lactoferricin-b and Melanoma

Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new

Topics: Adult; Animals; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Female; Humans; Lactoferrin; Lymphatic Metastasis; Melanoma; Mice; Mice, Inbred NOD; Pregnancy; Primary Cell Culture; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Xenograft Model Antitumor Assays

The aim of this study was to develop effective and specific anti-cancer drugs based on membrane active peptides. In previous studies we showed that human lactoferricin (hLFcin) derived peptides facilitate specific killing of cancer cells. These antitumor peptides were found by conventional melanoma two-dimensional (2D) cell cultures to induce apoptosis of cancer cells and to specifically target lipid phosphatidylserine located on the outside of cancer cell membranes. In order to have a more relevant in vitro model able to mimic the natural microenvironments of tumor tissues we established three-dimensional (3D) multicellular tumor spheroids (MCTS). We used a set of (retro) di-peptides derived from LF11, an 11 amino acid long fragment of hLFcin, which differed in peptide length, positive net charge and hydrophobicity and determined antitumor activity and non-specific toxicity on non-neoplastic cells using 2D and 3D model systems. 2D studies unveiled a correlation between length, positive net charge and hydrophobicity of peptides and their specific antitumor activity. (Retro) di-peptides as R-DIM-P-LF11-215 and DIM-LF11-322 with a net charge of +9 and moderate hydrophobicity exhibited the highest specific antitumor activity. Further evaluation of the peptides anticancer activity by 3D in vitro studies confirmed their higher activity and cancer specificity compared to their parent R-DIM-P-LF11, with the exception of DIM-LF11-339. This highly hydrophobic peptide caused cell death mainly at the border of tumor spheroids indicating that too high hydrophobicity may prevent peptides from reaching the center of the spheroids.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Humans; Lactoferrin; Melanoma; Peptides; Spheroids, Cellular; Tumor Microenvironment

Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Membrane; Humans; Lactoferrin; Melanoma; Membrane Potential, Mitochondrial; Microscopy, Confocal; Microscopy, Electron, Transmission; Mitochondria; Peptides; Structure-Activity Relationship

Malignant melanoma is the most aggressive and deadliest form of skin cancer due to its highly metastatic potential, which calls for new and improved therapies. Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line of defense against pathogens, and several CAPs have shown promising potential as novel anticancer agents. Structure-activity relationship studies on the CAP bovine lactoferricin allowed us to de novo design short chemically modified lytic anticancer peptides. In the present study, we investigated the in vivo antitumor effects of LTX-315 against intradermally established B16 melanomas in syngeneic mice. Intratumoral administration of LTX-315 resulted in tumor necrosis and the infiltration of immune cells into the tumor parenchyma followed by complete regression of the tumor in the majority of the animals. LTX-315 induced the release of danger-associated molecular pattern molecules such as the high mobility group box-1 protein in vitro and the subsequent upregulation of proinflammatory cytokines such as interleukin (IL) 1β, IL6 and IL18 in vivo. Animals cured by LTX-315 treatment were protected against a re-challenge with live B16 tumor cells both intradermally and intravenously. Together, our data indicate that intratumoral treatment with LTX-315 can provide local tumor control followed by protective immune responses and has potential as a new immunotherapeutic agent.

Topics: Animals; Antimicrobial Cationic Peptides; Blotting, Western; Cattle; Cells, Cultured; Cytokines; Female; Hemolysis; Humans; Inflammation; Inflammation Mediators; Lactoferrin; Lymphocytes, Tumor-Infiltrating; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Nude; Peptide Fragments

Despite favorable advancements in therapy cancer is still not curative in many cases, which is often due to inadequate specificity for tumor cells. In this study derivatives of a short cationic peptide derived from the human host defense peptide lactoferricin were optimized in their selective toxicity towards cancer cells. We proved that the target of these peptides is the negatively charged membrane lipid phosphatidylserine (PS), specifically exposed on the surface of cancer cells. We have studied the membrane interaction of three peptides namely LF11-322, its N-acyl derivative 6-methyloctanoyl-LF11-322 and its retro repeat derivative R(etro)-DIM-P-LF11-322 with liposomes mimicking cancerous and non-cancerous cell membranes composed of PS and phosphatidylcholine (PC), respectively. Calorimetric and permeability studies showed that N-acylation and even more the repeat derivative of LF11-322 leads to strongly improved interaction with the cancer mimic PS, whereas only the N-acyl derivative also slightly affects PC. Tryptophan fluorescence of selective peptide R-DIM-P-LF11-322 revealed specific peptide penetration into the PS membrane interface and circular dichroism showed change of its secondary structure by increase of proportion of β-sheets just in the presence of the cancer mimic. Data correlated with in vitro studies with cell lines of human melanomas, their metastases and melanocytes, revealing R-DIM-P-LF11-322 to exhibit strongly increased specificity for cancer cells. This indicates the need of high affinity to the target PS, a minimum length and net positive charge, an adequate but moderate hydrophobicity, and capability of adoption of a defined structure exclusively in presence of the target membrane for high antitumor activity.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Membrane Permeability; Humans; Lactoferrin; Melanocytes; Melanoma; Models, Molecular; Neoplasm Metastasis; Peptide Fragments; Phosphatidylserines; Protein Structure, Secondary