Lectin-anticancer peptide fusion demonstrates a significant cancer-cell-selective cytotoxic effect and inspires the production of "clickable" anticancer peptide in Escherichia coli.

Targeted killing of tumor cells while protecting healthy cells is the pressing priority in cancer treatment. Lectins that target a specific glycan marker abundant in cancer cells can be valuable new tools for selective cancer cell killing. The lectin Shiga-like toxin 1 B subunit (Stx1B) is an example that specifically binds globotriaosylceramide (CD77 or Gb3), which is overexpressed in certain cancers. In this study, a human lactoferricin-derived synthetic retro di-peptide R-DIM-P-LF11-215 with antitumor efficacy was fused to the lectin Stx1B to selectively target and kill Gb3+ cancer cells. We produced lectin-peptide fusion proteins in Escherichia coli, isolated them by Gb3-affinity chromatography, and assessed their ability to selectively kill Gb3+ cancer cells in a Calcein AM assay. Furthermore, to expand the applications of R-DIM-P-LF11-215 in developing therapeutic bioconjugates, we labeled R-DIM-P-LF11-215 with the unique reactive non-canonical amino acid Nε -((2-azidoethoxy)carbonyl)-L-lysine (AzK) at a selected position by amber stop codon suppression. The R-DIM-P-LF11-215 20AzK and the unlabeled R-DIM-P-LF11-215 parent peptide were produced as GST-fusion proteins for soluble expression in E. coli for the first time. We purified both variants by size-exclusion chromatography and analyzed their peptide masses. Finally, a cyanin 3 fluorophore was covalently conjugated to R-DIM-P-LF11-215 20AzK by strain-promoted alkyne-azide cycloaddition. Our results showed that the recombinant lectin-peptide fusion R-DIM-P-LF11-215-Stx1B killed >99% Gb3+ HeLa cells while Gb3-negative cells were unaffected. The peptides R-DIM-P-LF11-215 and R-DIM-P-LF11-215 20AzK were produced recombinantly in E. coli in satisfactory amounts and were tested functional by cytotoxicity and cell-binding assays, respectively.

FULL-MDS: Fluorescent Universal Lipid Labeling for Microfluidic Diffusional Sizing.

Microfluidic diffusional sizing (MDS) is a recent and powerful method for determining the hydrodynamic sizes and interactions of biomolecules and nanoparticles. A major benefit of MDS is that it can report the size of a fluorescently labeled target even in mixtures with complex, unpurified samples. However, a limitation of MDS is that the target itself has to be purified and covalently labeled with a fluorescent dye. Such covalent labeling is not suitable for crude extracts such as native nanodiscs directly obtained from cellular membranes. In this study, we introduce fluorescent universal lipid labeling for MDS (FULL-MDS) as a sparse, noncovalent labeling method for determining particle size. We first demonstrate that the inexpensive and well-characterized fluorophore, Nile blue, spontaneously partitions into lipid nanoparticles without disrupting their structure. We then highlight the key advantage of FULL-MDS by showing that it yields robust size information on lipid nanoparticles in crude cell extracts that are not amenable to other sizing methods. Furthermore, even for synthetic nanodiscs, FULL-MDS is faster, cheaper, and simpler than existing labeling schemes.

In Model, In Vitro and In Vivo Killing Efficacy of Antitumor Peptide RDP22 on MUG-Mel2, a Patient Derived Cell Line of an Aggressive Melanoma Metastasis.

