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.

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.

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.

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.

In vitro antiviral activity of a series of wild berry fruit extracts against representatives of Picorna-, Orthomyxo- and Paramyxoviridae.

Wild berry species are known to exhibit a wide range of pharmacological activities. They have long been traditionally applied for their antiseptic, antimicrobial, cardioprotective and antioxidant properties. The aim of the present study is to reveal the potential for selective antiviral activity of total methanol extracts, as well as that of the anthocyanins and the non-anthocyanins from the following wild berries picked in Bulgaria: strawberry (Fragaria vesca L.) and raspberry (Rubus idaeus L.) of the Rosaceae plant family, and bilberry (Vaccinium myrtillis L.) and lingonberry (Vaccinium vitis-idaea L) of the Ericaceae. The antiviral effect has been tested against viruses that are important human pathogens and for which chemotherapy and/or chemoprophylaxis is indicated, namely poliovirus type 1 (PV-1) and coxsackievirus B1 (CV-B1) from the Picornaviridae virus family, human respiratory syncytial virus A2 (HRSV-A2) from the Paramyxoviridae and influenza virus A/H3N2 of Orthomyxoviridae. Wild berry fruits are freeze-dried and ground, then total methanol extracts are prepared. Further the extracts are fractioned by solid phase extraction and the non-anthocyanin and anthocyanin fractions are eluted. The in vitro antiviral effect is examined by the virus cytopathic effect (CPE) inhibition test. The results reveal that the total extracts of all tested berry fruits inhibit the replication of CV-B1 and influenza A virus. CV-B1 is inhibited to the highest degree by both bilberry and strawberry, as well as by lingonberry total extracts, and influenza A by bilberry and strawberry extracts. Anthocyanin fractions of all wild berries strongly inhibit the replication of influenza virus A/H3N2. Given the obtained results it is concluded that wild berry species are a valuable resource of antiviral substances and the present study should serve as a basis for further detailed research on the matter.

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.