Antitumour activity of Helix hemocyanin against bladder carcinoma permanent cell lines

Abstract Hemocyanins are oxygen-transporting glycoproteins in molluscs and arthropods. In this study, we assayed the biomacromolecules from the molluscs Helix lucorum (HlH), Rapana venosa (RvH) and Megatura crenulata (KLH) including their functional units (FUs), for therapy of bladder cancer permanent cells. In vitro studies antitumour activities of these proteins were performed with bladder cancer permanent cell line CAL-29 and the normal urothelial cell line HL 10/29 in comparison to doxorubicin. The obtained results showed that the human tumour CAL-29 cell line is sensitive to the action of the tested hemocyanins and their isoforms. We observed a dose- and time-dependent inhibition of tumour cell growth after incubation with native HlH and two FUs (βc-HlH-a and FU βc-HlH-h); and of particular significance, FU βc-HlH-h showed a surprisingly stronger effect than that the doxorubicin-treated cells. Cells treated with βc-HlH-h, showed both, apoptotic and necrotic cells. In addition, two-dimensional polyacrylamide gel electrophoresis (PAGE) found for differential up-regulation of several proteins after hemocyanin treatment. Eight different down-regulated and two up-regulated proteins were identified, which may be associated with the apoptosis pathway. No inhibition of the normal urothelial cell line HL 10/29 was observed after treatment with HlH and its isoforms. The most effective inhibition of CAL-29 tumour cells was observed after treatment with βc-HlH-h, probably caused by a specific oligosaccharide structure of HlH with methylated hexoses. These results suggest that hemocyanin glycosylation plays an important role in its anticancer activity.

Hemocyanins are adjuvants for producing antibodies against different antigens in therapeutic vaccines against cancer [9][10][11] or against tumour antigens in generation of ex vivo autologous tumour cell lysate-loaded dendritic cells (DCs) to induce T-cell responses in cancer patients [9,12,13]. Hemocyanins have been used clinically as nonspecific immunostimulants to prevent the progression of superficial bladder cancer, suggesting them ideal for longterm ongoing treatment [9,13,14].
The relevance of the glycosylation of hemocyanin isoforms KLH1 and KLH2 of Megathura crenulata [15][16][17] and the clinical success in patients with bladder carcinoma are assumed to be caused by a disaccharide epitope Gal(b1-3)GalNAc, cross-reacting with an epitope on the bladder tumour cell surface [17]. As reviewed earlier [11], the versatile biomedical and clinical applications of KLH have led to growing interest in finding new alternative hemocyanins with similar or more potent immunomodulatory, antimicrobial, and antiviral properties [18][19][20][21].
Several hemocyanins from other species of mollusks have significant immunological and antitumour potential, including Concholepas concholepas (CCH) [22] and Fissurella latimarginata (FLH) [23,24]. In vitro studies on the mechanism of the antiproliferative activity of the hemocyanin from shrimp Litopenaeus vannamei (LvH) against the most commonly used human HeLa cell line the innate immunity by inducing different temporal patterns of proinflammatory cytokine expressions in macrophages [25].
Our previous studies demonstrated that isoforms and functional units of hemocyanins from Rapana venosa (RvH) and Helix lucorum (HlH) (previously called Helix vulgaris (HvH)) have antitumor activity and immunological properties [26][27][28]. The results showed that both hemocyanins may be useful for the development of new antiviral, antibacterial and antitumour vaccines since they seem to launch strong and specific immune response against conjugated antigens [27].
As was reported recently, the molluscan hemocyanins HlH and RvH change the gene expression in human bladder cancer cells [28]. Bladder cancer is the second most common urological cancer, clinically characterized by a high recurrence rate and poor prognosis [28,29]. Moreover, approximately 20% of recurring bladder cancer cases can develop into muscle-invasive tumours [30]. Therefore, bladder cancer remains a focus in cancer research and several novel bladder cancer biomarkers have been identified [31].
It was demonstrated that RvH and Helix aspersa (HaH) have a direct antiproliferative effect on CAL-29 and T-24 bladder cancer cell lines, and the antitumour properties of HlH are superior to KLH [31,32]. Native HlH and the structural subunits from RvH are alternative candidates for the treatment of human superficial bladder cancer [33]. These findings lay the ground for more extensive studies on Helix lucorum hemocyanins as potential therapeutic agents against bladder cancer [34]. Moreover, the structure of HlH is significantly different from that of KLH [35,36]. Native H. lucorum hemocyanin consists of three different structural subunits, bc-HlH, aD-HlH and aN-HlH. Each subunit, ranging from 350 to 450 kDa, includes eight globular-folded domains known as functional units (FUs) with molecular masses of about 50-60 kDa [9,35,36]. The aim of the present study was to assay the antitumour effect of molluscan hemocyanin from garden snail Helix lucorum (HlH) on the human bladder cancer cell line CAL-29 by proteomics analysis.