The host defense derived peptide was assessed in different model systems with increasing complexity employing the highly aggressive NRAS mutated melanoma metastases cell line MUG-Mel2. Amongst others, fluorescence microscopy and spectroscopy, as well as cell death studies were applied for liposomal, 2D and 3D in vitro models including tumor spheroids without or within skin models and in vivo mouse xenografts. Summarized, MUG-Mel2 cells were shown to significantly expose the negatively charged lipid phosphatidylserine on their plasma membranes, showing they are successfully targeted by RDP22. The peptide was able to induce cell death in MUG-Mel2 2D and 3D cultures, where it was able to kill tumor cells even inside the core of tumor spheroids or inside a melanoma organotypic model. In vitro studies indicated cell death by apoptosis upon peptide treatment with an LC50 of 8.5 µM and seven-fold specificity for the melanoma cell line MUG-Mel2 over normal dermal fibroblasts. In vivo studies in mice xenografts revealed effective tumor regression upon intratumoral peptide injection, indicated by the strong clearance of pigmented tumor cells and tremendous reduction in tumor size and proliferation, which was determined histologically. The peptide RDP22 has clearly shown high potential against the melanoma cell line MUG-Mel2 in vitro and in vivo.

MUG Mel3 Cell Lines Reflect Heterogeneity in Melanoma and Represent a Robust Model for Melanoma in Pregnancy.

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 BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options-especially during pregnancy.

Effect of L- to D-Amino Acid Substitution on Stability and Activity of Antitumor Peptide RDP215 against Human Melanoma and Glioblastoma.

The study investigates the antitumor effect of two cationic peptides, R-DIM-P-LF11-215 (RDP215) and the D-amino acid variant 9D-R-DIM-P-LF11-215 (9D-RDP215), targeting the negatively charged lipid phosphatidylserine (PS) exposed by cancer cells, such as of melanoma and glioblastoma. Model studies mimicking cancer and non-cancer membranes revealed the specificity for the cancer-mimic PS by both peptides with a slightly stronger impact by the D-peptide. Accordingly, membrane effects studied by DSC, leakage and quenching experiments were solely induced by the peptides when the cancer mimic PS was present. Circular dichroism revealed a sole increase in ß-sheet conformation in the presence of the cancer mimic for both peptides; only 9D-RDP215 showed increased structure already in the buffer. Ex vitro stability studies by SDS-PAGE as well as in vitro with melanoma A375 revealed a stabilizing effect of D-amino acids in the presence of serum, which was also confirmed in 2D and 3D in vitro experiments on glioblastoma LN-229. 9D-RDP215 was additionally able to pass a BBB model, whereupon it induced significant levels of cell death in LN-229 spheroids. Summarized, the study encourages the introduction of D-amino acids in the design of antitumor peptides for the improvement of their stable antitumor activity.

Cytokine-Mediated Inflammation in the Oral Cavity and Its Effect on Lipid Nanocarriers.

Topical drug administration to the oral mucosa proves to be a promising treatment alternative for inflammatory diseases. However, disease-related changes in the cell barrier must be considered when developing such delivery systems. This study aimed at investigating the changes in the lining mucosa caused by inflammation and evaluating the consequences on drug delivery systems such as nanostructured lipid carriers (NLC). For this, TR146 cells were treated with inflammatory cytokines and bacterial components. Cell viability and integrity, reactive oxygen species (ROS), and interleukin (IL)-8 release were used as endpoints to assess inflammation. Translocation of phosphatidylserine, cytoskeletal arrangement, opening of desmosomes, and cell proliferation were examined. Transport studies with NLC were performed considering active and passive pathways. The results showed that IL-1ß and tumor necrosis factor α induced inflammation by increasing IL-8 and ROS production (22-fold and 2-fold). Morphologically, loss of cell-cell connections and formation of stress fibers and hyperplasia were observed. The charge of the cell membrane shifted from neutral to negative, which increased the absorption of NLC due to the repulsive interactions between the hydrophobic negative particles and the cell membrane on the one hand, and interactions with lipophilic membrane proteins such as caveolin on the other.

Design of human lactoferricin derived antitumor peptides-activity and specificity against malignant melanoma in 2D and 3D model studies.

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.

Human melanoma brain metastases cell line MUG-Mel1, isolated clones and their detailed characterization.

Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life. Treatment opportunities have been restricted until now and new therapy options are urgently required. Our focus was to reveal the potential heterogeneity of melanoma brain metastasis. We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers. Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones. Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model. As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity. Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.

