Leptin Peptide Research Articles

Relationship between craving and plasma leptin concentrations in patients with cocaine addiction

BackgroundThere is robust evidence indicating an overlap between neurobiological circuitry and pathways that regulate addictions and those that regulate appetite and food intake. Rodent work suggests a role of the appetitive peptide leptin in cocaine-seeking behaviours. The goal of this study was to investigate the possible relationship between plasma leptin concentrations and cocaine craving and use in patients seeking treatment for cocaine dependence. MethodsPatients (N=43) with a DSM-IV diagnosis of cocaine dependence were studied before starting detoxification (baseline; T0) and then again 14days after (T1; only those patients who abstained from cocaine during the study). Blood samples for plasma leptin concentrations were collected and cocaine craving was assessed using the Brief Cocaine Craving Questionnaire (Brief-CCQ). Food craving was also assessed using a food Visual Analogue Scale (f-VAS). Barratt Impulsiveness Scale (BIS) was used to evaluate impulsivity. ResultsPlasma leptin concentrations at T0 significantly correlated with baseline Brief-CCQ scores (r=0.34, p<0.05). Furthermore, plasma leptin concentrations at T1 significantly correlated with the baseline amount of cocaine used (r=0.5, p<0.05). There were no significant correlations between plasma leptin concentrations and f-VAS scores either at T0 or T1 (p’s>0.05). ConclusionsThe present study suggests a potential relationship between plasma leptin concentrations and cocaine craving and use. Future mechanistic studies are needed to determine whether manipulations of leptin signalling may lead to novel pharmacological approaches to treat cocaine addiction.

Central injection of a synthetic chicken partial leptin peptide does not affect food intake in chicks.

Leptin is an adipose tissue-derived hormone in mammals that plays an important role in whole body energy balance via its inhibitory effects on food intake mediated through the hypothalamus. Chicken leptin has a low sequence homology to mammalian leptin and its role in appetite regulation is not reported; hence the objective of this study was to determine effects of central injection of chicken leptin on food and water intake and associated behaviors in chicks. Chicks were intracerebroventricularly injected with 0 (vehicle), 0.3, 1.0, or 3.0 nmol of a synthetic chicken leptin partial peptide and food and water intake were monitored. There were no effects observed and a second experiment was conducted to evaluate food and water intake at higher doses; after injection of 0, 2.5, 5.0, or 10.0 nmol leptin. Again, there were no effects on food or water intake. In the third experiment, behaviors were analyzed during the first 30 min post-injection of vehicle or 10 nmol leptin. At 5 min post-injection, vehicle-injected chicks spent more time sitting than leptin-injected chicks. A wide dose range was evaluated however, the absence of an effect on food intake or behavior suggests that the chicken leptin peptide that was tested does not mediate effects on appetite in the brain and that chicken leptin likely has a different physiological role in birds than in mammals.

Abstract 3540: Targeting obesity-related cancer progression with novel leptin receptor antagonists

