CCR2+ monocytes/macrophages drive steroid hormone imbalance-related prostatic fibrosis.

Benign Prostatic Hyperplasia (BPH) is a complex condition leading to Lower Urinary Tract Symptoms in aging men, characterized by cellular proliferation, smooth muscle dysfunction, inflammation, and fibrosis. While BPH is known to involve heightened macrophage infiltration, the specific contribution of infiltrating monocytes/macrophages to the disease mechanism remains uncertain. This research explores the impact of reducing circulating monocytes and subsequently limiting their tissue infiltration by using Ccr2 knockout (Ccr2-KO) mice. Ccr2-KO and wild type mice were implanted with testosterone and estradiol (T + E2, 25 mg + 2.5 mg) pellets. Urinary function was assessed via weekly void spot assays over 12 weeks, and prostatic macrophage levels were visualized and quantified in tissue sections using an F4/80 antibody. Additionally, Ki-67 staining was used to evaluate cell proliferation, and picrosirius red staining to assess collagen accumulation. Increased voiding frequency which developed in T + E2 mice, was significantly ameliorated in Ccr2-KO mice, however, both Ccr2-KO and wild type (WT) mice showed increased bladder weights after three month, representing a hypertrophic response to bladder outlet obstruction. T + E2 substantially increased the density of macrophages in WT but not Ccr2-KO mouse prostate. Proliferation rate, as indicated by Ki-67 positivity, was elevated in the vental and anterior prostate lobes but was only marginally reduced in Ccr2-KO mice. Most importantly, a significant prostatic collagen accumulation was observed in WT mice that was markedly reduced by Ccr2 deficiency post T + E2 treatment. The absence of Ccr2 mitigates urinary dysfunction and alters prostatic macrophage levels and collagen accumulation in steroid hormone imbalance. These findings suggest a crucial role for monocyte infiltration, giving rise to macrophages or other cell derivatives, to drive fibrosis.

PROSTATE CELL HETEROGENEITY AND CXCL17 UPREGULATION IN MOUSE STEROID HORMONE IMBALANCE.

Benign prostatic hyperplasia (BPH) is a prevalent age-related condition often characterized by debilitating urinary symptoms. Its etiology is believed to stem from hormonal imbalance, particularly an elevated estradiol-to-testosterone ratio and chronic inflammation. Our previous studies using a mouse steroid hormone imbalance model identified a specific increase in macrophages that migrate and accumulate in the prostate lumen where they differentiate into lipid-laden foam cells in mice implanted with testosterone and estradiol pellets, but not in sham animals. The current study focused on further characterizing the cellular heterogeneity of the prostate in this model as well as identifying the specific transcriptomic signature of the recruited foam cells. Moreover, we aimed to identify the epithelia-derived signals that drive macrophage infiltration and luminal translocation. Male C57BL/6J mice were implanted with slow-release testosterone and estradiol pellets (T+E2) and harvested the ventral prostates two weeks later for scRNA-seq analysis, or performed sham surgery. We identified Ear2+ and Cd72+ macrophages that were elevated in response to steroid hormone imbalance, whereas a Mrc1+ resident macrophage population did not change. In addition, an Spp1+ foam cell cluster was almost exclusively found in T+E2 mice. Further markers of foam cells were also identified, including Gpnmb and Trem2, and GPNMB was confirmed as a novel histological marker with immunohistochemistry. Foam cells were also shown to express known pathological factors Vegf, Tgfb1, Ccl6, Cxcl16 and Mmp12. Intriguingly, a screen for chemokines identified the upregulation of epithelial-derived Cxcl17, a known monocyte attractant, in T+E2 prostates suggesting that it might be responsible for the elevated macrophage number as well as their translocation to the lumen. Our study identified macrophage subsets that respond to steroid hormone imbalance as well as further confirmed a potential pathological role of luminal foam cells in the prostate. These results underscore a pathological role of the identified prostate foam cells and suggests CXCL17-mediated macrophage migration as a critical initiating event.

The Multifaceted Role of Osteopontin in Prostate Pathologies.

