Alastair J. S. Summerlee

H2 relaxin overexpression increases in vivo prostate xenograft tumor growth and angiogenesis

Our study reports a preliminary investigation into the role of human H2 relaxin in prostate tumor growth. A luciferase‐expressing human prostate cancer cell line, PC‐3, was generated and termed PC3‐Luc. PC3‐Luc cells were transduced with lentiviral vectors engineering the expression of either enhanced green fluorescent protein (eGFP) or both H2 relaxin and eGFP in a bicistronic format. These transduced cells were termed PC3‐Luc‐eGFP and PC3‐Luc‐H2/eGFP, respectively. To gauge effects, PC3‐Luc‐H2/eGFP and PC3‐Luc‐eGFP cells were injected into NOD/SCID mice and monitored over 6 weeks. PC‐3 tumor xenografts overexpressing H2 relaxin exhibited greater tumor volumes compared to control tumors. Circulating H2 relaxin levels in sera increased with the relative size of the tumor, with moderately elevated H2 relaxin levels in mice bearing PC3‐Luc‐H2/eGFP tumors compared to PC3‐Luc‐eGFP tumors. Zymographic analysis demonstrated that proMMP‐9 enzyme activity was significantly downregulated in H2 relaxin‐overexpressing tumors. An advanced angiogenic phenotype was observed in H2 relaxin‐overexpressing tumors indicated by greater intratumoral vascularization by immunohistochemical staining of endothelial cells with anti‐mouse CD31. Moreover, PC3‐Luc‐H2/eGFP tumors exhibited increased VEGF transcript by reverse‐transcription PCR, compared to basal levels in control animals. Taken together, our study provides the first account of a potential role of H2 relaxin in prostate tumor development.

Relaxin-like peptides in cancer

The members of the relaxin‐like hormone family, relaxin and INSL3, also known as relaxin‐like factor (RLF) or Leydig cell‐derived insulin‐like factor (LEY‐I‐L), are implicated in various mechanisms associated with tumor cell growth, differentiation, invasion and neovascularization. The recent discovery of the relaxin receptor LGR7 and the INSL3/relaxin receptor LGR8 has provided evidence of an auto/paracrine relaxin‐like action in tumor tissues and enables the elucidation of the cellular pathways involved in the proposed functions of relaxin in tumor biology. Our review summarizes our current knowledge of the expression of relaxin and INSL3 in human neoplastic tissues and discusses the etiological roles of these heterodimeric peptide hormones in cancer. Discussion of possible cellular cascades involved in actions linking relaxin‐like peptides and neoplasia include the role of relaxin‐like peptides in tumor cell growth and differentiation; the effect of relaxin in stimulating the synthesis of the vasodilatory and tumor cell cytostatic and antiapoptotic molecule, nitric oxide; the potential ability of relaxin to upregulate vascular endothelial growth factor to promote angiogenesis and neovascularization and the concerted fine‐tuned action of relaxin on the matrix metalloproteinases on the extracellular matrix to facilitate tumor cell attachment, migration and invasion.

Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth

Hormone antagonists can be effective tools to delineate receptor signaling pathways and their resulting downstream physiological actions. Mutation of the receptor binding domain (RBD) of human H2 relaxin (∆H2) impaired its biological function as measured by cAMP signaling. In a competition assay, ∆H2 exhibited antagonistic activity by blocking recombinant H2 relaxin from binding to receptors on THP‐1 cells. In a flow cytometry‐based binding assay, ∆H2 demonstrated weak binding to 293T cells expressing the LGR7 receptor in the presence of biotinylated H2 relaxin. When human prostate cancer cell lines (PC‐3 and LNCaP) were engineered to overexpress eGFP, wildtype (WT) H2, or ∆H2, and subsequently implanted into NOD/SCID mice, tumor xenografts overexpressing ∆H2 displayed smaller volumes compared to H2 and eGFP controls. Plasma osmolality readings and microvessel density and area assessment suggest that ∆H2 modulates physiological parameters in vivo. In a second murine model, intratumoral injections of len‐ tivectors engineered to express ∆H2/eGFP led to suppressed tumor growth compared to controls. This study provides further evidence supporting a role for H2 relaxin in prostate tumor growth. More importantly, we report how mutation of the H2 relaxin RBD confers the hormone derivative with antagonistic properties, offering a novel reagent for relaxin research.—Silvertown, J. D., Symes, J. C., Neschadim, A., Nonaka, T., Kao, J. C. H., Summerlee, A. J. S., Medin, J. A. Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth. FASEB J. 21, 754–765 (2007)