Materials
The hemolymph was collected from the foot of Bulgarian garden snail H. lucorum and the crude hemocyanin was isolated from the hemolymph as was described by Velkova et al. [36].

Purification of hemocyanins and FUs
The native H. lucorum hemocyanin was isolated from the hemolymph after centrifugation. The native of H. aspersa hemocyanin with a molecular mass of $9 MDa is present in the hemolymph as a-HaH and bc-HaH isoforms. bc-HaH is composed of only one subunit type (bc-subunit), and a-HaH is composed of two types of subunits (aN-HaH and aD-HaH). Three isoforms, one b-HlH and two a-HlH, were separated after precipitation or crystallisation during dialysis against sodium acetate buffer at low ionic strength as described by Velkova et al. [36]. The isoform bc-HaH was further analyzed by 5% native polyacrylamide gel electrophoresis, showing a single band with a mass of around 450 kDa [35].

Bladder cancer permanent cell lines
Experiments were carried out with commercially available permanent human CAL-29 tumour cell line (TCC), established from the primary lesion of a fatally invasive, metastatic TCC of the bladder (grade IV, stage T2) from an 80-year-old woman before treatment. After cultivation of CAL-29 cells in Dulbecco Modified Eagle's Medium (DMEM, Lonza, Austria), they were supplemented with 10% fetal bovine serum (Gibco, Austria), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1% non-essential amino acids in 75-mL tissue culture plastic flasks (Falcon). Cells were maintained in log growth phase at 37 C in humidified air containing 5% CO 2 .
Normal human urothelial cells were grown to confluence in complete keratinocyte serum-free medium (cKSFM) with human recombinant epithelial growth factor (5 ng/mL) bovine, pituitary extract (50 mg/mL; Gibco), supplemented with cholera toxin (30 ng/mL; List Biological Laboratories, Campbell, CA, USA) under standard conditions.

Determination of the antiproliferative activity of the tested hemocyanins
After washing with 0.1% EDTA/PBS (ethylenediaminetetraacetic acid/phosphate buffered saline) for 5 min, CAL-29 cells were trypsinized with TrypLE Express (Gibco), centrifuged for 5 min at 1500 g, and counted in a hemocytometer. Depending on the experimental need, cells were seeded at optimal inoculation density in well-plates or flasks and incubated overnight in expansion medium. Then cells were treated for 24, 48, and 72 h with various concentrations of the test substances glycosylated proteins: 150 lg/mL of the native of HlH (0.03 lmol/L), 150 and 300 lg/mL, with the structural subunit RvH1 (0.33 lmol/L and 0.66 lmol/L, respectively), and with the functional units (FUs) 150 and 300 lg/mL of b c -HlH-a (3.0 lmol/L and 6.0 lmol/L), 150 lg/mL of b c -HlH-h (3.0 lmol/L), 150 and 300 lg/ mL of b c -HlH-a (3.0 lmol/L and 6.0 lmol/L, respectively), 300 lg/mL RvH1-c (6.0 lmol/L) and compared to controls (¼ 100%), 150 lg/mL of KLH (0.03 lmol/L), and 0.1 lg/mL doxorubicin (DOX) (18.3 lmol/L) respectively. Doxorubicin served as positive control, medium as negative control. The effects of the substances on the cell viability were assessed by measuring the respiratory activity of mitochondria (WST-1assay) and DNA synthesis (BrdU assay; both kits from Roche Diagnostics) using an ELISA plate reader (Milenia Kinetic Analyzer, Diagnostic Products). These analyses were performed two times. The percentage of cell growth inhibition was calculated as follows:

