BACKGROUND
cancer is one of the major reasons for human death in the last decade following cardiovascular diseases, infectious diseases and ischemic heart diseases.
current therapies of cancer include chemotherapy and radiotherapy although they both have severe side effects.
each type of cancer requires specific treatment, which explains the need for development of highly specific targeted anti-cancer agents  <cit> .
the bcg  vaccine for tuberculosis that contains attenuated mycobacterium bovis is one of the most extensively used immunotherapeutics based on its strong non-specific immunostimulatory properties  <cit> .
bcg is widely administrated in cases of colorectal, lung cancers and melanoma.
in the last years a number of therapeutic antibodies have been approved for clinical treatment in cases of breast cancer , non-hodgkin  and hodgkin lymphoma , colorectal cancer , chronic lymphocyte leukemia , and acute myelogenous leukemia   <cit> .
immunotherapy with cytokines is another option for cancer treatment.
il- <dig> in metastatic melanoma and renal cell carcinoma and ifn-alpha in stage iii melanoma have been permitted for cancer therapy  <cit> .
different cancer vaccines have been developed for prevention and treatment of malignant diseases but only sipuleucel-t is approved for therapy of advanced prostate cancer in case hormonal treatment is ineffective  <cit> .
many of the developed vaccines include cancer carbohydrate antigens chemically conjugated to carrier protein with or without other adjuvant .
today natural products and their compounds derived from fungi, plants or microbes are of great interest for an anti-cancer research.
this huge variety of chemical structures provides different mechanisms of action and specific effects used for anti-tumor therapy.
marine compounds are known to have a serious potential as anti-cancer drugs.
most of them are small molecules, inhibitors of key enzymes for carcinogenesis like matrix metalloproteinazes , hifs, topoisomerase, protein kinase c  or transcripion factors like nfkb  <cit> .
special attention is payed on marine-derived anti-angiogenesis products, which suppress and prevent the succesful formation of vascular system, supporting tumor growth and invasion.
the majority of these substances act via inhibition of enzymes or factors, crucial for the process of angiogenesis  <cit> .
another group of potential anti-cancer agents are the hemocyanins  - oligomeric copper-containing glycoproteins that function as oxygen carriers in the hemolymph of several molluscs and arthropods.
molluscan hcs have been studied intensively for many years with respect to their structure and function  <cit> .
the huge molecular size  of molluscan hcs, their xenogenic character and carbohydrate content have been implicated in inducing strong immune response in mammals, which has led to the biomedical and therapeutic application of these proteins.
thus, keyhole limpet hemocyanin  isolated from marine gastropod megathura crenulata is a well-established immune stimulant, hapten carrier and tumor vaccine carrier  <cit> .
it is known that hcs stimulate the immune system non- specifically by interacting with macrophages, polymorphonuclears, cd4+ and cd8+ cells giving rise to potent humoral and cellular immune response.
one possibility for their anti-cancer effect is that they generally enhance statement of immune system that helps the immune cells to recognize self from non-self and to eliminate non-self cancerous cells.
klh and cch  are intensively studied for their anti-cancer properties in superficial bladder cancer  and in melanoma models  <cit> .
recently, we have demonstrated that the hc isolated from marine gastropod rapana thomasiana  possesses a high immunogenicity as a single model antigen and can be used as a protein carrier for viral peptides from influenza hemagglutinin  <cit> .
our results have shown that rth is acceptable as a potential bio-adjuvant for subunit bacterial and viral vaccines  <cit> .
compared to the numerous studies on the structural organization and protein stability of the hc isolated from garden snail helix pomatia , its immunological properties have been less investigated  <cit> .
these results point on the potential properties of rth and hph as anti-cancer agents.
c- <dig> murine model of colorectal carcinoma is classical tumor model, developed in  <dig> and determinated as an undifferentiated grade iv carcinoma  <cit> .
c- <dig> cells display high tumorigenicity and low tendency to metastasize mainly in the lungs.
inoculated in syngeneic balb/c mice c- <dig> cells cause high mortality.
this model is used more than 35 years for studying the process of carcinogenesis and anti-cancer therapy.
the aim of the present work was to develop an experimental murine model of colon carcinoma and to investigate the anti-tumor activity of rth and hph.