Abstract Obesity is a global health issue that has been identified as a risk factor for several types of cancer. High levels of body fat and circulating leptin are typical identifiers of obesity in humans and animals. Leptin is a 16kD protein hormone which is secreted by adipocytes, and maybe secreted from cancer cells, that functions to control satiation via leptin receptor binding. However, obese individuals often develop “leptin resistance”, which is a mechanism that leads to the accumulation of excess leptin. Increased binding of leptin to its receptor (OB-R) due to leptin resistance has been associated with disease progression and poor prognosis in human cancers. Our group has previously shown that leptin-mediated cancer cell proliferation is inhibited by the LPrA2 (leptin peptide receptor antagonist 2). We have prepared novel leptin antagonists and tested their ability to block leptin-induced survival and chemoresistance to Paclitaxel (TAX) and Gemcitabine (GEM) in cancer cells and derived tumorspheres (pancreatic: PANC-1, MiaPaca2 and triple negative breast cancer: MDA-MB231 and MDA-MB468). Western blot protein analyses showed the ability of the antagonists to specifically inhibit leptin-induced phosphorylation of STAT3, and expression of cyclin D, and Notch1 in cancer cells. Additionally, potential toxicity of antagonists was tested using MTT assay with concentrations up to 100X higher than the effective dose in non-malignant breast cells (MCF-10A). Data generated show no toxicity of the novel antagonists in vitro. Leptin-induced proliferation of breast cancer and pancreatic cancer cells (120-160%) was significantly inhibited by the novel antagonists. In addition, leptin-mediated progression of S-phase was also reduced by the antagonists. Leptin increased TAX and GEM chemoresistance in cells and tumorspheres that were efficiently inhibited by the antagonists. These data suggest that the new antagonists could be equally or more effective than LPrA2 for adjuvant treatment of cancer. Acknowledgements: This work has been supported by Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP, and facilities and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR 03034 and 1C06 RR18386). Citation Format: Crystal C. Lipsey, Adriana Harbuzariu, Ruben R. Gonzalez-Perez. Targeting obesity-related cancer progression with novel leptin receptor antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2017-3540

Hypoxia-Related Hormonal Appetite Modulation in Humans during Rest and Exercise: Mini Review.

Obesity is associated with numerous chronic ailments and represents one of the major health and economic issues in the modernized societies. Accordingly, there is an obvious need for novel treatment approaches. Recently, based on the reports of reduced appetite and subsequent weight loss following high-altitude sojourns, exposure to hypoxia has been proposed as a viable weight-reduction strategy. While altitude-related appetite modulation is complex and not entirely clear, hypoxia-induced alterations in hormonal appetite modulation might be among the key underlying mechanisms. The present paper summarizes the up-to-date research on hypoxia/altitude-induced changes in the gut and adipose tissue derived peptides related to appetite regulation. Orexigenic hormone ghrelin and anorexigenic peptides leptin, glucagon-like peptide-1, peptide YY, and cholecystokinin have to-date been investigated as potential modulators of hypoxia-driven appetite alterations. Current evidence suggests that hypoxia can, especially acutely, lead to decreased appetite, most probably via reduction of acylated ghrelin concentration. Hypoxia-related short and long-term changes in other hormonal markers are more unclear although hypoxia seems to importantly modulate leptin levels, especially following prolonged hypoxic exposures. Limited evidence also suggests that different activity levels during exposures to hypoxia do not additively affect hormonal appetite markers. Although very few studies have been performed in obese/overweight individuals, the available data indicate that hypoxia/altitude exposures do not seem to differentially affect appetite regulation via hormonal pathways in this cohort. Given the lack of experimental data, future well-controlled acute and prolonged studies are warranted to expand our understanding of hypoxia-induced hormonal appetite modulation and its kinetics in health and disease.

Oral delivery of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, synthetic peptide leptin mimetics: Immunofluorescent localization in the mouse hypothalamus

This study describes the localization of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, synthetic peptide leptin mimetics, in the hypothalamus of Swiss Webster and C57BL/6J wild-type mice, leptin-deficient ob/ob mice, and leptin-resistant diet-induced obese (DIO) mice. The mice were given [D-Leu-4]-OB3 or MA-[D-Leu-4]-OB3 in 0.3% dodecyl maltoside by oral gavage. Once peak serum concentrations were reached, the mice received a lethal dose of pentobarbital and were subjected to intracardiac perfusion fixation. The brains were excised, post-fixed in paraformaldehyde, and cryo-protected in sucrose. Free-floating frozen coronal sections were cut at 25-µm and processed for imaging by immunofluorescence microscopy. In all four strains of mice, dense staining was concentrated in the area of the median eminence, at the base and/or along the inner wall of the third ventricle, and in the brain parenchyma at the level of the arcuate nucleus. These results indicate that [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 cross the blood-brain barrier and concentrate in an area of the hypothalamus known to regulate energy balance and glucose homeostasis. Most noteworthy is the localization of [D-Leu-4]-OB3 immunoreactivity within the hypothalamus of DIO mice via a conduit that is closed to leptin in this rodent model, and in most cases of human obesity. Together with our previous studies describing the effects of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on energy balance, glucose regulation, and signal transduction pathway activation, these findings are consistent with a central mechanism of action for these synthetic peptide leptin mimetics, and suggest their potential usefulness in the management of leptin-resistant obesity and type 2 diabetes in humans.