Statistical analysis
The data were processed using Exel and GraphPad Prism 5 and are presented as means with standard deviation (SD). Significance was tested using one-way analysis of variance (ANOVA) with Bonferroni adjustment for multiple comparisons. p < .05 was considered to indicate a statistically significant difference (shown in the figures as Ã p < .05, ÃÃ p < .01 and ÃÃÃ p < .001 in comparison to a negative control, and^p < .05 and^p < .01, in comparison to a positive control). The experiments were performed in triplicate and repeated at least three times.

Pro-apoptotic activity of hemocyanins
The induction of apoptosis was assessed after 24 h cultivation of CAL-29 tumour cells with 500 lg/mL HlH (0.08 lmol/L) and 500 lg/mL b c -HlH-h (10 lmol/L) by the Annexin-V-FLUOS Kit (Roche, Diagnostics), and the results were compared with 0.1 mg/mL doxorubicin for positive and medium for the negative control. The cells were washed twice with PBS (Gibco) for 5 min and then covered with Annexin V labelling solution. After adding 1 mL of propidium iodide (PI) solution and 2 mL of fluorescein-annexin solution, the plates were incubated for 15 min at room temperature (15-20 C) and then analyzed by fluorescent microscopy.
The cells were distinguished based on the following staining: Positive Annexin-V/FITC-staining is characteristic for apoptotic cells; Positive Annexin-V/FITC-and PI-staining is observed for late apoptotic or necrotic cells; Specific PI-staining is typical for necrotic cells.

Extraction of proteins from CAL-29 cells
The cells were washed two times with a blank medium without serum, which was then substituted with serum-free medium (SFM, Thermo Fisher). The cells were incubated for a further period of 24-hours, and the supernatant was collected for 2D-gel electrophoreses. Ten milligrams of lyophilised CAL-29 cells without treatment and after treatment with HlH were suspended in 50 mmol/L Tris-HCl, pH 7.5, 0.1 mmol/L EDTA and 1 mmol/L b-mercaptoethanol, and the mixtures were stirred for 45 min in a cold room. Then they were homogenized using a glass homogenizer and the homogenate is centrifuged at 12 000 Â g for 20 min at 4 C. The supernatants were stabilized with protease inhibitors (cocktail Roche) and 40% proteins were precipitated by gradually adding sufficient solid (NH 4 ) 2 SO 4 (ultrapure reagent or enzyme grade). The precipitated protein was removed by centrifugation at 12 000 Â g for 10 min, at 4 C, and more (NH 4 ) 2 SO 4 was added gradually to the supernatant to yield 80% saturation. After centrifugation, the fractions of precipitated proteins between 40 and 80% saturation were resuspended gently in 10 mL of 20 mmol/L Tris-HCl buffer, pH 7.5, 20 mmol/L NaCl and 10 mmol/L MgCl 2 , and dialyzed against 3 L of the same buffer to remove residual (NH 4 ) 2 SO 4 . The dialyzed suspension was centrifuged at 12 000 Â g for 10 min, at 4 C and the supernatants were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).

