Immunotherapeutic Potential of Mollusk Hemocyanins in Murine Model of Melanoma.

The development of antitumor drugs and therapy requires new approaches and molecules, and products of natural origin provide intriguing alternatives for antitumor research. Gastropodan hemocyanins-multimeric copper-containing glycoproteins have been used in therapeutic vaccines and antitumor agents in many cancer models. MATERIALS AND METHODS: We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed. RESULTS: Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression. DISCUSSION: Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

Antitumor Properties of Epitope-Specific Engineered Vaccine in Murine Model of Melanoma.

Finding new effective compounds of natural origin for composing anti-tumor vaccines is one of the main goals of antitumor research. Promising anti-cancer agents are the gastropodan hemocyanins-multimeric copper-containing glycoproteins used so far for therapy of different tumors. The properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) upon their use as carrier-proteins in conjugated vaccines, containing ganglioside mimotope GD3P4 peptide, were studied in the developed murine melanoma model. Murine melanoma cell line B16F10 was used for solid tumor establishment in C57BL/6 mice using various schemes of therapy. Protein engineering, flow cytometry, and cytotoxicity assays were also performed. The administration of the protein-engineered vaccines RtH-GD3P4 or HaH-GD3P4 under the three different regimens of therapy in the B16F10 murine melanoma model suppressed tumor growth, decreased tumor incidence, and prolonged the survival of treated animals. The immunization of experimental mice induced an infiltration of immunocompetent cells into the tumors and generated cytotoxic tumor-specific T cells in the spleen. The treatment also generates significantly higher levels of tumor-infiltrated M1 macrophages, compared to untreated tumor-bearing control mice. This study demonstrated a promising approach for cancer therapy having potential applications for cancer vaccine research.

Structural, Thermal, and Storage Stability of Rapana Thomasiana Hemocyanin in the Presence of Cholinium-Amino Acid-Based Ionic Liquids.

Novel biocompatible compounds that stabilize proteins in solution are in demand for biomedical and/or biotechnological applications. Here, we evaluated the effect of six ionic liquids, containing mono- or dicholinium [Chol]1or2 cation and anions of charged amino acids such as lysine [Lys], arginine [Arg], aspartic acid [Asp], or glutamic acid [Glu], on the structure, thermal, and storage stability of the Rapana thomasiana hemocyanin (RtH). RtH is a protein with huge biomedicinal potential due to its therapeutic, drug carrier, and adjuvant properties. Overall, the ionic liquids (ILs) induce changes in the secondary structure of RtH. However, the structure near the Cu-active site seems unaltered and the oxygen-binding capacity of the protein is preserved. The ILs showed weak antibacterial activity when tested against three Gram-negative and three Gram-positive bacterial strains. On the contrary, [Chol][Arg] and [Chol][Lys] exhibited high anti-biofilm activity against E. coli 25213 and S. aureus 29213 strains. In addition, the two ILs were able to protect RtH from chemical and microbiological degradation. Maintained or enhanced thermal stability of RtH was observed in the presence of all ILs tested, except for RtH-[Chol]2[Glu].

Intensive therapy with gastropodan hemocyanins increases their antitumor properties in murine model of colon carcinoma.

Various natural compounds have been tested as anticancer therapeutics in clinical trials. Most promising direction for antitumor therapy is the use of substances which enhance the immune system response stimulating tumor-specific lymphocytes. Hemocyanins are large extracellular oxygen transport glycoproteins isolated from different arthropod and mollusk species which exhibit strong anticancer properties. Immunized in mammals they trigger Th1 immune response that promotes unspecific stimulation and adjuvant activity in experimental therapeutic vaccines for cancer and antibody development. In the present study we used two hemocyanins - one isolated from marine snail Rapana thomasiana (RtH) and another one, from the terrestrial snail Helix pomatia (HpH) which have been investigated by using different administration schedules (intensive and mild) in murine model of colon carcinoma. The treatment with RtH and HpH generated high levels of antitumor IgG antibodies, antibody-producing plasma cells and tumor-specific CTLs, stimulated secretion of proinflammatory cytokines, suppressed the manifestation of carcinoma symptoms as tumor growth and size, and prolonged the life span of treated mice. Our results showed a significant anti-cancer effect of RtH and HpH hemocyanins on a murine model of colon carcinoma with promising potential for immunotherapy in various schemes of administration based on cross-reactive tumor-associated epitopes.