Interaction of two antitumor peptides with membrane lipids - Influence of phosphatidylserine and cholesterol on specificity for melanoma cells.

R-DIM-P-LF11-322 and DIM-LF11-318, derived from the cationic human host defense peptide lactoferricin show antitumor activity against human melanoma. While R-DIM-P-LF11-322 interacts specifically with cancer cells, the non-specific DIM-LF11-318 exhibits as well activity against non-neoplastic cells. Recently we have shown that cancer cells expose the negatively charged lipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane, while non-cancer cells just expose zwitterionic or neutral lipids, such as phosphatidylcholine (PC) or cholesterol. Calorimetric and zeta potential studies with R-DIM-P-LF11-322 and cancer-mimetic liposomes composed of PS, PC and cholesterol indicate that the cancer-specific peptide interacts specifically with PS. Cholesterol, however, reduces the effectiveness of the peptide. The non-specific DIM-LF11-318 interacts with PC and PS. Cholesterol does not affect its interaction. The dependence of activity of R-DIM-P-LF11-322 on the presence of exposed PS was also confirmed in vitro upon PS depletion of the outer leaflet of cancer cells by the enzyme PS-decarboxylase. Further corresponding to model studies, cholesterol depleted melanoma plasma membranes showed increased sensitivity to R-DIM-P-LF11-322, whereas activity of DIM-LF11-318 was unaffected. Microscopic studies using giant unilamellar vesicles and melanoma cells revealed strong changes in lateral distribution and domain formation of lipids upon addition of both peptides. Whereas R-DIM-P-LF11-322 enters the cancer cell specifically via PS and reaches an intracellular organelle, the Golgi, inducing mitochondrial swelling and apoptosis, DIM-LF11-318 kills rapidly and non-specifically by lysis of the plasma membrane. In conclusion, the specific interaction of R-DIM-P-LF11-322 with PS and sensitivity to cholesterol seem to modulate its specificity for cancer membranes.

In vitro and in vivo cytotoxic activity of human lactoferricin derived antitumor peptide R-DIM-P-LF11-334 on human malignant melanoma.

Di-peptides derived from the human host defense peptide lactoferricin were previously described to specifically interact with the negatively charged lipid phosphatidylserine exposed by cancer cells. In this study one further derivative, namely R-DIM-P-LF11-334 is shown to exhibit even increased cancer toxicity in vitro and in vivo while non-neoplastic cells are not harmed. In liposomal model systems composed of phosphatidylserine mimicking cancerous and phosphatidylcholine mimicking non-cancerous membranes the specific interaction with the cancer marker PS was confirmed by specific induction of membrane perturbation and permeabilization in presence of the peptide. In vitro studies with cell lines of human malignant melanoma, such as A375, or primary cells of human melanoma metastases to the brain, as MUG Mel1, and non-neoplastic human dermal fibroblasts NHDF revealed high cytotoxic effect of R-DIM-P-LF11-334 on melanoma cells of A375 and MUG Mel1, whereas only minor effect on the dermal fibroblasts NHDF was observed, yielding an about 20-fold killing-specificity for A375 and MUG-Mel1. The LC50 values for melanoma A375 and MUG Mel1 were about 10 µM. Analysis of secondary structure of the peptide revealed an increase in the proportion of ß-sheets exclusively in presence of the cancer mimic. Stability studies further indicated a potential adequate stability in blood or under stringent conditions. Importantly the cytotoxic effect on cancer cells was also proven in vivo in mouse xenografts of human melanoma, where peptide treatment induced strong tumor regression and in average a tumor area reduction of 85% compared to tumors of control mice without peptide treatment.

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 µM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity.

Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells.

BACKGROUND AIMS: As angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells. METHODS: The usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs). RESULTS: Expression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities. CONCLUSIONS: The use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs.

Killing of melanoma cells and their metastases by human lactoferricin derivatives requires interaction with the cancer marker phosphatidylserine.

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 ß-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.