2-DE analysis of proteins in CAL-29 tumour cells after treatment with HlH
After determination of the protein concentration of supernatants with the Protein Assay Kit (BioRad), approximately 250 lg of protein was mixed with IPG rehydration buffer (8 mol/L urea, 2% w/v CHAPS, 0.3% dithiothreitol (DTT), final volume of 360 lL). The strips were allowed to rehydrate for 7 h and to focus (IEF) using a Multiphor II system (Amersham Biosciences) running the following program: 150 V (30 0 ), 150 V (120 0 ), 300 V (30 0 ), 300 V (45 0 ), 3500 V (90 0 ), 3500 V (540 0 ), 500 V (10 0 ), and held at 500 V. The temperature was kept at 18 C. After completion of the IEF program, the IPG strips were equilibrated in a 50 m mol/L Tris-HCl solution, pH 8.8, containing 6 mol/L urea, 30% glycerol, 2% SDS, and 1% DTT, for 10 min, after which the solution was replaced with the same solution, except that DTT was exchanged by 5% iodoacetamide. The strips were placed on home-casted vertical SDS-PAGE gels and subjected to electrophoresis at 10 mA/gel for 15 min, followed by a 9-h run at 20 mA/gel until the bromophenol blue front reached the bottom of the gel. Staining was performed using Coomassie Brilliant Blue G-250 Dye (CBB G-250, Thermo Fisher Scientific). The 2D-gel images were digitized using a GS-710 densitometer (BioRad) and analyzed with the accompanying PDQuest 7.1 software (BioRad).

Analyses of proteins after gel guanidination and trypsin digestion of spots
Guanidination was performed by adding 5 lL of Milli-Q (MQ) water, 11 lL of 7 N ammonium hydroxide (Merck, Darmstadt, Germany), and 3 lL of a freshly prepared 7.5 mol/L O-methylisourea hemisulfate solution (Acros, Geel, Belgium) to the excised spots. The samples were vortexed briefly and incubated at 65 C. After incubation for 2 h, the guanidinated samples were taken from the oven and the remainder of the solution was discarded. The gel pieces containing the guanidinated samples were desalted and destained in one step. Two washes using 150 lL of 200 mmol/L ammonium bicarbonate in 50% ACN/MQ (30 min at 30 C) were performed and subsequently the gel pieces were dried in a SpeedVac (Thermo Savant, Holbrook, NY).
To reduce and alkylate the protein mixture prior to apply to trypsin digestion, 10 lL of 10 mmol/L DTT was added for 100 lL of the sample (5 lg/lL). The Eppendorf tube was incubated for 30 min at 60 C. After being cooled down for 5 min to room temperature, 10 lL of 100 mmol/L iodoacetamide was added and the mixture was placed in the dark for 30 min. Then, 20 lL out of the 120 lL of the reduced and alkylated sample was digested. This sample was diluted in 60 lL of 0.1% SDS/50 mmol/L Tris (pH 7.4) and 20 lL of ACN and incubated overnight with 100 lL of 0.1 lg/ lL trypsin (Promega, Madison, WI). The peptide mixture was dried in a SpeedVac. The sample was then dissolved in 20 lL of 0.1% formic acid.
A volume of 8 lL digestion buffer (50 mmol/L ammonium bicarbonate, pH 7.8) containing modified trypsin per microliter (Promega) was added to the dried gel spots and the tubes were kept on ice for 45 min to allow the gel pieces to be completely soaked with the protease solution. Digestion was performed overnight at 37 C, the supernatants were recovered and the resulting peptides were extracted twice with 35 lL of 60% ACN/0.1% DIEA. The extracts were pooled and dried in the SpeedVac. The peptides were redissolved in 10 lL of 0.1% formic acid and matrix solution and spotted on the MALDI plate.