RESULTS
hemocyanins analysis
figure  <dig> presents the synopsis of our results from the structural studies of the hc isolated from marine snail r. thomasiana.
the electron micrograph shows typical for gastropodan hcs didecamers .
the quaternary structure of molluscan hcs is a hollow cylindrical molecule, about 35 nm in diameter, with an internal collar complex.
3-d reconstruction of the rth molecule is shown on figure 1b  <cit> .
molecules of molluscan hcs are structured as decamers  or didecamers  of subunits with molecular mass of 350–450 kda.
the polypeptide chains of subunits contain seven or eight globular functional units , depending on the species.
fus are generally termed , starting from the n-terminus of the respective subunit .
the fus have a binuclear copper active site, capable of binding reversibly one oxygen molecule.
the active site consists of two closely spaced copper ions  each coordinated by three histidine ligands .
figure 1d shows the crystal structure of a fu rth2-e  <cit> .
the different type of fus share sequence identities of around 45%.
being similar in primary, tertiary and quaternary structure, molluscan hcs differ in their carbohydrate content ) and monosaccharide composition  <cit> .
most probably the specific carbohydrate content and composition of molluscan hcs are the structural basis for the difference in their antigenicity.figure  <dig>
molecular structure of gastropod hcs.
tem of negatively stained rth molecules;  3-d reconstruction of the hollow cylindrical rth didecamer; side view and top view  <cit> ;  scheme of the subunit containing eight different functional units;  x-ray structure of functional unit rth2-e  <cit> .
hemocyanins increase tumor cells apoptosis in vitro
to study the apoptotic effect of gastropodan hcs on tumor cells we tested their ability to influence directly the c- <dig> cells by apoptosis assay.
c- <dig> carcinoma cells were incubated with different amounts of hph and rth .
the cells were further washed and the surface expression of phosphatidylserine was studied at the 24 h, 48 h and 72 h of incubation.
the levels of induced early and late apoptosis as well as necrosis were analyzed by annexin-v/propidium iodide staining and flow cytometry.
a significant increase in percentage of early apoptosis and a slight increase of late apoptosis were observed after 48 hours of incubation with all tested amounts of hcs.
in contrast, no obvious effect of the treatment was found on the 24 h of incubation .
high level of early apoptosis and considerable enhancement in late apoptosis was observed on the 72 h of incubation .
dose-dependant quantitative corelation between induced apoptosis and hcs added to the cells was not found at all tested time points.figure  <dig>
the gastropod hcs rth and hph induce apoptosis of c- <dig> carcinoma cell line
in vitro
.
the c- <dig> cells were incubated in the presence of different concentrations of hph and rth  for 72 hours at 37°С/5% co <dig>  at the end of the cultivation the cells were double-stained with annexin v-fitc /propidium iodide and were analyzed by flow cytometry.
percentage of stained cells is shown in each quadrant.
the panels represent the results from  <dig> independent experiments.
tumor model development
for selection of proper c- <dig> murine colon carcinoma model  <dig> groups of mice challenged with different cell numbers were monitored .
formation of solid tumors in all mice in the group as well as 0% survival within 3 months after cells challenge was the leading parameters for successful model establishment.
in the group inoculated with 5× <dig> cells palpable solid tumors were observed 2 weeks after c- <dig> challenge followed by 0% survival 3 months after cell challenge, which is acceptable for the model requested.
in the other groups mice developed tumors and died too early or survived for longer period, so they weren’t appropriate for further studies.
phenotyping of spleen and tumor suspensions
we have performed an immune system modulation analysis during tumor development when the tumor model was established to demonstrate the immune reaction to the tumor growth.
spleens from mice challenged with 5× <dig> c- <dig> carcinoma cells or pbs treated only were isolated and analyzed by flow cytometry.
we observed a decrease of cd4+, cd8+ , cd19+  as well as cd94+  and slight increase of cd14+ cells  compared to pbs treated balb/c mice .figure  <dig>
facs analysis of spleen and tumor suspensions.
spleens  and solid tumors  from representative number of animals  were excised and the cell suspensions were incubated with one of the following anti-mouse antibodies - anti-cd4-apc, anti-cd8-fitc, anti-cd19-pe, anti-cd94-pe or anti-cd14-apc.
ten thousands cells were analyzed from each sample by flow cytometry.
data are representative of at least  <dig> experiments.
a cell suspension prepared from solid tumors of the studied mice showed presence of cd4+, cd8+, cd19+, cd94+ as well as cd14+ cell populations in the tumor microenvironment .