The prostate gland, located beneath the bladder and surrounding the proximal urethra in men, plays a vital role in reproductive physiology and sexual health. Despite its importance, the prostate is vulnerable to various pathologies, including prostatitis, benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Osteopontin (OPN), a versatile protein involved in wound healing, inflammatory responses, and fibrotic diseases, has been implicated in all three prostate conditions. The role of OPN in prostatic pathophysiology, affecting both benign and malignant prostate conditions, is significant. Current evidence strongly suggests that OPN is expressed at a higher level in prostate cancer and promotes tumor progression and aggressiveness. Conversely, OPN is primarily secreted by macrophages and foam cells in benign prostate conditions and provokes inflammation and fibrosis. This review discusses the accumulating evidence on the role of OPN in prostatic diseases, cellular sources, and potential roles while also highlighting areas for future investigations.

Steroid hormone imbalance drives macrophage infiltration and Spp1/osteopontin+ foam cell differentiation in the prostate.

Benign prostatic hyperplasia (BPH) occurs progressively with aging in men and drives deteriorating symptoms collectively known as lower urinary tract symptoms (LUTS). Age-associated changes in circulating steroid hormones, and prostate inflammation have been postulated in the etiology of BPH/LUTS. The link between hormones and inflammation in the development of BPH/LUTS is conflicting because they may occur independently or as sequential steps in disease pathogenesis. This study aimed to decipher the prostatic immune landscape in a mouse model of lower urinary tract dysfunction (LUTD). Steroid hormone imbalance was generated by the surgical implantation of testosterone (T) and estradiol (E2) pellets into male C57BL/6J mice and gene expression analysis was performed on ventral prostates (VPs). These experiments identified an increase in the expression of macrophage markers and Spp1/osteopontin (OPN). Localization studies of OPN pinpointed that OPN+ macrophages travel to the prostate lumen and transition into lipid-accumulating foam cells. We also observed a significant increase in the number of tissue macrophages in the VP which was prevented in OPN-knockout (OPN-KO) mice. In contrast, mast cells, but not macrophages, were significantly elevated in the dorsal prostate of T + E2-treated mice which was diminished in OPN-KO mice. Steroid hormone implantation progressively increased urinary frequency, which was ameliorated in OPN-KO mice. Our study underscores the role of age-associated steroid hormone imbalances as a mechanism of expanding the prostatic macrophage population, their luminal translocation, and foam cell differentiation. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Synthesis of potent antagonists of receptors for growth hormone-releasing hormone with antitumor and anti-inflammatory activity.

The syntheses and biological evaluation of GHRH antagonists of AVR series with high anticancer and anti-inflammatory activities are described. Compared to our previously reported GHRH antagonist 602 of MIAMI series, AVR analogs contain additional modifications at positions 0, 6, 8, 10, 11, 12, 20, 21, 29 and 30, which induce greater antitumor activities. Five of nineteen tested AVR analogs presented binding affinities to the membrane GHRH receptors on human pituitary, 2-4-fold better than MIA-602. The antineoplastic properties of these analogs were evaluated in vitro using proliferation assays and in vivo in nude mice xenografted with various human cancer cell lines including lung (NSCLC-ADC HCC827 and NSCLC H460), gastric (NCI-N87), pancreatic (PANC-1 and CFPAC-1), colorectal (HT-29), breast (MX-1), glioblastoma (U87), ovarian (SK-OV-3 and OVCAR-3) and prostatic (PC3) cancers. In vitro AVR analogs showed inhibition of cell viability equal to or greater than MIA-602. After subcutaneous administration at 5 µg/day doses, some AVR antagonists demonstrated better inhibition of tumor growth in nude mice bearing various human cancers, with analog AVR-353 inducing stronger suppression than MIA-602 in lung, gastric, pancreatic and colorectal cancers and AVR-352 in ovarian cancers and glioblastoma. Both antagonists induced greater inhibition of GH release than MIA-602 in vitro in cultured rat pituitary cells and in vivo in rats. AVR-352 also demonstrated stronger anti-inflammatory effects in lung granulomas from mice with lung inflammation. Our studies demonstrate the merit of further investigation of AVR GHRH antagonists and support their potential use for clinical therapy of human cancers and other diseases.