Nanoparticle-linked antagonist for leptin signaling inhibition in breast cancer.

AIMTo develop a leptin peptide receptor antagonist linked to nanoparticles and determine its effect on viability of breast cancer cells.METHODSThe leptin antagonist, LPrA2, was coupled via EDAC [1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide] to iron oxide nanoparticles (IONP-LPrA2) to increase its efficacy. IONP-LPrA2 conjugation was confirmed by Western blot and nanoparticle tracking analysis. Human triple negative breast cancer (TNBC) MDA-MB-231, HCC1806 and estrogen receptor positive (ER+) MCF-7 cells were analyzed for the expression of the leptin receptor, Ob-R. The effects of leptin and antagonist on levels of leptin-induced STAT3 phosphorylation and cyclin D1, cell cycle progression, cell proliferation, and tumorsphere formation in breast cancer cells were determined. Doses of the chemotherapeutics [cisplatin (Cis), cyclophosphamide (CTX), doxorubicin (Dox) and paclitaxel (PTX)] to effectively reduce cell viability were calculated. The effects of combination treatments of IONP-LPrA2 and chemotherapeutics on cell viability were determined.RESULTSWestern blot analysis of coupling reaction products identified IONP-LPrA2 at approximately 100 kD. IONP-LPrA2 significantly decreased leptin-induced pSTAT3 levels in HCC1806 cells and drastically decreased cyclin D1 levels in all cell lines. IONP-LPrA2 significantly reduced leptin-induced S phase progression and cell proliferation in all breast cancer cell lines and the formation of tumorspheres in MDA-MB-231 cells. Also, IONP-LPrA2 showed an additive effect on the reduction of breast cancer cell survival with chemotherapeutics. Cis plus IONP-LPrA2 produced a significant reduction in the survival of MDA-MB-231 and HCC1806 cells. CTX plus IONP-LPrA2 caused a significant decrease in the survival of MDA-MB-231 cells. Dox plus IONP-LPrA2 caused a marked reduction in the survival of HCC1806 cells. Although, PTX plus IONP-LPrA2 did not have a major effect on the viability of the breast cancer cells when compared to PTX alone.CONCLUSIONPresent data indicate that IONP-LPrA2 may be a useful adjuvant for chemotherapeutic treatment of breast cancer, particularly for TNBC which lacks targeted therapeutic options.

Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin’s actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin’s actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin’s effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.

P2X4 Receptor Reporter Mice: Sparse Brain Expression and Feeding-Related Presynaptic Facilitation in the Arcuate Nucleus.