these results suggest a movement of immune cells populations to the developed tumors and a relevant decrease of the same populations in the spleen of the animals.
cytokine detection
tnfα, ifnγ, il <dig> and il <dig> levels were measured in mice sera using enzyme linked immunosorbent assay  kits.
challenge with 5× <dig> c- <dig> carcinoma cells and treatment with rth or hph did not result in a significant difference in the cytokine production between the groups .
detection of anti- c- <dig> antibodies formation
groups of balb/c mice were treated with hcs from gastropods r. thomasiana and h. pomatia.
two groups  were injected with rth or hph 2 weeks before tumor cells inoculation and after establishment of palpable solid tumors the mice were injected intratumoraly once weekly with rth or hph.
the other two groups  were inoculated with 5× <dig> cells/mouse of c- <dig> cells and after palpable solid tumor development the animals were injected once a week with rth or hph.
mice injected with c- <dig> cells only or pbs injected healthy mice were used as controls.
collected sera from all studied groups were tested for presence of anti-c- <dig> antibodies.
all groups developed anti-c- <dig> igg antibodies compared to the pbs treated control group .
the titer of these antibodies decreased in c- <dig> group without hemocyanin treatment during tumor development.figure  <dig>
detection of anti- c- <dig> antibodies and anti-c- <dig> antibody cross-reactivity.
anti- c- <dig> antibody levels in c- <dig> bearing-mice treated with rth or hph  were measured by elisa.
mice inoculated with c- <dig> cells without hcs or mice treated only with pbs were used as control groups.
the results are presented as relative units  calculated according to the titer of standard anti-c- <dig> antibodies used for elisa.
the data are presented as mean ± sd.
p values are calculated using the two-way anova test , in comparison to c- <dig> bearing mice.
elisa assays for anti-c- <dig> antibody cross-reactivity.
96-well elisa plates were coated with hph or rth  and after blocking the plates were incubated with diluted sera from mice inoculated with c- <dig> colon carcinoma cells only or pbs healthy controls.
the results are presented as relative units .
mean ± sd values were calculated for each group.
the level of anti-c- <dig> igg antibodies in the groups immunized with rth and hph without priming did not differ compared to the level of c- <dig> control group for the first 4 weeks after cell administration.
further treatment with rth or hph prolonged persistence of high levels of anti-c- <dig> antibodies in the sera of these animals.
a number of studies demonstrate the prolonged survival in case of enhanced anti-tumor antibody production  <cit> .
the administration of rth or hph to sensrth and senshph groups resulted in a presence of anti-c- <dig> antibodies even before cell inoculation which higher values were measured at the 4th week after c- <dig> injection .
to determine whether the recognition of c- <dig> by antibodies generated after priming with hcs is due to polyspecificity we tested the sera specifisity to foreign antigene .
we observed similar pattern of anibody titers to the pattern of anti-c- <dig> in all of the groups injected with hcs .
to examine the presence of cross-reactive epitopes in hc molecules and c- <dig> cells we set elisa with the hph and rth coated on the 96-well plates and incubated with sera from animals challenged with c- <dig> cells only.
the results showed high levels of crossreactive igg antibodies to hph and rth in the sera of animals after tumor cells inoculation .
these results suggest the presence of crossreactive epitopes within the hemocyanins and c- <dig> colon carcinoma cells.
the biochemical nature of these epitopes needs to be studied and determined  <cit> .
survival and tumor manifestation
the survival of all tested groups was compared with the survival of animals inoculated with c- <dig> cells only.
the results showed 15% survival of sensrth group on week  <dig>  30% survival of the senshph group and over 30% survival of the group treated with hph compared to the c- <dig> group while the group treated with rth did not show improved survival .figure  <dig>
tumor weight and survival.
survival curves of studied animal groups.
the dynamics of tumor growth was monitored in all hcs treated groups and compared to control group .
the data are presented as mean ± sd.
the p-values are calculated using the two-way anova test , in comparison to c- <dig> bearing mice.
the dynamics of tumor growth was monitored in all hcs treated groups and compared to the control group .
animals injected with rth and hph with or without priming developed small size tumors within the whole period of observation .
hph treated group with no priming even showed a decrease in the tumor growth at week 7th after c- <dig> challenge.
histology and organ comparison
splenomegaly is a characteristic feature of c- <dig> murine tumor model  <cit> .
representative numbers of mice from each group were sacrificed for organ comparison and examination.
we observed a more than 3-fold significant increase of the spleens in non treated c-26-bearing mice compared to hcs treated and mostly it was expressed in senshph and sensrth groups .figure  <dig>
organ comparison.
