Immunocytochemical Localization of Atrial Natriuretic Peptide, Vessel Dilator, Long-acting Natriuretic Peptide, and Kaliuretic Peptide in Human Pancreatic Adenocarcinomas
Departments of Biochemistry and Molecular Biology, Internal Medicine, Pathology, Physiology and Biophysics, University of South Florida Cardiac Hormone Center (SRS,AHG,LCC,WRG,DLV), and James A. Haley Veteran's Administration Medical Center (SRS,JS,WRG,DLV), Tampa, Florida
Correspondence to: David L. Vesely, MD, PhD, Director, Cardiac Hormone Center University of South Florida for Health Sciences, 13000 Bruce B. Downs Blvd., Tampa, FL 33612. E-mail: david.vesely{at}med.va.gov
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Summary |
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Key Words: atrial natriuretic peptides immunocytochemistry pancreatic adenocarcinomas
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Introduction |
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We have found that vessel dilator, LANP, kaliuretic peptide, and ANP decrease the number of human pancreatic adenocarcinoma cells in culture by 65%, 47%, 37%, and 34%, respectively, within 24 hr (Vesely et al. 2003). This decrease was sustained without any proliferation of the adenocarcinoma cells occurring in the 3 days after this decrease (Vesely et al. 2003
). The mechanism of these peptide hormones' decrease in cancer cell number and antiproliferative effects was a 83% or greater inhibition of DNA synthesis, but not owing to enhanced apoptosis (i.e., programmed cell death) (Vesely et al. 2003
). One of the known mediators of these peptide hormones' mechanisms of action (i.e., cGMP) inhibited DNA synthesis in these adenocarcinoma cells by 51% (Vesely et al. 2003
).
In vivo, vessel dilator (139 ng·min1·kg1 of body weight) infused for 14 d completely stopped the growth of the human pancreatic adenocarcinomas in athymic mice (n=14) with a decrease in their tumor volume, whereas the tumor volume increased 69-fold (p<0.001) in the placebo-treated mice (n=30) (Vesely et al. 2004). When the four peptide hormones synthesized the ANP gene (each at 1.4 µg·min1·kg1 body weight) were infused for 4 weeks, vessel dilator, LANP, and kaliuretic peptide, in addition to completely stopping the growth of this human pancreatic cancer, also decreased tumor volume after 1 week by 49%, 28%, and 11% with a 1- and 20-fold increase in the tumor volume in ANP- and placebo-treated mice (Vesely et al. 2004
).
The present investigation was designed to determine where these peptide hormones localize within human pancreatic cancers. Where these peptide hormones localize within any cancer has never been investigated.
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Materials and Methods |
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Immunohistochemistry
After deparaffinization and rehydration with PBS, pH 7.2, 3-µm sections were stained for vessel dilator, LANP, kaliuretic peptide, and ANP by the avidin-biotin peroxidase complex technique (Hsu et al. 1981) using a Vectastain kit (Vector Laboratories; Burlingame, CA). Sections were incubated for 1 hr at 24C in a humidity chamber with the respective primary antibody diluted (1:2001:1000) with PBS, and then, after rinsing, incubated again in the humidity chamber for 30 min with biotinylated antibody. The illustrations in this manuscript are all at the 1:800 dilution of the respective primary antibodies. The slides were then rinsed followed by a final incubation for 30 min in avidin-biotin peroxidase complex in a humidity chamber. Controls for the immunoperoxidase staining included: (a) substitution of the primary antibody with normal rabbit serum and (b) preincubation of the primary antibody with excess vessel dilator, LANP, kaliuretic peptide, or ANP in their respective immunoperoxidase assays for 24 hr at 37C. These respective human peptide hormone antisera for immunohistochemistry were from Peninsula Laboratories (Belmont, CA).
Cross-reactivity of Vessel Dilator, LANP, Kaliuretic Peptide, and ANP
Cross-reactivities of the various human antisera raised in rabbits to determine their specificity were determined by comparative radioimmunoassays using the human form of pure synthetic peptides vessel dilator, LANP, kaliuretic peptide, and ANP synthesized and assayed by Peninsula Laboratories, Inc. LANP antisera cross-reactivity with pure synthetic LANP was 100%, whereas its cross-reactivity with ANP and vessel dilator was 0%. Vessel dilator antisera had 100% cross-reactivity with pure synthetic vessel dilator, but 0% cross-reactivity with LANP or ANP. The antisera to ANP had 100% cross-reactivity with ANP, and 0% cross-reactivity with either LANP or vessel dilator. Kaliuretic peptide antisera had 100% cross-reactivity with human kaliuretic peptide, but 0% cross-reactivity with LANP, vessel dilator, or ANP.