P2X4 receptors are ATP-gated cation channels that are widely expressed in the nervous system. To identify P2X4 receptor-expressing cells, we generated BAC transgenic mice expressing tdTomato under the control of the P2X4 receptor gene (P2rx4). We found sparse populations of tdTomato-positive neurons in most brain areas with patterns that matched P2X4 mRNA distribution. tdTomato expression within microglia was low but was increased by an experimental manipulation that triggered microglial activation. We found surprisingly high tdTomato expression in the hypothalamic arcuate nucleus (Arc) (i.e., within parts of the neural circuitry controlling feeding). Immunohistochemistry and genetic crosses of P2rx4 tdTomato mice with cell-specific GFP reporter lines showed that the tdTomato-expressing cells were mainly AgRP-NPY neurons and tanycytes. There was no electrophysiological evidence for functional expression of P2X4 receptors on AgRP-NPY neuron somata, but instead, we found clear evidence for functional presynaptic P2X4 receptor-mediated responses in terminals of AgRP-NPY neurons onto two of their postsynaptic targets (Arc POMC and paraventricular nucleus neurons), where ATP dramatically facilitated GABA release. The presynaptic responses onto POMC neurons, and the expression of tdTomato in AgRP-NPY neurons and tanycytes, were significantly decreased by food deprivation in male mice in a manner that was partially reversed by the satiety-related peptide leptin. Overall, we provide well-characterized tdTomato reporter mice to study P2X4-expressing cells in the brain, new insights on feeding-related regulation of presynaptic P2X4 receptor responses, and the rationale to explore extracellular ATP signaling in the control of feeding behaviors. Cells expressing ATP-gated P2X4 receptors have proven problematic to identify and study in brain slice preparations because P2X4 expression is sparse. To address this limitation, we generated and characterized BAC transgenic P2rx4 tdTomato reporter mice. We report the distribution of tdTomato-expressing cells throughout the brain and particularly strong expression in the hypothalamic arcuate nucleus. Together, our studies provide a new, well-characterized tool with which to study P2X4 receptor-expressing cells. The electrophysiological studies enabled by this mouse suggest previously unanticipated roles for ATP and P2X4 receptors in the neural circuitry controlling feeding.

Abstract 1006: Novel adjuvants in triple negative breast cancer chemotherapy

Abstract Background: Obese patients develop more frequently triple negative breast cancer (TNBC) that has not target therapy and are commonly treated with chemotherapeutics. Leptin, whose levels are elevated in obesity, can induce TNBC proliferation, angiogenesis and resistance to chemotherapeutics. An antagonist for leptin receptor OB-R, leptin peptide receptor antagonist 2 (LPrA2), has been shown to block leptin signaling and decrease tumor progression. LPrA2 also countervails leptin induced resistance to chemotherapeutic agents. We used pegylation or iron oxide nanoparticle (IONPs) as adjuvants to increase LPrA2 solubility, stability and effectiveness. Hypothesis: Conjugated LPrA2 will decrease TNBC cells proliferation, expression of leptin target molecules and tumorigenesis. Methods: Human TNBC and murine breast adenocarcinoma derived cell line E0771 (progesterone and HER2 receptor negative) were used. The E0771 cell line was generated from an estrogen receptor positive (ER+) mammary adenocarcinoma isolated from a C57BL/6J mouse. E0771 was made insensitive to estrogen by treatment with drug Tamoxifen (TAM) mimicking TNBC. To specifically assess the role of RBP-Jk (CBS/CSL, an essential transcription factor for Notch signaling) the CRISPR/Cas9 system was used to disrupt RBP-JK gene expression in both wild-type and TAM treated E0771 cells. Cells were treated with chemotherapeutics (Paclitaxel, Doxorubicine, Ciclophosphamide) in presence of leptin and LPrA2-conjugates. The expression of leptin-targeted molecules, Breast Cancer Stem Cells (BCSC) and EMT markers were analyzed after cell treatment. Results: Chemotherapeutics effects on proliferation, survival and molecular markers were modified by the addition of LPrA2-conjugates. Notch molecules (receptors and ligands) were downregulated by LPrA2 treatment. Conclusion: The current data indicate that Leptin inhibition could be used as an adjuvant for chemotherapeutic treatments in TNBC patients. LPrA2-conjugates could increase the efficacy of chemotherapy. Acknowledgements: This work was partially supported by the National Institute of Health and National Cancer Institute Grant U54 CA118638, NIH/SBIR 1R41CA183399-01A1, and DOD Idea Award BC W81XWH-13-1-0382 to RRGP; and facilities, and support services at Morehouse School of Medicine (NIH RR03034 and 1C06 RR18386) and NIH/NCRR grant 1G12RR026250-03. Citation Format: Antonio Rampoldi, Adriana Harbuzariu, Tia L. Harmon, Ruben R. Gonzalez-Perez. Novel adjuvants in triple negative breast cancer chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1006.