: left panel: spleen size comparison of pbs healthy control , c- <dig> bearing mice , c- <dig> bearing rth treated animals , c- <dig> bearing hph treated animals , sens rth mice , sens hph mice .
right panel: the weights of the spleens from hcs treated animals compared to untreated mice.
the data are presented as mean ± sd.
the p-values are calculated using the two-way anova test .
comparison of metastatic manifestations in the lungs of pbs healthy control ; c- <dig> bearing mice , c- <dig> bearing rth treated animals , c- <dig> bearing hph treated animals , sens rth mice , sens hph mice .
data are representative of at least  <dig> independent experiments.
lungs from all studied groups were excised and the levels of metastatic manifestation were compared to the c- <dig> group.
the figure 6b shows large metastatic nodules formed in the lungs of the control mice and also in the rth-treated group compared to the markedly inhibited number of surface metastasis in the sensrth, senshph and in hph treated groups.
we have examined morphological and histological features of c- <dig> tumor model.
the paraffin sections of solid tumors from all studied groups were stained with heamatoxilin-eosin histological staining technique and were analyzed by light microscope.
the tumor necrosis indicates a poor prognosis for different cancers and is associated with the survival.
we observed differences into the necrosis sections within the tumor mass of the studied animals at 6th week of the treatment schedule.
in the sensrth and senshph groups the levels of necrosis were less than in the rth, hph groups and also in the group of animals bearing c- <dig> carcinoma cells only .
these results could be associated with the enhanced antibody titers that we have found in the sera of the hemocyanin-pretreated animals and with prolonged survival of these groups.
discussion
there is a great need for new natural products and compounds with anti-cancer effects.
the nature-derived substances can be formally separated into two groups i) targeted on the development of the tumor itself such as inhibitors of key for tumor growth molecules and ii) molecules enhancing the ability of immune cells to distinguish the non-self cancer cells and to induce adequate and potent anti-cancer response.
the majority of them have high specificities and limited adverse effects compared to common therapies.
the results from the current study confirm these statements.
molluscan hcs influence the apoptotic pathways of different cancer cell lines but the exact mechanisms are not known  <cit> .
mcfadden et al.
presumed that the tumor suppressive effect of klh could be mediated by alterations in apoptotic pathways.
they have demonstrated that klh has effect by increased apoptosis of seg- <dig> cell line  with intact p <dig> tumor suppressive gene.
on the other hand the klh shows no apoptotic effect on bic- <dig> cells  with mutated p <dig> gene.
it is one possible mechanism for induced pro-apoptotic effect of hcs on the colon carcinoma cell line c- <dig> but this has not been confirmed so far and further study needs to be done.
many authors performed proliferation assay  with hcs, but to study apoptotic effect of gastropodan hcs on tumor cells by facs is more precisely  <cit> .
in our hands we observed an increased apoptosis of tumor cells after hcs treatment in vitro, but always some cells remained unaffected.
the extensive proliferation of these unaffected cells could mask the hcs treatment results.
c- <dig> carcinoma cells challenged balb/c mice had a decrease of cd4+, cd8+, cd19+ as well as cd94+ cells in the spleen compared to pbs treated ones.
in contrast, we have observed a presence of tumor-infiltrating lymphocytes, which can explain the migration of these cell populations from the spleen.
the immunization with rth or hph did not result in a significant difference in the cytokine profiles  between the groups.
the presence of anti-c- <dig> antibodies generated after priming with rth or hph even before c- <dig> cell challenge could be explained with production of polyspecific antibodies recognizing hc molecules or with existence of crossreactive epitopes on the surfaces of c- <dig> carcinoma cells and hcs.
such epitope was already detected on the sbc cells and klh  <cit> .
it was observed a crossreactivity of anti-hc antibodies to the tetanus toxoid and c- <dig> cells.
this result reveals a broad spectrum of polyspecificity of generated antibodies after hc administration.
in the beginning of hcs administration some of the treated mice exhibited worse survival rate than the untreated ones.
finally, in the end-point hph treated groups had better performance and exhibited improved survival compared to the control group.
this observation illustrates that improvement of several clinical parameters  does not always prolong survival.
