Dickkopf-related protein 3 alters aerobic glycolysis in pancreatic cancer BxPC-3 cells, promoting CD4+ T-cell activation and function

Abstract

Background: To investigate the value of Dickkopf-related protein 3 (DKK3) on aerobic glycolysis in pancreatic cancer cells, where DKK3-overexpression is used to determine its effects on CD4⁺ T cells.

Methods: The BxPC-3-DKK3 cell line was constructed, and peripheral blood mononuclear cell (PBMC) was prepared. After isolated the CD4⁺ T cells, the lactic acid, glucose uptake ability, cellular viability, proliferation, apoptosis, and markers were detected by PCR and western blot, and the concentrations of multiple cytokines were determined using the ELISA method.

Results: After co-culture with pancreatic cancer cells overexpressing DKK3, the glucose uptake markedly, proliferation enhanced and apoptosis inhibited in CD4⁺ T cells. The co-culture model also revealed that DKK3-overexpression promotes the activation and regulates the metabolism and function of CD4⁺ T cells.

Conclusions: DKK3 alters the metabolic microenvironment of pancreatic cancer cells and further facilitates the function of CD4⁺ T cells which suggesting that DKK3 may have a therapeutic potential in pancreatic cancer.

Keywords: Aerobic glycolysis; CD4+ T cells; Dickkopf-related protein 3; Pancreatic cancer; β-catenin.

Fig. 1

DKK3 inhibits aerobic glycolysis. A Western blot analysis of epithelial–mesenchymal transition markers underhypoxic conditions. B Western blot analysis of aerobic glycolysis markers under hypoxic and normoxic conditions. Levels of C extracellular lactic acid, D glucose uptakeand, E ATP production. F OXPHOS protein expression in mitochondria isolated from pancreatic cancer cells. D ^#P < 0.01: compared with the BxPC-3 NC (–CoCl₂) group; ^*P < 0.01: compared with the BxPC-3 NC group. DKK3, dickkopf-related protein 3; OXPHOS, oxidative phosphorylation

Fig. 2

Effects of DKK3-overexpression in pancreatic cancer on CD4⁺ T-cell glucose levels, proliferation, and apoptosis. A Levels ofglucose uptake in CD4⁺ T cells. B Proliferative activity of CD4⁺ T cells. C, D Proportions of apoptotic cells by flow cytometric analysis. ^#P < 0.01: compared with groups B, C and D. ^*P < 0.01: compared with groups B and E. ^**P < 0.01 compared with groups F and I. DKK3, dickkopf-related protein 3

Fig. 3

Relative mRNA expression levels of A CD3, B CD28, C CD25, D CD69, E CD71 and F HLA-DR in inactivated CD4⁺ T cells of each group after 72 h of co-culture. DKK3, dickkopf-related protein 3; NC, negative control

Fig. 4

Flow cytometric detection of inactivated CD4⁺ T-cell surface markers. Proportions of A CD3⁺, B CD28⁺, C CD25⁺, D CD69⁺, E CD71⁺ and F HLA-DR⁺ cells. ^*P < 0.01: compared with the sodium sulphate-pretreated BxPC-3 NC group and BxPC-3-DKK3 group; ^** P < 0.01: compared with the BxPC-3-DKK3 group. Data are presented as the mean ± SE of three independent experiments. DKK3, dickkopf-related protein 3; NC, negative control

Fig. 5

Relative mRNA expression levels of A IFN-γ, B IL-2, C IL-4, D IL-10, E IL-17 and F ZAP70 in activated CD4⁺ T cells of each group after 72 h of co-culture. ^*P < 0.01: compared with the sodium sulphate-pretreated BxPC-3 NC group and BxPC-3-DKK3 group; ^**P < 0.01: compared with the BxPC-3-DKK3 group. DKK3, dickkopf-related protein 3; NC, negative control

Fig. 6

Concentrations of A IFN-γ, B IL-2, C IL-10, D IL-17 and E IL-4 in the supernatants of activated CD4⁺ T cells co-culturedfor 96 h, detected by ELISA. ^*P < 0.01: compared with the sodium sulphate-pretreated BxPC-3 NC group and BxPC-3-DKK3 group; ^**P < 0.01: compared with the BxPC-3-DKK3 group.DKK3, dickkopf-related protein 3; NC, negative control

Fig. 7

DKK3 inhibits aerobic glycolysis in pancreatic cancer BxPC-3 cells, which promotes glucose uptake, enhances proliferation, and inhibits apoptosis in CD4⁺ T cells