Date of Award
Master of Science
Jennifer A. Doll
Elizabeth Liedhegner, Claire de la Cova
cancer, hyperglycemia, progression, prostate, ZnT1
Background: Prostate cancer is the second leading cause of cancer in men worldwide. Many studies have shown that hyperglycemia is associated with an increased risk of prostate cancer progression. Furthermore, normal prostate glandular epithelium has a high zinc content that accommodates prostate glandular function for citrate secretion while malignant prostate tissues contain less zinc compared to normal prostate tissues. As shown in previous studies, accumulation of zinc appears to be detrimental in LNCaP and PC-3 prostate cancer cells. Zinc efflux transporter (ZnT1) has a crucial role in regulating intracellular zinc homeostasis by exporting zinc into the extracellular space, and it has a single N-glycosylation site at asparagine 299 (N299). Moreover, ZnT1 was found to be upregulated in prostate cancer. Interestingly, acute high glucose levels enhanced zinc secretion via ZnT1 in normal prostate epithelial cells and slightly in PC-3 cells. The exact mechanism by which acute and chronic high glucose might affect ZnT1 and intracellular zinc homeostasis is unknown. The object of this study is to determine the effect of high glucose level on ZnT1 expression, membrane localization, and glycosylation state in prostate cancer cells. Methods: Prostate cancer cell lines, LNCaP (androgen sensitive) and PC-3 (androgen resistant) cells were used in this study. LNCaP and PC-3 cells were maintained in low glucose media (5.5 mM), plated overnight, then incubated for 48 h in low glucose (L) media (5.5 mM; control) or in high glucose (H) media (25 mM). Total cell lysate (CL) was collected. Cell surface localization of ZnT1 was assessed using a cell surface biotinylation assay kit. CL samples and eluent samples containing cell surface protein were analyzed by Western blot using anti-ZnT1 antibody and GAPDH antibody as loading control for CL samples. Proliferation and viability of the cells were quantified by direct cell counts using trypan blue exclusion assay for viability. Densitometry was performed using ImageJ software, normalizing CL to GAPDH expression and biotinylated samples to cell number. Statistical significance was performed using a student t-test with a p-value cut of < 0.05. Results: In LNCaP and in PC-3 cells, no significant difference was observed in cell proliferation and viability in response to high glucose treatment compared to the control. In PC-3 cells, high glucose treatment significantly upregulated total ZnT1 expression (p = 0.032) and ZnT1 cell membrane expression (p = 0.007). In contrast, there was no significant difference in total ZnT1 level between high glucose treated LNCaP cells and the control. Analysis of the biotinylated ZnT1 in LNCaP showed that high glucose treatment significantly decreased nonglycosylated ZnT1 dimer (p = 0.034), and glycosylated monomer (p = 0.04) on the cell membrane. However, there was no significant difference in the total ZnT1, total glycosylated and ZnT1 dimer levels (glycosylated and nonglycosylated) on the cell membrane between high glucose-treated LNCaP and the control. Conclusion: Based on these data, high glucose levels may contribute to prostate cancer progression via ZnT1 upregulation as shown in PC-3 cells. Further studies are needed to determine the effect of high glucose on intracellular zinc concentration and glycosylation state of ZnT1 in normal prostate epithelium and prostate cancer cells.
Zayed, Huda Abdulla, "Hyperglycemia Regulation of Zinc Efflux Transporters, ZnT1, in Prostate Cancer Progression" (2023). Theses and Dissertations. 3377.
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