Protein identification by MS and MS/MS analyses
In all MALDI-MS and MS/MS applications, a 4800 plus MALDI TOF/TOF Analyzer (Applied Biosystems, Foster City, CA, USA) was used. Samples were prepared by mixing 0.7 lL of the sample with 0.7 lL matrix solution (7 mg/mL a-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN containing 0.1% TFA) and spotted on a stainless steel 192-well target plate. They were allowed to air dry at room temperature, and were then inserted in the mass spectrometer and subjected to mass analysis. The mass spectrometer was externally calibrated with a mixture of angiotensin I, Glu-fibrinopeptide B, ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and ACTH . For MS/MS experiments, the instrument was externally calibrated with fragments of Glu-fibrinopeptide B. The search included fixed modification of acetyl (N-terminal peptide), acetyl-lysine and cysteine carbamidomethylation, variable modifications of methionine oxidation and deamidation of glutamine and asparagine. Protein identification based on MS/MS spectra was made against the Swiss-Prot database using an in-house MASCOT server (Matrixscience, London, UK). Other restrictions to MASCOT were precursor ion m/z tolerance of ±0.3 Da, enzyme digestion with trypsin, up to one missed cleavage, and 1 þ , 2 þ and 3 þ charged precursor ions were considered. The error for matching the daughter ions in the MS/MS spectra was 0.5 Da.

Database searches
Mass spectral data were searched against different protein databases using an in-house MASCOT server (Matrixscience, London, UK). NCBI BLAST-search was done with the amino acid sequences revealed by manual interpretation of the MS/MS spectra. De novo determined peptide sequences were deduced manually and used for similarity searches using the FASTS, MS-BLAST and the MS-Homology algorithm.

Results and discussion
In our preliminary study, we examined the antitumour effect of the native molluscan hemocyanins from the marine snail R. venosa and the garden snail H. lucorum and their subunits on CAL-29 and T-24 human bladder cancer cell line in comparison to compounds routinely used clinically, such as KLH, doxorubicin hydrochloride and mitomycin-C [27].
The experiments with HlH, RvH, RvH1 and RvH2 showed a direct growth inhibitory effect on the tumour cells mainly after treatment with native HlH, RvH and the structural subunit RvH1, at a concentration of 500 mg/mL as observed for KLH. Moreover, we found a stronger antitumoural effect of the functional unit RvH1-c against these tumour cells after a 24 hour for treatment, probably due to specific oligosaccharide structures exposed on the surface of the glycoprotein [38]. The mechanism of antitumour activity of HlH, bc-HlH-h, and RvH2-g hemocyanins includes induction of apoptosis [28]. In addition to the antiproliferative effect, gene expression profiling of the CAL-29 and T-24 cell line treated with HlH is associated with immune system activation, transcriptional misregulation and programmed cell death. The downregulated genes are associated with responses to wounding, lipopolysaccharide and angiogenesis [28].
Based on these results, we performed in vitro experiments to analyze the antitumour activities of the native hemocyanin from H. lucorum against CAL-29 tumour cells in comparison to its isoforms and get some insight into its mechanism of action on a molecular level. The native HlH, structural subunit and functional unit, were purified. The primary protein structures as well as the oligosaccharide compositions of two FUs from HlH (bc-HlH-h and bc-HlH-a) and one FU (RvH1-c) from RvH are well known. Therefore, we analyzed their antitumour activities. The obtained isoforms were electrophoreticaly pure as confirmed by SDS-PAGE and MALDI-MS. The molecular mass of FU bc-HlH-h was determined to be 56625.6 [M þ H] þ as shown in the MALDI-MS spectrum (Figure 1). [13,26,36,38]. However, the mechanisms involved in facilitating cell death by HlH and other Hcs have not been investigated in detail. We therefore, explored the antitumour activities of the native hemocyanin from H. lucorum against CAL-29 tumour cells in comparison to its isoforms to get some insight into its mechanism of action on a molecular basis.