Abstract 2079: IONP-LPrA2 and PEG-LPrA2 therapies for triple negative breast cancer

Abstract Background: Breast cancer (BC) is an epidemic in the US. It is estimated that there will be over 230,000 new BC diagnoses in 2016. Triple Negative Breast Cancer (TNBC) comprises ∼15% of BC cases and lacks targeted therapeutic options. Obesity and high leptin levels are associated with higher TNBC incidence and poorer patient outcomes. Overexpression of leptin and its receptor, Ob-R, induce BC cell growth and angiogenesis; therefore leptin/Ob-R may serve as a TNBC therapeutic target. We have developed a Leptin Peptide Receptor Antagonist, LPrA2, which effectively inhibits leptin signaling. To increase its efficacy LPrA2 was coupled to iron oxide nanoparticles (IONPs) and polyethylene glycol (PEG), and subsequently tested in vitro and in vivo in obese mice hosting syngeneic TNBC-like mammary tumors. Methods: The conjugation of LPrA2 to IONPs and PEG was confirmed by immunoblotting analysis. E0771, mouse BC cells, which are progesterone receptor and HER2 negative were made insensitive to estrogen stimuli by long-term treatment with Tamoxifen (TAM). Cell cycle and MTT assays were performed to determine the effectiveness of leptin signaling inhibition by conjugated LPrA2 in vitro. C57BL/6 female mice were fed a 60% fat diet to induce obesity. The obese mice were injected with E0771-TAM cells in the mammary fat pad and treated with IONP-LPrA2 and PEG-LPrA2 after tumor development. Obese mice treated with IONP-LPrA2 Scramble (Sc) and PEG-LPrA2 Sc served as negative controls. Results and Conclusion: IONP-LPrA2 and PEG-LPrA2 attenuated leptin signaling in vitro and decreased tumor growth and progression in vivo in comparison to the controls. These findings indicate that conjugated LPrA2 may serve as a targeted therapy for TNBC. IONP-LPrA2 may be especially useful in treating this more aggressive form of BC due to its ability to capture multiple LPrA2 peptides as well as its small and uniform particle size. Citation Format: Tia L. Harmon, Adriana Harbuzariu, Antonio Rampoldi, Courtney Dill, Viola Lanier, Danielle Daley-Brown, Crystal Lipsey, Cynthia Tchio, Lily Yang, Ruben Rene Gonzalez-Perez. IONP-LPrA2 and PEG-LPrA2 therapies for triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2079.

Novel leptin OB3 peptide-induced signaling and progression in thyroid cancers: Comparison with leptin.

Obesity results in increased secretion of cytokines from adipose tissue and is a risk factor for various cancers. Leptin is largely produced by adipose tissue and cancer cells. It induces cell proliferation and may serve to induce various cancers. OB3-leptin peptide (OB3) is a new class of functional leptin peptide. However, its mitogenic effect has not been determined. In the present study, because of a close link between leptin and the hypothalamic-pituitary-thyroid axis, OB3 was compared with leptin in different thyroid cancer cells for gene expression, proliferation and invasion. Neither agent stimulated cell proliferation. Leptin stimulated cell invasion, but reduced adhesion in anaplastic thyroid cancer cells. Activated ERK1/2 and STAT3 contributed to leptin-induced invasion. In contrast, OB3 did not affect expression of genes involved in proliferation and invasion. In vivo studies in the mouse showed that leptin, but not OB3, significantly increased circulating levels of thyrotropin (TSH), a growth factor for thyroid cancer. In summary, OB3 is a derivative of leptin that importantly lacks the mitogenic effects of leptin on thyroid cancer cells.