Folate-conjugated Helix lucorum hemocyanin - preparation, stability, and cytotoxicity.

This is the first report on the modification of a hemocyanin from Helix lucorum (HlH), a large molluscan respiratory protein, with folic acid (FA). In a two-step synthetic reaction, we prepared samples of HlH conjugated with 20 and 50 FA residues denoted as FA-HlH-1 and FA-HlH-2, respectively. Comparison of the attenuated total reflectance-Fourier transform infrared spectra in the amide I band region showed a structural rearrangement in the HlH that is due to FA conjugation. The changes in the secondary structure were more noticeable for FA-HlH-2. The thermal stability of HlH was not significantly affected by the FA modification, which is consistent with the observed structural similarities with the native protein. Preliminary cytotoxicity assays showed that FA-HlH-1 and FA-HlH-2 stimulate fibroblast proliferation when applied in concentrations of 50 and 100 µg/well. A negligible reduction of fibroblast growth was observed only for FA-HlH-1 and FA-HlH-2, exposed to 200 µg/well for 48 h. We found that FA-HlH-2 exhibits a low to moderate cytotoxic effect on two breast cancer cell lines, which express folate receptors, a hormone-dependent (MCF-7) and a hormone-independent (MDA-MB-231). FA-HlH-2 protects nontransformed cells and affects only neoplastic cells, which could be an advantage, and the protein could have potential in combination with other chemotherapeutics.

Biophysical Properties and Cytotoxicity of Feruloylated Helix Lucorum Hemocyanin.

For the first time Helix lucorum hemocyanin (HlH) has been feruloylated. Two HlH conjugates with 40- and 120- ferulic acid residues were prepared, denoted as FA-HlH-1 and FA-HlH-2. Expectedly, the feruloylation of HlH induced a rearrangement of the protein molecule, a decrease in the ?-helical structure at the expense of ß-structures was observed. Besides, the FA-HlH conjugates were more prone to aggregation, which is probably due to the stabilization of the partially unfolded protein molecules by non-covalent bonding. Interestingly, the thermal stability of HlH was not affected by the modification. The native and feruloylated HlH were not toxic to normal fibroblasts (BJ cells). We observed a decrease in cell viability of breast cancer MCF-7 cells to about 66% after a 48h exposure to 70 µg/well of FA-HlH-2.

Calorimetric Study of Helix aspersa Maxima Hemocyanin Isoforms.

The thermal unfolding of hemocyanin isoforms, ß-HaH and αD+N -HaH, isolated from the hemolymph of garden snails Helix aspersa maxima, was studied by means of differential scanning calorimetry (DSC). One transition, with an apparent transition temperature (Tm ) at 79.88°C, was detected in the thermogram of ß-HaH in 20 mM HEPES buffer, containing 0.1 M NaCl, 5 mM CaCl2, and 5 mM MgCl2, pH 7.0, at scan rate of 1.0°C min-1. By means of successive annealing procedure, two individual transitions were identified in the thermogram of αD+N -HaH. Denaturation of both hemocyanins was found to be an irreversible process. The scan-rate dependence of the calorimetric profiles indicated that the thermal unfolding of investigated hemocyanins was kinetically controlled. The thermal denaturation of the isoforms ß-HaH and αD+N -HaH was described by the two-state irreversible model, and parameters of the Arrhenius equation were calculated.

Rapana thomasiana hemocyanin modified with ionic liquids with enhanced anti breast cancer activity.

