Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues

Abstract

Background: In prostate cancer, normal citrate-producing glandular secretory epithelial cells undergo a metabolic transformation to malignant citrate-oxidizing cells. m-Aconitase is the critical step involved in this altered citrate metabolism that is essential to prostate malignancy. The limiting m-aconitase activity in prostate epithelial cells could be the result of a decreased level of m-aconitase enzyme and/or the inhibition of existing m-aconitase. Earlier studies identified zinc as an inhibitor of m-aconitase activity in prostate cells; and that the depletion of zinc in malignant cells is an important factor in this metabolic transformation. However, a possibility remains that an altered expression and level of m-aconitase enzyme might also be involved in this metabolic transformation. To address this issue, the in situ level of m-aconitase enzyme was determined by immunohistochemical analysis of prostate cancer tissue sections and malignant prostate cell lines.

Results: The immunocytochemical procedure successfully identified the presence of m-aconitase localized in the mitochondrial compartment in PC-3, LNCaP, and DU-145 malignant prostate cell lines. The examination of prostate tissue sections from prostate cancer subjects demonstrated that m-aconitase enzyme is present in the glandular epithelium of normal glands, hyperplastic glands, adenocrcinomatous glands, and prostatic intraepithelial neoplastic foci. Quantitative analysis of the relative level of m-aconitase in the glandular epithelium of citrate-producing adenomatous glands versus the citrate-oxidizing adenocarcinomatous glands revealed no significant difference in m-aconitase enzyme levels. This is in contrast to the down-regulation of ZIP1 zinc transporter in the malignant glands versus hyperplastic glands that exists in the same tissue samples.

Conclusion: The results demonstrate the existence of m-aconitase enzyme in the citrate-producing glandular epithelial cells; so that deficient m-aconitase enzyme is not associated with the limiting m-aconitase activity that prevents citrate oxidation in these cells. The level of m-aconitase is maintained in the malignant cells; so that an altered enzyme level is not associated with the increased m-aconitase activity. Consequently, the elevated zinc level that inhibits m-aconitase enzyme is responsible for the impaired citrate oxidation in normal and hyperplastic prostate glandular epithelial cells. Moreover, the down-regulation of ZIP1 zinc transporter and corresponding depletion of zinc results in the increase in the activity of the existing m-aconitase activity in the malignant prostate cells. The studies now define the mechanism for the metabolic transformation that characterizes the essential transition of normal citrate-producing epithelial cells to malignant citrate-oxidizing cells.

Figure 1

Subcellular localization of m-aconitase. A-D. Immunofluorescent detection. LNCaP prostate cell line was probed with Mitotracker then fixed for immunofluorescence with Aconitase-2 Ab and visualized by Laser Scanning Confocal Microscope. A. no staining of either in nuclei (DAPI). B. red punctuate mitochondrial staining in cytoplasm (Mitotracker). C. green fluorescent of m-aconitase in cytoplasm (FITC). D. red and green fluorescence merge shows co-localization of m-aconitase in mitochondria. E. Immunocytochemical detection of m-aconitase. Note the dark brown punctuate mitochondrial staining in cytoplasm; and well defined clear nucleus.

Figure 2

Representative m-aconitase immunohistochemistry of human prostate tissue sections. Left panel. A. Negative control. B. Field that shows adjacent BPH and malignant foci. C. Field showing adjacent PIN and malignant glands. Right panel. A-D are H&E staining; a-d are immunohistochemical staining. Note strong brown positive staining of glandular epithelium in all cases; and contrasting less staining of stroma.

Figure 3

Western blot analysis of m-aconitase levels in human prostate cancer cell lines. The cells were exposed for 24 hours to medium supplemented with 15 uM zinc and to unsupplemented medium.