Abstract B15: Development of an innovative treatment for obesity-related triple-negative breast cancer

Abstract Background: Breast cancer is the second leading cause of cancer deaths in women in the US. Although Caucasian women have an increased risk of developing breast cancer, more African American women die from this disease. Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer which comprises ~18 % of breast cancer cases and is characterized by the lack of expression of the estrogen receptor (ER), progesterone receptor (PgR), and the human epidermal growth factor receptor B2 (Her2). Due to the lack of receptor expression there is no targeted therapy for this more aggressive form of breast cancer mainly affecting women under the age of 50, minorities, and those with a BRCA1 gene mutation. There are several risk factors for breast cancer, notably being overweight or obese. Obesity is associated with high levels of leptin, a cytokine produced by adipose tissue. Leptin upregulates Notch, interleukin 1 (IL-1), vascular endothelial growth factor (VEGF), and its receptor VEGFR2 which promote survival and angiogenesis. Leptin induces expression of Notch and VEGFR2 via the activation of the JAK2/STAT3 pathway. We have developed leptin peptide receptor antagonists linked to Iron Oxide Nanoparticles (IONP-LPrA2). IONP-LPrA2 has been shown to inhibit leptin signaling and decrease expression of its targets in cancer cells. Methods: Human breast cancer cells were cultured in MEM with 10% fetal bovine serum and maintained in an incubator at 37⁰C with 5% CO2. Cells were cultured in 6 well culture plates and grown to 70-80% confluence. They were then treated with leptin and IONP-LPrA2. Subsequently, the cells were analyzed for leptin target expression with the Cellometer Vision Image Cytometer ® and by Immunoblotting analysis. Results: IONP-LPrA2 was shown to abrogate leptin-induced JAK2/STAT3, Notch 1, and VEGFR2 expression in the breast cancer cell lines. Conclusion: IONP-LPrA2 inhibits leptin signaling and decreases expression of leptin-induced target proteins. This data suggests that IONP-LPrA2 may serve as a treatment for breast cancer patients by minimizing the harmful effects of leptin signaling. IONP-LPrA2 may also act as a targeted therapy for those suffering from TNBC. Acknowledgements: This work was partially supported by the DOD W81XWH-13-1-0382; NIH/SBIR 1R41CA183399-01A1; Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center Partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP, and facilities and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR03034 and 1C06 RR18386). Citation Format: Tia Harmon, Viola Lanier, Adriana Harbuzariu, Lily Yang, Ruben R. Gonzalez-Perez. Development of an innovative treatment for obesity-related triple-negative breast cancer. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B15.

Abstract 3909: Iron oxide nanoparticle-leptin receptor antagonist: A novel targeted adjuvant therapy for triple negative breast cancer

Abstract Background: Leptin is a cytokine secreted by adipose tissue that binds its receptor, OB-R, leading to increased growth and survival of cancer cells. High levels of leptin have been correlated with poorer outcomes in breast cancer patients, particularly those afflicted with triple negative breast cancer (TNBC). We have created a leptin peptide receptor antagonist, LPrA2, which has been shown to block leptin signaling in several cancer types. To increase its effectiveness, LPrA2 was coupled to iron oxide nanoparticles (IONPs). We aim to determine if IONP-LPrA2 will serve as an adjuvant treatment, when combined with chemotherapeutics, for human TNBC and mouse TNBC-like (Tamoxifen-resistant) breast cancer cells E0771-TAM. Methods: Conjugation of LPrA2 to IONPs was confirmed by Western Blot and inhibition assays. To evaluate the effects of IONP-LPrA2 in mouse TNBC-like cells, the E0771 cell line (progesterone receptor and HER2 negative) was made insensitive to estrogen stimuli by long-term treatment with Tamoxifen (TAM). Human TNBC and E0771 cells, both responsive and unresponsive to estrogen (E0771-TAM), were treated with recombinant leptin and culture medium containing fetal bovine serum plus chemotherapeutics and IONP-LPrA2. Cell cycle progression, cell viability and apoptosis were analyzed using the Cellometer Vision Image Cytometer ®. Results: IONP-LPrA2 was found to cause significant decrease in DNA synthesis during the S phase of the cell cycle in TNBC and TNBC-like cells when compared to E0771 cells. Additionally, IONP-LPrA2 showed additive effects on reduction of cell viability and apoptosis when combined with chemotherapeutics. Conclusion: These findings indicate that IONP-LPrA2 may be useful in the treatment and prevention of TNBC. Further, IONP-LPrA2 treatment may increase the effectiveness and allow for reduction of the dosage of chemotherapeutics. This will in turn attenuate undesired side effects of chemotherapy. These results may be beneficial for breast cancer patients, particularly those with TNBC who are overweight or obese lacking targeted therapy thus suffering the poorest outcomes. Citation Format: Tia L. Harmon, Adriana Harbuzariu, Lily Yang, Ruben R. Gonzalez-Perez. Iron oxide nanoparticle-leptin receptor antagonist: A novel targeted adjuvant therapy for triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3909. doi:10.1158/1538-7445.AM2015-3909