Current research and future directions in non-malignant urologic research - proceedings of the annual CAIRIBU meeting.

The Annual Collaborating for the Advancement of Interdisciplinary Research (CAIRIBU) Meeting in 2022 highlighted basic, translational, and clinical non-malignant urology research within five main areas affecting the urinary tract: urinary dysfunction due to prostate disease, microbes and infection, bladder function and physiology, neurology and neuromuscular influences and calculi and obstruction. In this paper, we summarize main findings and future directions outlined by CAIRIBU-affiliated scientists who presented as part of the scientific sessions.

Osteopontin Deficiency Ameliorates Prostatic Fibrosis and Inflammation.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

Increased nuclear factor I/B expression in prostate cancer correlates with AR expression.

BACKGROUND: Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that modify AR function. We have shown that nuclear factor I/B (NFIB) regulates AR activity in androgen-dependent prostate cancer cells in vitro. However, the status of NFIB in prostate cancer was unknown. METHODS: We immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We interrogated publically available data sets in cBioPortal to correlate NFIB expression and AR and neuroendocrine prostate cancer (NEPCa) activity scores. We analyzed prostate cancer cell lines for NFIB expression via Western blot analysis and used nuclear and cytoplasmic fractionation to assess where NFIB is localized. We performed co-immunoprecipitation studies to determine if NFIB and AR interact. RESULTS: NFIB increased in the nucleus and cytoplasm of prostate cancer samples versus matched normal controls, independent of Gleason score. Similarly, cytoplasmic AR and synaptophysin increased in primary prostate cancer. We observed strong NFIB staining in primary small cell prostate cancer. The ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence in prostate cancer, once adjusted for tumor margin status. Cytoplasmic AR was an independent predictor of biochemical recurrence. There was no statistically significant difference between NFIB and synaptophysin expression in primary and castration-resistant prostate cancer, but cytoplasmic AR expression was increased in castration-resistant samples. In primary prostate cancer, nuclear NFIB expression correlated with cytoplasmic NFIB and nuclear AR, while cytoplasmic NFIB correlated with synaptophysin, and nuclear and cytoplasmic AR. In castration-resistant prostate cancer samples, NFIB expression correlated positively with an AR activity score, and negatively with the NEPCa score. In prostate cancer cell lines, NFIB exists in several isoforms. We observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant cell lines. We observed an interaction between AR and NFIB through co-immunoprecipitation experiments. CONCLUSION: We have described the expression pattern of NFIB in primary and castration-resistant prostate cancer and its positive correlation with AR. We have also demonstrated AR interacts with NFIB.

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia.

BACKGROUND: Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. METHODS: Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1ß and TGF-ß1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN. RESULTS: OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-ß1 stimulated OPN secretion by NHPrE-1 cells and both IL-1ß and TGF-ß1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines. CONCLUSIONS: OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

Agonists of growth hormone-releasing hormone (GHRH) inhibit human experimental cancers in vivo by down-regulating receptors for GHRH.

The effects of the growth hormone-releasing hormone (GHRH) agonist MR409 on various human cancer cells were investigated. In H446 small cell lung cancer (SCLC) and HCC827 and H460 (non-SCLC) cells, MR409 promoted cell viability, reduced cell apoptosis, and induced the production of cellular cAMP in vitro. Western blot analyses showed that treatment of cancer cells with MR409 up-regulated the expression of cyclins D1 and D2 and cyclin-dependent kinases 4 and 6, down-regulated p27kip1, and significantly increased the expression of the pituitary-type GHRH receptor (pGHRH-R) and its splice-variant (SV1). Hence, in vitro MR409 exerts agonistic action on lung cancer cells in contrast to GHRH antagonists. However, in vivo, MR409 inhibited growth of lung cancers xenografted into nude mice. MR409 given s.c. at 5 µg/day for 4 to 8 weeks significantly suppressed growth of HCC827, H460, and H446 tumors by 48.2%, 48.7%, and 65.6%, respectively. This inhibition of tumor growth by MR409 was accompanied by the down-regulation of the expression of pGHRH-R and SV1 in the pituitary gland and tumors. Tumor inhibitory effects of MR409 in vivo were also observed in other human cancers, including gastric, pancreatic, urothelial, prostatic, mammary, and colorectal. This inhibition of tumor growth parallel to the down-regulation of GHRH-Rs is similar and comparable to the suppression of sex hormone-dependent cancers after the down-regulation of receptors for luteinizing hormone-releasing hormone (LHRH) by LHRH agonists. Further oncological investigations with GHRH agonists are needed to elucidate the underlying mechanisms.