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Results |
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Kaliuretic peptide immunoperoxidase staining in the adenocarcinomas infused with kaliuretic peptide had a decreased intensity compared with the other peptide hormones (Table 1 and Figure 1C). Kaliuretic peptide, however, localized to the same structures within the human pancreatic adenocarcinomas (Figure 1C). Thus kaliuretic peptide immunoperoxidase staining (+) localized to the nucleus of the cancer cells similar to that observed with vessel dilator and LANP (Figure 1C). Staining of the cytoplasm (++) of the human pancreatic adenocarcinoma cells with kaliuretic peptide was less than that observed with vessel dilator, but there was definite localization of kaliuretic peptide to the cytoplasm. Kaliuretic peptide also localized to the endothelium of the small capillaries and to fibroblasts (Figure 1C), with an intensity similar to that of its localization to cytoplasm.
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There was not any immunoperoxidase staining with vessel dilator, LANP, kaliuretic peptide, or ANP in the human adenocarcinomas when their respective primary antisera were either substituted with normal rabbit serum (Figure 1D) or when the primary antibody was preincubated with an excess of vessel dilator, LANP, kaliuretic peptide, or ANP in their respective immunoperoxidase assays for 24 hr at 37C.
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Discussion |
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Vessel dilator, LANP, kaliuretic peptide, and ANP had a very strong localization to the cytoplasm of these cancer cells. We had hypothesized that if these peptide hormones were reaching the nucleus to inhibit DNA synthesis, they should also be abundantly present in the cytoplasm after binding to the cell surface receptors on plasma membranes of the cancer cells. These peptides are known to bind to specific receptors in the plasma membranes of normal and cancer cells (Vesely et al. 1987,1990
,1992
). After ANP binds to its receptor, the receptor internalizes and ANP was then thought to be degraded, with the receptors recycling to the plasma membrane (Hirata et al. 1985
; Napier et al. 1986
; Hughes et al. 1987
; Morel and Heisler 1988
). Part of the cytoplasmic demonstration of these peptide hormones within the cancer cells may be the ANPs attached to their receptors that are being internalized. However, the intense cytochemical localization throughout the cytoplasm and the new knowledge of the present investigation that these peptides also localize strongly to the perinuclear area and are in the nucleus suggests that these peptides are not all being degraded in the cytoplasm as previously thought, but rather are traveling through the cytoplasm to reach the nucleus to directly inhibit DNA synthesis.
There is evidence that other peptides are transported from the external plasma membrane to the nucleus (Burack and Shaw 2000). One example of this is extracellular receptor kinase (ERK), which is transported to the nucleus to cause proliferation (i.e., a growth-promoting protein) (Burack and Shaw 2000
). Proteins such as ERK are thought to move from the plasma membrane to the nucleus via attaching to a scaffolding protein, which moves them to the nucleus (Burack and Shaw 2000
). One could envision a similar type of scaffolding protein to transport the four growth-inhibiting proteins of the present investigation, but this protein(s) has not been defined.
These peptide hormones do have an intracellular mediator within the cytoplasm (i.e., cGMP) (Brenner et al. 1990; Vesely 1997
). cGMP inhibits DNA synthesis in these cancer cells, but only to about half of the extent of vessel dilator (Vesely et al. 2003
). The new knowledge of the present investigation that the peptide hormones are reaching the nucleus via the cytoplasm suggest that these peptide hormones may be directly inhibiting DNA synthesis and decreasing cell-cycle progression (with 62% less cells in the synthetic phase) and indirectly inhibiting DNA synthesis via the intracellular messenger cGMP.
It is important to note that all of the previously reported peptide hormones that localize to the nucleus (i.e., insulin, epidermal growth factor, nerve growth factor, platelet-derived growth factor, luteinizing hormone releasing hormone, and human chronic gonadotrophin) (Burwen and Jones 1987) and ERK (Burack and Shaw 2000
) have been growth-promoting hormones. The present investigation is the first demonstration we are aware of that growth-inhibiting peptide hormones localize to the nucleus, where they can directly interact to inhibit the effects of the growth-stimulating hormones and act via their demonstrated direct decrease of DNA synthesis in the nucleus (Vesely et al. 2003
).
In the present investigation, these peptide hormones also localized to the endothelium of the blood vessels growing into this cancer. This finding is similar to previous finding (Figure 3)
that these peptide hormones localized to the endothelium of blood vessels of normal kidneys (Ramirez et al. 1992; Saba et al. 1993
). In normal tissues, these peptides localize to the endothelium of larger arteries and veins (Ramirez et al. 1992
; Saba et al. 1993
), but in the present investigation, when examined by H & E staining (Figure 2), there were not any large blood vessels growing into these tumors, but rather only capillaries that were invading this cancer. Within the capillaries, it was the endothelium that had immunoperoxidase staining of these peptide hormones (Figure 1). That no large blood vessels had grown into these adenocarcinomas helps explain the observation on H & E examination that the centers of each of these tumors were necrotic. This finding is apparently the result of these tumors outgrowing their blood supply (i.e., no large blood vessels were present) rather than from an effect of these peptide hormones per se, because the untreated adenocarcinomas had equally as much necrosis in the center of their tumors as the vessel dilator, LANP-, kaliuretic peptide, and ANP-treated tumors.
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Acknowledgments |
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Footnotes |
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