The Emerging Concept of Relaxn as a Centrally Actng Peptide Hormone with Hemodynamic Actions

The novel finding that relaxin has an action on the brain was first published in 1984. Since then, it has been shown that exogenous relaxin affects the release of a number of hypothalamo‐pituitary hormones and has a robust pressor action. In this paper, we review the accumulating evidence that relaxin affects the release of oxytocin and vasopressin by an action at the level of the brain. The potential mechanisms of this central action are discussed and the evidence presented for the interaction between relaxin and the forebrain angiotensin‐ll system. Furthermore, we articulate the possible physiological influences of relaxin on the changes in cardiovascular function that occur during pregnancy.

Mechanism of the haemotensive action of porcine relaxin in anaesthetized rats.

Experiments were done to examine the pressor effect of iv porcine relaxin in anaesthetized rats. Acute injections of relaxin caused consistent and sustained rises in systemic blood pressure that were dose‐dependent within the physiological range. Pretreatment of rats with a specific vasopressin (V1) receptor antagonist, but not an α‐adrenoreceptor antagonist, substantially reduced the pressor effect of relaxin. After the vasopressin receptor antagonist, small rises in blood pressure occurred after a longer latent period, compared with the responses in intact animals. The data clearly indicate that acute injections of relaxin cause a pressor response that is predominantly affected via the release of vasopressin. The possible sources of the persistent hypertensive component are discussed and it is suggested that relaxin might act through the central angiotensinergic systems to release vasopressin and cause a pressor response.

Central administration of porcine relaxin stimulates drinking behaviour in rats: an effect mediated by central angiotensin II

Central injection of porcine relaxin into the lateral ventricle of water-replete rats caused a marked drinking response. Relaxin in 2 µL 0.9% saline caused a dose-dependent (range 10–50 ng), significant (P<0.01) dipsogenesis compared with saline-treated controls. There was no drinking response to <10 ng relaxin. At 10 ng relaxin ICV rats drank 4.2 ± 0.2 mL water within 15 min of injection. The amount of water taken increased with increasing dose and plateaued at 50 ng ICV (10.2 ± 1.3 mL) thereafter; increasing the dose of relaxin did not significantly increase the total volume of water consumed. In contrast, there was no significant increase in water consumed in rats treated with a deactivated form of porcine relaxin, or with insulin. Rats appeared to compensate for the period of hyperdipsia, as there was no significant difference in the water consumed in control (saline-injected) and relaxin-treated rats in the 23 h period after testing.The effect of blocking the central action of angiotensin II on the dipsogenic effects of relaxin was tested by infusing of a specific angiotensin II receptor antagonist into the lateral ventricle before treatment with relaxin. Antagonism of the central angiotensin II system, confirmed by lack of a dipsogenic response to ICV exogenous angiotensin II (10 ng), completely blocked the dipsogenic response of relaxin (50 ng in 1 µL) in female rats.These data demonstrate that exogenous porcine relaxin is dipsogenic in the rat and that the mechanism of action appears to be through the central angiotensin II system. It is possible that relaxin may affect water intake during pregnancy when relaxin levels are detectable in the plasma and the hormone may be implicated in the regulation of cardiovascular function in pregnancy.

Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues.

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.