This is the first study on the surface modification of a hemocyanin from marine snail Rapana thomasiana (RtH) with series of imidazolium-based amino acid ionic liquids [emim][AA]. We monitored the induced by [emim][AA] conformational changes in RtH molecule and evaluated the effect of these ionic liquids (ILs) on the protein thermal stability. The cytotoxicity of all obtained RtH-[emim][AA] complexes was assessed toward breast cancer cells (MCF-7) and murine fibroblasts (3T3). As a whole, even small amounts of the tested ILs altered the secondary structure of RtH. The thermal denaturation of RtH in presence of [emim][AA] displayed multi-component transitions, which were shifted toward lower temperatures in comparison to those estimated for the native RtH. The profiles of the RtH-IL calorimetric curves show a clear dependence on the structure of the added salts. In addition, all RtH-[emim][AA] complexes exhibited an enhanced antiprofilerative activity of toward MCF-7 cells in comparison to that of the native RtH. The best results are observed for RtH-[emim][Leu], RtH-[emim][Trp] or RtH-[emim][Ile], which applied in concentration of 700 µg/mL inhibited the MCF-7 cell viability (for 24h) by 66, 63 and 53%, respectively. In addition, these IL-RtH complexes were less cytotoxic to 3T3 cells, i.e. they exhibited some cell specificity.

Helix pomatia hemocyanin - a novel bio-adjuvant for viral and bacterial antigens.

BACKGROUND: New generated subunit vaccines are characterized by increased safety and lack of side effects, however they suffer from weak immunogenicity. The adjuvants are substances that have the ability to enhance the magnitude and duration of the immune response and to increase vaccine efficacy, but the different vaccines may require diverse adjuvants. The urgent need of novel adjuvant formulations occurs, thus ensuring protective cellular and humoral responses against infectious pathogens. The hemocyanins, oxygen binding copper proteins in the hemolymph of molluscs and arthropods, are widely used as peptide carriers and vaccine adjuvants. RESULTS: In the present study we promote the hemocyanin isolated from the terrestrial gastropod Helix pomatia (HPH) as bio-adjuvant, combined with standard antigens. The purified HPH combined with influenza virus hemagglutinin intersubunit peptide (IP) or with tetanus toxoid (TT) were used for immunization. Administration of tetanus toxoid combined with HPH in mice resulted in an increased number of anti-TT IgG producing plasmocytes and induced a significant increase of B and T cell proliferation. The level of the anti-TT IgG antibodies in mice sera was comparable to the group administered with TT+Al(OH)3. An immunization of experimental animals with IP combined with H. pomatia hemocyanin led to generation of strong anti-influenza cytotoxic response. CONCLUSION: The vaccination of mice demonstrates that the HPH is acceptable as a potential bio-adjuvant for subunit vaccines and it could be used as a natural adjuvant or protein carrier.

Anti-cancer properties of gastropodan hemocyanins in murine model of colon carcinoma.

BACKGROUND: Various immunotherapeutic approaches have been used for the treatment of cancer. A number of natural compounds are designed to repair, stimulate, or enhance the immune system response. Among them are the hemocyanins (Hcs) - extracellular copper proteins isolated from different arthropod and mollusc species. Hcs are oxygen transporter molecules and normally are freely dissolved in the hemolymph of these animals. Hemocyanins are very promising class of anti-cancer therapeutics due to their immunogenic properties and the absence of toxicity or side effects. KLH (Megathura crenulata hemocyanin) is the most studied molecule of this group setting a standard for natural carrier protein for small molecules and has been used in anti-tumor clinical trials. RESULTS: The Hcs isolated from marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix pomatia (HpH) express strong in vivo anti-cancer and anti-proliferative effects in the developed by us murine model of colon carcinoma. The immunization with RtH and HpH prolonged the survival of treated animals, improve humoral anti-cancer response and moderate the manifestation of C-26 carcinoma symptoms as tumor growth, splenomegaly and lung metastasis appearance. CONCLUSION: Hemocyanins are used so far for therapy of superficial bladder cancer and murine melanoma models. Our findings demonstrate a potential anti-cancer effect of hemocyanins on a murine model of colon carcinoma suggesting their use for immunotherapy of different types of cancer.

Radioprotective effect of Rapana thomasiana hemocyanin in gamma induced acute radiation syndrome.