Cloning, tissue distribution and effects of fasting on mRNA expression levels of leptin and ghrelin in red-bellied piranha (Pygocentrus nattereri)

cDNAs encoding the appetite regulating peptides leptin and ghrelin were isolated in red-bellied piranha (Characiforme, Serrasalmidae) and their mRNA tissue and brain distributions examined. When compared to other fish, the sequences obtained for all peptides were most similar to that of other Characiforme fish and Siluriformes. All peptides were widely expressed within the brain and in several peripheral tissues, including gastrointestinal tract. In order to better understand the role of these peptides in the regulation of feeding of red-bellied piranha, the mRNA expression levels of leptin and ghrelin were examined in both brain and intestine, in fed and 7-day fasted fish. No significant differences in expression were seen in whole brain for either peptide. Within the intestine, there was a decrease in leptin mRNA expression and an increase in ghrelin mRNA expression in fasted fish, compared to fed fish. The results suggest that leptin and ghrelin might play a major role in the regulation of feeding and energy homeostasis of red-bellied piranha and this role might be more prominent in the intestine than in the brain.

Abstract P5-05-10: Leptin peptide receptor antagonist linked to nanoparticles: A novel adjuvant therapy for triple negative breast cancer

Abstract Background: Obesity and high leptin levels are strongly associated with breast cancer, relapse, drug resistance, and poor patient outcomes. Over expression of leptin and its receptor, Ob-R, induce cell proliferation, angiogenesis, and metastasis in Triple Negative Breast Cancer (TNBC). This aggressive form of the disease has no targeted therapy and chemotherapeutics show several undesirable side effects. We have created a Leptin Peptide Receptor Antagonist, LPrA2, which has been shown to effectively prevent leptin signaling. LPrA2 was coupled to iron oxide nanoparticles (IONPs) and used to determine its potential use as an adjuvant to chemotherapeutics. Methods: IONPs, bound to LPrA2, were confirmed by Western Blot. TNBC cells were then treated with IONP-LPrA2 plus Cisplatin, Doxorubicin, Paclitaxel, Cyclophosphamide, and Sunitinib. Subsequently, the TNBC cells were analyzed for proliferation, cell cycle progression, and apoptosis with the Cellometer Vision Image Cytometer ®. Results: IONP-LPrA2 was found to cause a greater decrease in DNA synthesis during the S phase of the cell cycle in TNBC cells than LPrA2 alone. Additionally, IONP-LPrA2 when combined with chemotherapeutics or anti-angiogenic drugs showed synergistic effects on cell proliferation and apoptosis. Conclusion: These findings indicate that IONP-LPrA2 may be useful in the prevention and treatment of TNBC. Further IONP-LPrA2 treatment may increase the efficiency of chemotherapeutics. These results could be particularly relevant for obese patients, whom show high incidence and the poorest outcome of TNBC. Acknowledgements: This work was partially supported by the National Institutes of Health and National Cancer Institute Grant 1SC1CA138658-05 and U54 CA118638, and DOD Idea Award BC 123427 to RRGP; and facilities, and support services at Morehouse School of Medicine (NIH RR03034 and 1C06 RR18386) and NIH/NCRR grant 1G12RR026250-03. Citation Format: Tia L Harmon, Adriana Harbuzariu, Courtney D Dill, Lily Yang, Ruben R Gonzalez-Perez. Leptin peptide receptor antagonist linked to nanoparticles: A novel adjuvant therapy for triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-10.

