Petra Popovics | University of Wisconsin-Madison (original) (raw)
Papers by Petra Popovics
Proceedings of the National Academy of Sciences, 2013
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Histochemistry and cell biology, Jan 15, 2015
Neurite growth is central to the formation and differentiation of functional neurons, and recentl... more 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 compa...
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Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2015
Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote grow... more 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 β-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 o...
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Oncotarget, Jan 14, 2015
We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective f... more 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. 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...
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INTRODUCTION: Increasing evidence suggests that prostate cancer cells undergo unique metabolic r... more 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.
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http://www.tandfonline.com/doi/full/10.4161/15384101.2015.945879
Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent ... more 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.
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Peptides, Feb 2014
In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on... more In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr(1)-JI-38), MR-361(N-Me-Tyr(1), D-Ala(2)-JI-38) and MR-367(N-Me-Tyr(1), D-Ala(2), Asn(8)-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr(1), showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr(1) and Arg(29)-NHCH3 as in MR-403 (N-Me-Tyr(1), D-Ala(2), Arg(29)-NHCH3-JI-38), MR-406 (N-Me-Tyr(1), Arg(29)-NHCH3-JI-38) and MR-409 (N-Me-Tyr(1), D-Ala(2), Asn(8), Arg(29)-NHCH3-JI-38), and MR-410 (N-Me-Tyr(1), D-Ala(2), Thr(8), Arg(29)-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa(30)-NH2 such as MR-326 (N-Me-Tyr(1), D-Ala(2), Arg(29), Apa(30)-NH2-JI-38), and with Gab(30)-NH2, as MR-502 (D-Ala(2), 5F-Phe(6), Ser(28), Arg(29),Gab(30)-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.
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Oncoscience. 2014 October 30; 1(10):665 -673.
Introduction: This study evaluated the effects of an ntagonistic analog of growth hormone-releasi... more Introduction: This study evaluated the effects of an ntagonistic analog of growth hormone-releasing hormone, MIA-602, on tumor growth, response to doxorubicin, expression of drug resistance genes, and efflux pump function in human triple negative breast cancers.
Methods: HCC1806 (doxorubicin-sensitive) and MX-1 doxorubicin-resistant), cell lines were xenografted into nude mice and treated with MIA-602, doxorubicin, or their combination. Tumors were evaluated for changes in volume and the expression of the drug resistance genes MDR1 and NANOG. In-vitro cell culture assays were used to analyze the effect of MIA-602 on efflux pump function.
Results: Therapy with MIA-602 significantly reduced tumor growth and enhanced the efficacy of doxorubicin in both cell lines. Control HCC1806 tumors grew by 435%, while the volume of tumors treated with MIA-602 enlarged by 172.2% and with doxorubicin by 201.6%. Treatment with the combination of MIA-602 and doxorubicin resulted in an increase in volume of only 76.2%. Control MX-1 tumors grew by 907%, while tumors treated with MIA-602 enlarged by 434.8% and with doxorubicin by 815%. The combination of MIA-602 and doxorubicin reduced the increase in tumor volume to 256%. Treatment with MIA-602 lowered the level of growth hormonereleasing hormone and growth hormone-releasing hormone receptors and significantly reduced the expression of multidrug resistance (MDR1) gene and the drug resistance
regulator NANOG. MIA-602 also suppressed efflux pump function in both cell lines.
Conclusions: We conclude that treatment of triple negative breast cancers with growth hormone-releasing hormone antagonists reduces tumor growth and potentiates the effects of cytotoxic therapy by nullifying drug resistance.
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World J Clin Urol. 2014 November 24; 3(3): 184-194. , Nov 24, 2014
Benign prostatic hyperplasia (BPH) is a pathologic condition of the prostate described as a subst... more Benign prostatic hyperplasia (BPH) is a pathologic condition of the prostate described as a substantial increase in its number of epithelial and stromal cells. BPH may significantly reduce the quality of life due to the initiation of bladder outlet obstruction and lower urinary tract syndromes. Current medical therapies mostly consist of inhibitors of 5α-reductase or α1-adrenergic blockers; their efficacy is often insufficient. Antagonistic analogs of neuropeptide hormones are novel candidates for the management of BPH. At first, antagonists of luteinizing hormone-releasing hormone (LHRH) have been introduced to the therapy aimed to reduce serum testosterone levels. However, they have also been found to produce an inhibitory activity on local LHRH receptors in the prostate as well as impotence and other related side effects. Since then, several preclinical and clinical studies reported the favorable effects
of LHRH antagonists in BPH. In contrast, antagonists of growth hormone-releasing hormone (GHRH) and gastrin-releasing peptide (GRP) have been tested only in preclinical settings and produce significant reduction in prostate size in experimental models of BPH. They act at least in part, by blocking the action of respective ligands produced locally on prostates through their respective receptors in the prostate, and by inhibition
of autocrine insulin-like growth factors-Ⅰ/Ⅱ and epidermal growth factor production. GHRH and LHRH antagonists were also tested in combination resulting in a cumulative effect that was greater than that of each alone. This article will review the numerous studies that demonstrate the beneficial effects of antagonistic analogs of LHRH, GHRH and GRP in BPH, as well as
suggesting a potential role for somatostatin analogs in experimental therapies.