The radioprotective effect of Rapana thomasiana hemocyanin (RtH) against radiation-induced injuries (stomach ulcers, survival time and endogenous haemopoiesis) and post-radiation recovery was investigated in male albino mice (C3H strain). Radiation course was in a dose of 7.5 Gy (LD 100/30 - dose that kills 100% of the mice at 30 days) from 137Cs with a dose of 2.05 Gy/min. Radiation injuries were manifested by inducing а hematopoietic form of acute radiation syndrome. RtH was administered intraperitoneally in a single dose of 50, 100, 150 and 200 mg/kg body weight (b. w.) once a day for five consecutive days before irradiation. The results obtained showed that radiation exposure led to (1) 100% mortality rate, (2) ulceration in the stomach mucosa and (3) decrease formation of spleen colonies as a marker of endogenous haemopoiesis. Administration of RtH at a dose of 200 mg/kg provided better protection against radiation-induced stomach ulceration, mitigated the lethal effects of radiation exposure and recovered endogenous haemopoiesis versus irradiated but not supplemented mice. It could be expected that RtH will find a use in mitigating radiation induced injury and enhanced radiorecovery.

Phenoloxidase activity of Helix aspersa maxima (garden snail, gastropod) hemocyanin.

The oxygen-transporting protein, hemocyanin (Hc), of the garden snail Helix aspersa maxima (HaH) was isolated and kinetically characterized. Kinetic parameters of the reaction of catalytic oxidation of catechol to quinone, catalyzed by native HaH were determined: the V max value amounted to 22 nmol min(-1) mg(-1), k cat to 1.1 min(-1). Data were compared to those reported for other molluscan Hcs and phenoloxidases (POs). The o-diphenoloxidase activity of the native HaH is about five times higher than the activity determined for the Hcs of the terrestrial snail Helix pomatia and of the marine snail Rapana thomasiana (k cat values of 0.22 and 0.25 min(-1), respectively). The K m values obtained for molluscan Hcs from different species are comparable to those for true POs, but the low catalytic efficiency of Hcs is probably related to inaccessibility of the active sites to potential substrates. Upon treatment of HaH with subtilisin DY, the enzyme activity against substrate catechol was considerably increased. The relatively high proteolytically induced o-diPO activity of HaH allowed using it for preparation of a biosensor for detection of catechol.

Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin.

The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K(m) value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k(cat) is highest for catechol (2.44 min(-1) versus 0.67 min(-1) for l-Dopa and 0.71 min(-1) for dopamine). On treatment with 4mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T(m)~80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T(m)~92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.

Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin.

Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV-vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60°C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80°C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal ß-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90°C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.

Marine gastropod hemocyanins as adjuvants of non-conjugated bacterial and viral proteins.

Killed viral vaccines and bacterial toxoids are weakly immunogenic. Numerous compounds are under evaluation as immunological adjuvants and peptide-carriers to improve the immune response. The hemocyanins, giant extracellular copper proteins in the blood of many mollusks, are widely used as immune stimulants. In the present study we investigated the adjuvant properties of hemocyanins isolated from marine gastropods Rapana thomasiana and Megathura crenulata. An immunization with Influenza vaccine or tetanus toxoid combined with Rapana thomasiana hemocyanin (RtH) and Keyhole limpet hemocyanin (KLH) in mice induced an anti-influenza cytotoxic response lasting at least 5 months and an antibody response to viral proteins. The IgG antibody response to the tetanus toxoid (TT) combined with RtH or KLH was comparable to the response of the toxoid in complete Freund's adjuvant. The results obtained demonstrate that the both hemocyanins are acceptable as potential bio-adjuvants for subunit vaccines.

Influence of limited proteolysis, detergent treatment and lyophilization on the phenoloxidase activity of Rapana thomasiana hemocyanin.

The intrinsic and inducible phenoloxidase (PO) activity of Rapana thomasiana hemocyanin (RtH) and its substructures were studied. With catechol as substrate, a weak o-diPO activity was measured for the didecameric RtH and its subunits. Some activation of the o-diPO activity of RtH was achieved by limited treatment with subtilisin and by incubation of RtH with 2.9 mM sodium dodecyl sulphate (SDS), suggesting an enhanced substrate access to the active sites. The highest artificial induction of o-diPO activity in RtH, however, was obtained by lyophilization of the protein. This is ascribed to conformational changes during the lyophilization process of the didecameric RtH molecules, affecting the accessibility of the active sites. These conformational changes must be very small, since Fourier-transform infrared and circular dichroism spectroscopies did not reveal any changes in secondary structure of lyophilized RtH. The difference in accessibility of the copper containing active site for substrates between catechol oxidase and functional unit RtH2-e was demonstrated by molecular modeling and surface area accessibility calculations. The low level of intrinsic PO activity in the investigated hemocyanin is related to the inaccessibility of the binuclear copper active sites to the substrates.