Hemocyanins (Hcs) may act as anti-proliferative agents on a variety of mammalian cells in vitro and in vivo
Treatment of CAL-29 tumour cells with Hcs reduced the viability of the cells as determined by WST-assay ( Figure 2). The inhibition of cell viability was doseand time-dependent. After 72 hours of incubation, the respiratory activity of the cells was reduced to 67% with 300 lg/mL of HlH (0.06 lmol/L) and to 41% with 150 lg/mL of subunit RvH1 (0.33 lmol/L), compared to controls (100%). The cell viability drastically decreased after treatment of the tumour cells with 150 lg/mL of bc-HlH-h (3.0 lmol/L) (20.3%) and with 300 lg/mL of bc-HlH-a (6.0 lmol/L) (17.47%). For both FUs, bc-HlH-h and bc-HlH-a, this inhibition was stronger compared to the effect of native HlH and the structural subunit RvH1. HlH and RvH1 are more active than KLH (87%).
There were alterations of the CAL-29 cells' morphology after 24 and 48 h of treatment with 150 lg/mL (3.0 lmol/L) of both b c -HlH-h and RvH1-c. Treatment with doxorubicin (0.1 mg/mL) served as control ( Figure 3).
Comparison of the actions of the native molecules with the FUs from HlH and RvH showed that treatment with FUs more effective. FUs were purified and analyzed by 10% SDS-PAGE (data not shown). They induced significant inhibition on tumour cell growth and at the same time caused comparatively low effects on the metabolism and proliferation of normal cells (Figure 4(A)). This effect is probably due to specific oligosaccharide structures of these molecules, which are more accessible in the FUs of HlH [37] than the native molecules. The oligosaccharide structures of HlH are known and a mechanism of inhibition of tumour cell growth is suggested specific glycosylation sites of HlH and its FUs to interact with the target cells.
There are several reports on the interaction of glycosylation moieties of molluscan hemocyanins with the target cells. It is assumed that the clinical success of intravesical administration of KLH to patients with bladder carcinoma is based on the cross-reactivity of the disaccharide epitope Gal (b1-3)GalNAc with corresponding epitope on the bladder tumour cell surface [17]. To evaluate and compare the effect of the tested hemocyanins on normal urothelial cells HL 10/29, the tests were carried out with the lowest concentration of samples effective in arresting tumour cell growth. The data in Figure 3 (Figure 4(B)).

Analysis of cell death by fluorescence microscopy
The objective of anti-cancer therapy is to eliminate cancer cells from the body and also to prevent them from spreading to other parts of the organism. There are two major approaches: to limit tumour growth, i.e. limit the proliferation of tumour cells, or to induce apoptosis, the controlled death of tumour cells. The study of these two processes, apoptosis and proliferation of tumour cells, is the basis for the developments of new drugs and therapeutic approaches. Representative fluorescent morphologies of CAL-29 transitional carcinoma cell line after 24-h incubation with doxorubicin, HlH and b c -HlH-h, are shown in     Annexin-V and the nuclei were co-stained with PI after 24-h incubation with 0.5 mg/mL of both samples. The adherent cells were detected by phase contrast microscopy ( Figure 5). In the untreated controls, only very few cells were stained with Annexin-V, thus delivering a weak green signal. Moreover, red nuclear signals indicating dead cells were not observed for control cells (Figure 5(A)). In contrast, the doxorubicintreated culture showed massive detachment and loss of cells in the phase contrast micrographs, strong binding of Annexin-V to most of the remaining cell membranes, but a low red nuclear PI signal, indicating that the cellular structure was dissolved to large parts and cells had probably undergone apoptosis ( Figure 5(B)). A comparable effect was observed after treatment of CAL-29 tumour cells with Hcs. Strong binding of Annexin-V was detected after treatment of the cells with HlH, while the red fluorescence of PI remained low in most cells ( Figure 5(C)). PI-positive nuclei correlated with round cells observed by phase contrast microscopy, indicating that most cells were undergoing apoptosis (Figure 5(C)). Again, mostly apoptotic and a few necrotic cells were observed after treatment of CAL-29 with FU b c -HlH-h as well ( Figure 5(D)). Zheng et al. [25] described an in vitro effect of hemocyanin from L. vannamei against HeLa cell growth and the molecular mechanisms underpinning the antiproliferative effect of FLH was suggested to be a mitochondria-mediated apoptosis pathway.
These reports were supported by our data obtained from the tests for cytotoxic activity of hemocyanins and their isoforms. We observed a significant influence of Hcs on cell viability, which is in correlation with the processes of cell death, and it is possible that hemocyanins could be involved in the induction of cell death via different pathways.   insight into the mechanism of HlH inhibition on CAL-29 tumour cells, we performed the proteomic analysis. The proteins were extracted from untreated CAL-29 tumour cells and cells treated with HlH and were analyzed by 2D-gel SDS PAGE using a pH gradient from 4-7. A comparison of the proteins extracted from CAL-29 tumour cells (Figure 6(A)) with the proteins extracted after treatment with HlH ( Figure 6(B)) revealed in a number of different protein spots. In addition, 2D PAGE detected differential upregulation (spots B2, B3) and downregulation (spots A1, A2, A3, A4, A6, A7, A8) of several proteins after hemocyanin treatment.