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Journal of cellular biochemistry, 2013
Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing... more Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ∼1 µM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca2+. The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ∼1 µM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca2+. The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
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Proceedings of the National Academy of Sciences of the United States of America, 2014
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Proceedings of the National Academy of Sciences of the United States of America, 2013
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Peptides, 2013
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The Journal of endocrinology, 2011
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Journal of neurochemistry, 2013
Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of... more Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca²⁺ signals by stimulating further Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLCη2 expression increased two-fold during a 4-day differentiation period. Stable expression of PLCη2-targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA-treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two-hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co-localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA-induced phosphorylation of LIMK1 and cAMP-responsive element-binding protein was reduced in PLCη2 knock-down cells. The phosphoinositide-binding properties of the PLCη2 PH domain, assessed using a FRET-based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P₃. Immunostaining of PLCη2 together with PtdIns(3,4,5)P₃ in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P₃ levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.
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Journal of Alzheimer's disease : JAD, 2012
Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) is assoc... more Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) is associated with altered neuronal Ca²⁺ homeostasis. Ca²⁺ is known to accumulate in AD-affected neurons leading to deficits in neurological activity that are characteristic of the disease. This has led to the coinage of the term &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;calciumopathy&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;. However, the mechanisms of how and why Ca²⁺ levels are increased in the AD-affected brain remain unknown. Identifying these mechanisms is crucial for our ability to treat and understand the disease processes that are occurring. Recent work has revealed the existence of a novel signaling pathway that may contribute toward this calciumopathy. Phospholipase C-η enzymes have recently been implicated in the modulation and amplification of Ca²⁺ signals and are known to be expressed in neuronal regions of the brain associated with cognition and memory. In this article their potential impact on neuronal Ca²⁺ signaling and AD pathogenesis is discussed.
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Biochemical Society transactions, 2012
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Cellular Signalling, 2011
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Experientia, 2011
GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subf... more GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn2+ has been shown to be a potent stimulator of GPR39 activity via the Gαq, Gα12/13 and Gαs pathways. The potency and specificity of Zn2+ in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn2+ signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.
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Proceedings of the National Academy of Sciences, 2013
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Histochemistry and cell biology, Jan 15, 2015
Neurite growth is central to the formation and differentiation of functional neurons, and recentl... more 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 compa...
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Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2015
Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote grow... more 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 β-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 o...
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Oncotarget, Jan 14, 2015
We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective f... more 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. 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...
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INTRODUCTION: Increasing evidence suggests that prostate cancer cells undergo unique metabolic r... more 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.
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http://www.tandfonline.com/doi/full/10.4161/15384101.2015.945879
Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent ... more 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.
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Peptides, Feb 2014
In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on... more In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr(1)-JI-38), MR-361(N-Me-Tyr(1), D-Ala(2)-JI-38) and MR-367(N-Me-Tyr(1), D-Ala(2), Asn(8)-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr(1), showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr(1) and Arg(29)-NHCH3 as in MR-403 (N-Me-Tyr(1), D-Ala(2), Arg(29)-NHCH3-JI-38), MR-406 (N-Me-Tyr(1), Arg(29)-NHCH3-JI-38) and MR-409 (N-Me-Tyr(1), D-Ala(2), Asn(8), Arg(29)-NHCH3-JI-38), and MR-410 (N-Me-Tyr(1), D-Ala(2), Thr(8), Arg(29)-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa(30)-NH2 such as MR-326 (N-Me-Tyr(1), D-Ala(2), Arg(29), Apa(30)-NH2-JI-38), and with Gab(30)-NH2, as MR-502 (D-Ala(2), 5F-Phe(6), Ser(28), Arg(29),Gab(30)-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.
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Oncoscience. 2014 October 30; 1(10):665 -673.
Introduction: This study evaluated the effects of an ntagonistic analog of growth hormone-releasi... more Introduction: This study evaluated the effects of an ntagonistic analog of growth hormone-releasing hormone, MIA-602, on tumor growth, response to doxorubicin, expression of drug resistance genes, and efflux pump function in human triple negative breast cancers.
Methods: HCC1806 (doxorubicin-sensitive) and MX-1 doxorubicin-resistant), cell lines were xenografted into nude mice and treated with MIA-602, doxorubicin, or their combination. Tumors were evaluated for changes in volume and the expression of the drug resistance genes MDR1 and NANOG. In-vitro cell culture assays were used to analyze the effect of MIA-602 on efflux pump function.
