Julia Olah

Efficacy of a leptin receptor antagonist peptide in a mouse model of triple-negative breast cancer

Triple-negative breast cancers, which represent 10-20% of all mammary tumours, are characterised by the aggressive phenotype, are often found in younger women and have been associated with poor prognosis. Obesity increases the risk for triple-negative breast cancer development. Because triple-negative breast cancer patients are unresponsive to current targeted therapies and other treatment options are only partially effective, new pharmacological modalities are urgently needed. Here we examined if the leptin (obesity hormone) receptor is a viable target for the treatment of this cancer subtype. In human triple-negative breast cancer tissues, the leptin receptor was expressed in 92% (64/69) and leptin in 86% (59/69) of cases. In a model triple-negative breast cancer cell line MDA-MB-231, the leptin receptor antagonist peptide Allo-aca inhibited leptin-induced proliferation at 50 pM concentration. In an MDA-MB-231 orthotopic mouse xenograft model, Allo-aca administered subcutaneously significantly extended the average survival time from 15.4 days (untreated controls) to 24 and 28.1 days at 0.1 and 1mg/kg/day doses, respectively. In parallel, conventional treatment with 1mg/kg/day intraperitoneal cisplatin prolonged the average survival time to 18.6 days, while administration of 20mg/kg/day oral Tamoxifen (negative control) had no significant survival effects relative to controls. In normal CD-1 mice, Allo-aca produced no systemic toxicity up to the highest studied subcutaneous bolus dose of 50mg/kg, while, as expected, it induced a modest 6-10% body weight increase. Our results indicate that leptin receptor antagonists could become attractive options for triple-negative breast cancer treatment, especially in the obese patient population.

Thioacetamide-induced hepatic fibrosis in transforming growth factor beta-1 transgenic mice

Objective Transforming growth factor beta-1 (TGF-β1) is thought to be one of the most important factors affecting the development of fibrotic processes in the liver. Aim To discover whether endogenously higher TGF-β1 production influences the progression and reversibility of liver fibrosis in mice. Method We compared thioacetamide-induced liver fibrosis between wild-type and transgenic mice overexpressing active TGF-β1 in the liver. Hepatic fibrosis was detected on histological sections, and fibrotic areas were measured by means of morphometric analysis. We also performed Northern blot hybridisation and gelatine zymography to improve our understanding of the process. Results The fibrotic process was faster in the transgenic animals, and regression after the withdrawal of the fibrogenic agent was slower. Fibrosis did not disappear completely from the TGF-β1 overexpressing mice, even at the endpoint of the experiment. Conclusion Since the increased TGF-β1 production in the liver slowed down the regression of the liver fibrosis, the behaviour of these transgenic mice is more similar to the human situation, where cirrhosis is irreversible. We propose that this transgenic model is more suitable for investigating fibrotic liver diseases than the experiments done previously on wild-type rodents.

Peptide-based leptin receptor antagonists for cancer treatment and appetite regulation

Leptin, a multifunctional hormone, controls various processes in both the central nervous system and in peripheral tissues. Because of the presence of multiple leptin/receptor (ObR) interaction sites and diverse leptin activities, the literature lacks truly monofunctional leptin protein derivatives or fragments. To date, selective ObR antagonists have not been reported. We developed short, pharmacologically advantageous peptide analogs of ObR‐binding site III of leptin that acted as selective ObR inhibitors without any partial agonistic activity. These reduced leptin‐dependent growth and signaling in cancer cell lines at picomolar and low nanomolar concentrations. In immunocompromised mice the peptides suppressed the growth of rapidly proliferating orthotopic human breast cancer xenografts by 50% when administered either intraperitoneally (i.p.) or subcutaneously (s.c.) for 38 days at a 0.1 mg/kg/day dose. The peptides were distributed to the brain, and when added to growing C57BL/6 normal mice i.p., s.c., or orally, the lead antagonist accelerated normal weight increase without producing any toxic effects. Weight gain increases could not be observed after 10–12 days of treatment indicating that the mice became resistant to the central nervous system activity of leptin antagonists. However, in normal growing rats the intranasal administration at 0.1 mg/kg/day for 20 days resulted in a 2% net total body weight gain without signs of resistance induction. In addition to the potential of these peptides in drug development against primary and metastatic tumors and cachexia, our data confirm that resistance to leptin resides at the blood‐brain barrier.

Leptin-based glycopeptide induces weight loss and simultaneously restores fertility in animal models

Aim: To design, manufacture and test a second generation leptin receptor (ObR) agonist glycopeptide derivative. The major drawback to current experimental therapies involving leptin protein is the appearance of treatment resistance. Our novel peptidomimetic was tested for efficacy and lack of resistance induction in rodent models of obesity and appetite reduction.