Growth hormone-releasing hormone antagonists reduce prostatic enlargement and inflammation in carrageenan-induced chronic prostatitis.

BACKGROUND: Inflammation plays a key role in the etiology of benign prostatic hyperplasia (BPH) through multiple pathways involving the stimulation of proliferation by cytokines and growth factors as well as the induction of the focal occurrence of epithelial-to-mesenchymal transition (EMT). We have previously reported that GHRH acts as a prostatic growth factor in experimental BPH and in autoimmune prostatitis models and its blockade with GHRH antagonists offer therapeutic approaches for these conditions. Our current study was aimed at the investigation of the beneficial effects of GHRH antagonists in λ-carrageenan-induced chronic prostatitis and at probing the downstream molecular pathways that are implicated in GHRH signaling. METHODS: To demonstrate the complications triggered by recurrent/chronic prostatic inflammation in Sprague-Dawley rats, 50 µL 3% carrageenan was injected into both ventral prostate lobes two times, 3 weeks apart. GHRH antagonist, MIA-690, was administered 5 days after the second intraprostatic injection at 20 µg daily dose for 4 weeks. GHRH-induced signaling events were identified in BPH-1 and in primary prostate epithelial (PrEp) cells at 5, 15, 30, and 60 min with Western blot. RESULTS: Inflammation induced prostatic enlargement and increased the area of the stromal compartment whereas treatment with the GHRH antagonist significantly reduced these effects. This beneficial activity was consistent with a decrease in prostatic GHRH, inflammatory marker COX-2, growth factor IGF-1 and inflammatory and EMT marker TGF-ß1 protein levels and the expression of multiple genes related to EMT. In vitro, GHRH stimulated multiple pathways involved in inflammation and growth in both BPH-1 and PrEp cells including NFκB p65, AKT, ERK1/2, EGFR, STAT3 and increased the levels of TGF-ß1 and Snail/Slug. Most interestingly, GHRH also stimulated the transactivation of the IGF receptor. CONCLUSIONS: The study demonstrates that GHRH antagonists could be beneficial for the treatment of prostatic inflammation and BPH in part by inhibiting the growth-promoting and inflammatory effects of locally produced GHRH.

A new approach to the treatment of acute myeloid leukaemia targeting the receptor for growth hormone-releasing hormone.

Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and acts on the pituitary gland to stimulate the release of growth hormone (GH). GHRH can also be produced by human cancers, in which it functions as an autocrine/paracrine growth factor. We have previously shown that synthetic antagonistic analogues of GHRH are able to successfully suppress the growth of 60 different human cancer cell lines representing over 20 cancers. Nevertheless, the expression of GHRH and its receptors in leukaemias has never been examined. Our study demonstrates the presence of GHRH receptor (GHRH-R) on 3 of 4 human acute myeloid leukaemia (AML) cell lines-K-562, THP-1, and KG-1a-and significant inhibition of proliferation of these three cell lines in vitro following incubation with the GHRH antagonist MIA-602. We further show that this inhibition of proliferation is associated with the upregulation of pro-apoptotic genes and inhibition of Akt signalling in leukaemic cells. Treatment with MIA-602 of mice bearing xenografts of these human AML cell lines drastically reduced tumour growth. The expression of GHRH-R was further confirmed in 9 of 9 samples from patients with AML. These findings offer a new therapeutic approach to this malignancy and suggest a possible role of GHRH-R signalling in the pathology of AML.