Results: Therapy with MIA-602 significantly reduced tumor growth and enhanced the efficacy of doxorubicin in both cell lines. Control HCC1806 tumors grew by 435%, while the volume of tumors treated with MIA-602 enlarged by 172.2% and with doxorubicin by 201.6%. Treatment with the combination of MIA-602 and doxorubicin resulted in an increase in volume of only 76.2%. Control MX-1 tumors grew by 907%, while tumors treated with MIA-602 enlarged by 434.8% and with doxorubicin by 815%. The combination of MIA-602 and doxorubicin reduced the increase in tumor volume to 256%. Treatment with MIA-602 lowered the level of growth hormonereleasing hormone and growth hormone-releasing hormone receptors and significantly reduced the expression of multidrug resistance (MDR1) gene and the drug resistance
regulator NANOG. MIA-602 also suppressed efflux pump function in both cell lines.
Conclusions: We conclude that treatment of triple negative breast cancers with growth hormone-releasing hormone antagonists reduces tumor growth and potentiates the effects of cytotoxic therapy by nullifying drug resistance.
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World J Clin Urol. 2014 November 24; 3(3): 184-194. , Nov 24, 2014
Benign prostatic hyperplasia (BPH) is a pathologic condition of the prostate described as a subst... more Benign prostatic hyperplasia (BPH) is a pathologic condition of the prostate described as a substantial increase in its number of epithelial and stromal cells. BPH may significantly reduce the quality of life due to the initiation of bladder outlet obstruction and lower urinary tract syndromes. Current medical therapies mostly consist of inhibitors of 5α-reductase or α1-adrenergic blockers; their efficacy is often insufficient. Antagonistic analogs of neuropeptide hormones are novel candidates for the management of BPH. At first, antagonists of luteinizing hormone-releasing hormone (LHRH) have been introduced to the therapy aimed to reduce serum testosterone levels. However, they have also been found to produce an inhibitory activity on local LHRH receptors in the prostate as well as impotence and other related side effects. Since then, several preclinical and clinical studies reported the favorable effects
of LHRH antagonists in BPH. In contrast, antagonists of growth hormone-releasing hormone (GHRH) and gastrin-releasing peptide (GRP) have been tested only in preclinical settings and produce significant reduction in prostate size in experimental models of BPH. They act at least in part, by blocking the action of respective ligands produced locally on prostates through their respective receptors in the prostate, and by inhibition
of autocrine insulin-like growth factors-Ⅰ/Ⅱ and epidermal growth factor production. GHRH and LHRH antagonists were also tested in combination resulting in a cumulative effect that was greater than that of each alone. This article will review the numerous studies that demonstrate the beneficial effects of antagonistic analogs of LHRH, GHRH and GRP in BPH, as well as
suggesting a potential role for somatostatin analogs in experimental therapies.
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Journal of cellular biochemistry, 2013
Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing... more Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ∼1 µM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca2+. The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ∼1 µM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca2+. The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
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Proceedings of the National Academy of Sciences of the United States of America, 2014
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Proceedings of the National Academy of Sciences of the United States of America, 2013
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Peptides, 2013
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The Journal of endocrinology, 2011
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Journal of neurochemistry, 2013
Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of... more Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca²⁺ signals by stimulating further Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLCη2 expression increased two-fold during a 4-day differentiation period. Stable expression of PLCη2-targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA-treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two-hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co-localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA-induced phosphorylation of LIMK1 and cAMP-responsive element-binding protein was reduced in PLCη2 knock-down cells. The phosphoinositide-binding properties of the PLCη2 PH domain, assessed using a FRET-based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P₃. Immunostaining of PLCη2 together with PtdIns(3,4,5)P₃ in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P₃ levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.
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Journal of Alzheimer's disease : JAD, 2012
Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) is assoc... more Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) is associated with altered neuronal Ca²⁺ homeostasis. Ca²⁺ is known to accumulate in AD-affected neurons leading to deficits in neurological activity that are characteristic of the disease. This has led to the coinage of the term &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;calciumopathy&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;. However, the mechanisms of how and why Ca²⁺ levels are increased in the AD-affected brain remain unknown. Identifying these mechanisms is crucial for our ability to treat and understand the disease processes that are occurring. Recent work has revealed the existence of a novel signaling pathway that may contribute toward this calciumopathy. Phospholipase C-η enzymes have recently been implicated in the modulation and amplification of Ca²⁺ signals and are known to be expressed in neuronal regions of the brain associated with cognition and memory. In this article their potential impact on neuronal Ca²⁺ signaling and AD pathogenesis is discussed.
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Biochemical Society transactions, 2012
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Cellular Signalling, 2011
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Experientia, 2011
GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subf... more GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn2+ has been shown to be a potent stimulator of GPR39 activity via the Gαq, Gα12/13 and Gαs pathways. The potency and specificity of Zn2+ in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn2+ signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.
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