CONCLUSIONS
the hcs - treated animals had prolonged survival and inhibited tumor growth, splenomegaly and lung metastasis appearance.
these huge proteins can induce a vigorous anti-tumor adaptive immune response.
taken together our findings demonstrate that the hcs isolated from different arthropod and mollusc species could be used as pro-apoptotic agents able to improve humoral anti-cancer response and moderate the manifestation of c- <dig> carcinoma symptoms.

 cancer is one of the major reasons for human death in the last decade following cardiovascular diseases, infectious diseases and ischemic heart diseases. current therapies of cancer include chemotherapy and radiotherapy although they both have severe side effects. each type of cancer requires specific treatment, which explains the need for development of highly specific targeted anti-cancer agents. the bcg vaccine for tuberculosis that contains attenuated mycobacterium bovis is one of the most extensively used immunotherapeutics based on its strong non-specific immunostimulatory properties. bcg is widely administrated in cases of colorectal, lung cancers and melanoma. in the last years a number of therapeutic antibodies have been approved for clinical treatment in cases of breast cancer, non-hodgkin and hodgkin lymphoma, colorectal cancer, chronic lymphocyte leukemia, and acute myelogenous leukemia. immunotherapy with cytokines is another option for cancer treatment. il- in metastatic melanoma and renal cell carcinoma and ifn-alpha in stage iii melanoma have been permitted for cancer therapy. different cancer vaccines have been developed for prevention and treatment of malignant diseases but only sipuleucel-t is approved for therapy of advanced prostate cancer in case hormonal treatment is ineffective. many of the developed vaccines include cancer carbohydrate antigens chemically conjugated to carrier protein with or without other adjuvant. today natural products and their compounds derived from fungi, plants or microbes are of great interest for an anti-cancer research. this huge variety of chemical structures provides different mechanisms of action and specific effects used for anti-tumor therapy. marine compounds are known to have a serious potential as anti-cancer drugs. most of them are small molecules, inhibitors of key enzymes for carcinogenesis like matrix metalloproteinazes, hifs, topoisomerase, protein kinase c or transcripion factors like nfkb. special attention is payed on marine-derived anti-angiogenesis products, which suppress and prevent the succesful formation of vascular system, supporting tumor growth and invasion. the majority of these substances act via inhibition of enzymes or factors, crucial for the process of angiogenesis. another group of potential anti-cancer agents are the hemocyanins - oligomeric copper-containing glycoproteins that function as oxygen carriers in the hemolymph of several molluscs and arthropods. molluscan hcs have been studied intensively for many years with respect to their structure and function. the huge molecular size of molluscan hcs, their xenogenic character and carbohydrate content have been implicated in inducing strong immune response in mammals, which has led to the biomedical and therapeutic application of these proteins. thus, keyhole limpet hemocyanin isolated from marine gastropod megathura crenulata is a well-established immune stimulant, hapten carrier and tumor vaccine carrier. it is known that hcs stimulate the immune system non- specifically by interacting with macrophages, polymorphonuclears, cd4+ and cd8+ cells giving rise to potent humoral and cellular immune response. one possibility for their anti-cancer effect is that they generally enhance statement of immune system that helps the immune cells to recognize self from non-self and to eliminate non-self cancerous cells. klh and cch are intensively studied for their anti-cancer properties in superficial bladder cancer and in melanoma models. recently, we have demonstrated that the hc isolated from marine gastropod rapana thomasiana possesses a high immunogenicity as a single model antigen and can be used as a protein carrier for viral peptides from influenza hemagglutinin. our results have shown that rth is acceptable as a potential bio-adjuvant for subunit bacterial and viral vaccines. compared to the numerous studies on the structural organization and protein stability of the hc isolated from garden snail helix pomatia, its immunological properties have been less investigated. these results point on the potential properties of rth and hph as anti-cancer agents. c- murine model of colorectal carcinoma is classical tumor model, developed in and determinated as an undifferentiated grade iv carcinoma. c- cells display high tumorigenicity and low tendency to metastasize mainly in the lungs. inoculated in syngeneic balb/c mice c- cells cause high mortality. this model is used more than 35 years for studying the process of carcinogenesis and anti-cancer therapy. the aim of the present work was to develop an experimental murine model of colon carcinoma and to investigate the anti-tumor activity of rth and hph.  hemocyanins analysis figure  presents the synopsis of our results from the structural studies of the hc isolated from marine snail r. thomasiana. the electron micrograph shows typical for gastropodan hcs didecamers. the quaternary structure of molluscan hcs is a hollow cylindrical molecule, about 35 nm in diameter, with an internal collar complex. 