Combinatorial gene construct and non-viral delivery for anti-obesity in diet-induced obese mice

The combinatorial peptidergic therapy of islet amyloid polypeptide (IAPP) and leptin (LEP) analogues was once an optimistic option in treating obese animals and patients. However, the need for frequent administrations and its negative side effects prevent it from being a viable choice. Here, we developed a combinatorial gene therapy of IAPP and LEP, where two genes are inserted into a single plasmid with self-cleaving furin and 2A sites to treat diet-induced obese (DIO) mice. The developed plasmid DNA (pDNA) individually produced both IAPP and LEP peptides in vitro and in vivo. The pDNA was delivered with a non-viral polymeric carrier, and its once-a-week administrations demonstrated a synergistic loss of body weight and significant reductions of fat mass, blood glucose, and lipid levels in DIO mice. The results suggest that the combinatorial gene therapy would have higher potential than the peptidergic approach for future translation due to its improved practicability.

Leptin And Ghrelin – The New Old Players In Obesity And Depression

Summary The participation of the anorexigenic peptide leptin and the orexigenic peptide ghrelin in the pathogenesis of metabolic syndrome and obesity is well studied. In this review, we are taking a look at the structure, anatomical expression, regulation, receptors and physiological functions of these two neuropeptides. Leptin is produced almost exclusively in adipose tissue. It acts on the brain and is a key element in long-term regulation of energy balance. Leptin suppresses appetite and reduces body weight. Besides its central effects, important aspects of its action on peripheral tissues have been discovered recently: direct regulation of immune cells, pancreatic beta cells, adipocytes and muscle cells. Ghrelin is an endogenous ligand for the active form of the growth hormone receptor (GHS-R1a) and stimulates food intake and growth hormone secretion. We focus on the role of leptin and ghrelin in central nervous system neural mechanisms that are associated with depression. Studying new aspects of these two neuropeptides aims to expand our knowledge of the pathogenesis and therapeutic approaches to diseases with which they are associated: obesity, depression, type 2 diabetes, essential hypertension, and more.

ChemInform Abstract: Therapeutic Potential of Leptin Receptor Modulators

AbstractReview: 95 refs.

Appetite Response among Those Susceptible or Resistant to Obesity

An alternative approach in determining cause, treatment, and prevention of obesity is to study those who appear resistant to the obesogenic environment. We examined appetite responses in 33 obesity resistant individuals (ORI) versus 28 obesity susceptible individuals (OSI). Fingerprick blood samples to measure ghrelin, total peptide YY (PYY), leptin, glucose, and insulin along with appetite ratings were collected at baseline and 15, 30, 60, 120, and 180 min following consumption of a standardized meal. Fasting, area under the curve (AUC), peak/nadir, and time to peak/nadir were compared. Participants completed the three factor eating questionnaire (TFEQ). No significant differences were observed for ghrelin or PYY. Higher leptin concentrations in the OSI disappeared after controlling for percent body fat (%BF). Significant differences in appetite ratings included a lower hunger nadir among OSI compared with ORI (P = 0.017). Dietary restraint (P < 0.001) and disinhibition (P < 0.001) were lower in ORI compared with OSI, with and without adjustment for %BF. Given the differential body weight of the study groups, similar observed ghrelin concentrations were unexpected, perhaps indicating OSI and ORI respond differently to the same ghrelin concentration. Also ORI response to hunger appears different as they exhibit lower levels of dietary restraint and disinhibition compared with OSI.

Peptide YY, acylated ghrelin and leptin concentrations following moderate and high intensity of exercise in males

Peptide YY, acylated ghrelin and leptin concentrations following moderate and high intensity of exercise in males