Chapter 16 Relaxin and drinking in pregnant rats

Work reported in this chapter describes the potential role of relaxin in resetting cardiovascular thresholds in pregnant rats. Relaxin, a polypeptide produced primarily by the ovary in pregnant animals in many species, is also produced in the brain. Exogenous administration of relaxin into the brain causes a profound drinking response which is negated by pretreatment with a specific monoclonal antibody to rat relaxin when the antibody is injected into the brain. Neutralizing the action of endogenous brain relaxin in pregnant rats also blocks the normal increase in drinking that is observed in rats at night during the second half of pregnancy. Relaxin acts through the forebrain angiotensin system at the level of the subfornical organ (an important interface between the blood, the brain and the cerebrospinal fluid) as blockade of the angiotensin II receptor action negates several central actions of relaxin. Expression of angiotensin II AT1 receptors in the subfornical organ increases in parallel with the increase in circulating relaxin seen in the second half of pregnancy. Neutralizing the effects of endogenous brain relaxin, using central injections of the monoclonal antibody, blocks this increase in the expression of angiotensin II AT1 receptors in subfornical organ. These data imply that relaxin in the brain may act to affect central cardiovascular thresholds in rats and this may be important for the normal physiology of pregnancy.

Relaxin induces rapid, transient vasodilation in the microcirculation of hamster skeletal muscle

Relaxin produces a sustained decrease in total peripheral resistance, but the effects of relaxin on skeletal muscle arterioles, an important contributor to systemic resistance, are unknown. Using the intact, blood-perfused hamster cremaster muscle preparation in situ, we tested the effects of relaxin on skeletal muscle arteriolar microvasculature by applying 10(-10) M relaxin to second-, third- and fourth-order arterioles and capillaries. The mechanisms responsible for relaxin-induced dilations were explored by applying 10(-10) M relaxin to second-order arterioles in the presence of 10(-5) M N(G)-nitro-l-arginine methyl ester (l-NAME, nitric oxide (NO) synthase inhibitor), 10(-5) M glibenclamide (GLIB, ATP-dependent potassium (K(+)) channel inhibitor), 10(-3) M tetraethylammonium (TEA) or 10(-7) M iberiotoxin (IBTX, calcium-associated K(+) channel inhibitor). Relaxin caused second- (peak change in diameter: 8.3 ± 1.7 μm) and third (4.5 ± 1.1 μm)-order arterioles to vasodilate transiently while fourth-order arterioles did not (0.01 ± 0.04 μm). Relaxin-induced vasodilations were significantly inhibited by l-NAME, GLIB, TEA and IBTX. Relaxin stimulated capillaries to induce a vasodilation in upstream fourth-order arterioles (2.1 ± 0.3 μm), indicating that relaxin can induce conducted responses vasodilation that travels through blood vessel walls via gap junctions. We confirmed gap junction involvement by showing that gap junction uncouplers (18-β-glycyrrhetinic acid (40 × 10(-6) M) or 0.07% halothane) inhibited upstream vasodilations to localised relaxin stimulation of second-order arterioles. Therefore, relaxin produces transient NO- and K(+) channel-dependent vasodilations in skeletal muscle arterioles and stimulates capillaries to initiate conducted responses. The transient nature of the arteriolar dilation brings into question the role of skeletal muscle vascular beds in generating the sustained systemic haemodynamic effects induced by relaxin.

Iron-deficiency anaemia in rural Cambodia: community trial of a novel iron supplementation technique

BACKGROUND More than 3.5 billion people are affected by iron-deficiency anaemia (IDA). Previous studies have shown that the use of iron pots in daily cooking ameliorates IDA. We report a study on the use of a novel treatment to address IDA in rural women in Cambodia, where the use of iron pots is not common. METHODS A community-wide randomized controlled trial was conducted in the village of Preak Ruessei, Kandal Province, Cambodia. Rural women (n = 189) were enrolled and randomly assigned by household to one of three groups: (i) control, (ii) iron treatment with no follow-up and (iii) iron treatment with follow-up visits to provide IDA education. Haemoglobin, serum iron and C-reactive protein concentrations were measured at baseline, 3 and 6 months. A reusable fish-shaped iron ingot was distributed to the two treatment groups and participants were directed to use them daily for cooking. We hypothesized that iron from the ingot would leach iron into food providing an effective iron source. RESULTS Blood iron levels were higher in women in the iron fish plus follow-up at 3 months compared with controls, but this was not maintained. At 6 months, haemoglobin and serum iron had fallen in all groups and the proportion of anaemic women had increased. CONCLUSIONS This study shows that the iron ingot was effective in the short but not longer-term against IDA. Though a novel treatment option, further research is warranted to determine bioavailability of leached iron and whether or not the surface area is large enough for sufficient iron leaching.