Modulation of the immune response using Rapana thomasiana hemocyanin.

We have investigated the non-specific immunostimulatory and specific immunomodulatory effects of hemocyanin from marine gastropod Rapana thomasiana (RtH). The purified RtH, its structural subunits RtH1 and RtH2 and a construct with influenza virus hemagglutinin intersubunit peptide (IP) were used in immunization protocols of Balb/c mice. Antibody formation against RtH, RtH1, RtH2, RtH-IP as well as anti-RtH IgG antibody isotypes were determined by ELISA. The immune homology between both subunits and the whole RtH molecule was investigated by cross-blotting technique. The retaining of the B-cell epitope of IP, coupled to the RtH was recognised by Western blot. The results obtained demonstrate that the immunization with RtH or its subunits in experimental models resulted in strong immune response in vivo. Common epitope of influenza A virus hemagglutinin jointed to RtH results in generation of molecule with increased immunogenicity. Our results are the first demonstration that RtH and/or its subunits could be used in different immunization protocols as an adjuvant or as a protein-carrier.

Spectroscopic properties and conformational stability of Concholepas concholepas hemocyanin.

The structure in solution and conformational stability of the hemocyanin from the Chilean gastropod mollusk Concholepas concholepas (CCH) and its structural subunits, CCH-A and CCH-B, were studied using fluorescence spectroscopy and differential scanning calorimetry (DSC). The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Besides tryptophan residues buried in the hydrophobic interior of the protein molecule also exposed fluorophores determine the fluorescence emission of the oxy- and apo-forms of the investigated hemocyanins. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-forms of CCH and its subunits. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophan residues in the respective apo-forms. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the CCH and its subunits exist in different conformations. The thermal denaturation of the hemocyanin is an irreversible process, under kinetic control. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Arrhenius equation parameter for the two-state irreversible model of the thermal denaturation of oxy-CCH at pH 7.2 was estimated. Both factors, oligomerization and the copper-dioxygen system at the active site, are important for stabilizing the structure of the hemocyanin molecule.

Involvement of glycan chains in the antigenicity of Rapana thomasiana hemocyanin.

Functional unit (FU) RtH2-e from Rapana thomasiana hemocyanin (Hc) was degraded into small fragments with chymotrypsin. The glycopeptides were separated from the non-glycosylated peptides by chromatography on Concanavalin-A-Sepharose and characterized by mass spectrometry. The glycan part of the glycopeptides (all with common peptide stretch of 14 amino acids) consists of the classical trimannosyl-N,N-diacetylchitobiose core for N-glycosylation, predominantly extended with a unique tetrasaccharide that is branched on fucose. In inhibition ELISA experiments, the glycopeptides interfered in the complex formation between FU RtH2-e and rabbit antibodies against Rapana Hc (about 30% of inhibition). The inhibition also was retained after treatment of the glycopeptides with pronase in order to completely destroy the peptide part. The inhibitory effect of the non-glycosylated peptides, on the other hand, was very low. This study thus demonstrates that the glycans attached to FU RtH2-e contribute to the antigenicity of Rapana Hc.

Conformational stabilization at the active site of molluskan (Rapana thomasiana) hemocyanin by a cysteine-histidine thioether bridge A study by mass spectrometry and molecular modeling.

In some type-3 copper proteins (molluskan hemocyanin, catechol oxidase and fungal tyrosinase) one of the histidine residues, liganding the Cu(A) atom of the dinuclear copper active site, is covalently linked to a cysteine residue by a thioether bridge. The purpose of this study was to disclose the function of this bridge. Mass spectral analysis of a peptide, isolated from Rapana thomasiana (gastropodan mollusk) hemocyanin, indicated a stabilization of the peptide structure in the region of the bridge. Molecular modeling of three thioether containing type-3 copper proteins using the dead-end elimination method showed that the concerned histidine would be very flexible if not linked to the cysteine. Also, the side chain orientation of the histidine is rather exceptional, as evidenced by statistical data from the protein databank. It is suggested that the role of the bridge is to fix the histidine in an orientation that is optimal for coordination of the Cu(A) atom.