3-d reconstruction of the rth molecule is shown on figure 1b. molecules of molluscan hcs are structured as decamers or didecamers of subunits with molecular mass of 350–450 kda. the polypeptide chains of subunits contain seven or eight globular functional units, depending on the species. fus are generally termed, starting from the n-terminus of the respective subunit. the fus have a binuclear copper active site, capable of binding reversibly one oxygen molecule. the active site consists of two closely spaced copper ions each coordinated by three histidine ligands. figure 1d shows the crystal structure of a fu rth2-e. the different type of fus share sequence identities of around 45%. being similar in primary, tertiary and quaternary structure, molluscan hcs differ in their carbohydrate content ) and monosaccharide composition. most probably the specific carbohydrate content and composition of molluscan hcs are the structural basis for the difference in their antigenicity.figure molecular structure of gastropod hcs. tem of negatively stained rth molecules; 3-d reconstruction of the hollow cylindrical rth didecamer; side view and top view; scheme of the subunit containing eight different functional units; x-ray structure of functional unit rth2-e. hemocyanins increase tumor cells apoptosis in vitro to study the apoptotic effect of gastropodan hcs on tumor cells we tested their ability to influence directly the c- cells by apoptosis assay. c- carcinoma cells were incubated with different amounts of hph and rth. the cells were further washed and the surface expression of phosphatidylserine was studied at the 24 h, 48 h and 72 h of incubation. the levels of induced early and late apoptosis as well as necrosis were analyzed by annexin-v/propidium iodide staining and flow cytometry. a significant increase in percentage of early apoptosis and a slight increase of late apoptosis were observed after 48 hours of incubation with all tested amounts of hcs. in contrast, no obvious effect of the treatment was found on the 24 h of incubation. high level of early apoptosis and considerable enhancement in late apoptosis was observed on the 72 h of incubation. dose-dependant quantitative corelation between induced apoptosis and hcs added to the cells was not found at all tested time points.figure the gastropod hcs rth and hph induce apoptosis of c- carcinoma cell line in vitro. the c- cells were incubated in the presence of different concentrations of hph and rth for 72 hours at 37°С/5% co at the end of the cultivation the cells were double-stained with annexin v-fitc /propidium iodide and were analyzed by flow cytometry. percentage of stained cells is shown in each quadrant. the panels represent the results from independent experiments. tumor model development for selection of proper c- murine colon carcinoma model groups of mice challenged with different cell numbers were monitored. formation of solid tumors in all mice in the group as well as 0% survival within 3 months after cells challenge was the leading parameters for successful model establishment. in the group inoculated with 5× cells palpable solid tumors were observed 2 weeks after c- challenge followed by 0% survival 3 months after cell challenge, which is acceptable for the model requested. in the other groups mice developed tumors and died too early or survived for longer period, so they weren’t appropriate for further studies. phenotyping of spleen and tumor suspensions we have performed an immune system modulation analysis during tumor development when the tumor model was established to demonstrate the immune reaction to the tumor growth. spleens from mice challenged with 5× c- carcinoma cells or pbs treated only were isolated and analyzed by flow cytometry. we observed a decrease of cd4+, cd8+, cd19+ as well as cd94+ and slight increase of cd14+ cells compared to pbs treated balb/c mice.figure facs analysis of spleen and tumor suspensions. spleens and solid tumors from representative number of animals were excised and the cell suspensions were incubated with one of the following anti-mouse antibodies - anti-cd4-apc, anti-cd8-fitc, anti-cd19-pe, anti-cd94-pe or anti-cd14-apc. ten thousands cells were analyzed from each sample by flow cytometry. data are representative of at least experiments. a cell suspension prepared from solid tumors of the studied mice showed presence of cd4+, cd8+, cd19+, cd94+ as well as cd14+ cell populations in the tumor microenvironment. these results suggest a movement of immune cells populations to the developed tumors and a relevant decrease of the same populations in the spleen of the animals. cytokine detection tnfα, ifnγ, il and il levels were measured in mice sera using enzyme linked immunosorbent assay kits. challenge with 5× c- carcinoma cells and treatment with rth or hph did not result in a significant difference in the cytokine production between the groups. detection of anti- c- antibodies formation groups of balb/c mice were treated with hcs from gastropods r. thomasiana and h. pomatia. two groups were injected with rth or hph 2 weeks before tumor cells inoculation and after establishment of palpable solid tumors the mice were injected intratumoraly once weekly with rth or