The antiproliferative action of a melphalan hexapeptide with collagenase-cleavable site

Purpose: The objective of the present study was to examine the relevance of collagenase in the antitumor action of a melphalan peptide (MHP) with a collagenase-cleavable sequence. The question was addressed as to whether collagenase may act as an activator or a target in the antiproliferative mechanism of MHP. Methods: Melphalan was inserted into peptides representing the sequence Pro-Gln-Gly-Ile-Ala.Gly of the collagenase-cleavable site in collagens. Changes in growth and collagenase IV activities of HT-1080, HT-29, HT-168, and MCF-7 cell cultures were investigated. Results: The present investigations provide data indicating that Pro-Gln-Gly-Ile-Mel-Gly (melphalan hexapeptide, MHP) is a substrate for both bacterial and 72-kDa type IV collagenases and that in this way it can generate Ile-Mel-Gly (melphalan tripeptide, MTP) of higher cytotoxic potency. Indeed, the formation of MTP was detected in the conditioned medium of HT-1080, a collagenase IV-producing human fibrosarcoma. In a comparison of equimolar concentrations of melphalan and its two peptide derivatives (MHP and MTP), superior antiproliferative action of MTP was seen in HT-29, HT-1080, and HT-168 tumor cell cultures. However, the relatively modest cytostatic actions of MHP were increased when bacterial collagenase was added to the cell cultures. After melphalan treatment, reduced levels of both 92 and 72-kDa type IV collagenases were seen in the HT-1080 cell cultures. However, the reduction of collagenase activity and the cell counts did not run parallel in the MTP- or MHP-treated cultures; indeed, collagenase activity related to cell numbers showed an elevated level. Conclusions: As the conversion of MHP to the more toxic MTP was detected in the presence of collagenases, it is possible that collagenase-directed activation of prodrugs may be a promising approach for the development of more selective cytostatic drugs against malignant tumors with high collagenase activities.

Role of the Basement Membrane in Tumor Cell Dormancy and Cytotoxic Resistance

Objectives and Methods: Tumor dormancy and resistance to cytotoxic agents are key limiting events in the treatment of malignant diseases. To determine whether both are influenced by the extracellular milieu in which tumors reside, HT1080 human fibrosarcoma, MCF-7 breast carcinoma and OSCORT osteosarcoma cell proliferation, viability, apoptosis and cytoreductive-treatment-induced death were investigated in the presence or absence of extracellular matrix (ECM). Results: ECM-adherent, but not plastic-adherent HT1080 cells formed a multicellular network accompanied by reduced proliferation and lowered DNA synthetic capacity. The number of cells in S-phase was dramatically reduced. Viable cells entered a state of dormancy reminiscent of that observed in the step of metastasis after extravasation, i.e. prior to the initiation of progressive growth. Such ECM-induced dormancy could be reversed by plating cells on plastic, but only after a 48-hour lag period. No difference was indicated in clonogenicity of HT1080 cells originated from plastic or ECM gel. However, the cells released from ECM gel showed significantly reduced migration ability. The resistance of anchored cells against cytotoxic damage was increased by ECM gel. Examination of cytoreductive treatment revealed that ECM adherence at the time of injury is partially protective, a property which was also moderately apparent when injured cells were transferred to the basement membrane. Conclusions: Taken together, these results suggest that the ECM plays a key role in tumor dormancy and cytotoxic resistance, both explorable at the molecular level using our in vitro model system.

Development of second generation peptides modulating cellular adiponectin receptor responses.

The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM—low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10–1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

Optimization of adiponectin‐derived peptides for inhibition of cancer cell growth and signaling

Adiponectin, an adipose tissue‐excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti‐cancer activities, partially by interfering with leptin‐induced signaling. Previously we identified the active site in the adiponectin protein, and generated both a nanomolar monomeric agonist of the adiponectin receptor (10‐mer ADP355) and an antagonist (8‐mer ADP400) to modulate various adiponectin receptor‐mediated cellular functions. As physiologically circulating adiponectin forms multimeric complexes, we also generated an agonist dimer with improved biodistribution and in vitro efficacy. In the current report, we attempted to optimize the monomeric agonist structure. Neither extension of the peptide up to 14‐mer analogs nor reinstallation of native residues in permissible positions enhanced significantly the activity profile. The only substitutions that resulted in 5–10‐fold improved agonistic activity were the replacement of turn‐forming Gly4 and Tyr7 residues with Pro and Hyp, respectively, yielding the more active native β‐sheet structure. All peptides retained good stability in human serum exhibiting half‐lives >2 h. The cellular efficacy and stability rankings among the peptides followed expected structure–activity relationship trends. To investigate whether simultaneous activation of adiponectin pathways and inhibition of leptin‐induced signals can result in cytostatic and anti‐oncogenic signal transduction processes, we developed a chimera of the leptin receptor antagonist peptide Allo‐aca (placed to the N‐terminus) and ADP355 (at the C‐terminus). The in vitro anti‐tumor activity and intracellular signaling of the chimera were dominated by the more active Allo‐aca component. The ADP355 part, however, reversed unfavorable in vivo metabolic effects of the leptin receptor antagonist.