Nickel's Role in Pancreatic Ductal Adenocarcinoma: Potential Involvement of microRNAs

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types with a limited overall survival rate due to the asymptomatic progression of symptoms in metastatic stages of the malignancy and the lack of an early reliable diagnostic biomarker. MicroRNAs (miRs/miRNAs) are small (~18-24 nucleotides), endogenous, non-coding RNAs, which are closely linked to the development of numerous malignancies comprising PDAC. Recent studies have described the role of environmental pollutants such as nickel (Ni) in PDAC, but the mechanisms of Ni-mediated toxicity in cancer are still not completely understood. Specifically, Ni has been found to alter the expression and function of miRs in several malignancies, leading to changes in target gene expression. In this study, we found that levels of Ni were significantly higher in cancerous tissue, thus implicating Ni in pancreatic carcinogenesis. Hence, in vitro studies followed by using both normal and pancreatic tumor cell lines and increasing Ni concentration increased lethality. Comparing LC50 values, Ni-acetate groups demonstrated lower values needed than in NiCl₂ groups, suggesting greater Ni-acetate. Panc-10.05 cell line appeared the most sensitive to Ni compounds. Exposure to Ni-acetate resulted in an increased phospho-AKT, and decreased FOXO1 expression in Panc-10.05 cells, while NiCl₂ also increased PTEN expression in Panc-10.05 cells. Specifically, following NiCl₂ exposure to PDAC cells, the expression levels of miR-221 and miR-155 were significantly upregulated, while the expression levels of miR-126 were significantly decreased. Hence, our study has suggested pilot insights to indicate that the environmental pollutant Ni plays an important role in the progression of PDAC by promoting an association between miRs and Ni exposure during PDAC pathogenesis.

Keywords: apoptosis; environmental toxins; microRNAs; nickel; non-coding RNAs; pancreatic ductal adenocarcinoma.

Figure 1

Schematic representing the relationship between proteins associated with apoptosis. Abbreviations: PIP2—phosphatidylinositol 4,5-bisphosphate; PIP3—phosphatidylinositol 3,4,5-trisphosphate; PTEN—phosphatase and tensin homolog; AKT—Ak strain transforming; GSK3β—glycogen synthase kinase 3 beta; IKK-ɑ—IkappaB kinase; MDM2—mouse double minute 2 homolog or E3 ubiquitin-protein ligase; PARP—poly (ADP-ribose) polymerase; mTOR—mechanistic target of rapamycin; Bad—Bcl-2-associated death promoter. Green arrows represent stimulation and red arrows represent inhibition.

Figure 2

Levels of Ni in human pancreatic tissue in the investigated population. Boxes represent interquartile intervals (25–75%), while lines inside boxes represent medians. The box represents the interquartile range (25–75th percentile), the line within the box represents median value and ends of the whiskers represent the minimum and maximum values within the group. The asterisk indicates a statistically significant difference from the control levels (Mann–Whitney U test, p < 0.001).

Figure 3

LC50 curves for NiCl₂ in PDAC cell lines. Cells were exposed to increasing concentrations of NiCl₂ (0–10 mM) for 48 h and cell viability was measured using the MTT assay. Curve shapes best fitted a single site model (three-parameter) (A). The rank order for NiCl₂ LC50 values was AsPC-1 < HPNE < BxPC-3 = Panc-1 << Panc-10.05 = MiaPaCa-2 (B). Comparing lethality at 10 mM (C), with viability expressed as percent of control values, the BxPC-3 was the most sensitive cell line, whereas the Panc-10.05 and MiaPaCa-2 cells were the least sensitive. Data are expressed as the mean ±SD of eight assays performed in duplicate (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 4

LC50 curves for Ni-acetate in PDAC cell lines. Cells were exposed to increasing concentrations of NiCl₂ (0–10 mM) for 48 h and cell viability was measured using the MTT assay. Curve shapes were best fitted to a single site model (three-parameter) (A). The rank order for Ni-acetate LC50 values was AsPC-1 < MiaPaCa-2 ≤ HPNE ≤ Panc-10.05 < BxPC-3 << Panc-1. (B). Except for the Panc-1 cells, all the PDAC cell lines exhibited lower LC50 values after exposure to Ni-acetate compared to NiCl₂. Comparing lethality at 10 mM (C), the BxPC-3 was the most sensitive of the cell lines whereas AsPC-1 and Panc-10.05 cells were least sensitive. Data are expressed as the mean ± SD of eight assays performed in duplicate (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 5

Quantification and expression of key cell function proteins after 48 h exposure to 50 μM NiCl₂ or Ni-acetate in PDAC cell lines. These assays quantified the amount of β-catenin (A), phospho-AKT (B), p53 (C), and FOXO-1 (D). The predominant difference was in the basal levels of each protein and the differences between the cell lines. The expressions of phospho-AKT (B) and p53 (C) typify this response. Treatment with either NiCl₂ or Ni-acetate had little effect after 48 h, except for phospho-AKT (B) and FOXO-1 (D), in which NiCl₂ and Ni-acetate increased phospho-AKT expression and decreased FOXO-1 expression in Panc-10.05 cells. In BxPC-3 cells, exposure to NiCl₂ increased phospho-AKT expression 146% compared to vehicle control. Data are expressed as the mean ± SD of 4 assays performed in duplicate (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 6

Quantification and expression of key cell-function proteins after 48 h exposure to 50 μM NiCl₂ or Ni-acetate in PDAC cell lines. These assays quantified the amount of PTEN (A), PARP (B), caspase 3/7 activity (C), and cleaved (active) caspase 3 expression (D). The major difference observed between groups was dependent on the cell line, with each of the PDAC cell lines expressing different protein levels. Treatment effects were evident in PTEN expression in the Panc-10.05 cell line, in which NiCl₂ exposure resulted in a significant (p < 0.01) increase in PTEN expression. Caspase 3/7 activity was significantly (p < 0.01) reduced after exposure to Ni-acetate. Data are expressed as the mean ±SD of four (protein expression) or eight (caspase 3/7 activity) assays performed in duplicate (** p < 0.01; *** p < 0.001).

Figure 7

NiCl₂ treatment-mediated effects on miR expression levels in PDAC cells. (A–C): Effects of NiCl₂ in the Panc-1 cell line: miR-221 relative expression levels (A); miR-155 relative expression levels (B); miR-126 relative expression levels (C). (D–F): Effects of NiCl₂ in the MiaPaCa-2 cell line: miR-221 relative expression levels (D); miR-155 relative expression levels (E); miR-126 relative expression levels (F). NiCl₂ treatment effects were evident in the expression levels of miR-221 (p < 0.001; Panc-1, p < 0.05; MiaPaCa-2), and miR-155 (p < 0.05 for all) compared to the control cells, while the expression levels of miR-126 were significantly downregulated (p < 0.0001 for all). The column graphic represents the average of three replicates of RNA isolated from the Panc-1 and MiaPaCa-2 cell lines. Data normalized according to RNU6 expression by fold analysis (n = 3, p < 0.05); exact p-values are indicated (* p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001); error bars indicate SD.