N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill bacteria by interacting with bacterial membrane lipids but only N-acylated peptides interact with both charged cardiolipin and zwitterionic phosphatidylethanolamine resulting in remodeling of the natural phospholipid domains in the E. coli membrane that leads to defects in cell division.

In toto differentiation of human amniotic membrane towards the Schwann cell lineage.

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100ß, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100ß was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.

Catalytic and structural role of a conserved active site histidine in berberine bridge enzyme.

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1-C2=O locus via interaction with the hydroxyl group at C2' of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.

Membrane-active host defense peptides--challenges and perspectives for the development of novel anticancer drugs.

Although much progress has been achieved in the development of cancer therapies in recent decades, problems continue to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs. Host defense peptides as effector molecules of innate immunity represent a novel strategy for the development of alternative anticancer drug molecules. These cationic amphipathic peptides are able to discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as phosphatidylserine (PS), sialic acid or heparan sulfate, which differ between cancer and non-cancer cells. Furthermore, an increased number of microvilli has been found on cancer cells leading to an increase in cell surface area, which may in turn enhance their susceptibility to anticancer peptides. Thus, part of this review will be devoted to the differences in membrane composition of non-cancer and cancer cells with a focus on the exposure of PS on the outer membrane. Normally, surface exposed PS triggers apoptosis, which can however be circumvented by cancer cells by various means. Host defense peptides, which selectively target differences between cancer and non-cancer cell membranes, have excellent tumor tissue penetration and can thus reach the site of both primary tumor and distant metastasis. Since these molecules kill their target cells rapidly and mainly by perturbing the integrity of the plasma membrane, resistance is less likely to occur. Hence, a chapter will also describe studies related to the molecular mechanisms of membrane damage as well as alternative non-membrane related mechanisms. In vivo studies have demonstrated that host defense peptides display anticancer activity against a number of cancers such as e.g. leukemia, prostate, ascite and ovarian tumors, yet so far none of these peptides has made it on the market. Nevertheless, optimization of host defense peptides using various strategies to enhance further selectivity and serum stability is expected to yield novel anticancer drugs with improved properties in respect of cancer cell toxicity as well as reduced development of drug resistance.

In search of a novel target - phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy.

This study was performed in the aim to identify potential targets for the development of novel therapy to treat cancer with poor outcome or treatment efficacy. We show that the negatively charged phospholipid phosphatidylserine (PS) is exposed in the outer leaflet of their plasma membrane not only in tumor cell lines, but also in metastases and primary cultures thereof, which contrasts with a lack of PS exposure by differentiated non-tumorigenic counterparts. Studied tumor cell lines were derived from non-tumorigenic and malignant melanomas, prostate- and renal cancer, glioblastoma and a rhabdomyosarcoma. Importantly, also metastases of melanoma expose PS and there is a correlation between malignancy of melanoma cell lines from different stages of tumor progression and PS exposure. The PS exposure we found was neither of apoptotic nor of experimental artificial origin. Finally potentially malignant and non-malignant cells could be differentiated by sorting of a primary cell culture derived from a glioblastoma based on PS exposure, which has so far not been possible within one culture due to lack of a specific marker. Our data provide clear evidence that PS could serve as uniform marker of tumor cells and metastases as well as a target for novel therapeutic approaches based on e.g. PS-specific host defense derived peptides.

Structural and mechanistic studies reveal the functional role of bicovalent flavinylation in berberine bridge enzyme.

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8alpha-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8alpha-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.

A concerted mechanism for berberine bridge enzyme.

Berberine bridge enzyme catalyzes the conversion of (S)-reticuline to (S)-scoulerine by formation of a carbon-carbon bond between the N-methyl group and the phenolic ring. We elucidated the structure of berberine bridge enzyme from Eschscholzia californica and determined the kinetic rates for three active site protein variants. Here we propose a catalytic mechanism combining base-catalyzed proton abstraction with concerted carbon-carbon coupling accompanied by hydride transfer from the N-methyl group to the N5 atom of the FAD cofactor.