hph. the other two groups were inoculated with 5× cells/mouse of c- cells and after palpable solid tumor development the animals were injected once a week with rth or hph. mice injected with c- cells only or pbs injected healthy mice were used as controls. collected sera from all studied groups were tested for presence of anti-c- antibodies. all groups developed anti-c- igg antibodies compared to the pbs treated control group. the titer of these antibodies decreased in c- group without hemocyanin treatment during tumor development.figure detection of anti- c- antibodies and anti-c- antibody cross-reactivity. anti- c- antibody levels in c- bearing-mice treated with rth or hph were measured by elisa. mice inoculated with c- cells without hcs or mice treated only with pbs were used as control groups. the results are presented as relative units calculated according to the titer of standard anti-c- antibodies used for elisa. the data are presented as mean ± sd. p values are calculated using the two-way anova test, in comparison to c- bearing mice. elisa assays for anti-c- antibody cross-reactivity. 96-well elisa plates were coated with hph or rth and after blocking the plates were incubated with diluted sera from mice inoculated with c- colon carcinoma cells only or pbs healthy controls. the results are presented as relative units. mean ± sd values were calculated for each group. the level of anti-c- igg antibodies in the groups immunized with rth and hph without priming did not differ compared to the level of c- control group for the first 4 weeks after cell administration. further treatment with rth or hph prolonged persistence of high levels of anti-c- antibodies in the sera of these animals. a number of studies demonstrate the prolonged survival in case of enhanced anti-tumor antibody production. the administration of rth or hph to sensrth and senshph groups resulted in a presence of anti-c- antibodies even before cell inoculation which higher values were measured at the 4th week after c- injection. to determine whether the recognition of c- by antibodies generated after priming with hcs is due to polyspecificity we tested the sera specifisity to foreign antigene. we observed similar pattern of anibody titers to the pattern of anti-c- in all of the groups injected with hcs. to examine the presence of cross-reactive epitopes in hc molecules and c- cells we set elisa with the hph and rth coated on the 96-well plates and incubated with sera from animals challenged with c- cells only. the results showed high levels of crossreactive igg antibodies to hph and rth in the sera of animals after tumor cells inoculation. these results suggest the presence of crossreactive epitopes within the hemocyanins and c- colon carcinoma cells. the biochemical nature of these epitopes needs to be studied and determined. survival and tumor manifestation the survival of all tested groups was compared with the survival of animals inoculated with c- cells only. the results showed 15% survival of sensrth group on week 30% survival of the senshph group and over 30% survival of the group treated with hph compared to the c- group while the group treated with rth did not show improved survival.figure tumor weight and survival. survival curves of studied animal groups. the dynamics of tumor growth was monitored in all hcs treated groups and compared to control group. the data are presented as mean ± sd. the p-values are calculated using the two-way anova test, in comparison to c- bearing mice. the dynamics of tumor growth was monitored in all hcs treated groups and compared to the control group. animals injected with rth and hph with or without priming developed small size tumors within the whole period of observation. hph treated group with no priming even showed a decrease in the tumor growth at week 7th after c- challenge. histology and organ comparison splenomegaly is a characteristic feature of c- murine tumor model. representative numbers of mice from each group were sacrificed for organ comparison and examination. we observed a more than 3-fold significant increase of the spleens in non treated c-26-bearing mice compared to hcs treated and mostly it was expressed in senshph and sensrth groups.figure organ comparison.: left panel: spleen size comparison of pbs healthy control, c- bearing mice, c- bearing rth treated animals, c- bearing hph treated animals, sens rth mice, sens hph mice. right panel: the weights of the spleens from hcs treated animals compared to untreated mice. the data are presented as mean ± sd. the p-values are calculated using the two-way anova test. comparison of metastatic manifestations in the lungs of pbs healthy control; c- bearing mice, c- bearing rth treated animals, c- bearing hph treated animals, sens rth mice, sens hph mice. data are representative of at least independent experiments. lungs from all studied groups were excised and the levels of metastatic manifestation were compared to the c- group. the figure 6b shows large metastatic nodules formed in the lungs of the control mice and also in the rth-treated group compared to the markedly inhibited number of surface metastasis in the sensrth, senshph and in hph treated groups. we have examined morphological and histological features of c- tumor model. the paraffin sections of solid tumors from all studied groups were stained with heamatoxilin-eosin