Proteomic analysis of untreated CAL-29 cells and cells treatmed with HlH
All differential spots were subjected to mass spectrometric analysis. The peptide mass fingerprint and MS/MS database search against the public protein databases did not facilitate an identification of the proteins. Therefore, they were identified by MS/MS analyses, Peaks studio and Mascot. The quality of 10 MS/MS spectra of tryptic peptides was sufficient to reveal 10 protein fragment sequences. Figure 7(A) shows an MS spectrum of the peptides obtained after trypsinolysis of proteins from spot A2 of the 2D-PAGE. The peptide at m/z 1824.84 was analyzed by MS/MS and the sequence determined as follows: ETNLDSLPLVDTHSKR (Figure 7(B)). Five different peptides extracted from spot A2 were identified by their MS/MS spectra and their sequences are presented in Table 1. A search in the protein sequence using NCBI BLAST showed amino acid sequences highly similar to the protein vimentin (VIME_HUMAN) with a mass of 53676 Da [39]. By this method, 10 different proteins were successfully identified: spot A1: serum albumin from urine samples [40], spot A2: vimentin [Homo sapiens] [41]; spot A3: ATP-phosphoribo-syltransferase [42]; spot A4: alpha-enolase [Homo sapiens] [43]; spot A5: Glyceraldehyde-3-phosphate dehydrogenase (Homo sapiens) [44]; spot A6: alpha-1-antitrypsin (A1AT_HUMAN) [45]; spot A7: carbonic anhydrase 1 (CAH1_HUMAN) [46]; and spot A8: human alpha 2-HSglycoprotein [47]. Figure 6(B) shows an increase in the expression of two proteins: B3 spot: glutathione Stransferase P1 [48] and Spot B2: apolipoprotein A-I [Homo sapiens (human)] [48,49]. Table 2 lists the identified proteins grouped by function in the main known metabolic pathways.

Conclusions
The observed results demonstrate that HlH, unlike KLH, shows potent antitumour activity on CAL-29 tumour cells. Moreover, the treatment with the FUs b c -HlH-h and RvH1-c is more effective in comparison to native HlH. The mechanism of antitumour activity of HlH, b c -HlH-h, and RvH2-c hemocyanins includes induction of apoptosis. The present study is the first report of protein expression in CAL-29 human cells under the influence of H. lucorum hemocyanin. Thus, the data provide an initial roadmap for further indepth investigations into the mechanism of action of these proteins, thus supporting their application, especially as potential therapeutic agents against bladder cancer.

Disclosure statement
No potential conflict of interest was reported by the authors.