Antagonists of growth hormone-releasing hormone inhibit proliferation induced by inflammation in prostatic epithelial cells.

The etiology of benign prostatic hyperplasia (BPH) is multifactorial, and chronic inflammation plays a pivotal role in its pathogenesis. Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide that has been shown to act as paracrine/autocrine factor in various malignancies including prostate cancer. GHRH and its receptors are expressed in experimental models of BPH, in which antagonists of GHRH suppressed the levels of proinflammatory cytokines and altered the expression of genes related to epithelial-to-mesenchymal transition (EMT). We investigated the effects of GHRH antagonist on prostatic enlargement induced by inflammation. Autoimmune prostatitis in Balb/C mice was induced by a homogenate of reproductive tissues of male rats. During the 8-wk induction of chronic prostatitis, we detected a progressive increase in prostatic volume reaching 92% at week 8 compared with control (P < 0.001). Daily treatment for 1 mo with GHRH antagonist MIA-690 caused a 30% reduction in prostate volume (P < 0.05). Conditioned medium derived from macrophages increased the average volume of spheres by 82.7% (P < 0.001) and elevated the expression of mRNA for N-cadherin, Snail, and GHRH GHRH antagonist reduced the average volume of spheres stimulated by inflammation by 75.5% (P < 0.05), and TGF-ß2 by 91.8% (P < 0.01). The proliferation of primary epithelial cells stimulated by IL-17A or TGF-ß2 was also inhibited by 124.1% and 69.9%, respectively. GHRH stimulated the growth of BPH-1 and primary prostate spheres. This study provides evidence that GHRH plays important roles in prostatic inflammation and EMT and suggests the merit of further investigation to elucidate the effects of GHRH antagonists in prostatitis and BPH.

Synthesis and structure-activity studies on novel analogs of human growth hormone releasing hormone (GHRH) with enhanced inhibitory activities on tumor growth.

The syntheses and biological evaluations of new GHRH analogs of Miami (MIA) series with greatly increased anticancer activity are described. In the design and synthesis of these analogs, the following previous substitutions were conserved: D-Arg2, Har9, Abu15, and Nle27. Most new analogs had Ala at position 8. Since replacements of both Lys12 and Lys21 with Orn increased resistance against enzymatic degradation, these modifications were kept. The substitutions of Arg at both positions 11 and 20 by His were also conserved. We kept D-Arg28, Har29 -NH2 at the C-terminus or inserted Agm or 12-amino dodecanoic acid amide at position 30. We incorporated pentafluoro-Phe (Fpa5), instead of Cpa, at position 6 and Tyr(Me) at position 10 and ω-amino acids at N-terminus of some analogs. These GHRH analogs were prepared by solid-phase methodology and purified by HPLC. The evaluation of the activity of the analogs on GH release was carried out in vitro on rat pituitaries and in vivo in male rats. Receptor binding affinities were measured in vitro by the competitive binding analysis. The inhibitory activity of the analogs on tumor proliferation in vitro was tested in several human cancer cell lines such as HEC-1A endometrial adenocarcinoma, HCT-15 colorectal adenocarcinoma, and LNCaP prostatic carcinoma. For in vivo tests, various cell lines including PC-3 prostate cancer, HEC-1A endometrial adenocarcinoma, HT diffuse mixed ß cell lymphoma, and ACHN renal cell carcinoma cell lines were xenografted into nude mice and treated subcutaneously with GHRH antagonists at doses of 1-5µg/day. Analogs MIA-602, MIA-604, MIA-610, and MIA-640 showed the highest binding affinities, 30, 58, 48, and 73 times higher respectively, than GHRH (1-29) NH2. Treatment of LNCaP and HCT-15 cells with 5µM MIA-602 or MIA-690 decreased proliferation by 40%-80%. In accord with previous tests in various human cancer lines, analog MIA-602 showed high inhibitory activity in vivo on growth of PC-3 prostate cancer, HT-mixed ß cell lymphoma, HEC-1A endometrial adenocarcinoma and ACHN renal cell carcinoma. Thus, GHRH analogs of the Miami series powerfully suppress tumor growth, but have only a weak endocrine GH inhibitory activity. The suppression of tumor growth could be induced in part by the downregulation of GHRH receptors levels.

