Generation of glyceraldehyde-derived advanced glycation end-products in pancreatic cancer cells and the potential of tumor promotion

Abstract

Aim: To determine the possibility that diabetes mellitus promotes pancreatic ductal adenocarcinoma via glyceraldehyde (GA)-derived advanced glycation-end products (GA-AGEs).

Methods: PANC-1, a human pancreatic cancer cell line, was treated with 1-4 mmol/L GA for 24 h. The cell viability and intracellular GA-AGEs were measured by WST-8 assay and slot blotting. Moreover, immunostaining of PANC-1 cells with an anti-GA-AGE antibody was performed. Western blotting (WB) was used to analyze the molecular weight of GA-AGEs. Heat shock proteins 90α, 90β, 70, 27 and cleaved caspase-3 were analyzed by WB. In addition, PANC-1 cells were treated with GA-AGEs-bovine serum albumin (GA-AGEs-BSA), as a model of extracellular GA-AGEs, and proliferation of PANC-1 cells was measured.

Results: In PANC-1 cells, GA induced the production of GA-AGEs and cell death in a dose-dependent manner. PANC-1 cell viability was approximately 40% with a 2 mmol/L GA treatment and decreased to almost 0% with a 4 mmol/L GA treatment (each significant difference was P < 0.01). Cells treated with 2 and 4 mmol/L GA produced 6.4 and 21.2 μg/mg protein of GA-AGEs, respectively (P < 0.05 and P < 0.01). The dose-dependent production of some high-molecular-weight (HMW) complexes of HSP90β, HSP70, and HSP27 was observed following administration of GA. We considered HMW complexes to be dimers and trimers with GA-AGEs-mediated aggregation. Cleaved caspase-3 could not be detected with WB. Furthermore, 10 and 20 μg/mL GA-AGEs-BSA was 27% and 34% greater than that of control cells, respectively (P < 0.05 and P < 0.01).

Conclusion: Although intracellular GA-AGEs induce pancreatic cancer cell death, their secretion and release may promote the proliferation of other pancreatic cancer cells.

Keywords: Glyceraldehyde-derived advanced glycation-end products; Pancreatic ductal adenocarcinoma; Tumor promotion.

Figure 1

Analysis of cell viability, quantity of glyceraldehyde-derived advanced glycation-end products, immunostaining of glyceraldehyde-derived advanced glycation-end products, and molecular weight of glyceraldehyde-derived advanced glycation-end products in PANC-1 cells treated with glyceraldehyde for 24 h. A: Cell viability was assessed by the WST-8 assay. This assay was performed for three independent experiments. One assay was performed for n = 7. Data are shown as mean ± SD (n = 7); B: Slot blotting analysis of intracellular glyceraldehyde (GA)-derived advanced glycation-end products (GA-AGEs). Cell lysates (2.0 μg of protein/lane) were blotted onto polyvinylidene difluoride (PVDF) membranes. The amount of GA-AGEs was calculated based on a standard curve for GA-AGEs-BSA. Slot blotting was performed for three independent experiments. Data are shown as mean ± SD (n = 3); C: Immunostaining of GA-AGEs in PANC-1 cells. Cells were treated with 0, 1, 2 and 4 mmol/L GA. The arrow indicates the area stained by the anti-GA-AGE antibody. The scale bar represents 200 μm; D: Western blotting analysis of intracellular GA-AGEs in PANC-1 cells. Cell lysates (15 μg of proteins/lane) were loaded on a 40-150 g/L polyacrylamide gradient gel. Proteins on the PVDF membrane were probed with anti-GA-AGE and anti-GA-3-phosphate dehydrogenase (GAPDH) antibodies. The molecular weight of GA-AGEs was calculated based on a single logarithmic chart used by the molecular marker. GAPDH was used as the loading control. WB was performed for two independent experiments. A and B: P values were based on Dunnett’s test. ^aP < 0.05, ^bP < 0.01 vs control.

Figure 2

Western blotting analysis of HSP90α and HSP90β. PANC-1 cell lysates (15 μg of proteins/lane) were loaded on a 40-150 g/L polyacrylamide gradient gel. A: Proteins on the polyvinylidene difluoride (PVDF) membrane were probed with anti-HSP90α and anti-GAPDH antibodies; B: Expression levels of HSP90α were normalized with GAPDH; C: Proteins on the PVDF membrane were probed with anti-HSP90β and anti-GAPDH antibodies. A band of 173 kDa HSP90β only appeared in PANC-1 cells treated with GA; D: Expression levels of the monomer HSP90β were normalized with GAPDH; E: The 173 kDa HSP90β/total HSP90β ratio. A and C: Western blotting was performed for three independent experiments. GAPDH was used as the loading control; B, D and E: Data are shown as mean ± SD (n = 3). P values were based on Dunnett’s test. ^aP < 0.05, ^bP < 0.01 vs control. GA: Glyceraldehyde.

Figure 3

Western blotting analysis of HSP70 and HSP27. PANC-1 cell lysates (15 μg of proteins/lane) were loaded on a 40-150 g/L polyacrylamide gradient gel. A: Proteins on the polyvinylidene difluoride (PVDF) membrane were probed with anti-HSP70 and anti-GA-3 phosphate dehydrogenase (anti-GAPDH) antibodies. We found four high-molecular-weight (HMW) complexes of HSP70s only in PANC-1 cells treated with GA. Their MWs were 138, 155, 184 and 244 kDa; B: Expression levels of the monomer HSP70 were normalized with GAPDH; C-F: The 138 kDa HSP70/total HSP70, 155 kDa HSP70/total HSP70, 184 kDa HSP70/total HSP70, and 244 kDa HSP70/total HSP70 ratios; G: Proteins on the PVDF membrane were probed with anti-HSP27 and anti-GAPDH antibodies. A HMW complex with a MW of 54 kDa appeared only in PANC-1 cells treated with GA; H: Expression levels of the monomer HSP27 were normalized with GAPDH; I: The 54 kDa HSP27/total HSP27 ratio; A and G: WB was performed for three independent experiments. GAPDH was used as a loading control. B-F, H, I: Data are shown as mean ± SD (n = 3). P values were based on Dunnett’s test. ^aP < 0.05, ^bP < 0.01 vs control. GA: Glyceraldehyde.

Figure 4

Western blotting analysis of cleaved caspase-3. PANC-1 cell lysates (15 μg of protein/lane) and Jurkat cell lysates (10 μg of protein/lane) were loaded on a 40-150 g/L polyacrylamide gradient gel. Proteins on the PVDF membrane were probed with anti-cleaved caspase-3 and anti-GA-3 phosphate dehydrogenase (anti-GAPDH) antibodies. U: The lysate of Jurkat cells not treated with cytochrome c. T: The lysate of Jurkat cells treated with cytochrome c. Western blotting was performed for three independent experiments. GAPDH was used as a loading control. GA: Glyceraldehyde.

Figure 5

Analysis of the proliferation of PANC-1 cells treated with glyceraldehyde-derived advanced glycation-end products-bovine serum albumin. PANC-1 cells were treated with 10 and 20 μg/mL of non-glycated control BSA and glyceraldehyde-derived advanced glycation-end products-bovine serum albumin (GA-AGEs-BSA) to then be incubated for 24 h. This assay was performed for two independent experiments. One assay was performed for n = 7. Cell proliferation was assessed by the WST-8 assay. Data are shown as mean ± SD (n = 7). P values were based on the Student’s t-test. ^aP < 0.05, ^bP < 0.01 vs control.