histological staining technique and were analyzed by light microscope. the tumor necrosis indicates a poor prognosis for different cancers and is associated with the survival. we observed differences into the necrosis sections within the tumor mass of the studied animals at 6th week of the treatment schedule. in the sensrth and senshph groups the levels of necrosis were less than in the rth, hph groups and also in the group of animals bearing c- carcinoma cells only. these results could be associated with the enhanced antibody titers that we have found in the sera of the hemocyanin-pretreated animals and with prolonged survival of these groups. discussion there is a great need for new natural products and compounds with anti-cancer effects. the nature-derived substances can be formally separated into two groups i) targeted on the development of the tumor itself such as inhibitors of key for tumor growth molecules and ii) molecules enhancing the ability of immune cells to distinguish the non-self cancer cells and to induce adequate and potent anti-cancer response. the majority of them have high specificities and limited adverse effects compared to common therapies. the results from the current study confirm these statements. molluscan hcs influence the apoptotic pathways of different cancer cell lines but the exact mechanisms are not known. mcfadden et al. presumed that the tumor suppressive effect of klh could be mediated by alterations in apoptotic pathways. they have demonstrated that klh has effect by increased apoptosis of seg- cell line with intact p tumor suppressive gene. on the other hand the klh shows no apoptotic effect on bic- cells with mutated p gene. it is one possible mechanism for induced pro-apoptotic effect of hcs on the colon carcinoma cell line c- but this has not been confirmed so far and further study needs to be done. many authors performed proliferation assay with hcs, but to study apoptotic effect of gastropodan hcs on tumor cells by facs is more precisely. in our hands we observed an increased apoptosis of tumor cells after hcs treatment in vitro, but always some cells remained unaffected. the extensive proliferation of these unaffected cells could mask the hcs treatment results. c- carcinoma cells challenged balb/c mice had a decrease of cd4+, cd8+, cd19+ as well as cd94+ cells in the spleen compared to pbs treated ones. in contrast, we have observed a presence of tumor-infiltrating lymphocytes, which can explain the migration of these cell populations from the spleen. the immunization with rth or hph did not result in a significant difference in the cytokine profiles between the groups. the presence of anti-c- antibodies generated after priming with rth or hph even before c- cell challenge could be explained with production of polyspecific antibodies recognizing hc molecules or with existence of crossreactive epitopes on the surfaces of c- carcinoma cells and hcs. such epitope was already detected on the sbc cells and klh. it was observed a crossreactivity of anti-hc antibodies to the tetanus toxoid and c- cells. this result reveals a broad spectrum of polyspecificity of generated antibodies after hc administration. in the beginning of hcs administration some of the treated mice exhibited worse survival rate than the untreated ones. finally, in the end-point hph treated groups had better performance and exhibited improved survival compared to the control group. this observation illustrates that improvement of several clinical parameters does not always prolong survival.  the hcs - treated animals had prolonged survival and inhibited tumor growth, splenomegaly and lung metastasis appearance. these huge proteins can induce a vigorous anti-tumor adaptive immune response. taken together our findings demonstrate that the hcs isolated from different arthropod and mollusc species could be used as pro-apoptotic agents able to improve humoral anti-cancer response and moderate the manifestation of c- carcinoma symptoms. 
the immunization with rth and hph prolonged the survival of treated animals, improve humoral anti-cancer response and moderate the manifestation of c- carcinoma symptoms as tumor growth, splenomegaly and lung metastasis appearance. klh 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. among them are the hemocyanins - extracellular copper proteins isolated from different arthropod and mollusc species. hemocyanins are used so far for therapy of superficial bladder cancer and murine melanoma models. the hcs isolated from marine snail rapana thomasiana and the terrestrial snail helix pomatia express strong in vivo anti-cancer and anti-proliferative effects in the developed by us murine model of colon carcinoma. 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.
 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 - 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 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.  the hcs isolated from marine snail rapana thomasiana and the terrestrial snail helix pomatia 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- carcinoma symptoms as tumor growth, splenomegaly and lung metastasis appearance.  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. keywords c- carcinomamurine cancer modelrapana thomasianahelix pomatiahemocyaninsanti-cancer activityissue-copyright-statement© the author 2014 