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis.

Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocin-induced NOD/SCID mice.

Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote growth, function, and engraftment of islet cells following transplantation. Here we evaluated recently synthesized GHRH agonists on the proliferation and biological functions of rat pancreatic ß-cell line (INS-1) and islets. In vitro treatment of INS-1 cells with GHRH agonists increased cell proliferation, the expression of cellular insulin, insulin-like growth factor-1 (IGF1), and GHRH receptor, and also stimulated insulin secretion in response to glucose challenge. Exposure of INS-1 cells to GHRH agonists, MR-356 and MR-409, induced activation of ERK and AKT pathways. Agonist MR-409 also significantly increased the levels of cellular cAMP and the phosphorylation of cAMP response element binding protein (CREB) in INS-1 cells. Treatment of rat islets with agonist, MR-409 significantly increased cell proliferation, islet size, and the expression of insulin. In vivo daily s.c. administration of 10 µg MR-409 for 3 wk dramatically reduced the severity of streptozotocin (STZ)-induced diabetes in nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice. The maximal therapeutic benefits with respect to the efficiency of engraftment, ability to reach normoglycemia, gain in body weight, response to high glucose challenge, and induction of higher levels of serum insulin and IGF1 were observed when diabetic mice were transplanted with rat islets preconditioned with GHRH agonist, MR-409, and received additional treatment with MR-409 posttransplantation. This study provides an improved approach to the therapeutic use of GHRH agonists in the treatment of diabetes mellitus.

New therapeutic approach to heart failure due to myocardial infarction based on targeting growth hormone-releasing hormone receptor.

BACKGROUND: We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair. METHODS AND RESULTS: H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF-α compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and pro-fibrotic systems, and by elevation of bone morphogenetic proteins. CONCLUSIONS: Treatment with GHRH agonists appears to reduce the inflammatory responses post-MI and may consequently improve mechanisms of healing and cardiac remodeling by regulating pathways involved in fibrosis, apoptosis and cardiac repair. Patients with cardiac dysfunction could benefit from treatment with novel GHRH agonists.

Targeting the 5'-AMP-activated protein kinase and related metabolic pathways for the treatment of prostate cancer.

INTRODUCTION: Increasing evidence suggests that prostate cancer cells undergo unique metabolic reprogramming during transformation. A master regulator of cellular homeostasis, 5'-AMP-activated protein kinase (AMPK), directs metabolic adaptation that supports the growth demands of rapidly dividing cancer cells. The utilization of AMPK as a therapeutic target may therefore provide an effective strategy in the treatment of prostate cancer. AREAS COVERED: Our review describes the regulation of AMPK by androgens and upstream kinases including the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in prostate cancer. Oncogenic, AMPK-regulated pathways that direct various metabolic processes are also addressed. Furthermore, we discuss the role of AMPK in growth arrest and autophagy as a potential survival pathway for cancer cells. In addition, by regulating non-metabolic pathways, AMPK may stimulate migration and mitosis. Finally, this review summarizes efforts to treat prostate cancer with pharmacological agents capable of modulating AMPK signaling. EXPERT OPINION: Current research is primarily focused on developing drugs that activate AMPK as a treatment for prostate cancer. However, oncogenic aspects of AMPK signaling calls for caution about employing such therapies. We think that inhibitors of CaMKK2 or AMPK, or perhaps the modulation of downstream targets of AMPK, will gain importance in the clinical management of prostate cancer.

Novel GHRH antagonists suppress the growth of human malignant melanoma by restoring nuclear p27 function.

Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitro using the MTS assay. The effect of MIA-690 (5 µg/day 28 d) was further evaluated in vivo in nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 µM, and 19.2% at 5 µM, P < 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P < 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P < 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitro and in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.