Figure 4

Expression and activity of the AMP-metabolizing enzyme CD73 in gastric cancer stem cells. (A) Total, (B) CD73, and (C) non-CD73 AMPase activity were evaluated in GCSCs and non-GCSCs derived from the MKN-74 cell line, as described in Section 4.9. (D) Relative transcript levels of the 5NTE gene in GCSCs and non-GCSCs. Values were normalized to ACTB mRNA expression. (E) Representative Western blot of CD73 and β-actin in GCSCs and non-GCSCs. (F) The graph represents the quantification of the CD73 signal in Western blot normalized against the β-actin signal. The graphs represent the means ± SD. * p < 0.05. n = 6.

Figure 5

ENTs regulate adenosine transport in both GCSCs and non-GCSCs. Adenosine accumulation assays were conducted as outlined for adenosine in Section 4.8. (A) Extracellular adenosine levels were determined in Tyrode’s medium both with and without NaCl. (B) Extracellular adenosine levels were measured in Tyrode’s medium without NaCl. (C) Extracellular adenosine levels were measured in Tyrode’s medium with NaCl. (D) Extracellular adenosine levels were quantified in culture medium exposed to 1 µM NBTI (to inhibit ENT1) or 10 µM NBTI (to inhibit ENT1 and ENT2). The plots represent the means ± SD. * p < 0.05. n = 5.

Figure 6

Transcript levels of genes encoding CNTs and ENTs in GCSCs and non-GCSCs. MKN-74 cells were subjected to different culture conditions to generate GCSCs and non-GCSCs as described in the Methods section. qRT-PCR of (A) SLC28A1, (B) SLC28A2, (C) SLC28A3, (D) SLC29A1, (E) SLC29A2, (F) SLC29A3 and (G) SLC29A4 in GCSCs and non-GCSCs. Values were normalized to ACTB mRNA. The plots represent the means ± SD. * p < 0.05. n = 5.

Figure 7

ENT1 expression in gastric cancer stem cells (GCSCs). (A) Representative images of spheroids derived from MKN-74 cells, compared to adherent MKN-74 cells. ENT1 expression was visualized using a green fluorescent dye, while cell nuclei were counterstained with DAPI. Scale bar: 50 μm. (B) Quantification of mean fluorescence intensity (MFI) of ENT1 signal in both cell types. Data represent the analysis of six representative images from distinct fields per condition, obtained from three independent experiments. Graphs show means ± SD; n = 6. (C) Representative Western blot of ENT1 and β-actin in GCSCs and non-GCSCs. (D) Quantification of ENT1 signal from Western blot normalized to β-actin. Bars represent means ± SD. * p < 0.05; n = 5.

Figure 8

GCSCs exhibit higher levels of ENT2 compared to non-GCSCs. (A) Representative images of spheroids derived from MKN-74 cells, compared to adherent MKN-74 cells. ENT2 expression was visualized using a green fluorescent dye, while cell nuclei were counterstained with DAPI. Scale bar: 50 μm. (B) Quantification of mean fluorescence intensity (MFI) of ENT2 signal in both cell types. Data represent the analysis of six representative images from distinct fields per condition, obtained from three independent experiments. Graphs show means ± SD; n = 6. (C) Representative Western blot of ENT2 and β-actin in GCSCs and non-GCSCs. (D) Quantification of ENT2 signals from Western blot normalized to β-actin. Bars represent means ± SD. * p < 0.05; n = 3.