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. 2003 Jun 13;3(1):8.
doi: 10.1186/1475-2867-3-8.

Epithelial Na, K-ATPase expression is down-regulated in canine prostate cancer; a possible consequence of metabolic transformation in the process of prostate malignancy

Ali Mobasheri  1 , Richard Fox, Iain Evans, Fay Cullingham, Pablo Martín-Vasallo, Christopher S Foster
Affiliations

Epithelial Na, K-ATPase expression is down-regulated in canine prostate cancer; a possible consequence of metabolic transformation in the process of prostate malignancy

Ali Mobasheri et al. Cancer Cell Int. .

Abstract

BACKGROUND: An important physiological function of the normal prostate gland is the synthesis and secretion of a citrate rich prostatic fluid. In prostate cancer, citrate production levels are reduced as a result of altered cellular metabolism and bioenergetics. Na, K-ATPase is essential for citrate production since the inward Na+ gradients it generates are utilized for the Na+ dependent uptake of aspartate, a major substrate for citrate synthesis. The objective of this study was to compare the expression of previously identified Na, K-ATPase isoforms in normal canine prostate, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma (PCa) using immunohistochemistry in order to determine whether reduced citrate levels in PCa are also accompanied by changes in Na, K-ATPase expression. RESULTS: Expression of Na, K-ATPase alpha1 and beta1 isoforms was observed in the lateral and basolateral plasma membrane domains of prostatic epithelial cells in normal and BPH prostates. Canine kidney was used as positive control for expression of Na, K-ATPase alpha1 and gamma isoforms. The alpha1 isoform was detected in abundance in prostatic epithelial cells but there was no evidence of alpha2, alpha3 or gamma subunit expression. In advanced PCa, Na, K-ATPase alpha1 isoform expression was significantly lower compared to normal and BPH glands. The abundant basolateral immunostaining observed in normal and BPH tissue was significantly attenuated in PCa. CONCLUSION: The loss of epithelial structure and function and the transformation of normal epithelial cells to malignant cells in the canine prostate have important implications for cellular metabolism and are accompanied by a down regulation of Na, K-ATPase.

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Figures

Figure 1
Figure 1
Histological appearance of normal (A), BPH (B) and PCa canine prostate tissues incorporated in this study. In the normal prostate continuous sheets of epithelial cells (ec) are separated by connective tissue stroma (S) consisting of blood capillaries (indicated by closed arrows), smooth muscle and neuroendocrine cells. BPH glands display smaller lumens (L) compared to normal tissue with evidence of tufting and micropapillae. PCa glands are lined with neoplastic cells exhibiting cellular and nuclear pleomorphisms and numerous mitotic figures (open arrows). The basal cells (bc) were irregular in contour but the basement membrane appears confluent. Original magnifications: × 400.
Figure 2
Figure 2
Immunohistochemical staining of Na, K-ATPase α1 and β1 subunits in canine normal, BPH and PCa tissues. Sites of Na, K-ATPase immunoreactivity are stained red (using Fast-Red TR/Naphthol AS-MX as precipitating substrate for the alkaline phosphatase conjugated secondary antibody). In normal and BPH sections Na, K-ATPase immunostaining is clearly visible in the basolateral membrane domain of epithelial cells (arrows). In three different PCa specimens Na, K-ATPase expression levels appear to be significantly lower than in normal or BPH tissues and Na, K-ATPase is diffusely spread within focal areas of the neoplasm. Nuclei were counterstained with haematoxylin.
Figure 3
Figure 3
Comparing the immunohistochemical localization of Na, K-ATPase "α" subunits in normal, BPH and PCa tissues using a pan α monoclonal antibody (mAb9A7) that recognizes all known α isoforms. The pan α monoclonal antibody produces a staining pattern which is identical to the α1 specific antibodies providing further evidence for the predominance of α1 expression in the prostate and the downregulation of Na, K-ATPase in PCa.
Figure 5
Figure 5
Quantitative analysis of Na, K-ATPase immunoreactivity in normal, BPH and PCa canine prostate. Panel A; results of image analysis experiments performed in triplicate comparing Na, K-ATPase expression levels in tissue sections immunoassayed with a polyclonal antibody to the α1 subunit of Na, K-ATPase under identical experimental conditions. Panels B and C; representative sections used for analysis. Panels D and E; plot profiles of Na, K-ATPase abundance in the BPH and PCa sections.
Figure 4
Figure 4
Panel A: Absence of Na, K-ATPase α2, α3 and γ isoform expression and comparison with α1 expression in canine prostate. Original magnifications: × 800. Panel B: Evidence to confirm that the γ C33 polyclonal raised against the rodent γ isoform recognizes the canine γ isoform protein in the canine kidney. The expression of the γ isoform is not detected in all nephron segments (i.e. medullary and papillary collecting ducts) but it is present in low levels in proximal convoluted tubules and in high levels in the medullary thick ascending limbs, and the distal nephron.
Figure 6
Figure 6
Proposed scheme for the role of Na, K-ATPase in maintaining prostatic epithelial cell polarity and citrate-related energy metabolism (adapted from a concept first proposed by Costello and Franklin, 2000[4]). Normal prostate cells abundantly express basolateral Na, K-ATPase to maintain cell homeostasis and epithelial polarity. These cells contain specific transport mechanism for the accumulation of high levels of zinc and aspartate for optimal citrate biosynthesis. Aspartate, is transported into prostate epithelial cells on a high-affinity Na+-dependent carrier which resides in the basal cell membrane and is dependant on Na, K-ATPase activity [3]. The citrate transporter itself has yet to be identified but has been proposed to be localized in the apical membrane where it contributes to the accumulation of a citrate rich prostatic fluid [2,1]. The genetic transformation of a normal prostate cell to a neoplastic cell has been proposed to be accompanied by an impaired ability to accumulate high levels of citrate. The neoplastic cell begins a metabolic transformation to a citrate-oxidizing cell, losing the ability to accumulate important factors such as zinc. Thus begins a metabolic conversion to a pre-malignant or malignant citrate-oxidizing cell. The loss of basolateral Na, K-ATPase appears to be one of a number of phenotypic changes that accompany this transformation. This alteration will no doubt influence the membrane potential, homeostatic responses to ionic perturbations mediated by ion channels and secretory functions.
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