Enhancement of Cancericidal Activity of Bamlet Complexes by Enzymatic Treatment

doi:10.21203/rs.3.rs-2082257/v1

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Enhancement of Cancericidal Activity of Bamlet Complexes by Enzymatic Treatment

https://doi.org/10.21203/rs.3.rs-2082257/v1

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BAMLET (Bovine α-Lactalbumin Made LEthal to Tumors) is a tumoricidal molecular complex of partially unfolded bovine α-lactalbumin and multiple oleic acid molecules. Although this class of molecules was shown to be effective in many cancer types, their effectivity was not promising compared to synthetic drug products. In this study, the α-lactalbumin was purified from the skimmed cow milk, and BAMLET was produced with oleic acid. The cytotoxic character and the anti-cancer activity of BAMLET and of its hydrolysed form were comparatively analyzed in vitro focusing on breast cancer (MCF7) and prostate cancer cell (DU145) lines. The results showed that the most effective dose of the untreated form of BAMLET decreased the viability of MCF7 and DU145 by 89.2% at 10 µg/mL and 48.0% at 2.14 µg/mL after 24 hours, respectively. After the hydrolyses, the most effective doses were altered, but the anti-cancer effect was improved to 21.9% for MCF7 (6.38 µg/mL) and 32.2% for DU145(6.38 µg/mL) under the same condition. Interestingly, the untreated BAMLET demonstrated cytotoxic effect on fibroblasts above the concentration of 2.1 µg/mL, but this detrimental effect was vanished after the enzyme treatment of BAMLET. The cell viability was supported by 2.7-fold at 6.38 µg/mL hydrolised BAMLET. As conclusion, BAMLET produced from the hydrolysed form of the α-lactalbumin was found to be more effective against the cancer cells than its non-hydrolysed form. The hydrolysed BAMLET was found to be a promising natural anti-cancer product without any toxic effect on fibroblasts.

Bovine α-lactalbumin

BAMLET

Breast cancer

Prostate cancer

Enzymatic hydrolysis

Protein purification

Complexes formed with oleic acid (OA) and α-Lactalbumin (α-LA) have attracted attention due to their therapeutic potential. Many studies have been carried out for years about this complex, which was discovered serendipitously by the Svanborg group (Håkansson et al., 1995; Svensson et al., 2003; Svenssonet al., 2000). α-LA is an acidic (pI around 4.5), (Permyakov & Berliner, 2000), globular protein composed of 123 amino acid residues found in milk. The molecular weight of this acidic calcium-binding protein in its native conformation is 14.2 kDa (Svensson et al., 2003). The three-dimensional structure of α-LA consists of two domains: the α-domain (residues 1–34 and 86–123), consisting of four α-helices and two short 3₁₀-helices; and the smaller β-domain (residues 35–85), (Casbarra, et al., 2004; Spolaore, et al., 2010). The native conformation is stabilized by four disulfide bonds (amino acids 6-120, 61–77, 73–91 and 28–111), and the two domains are linked by the calcium-binding loop (Pettersson et al., 2006). Either by heating, by the interaction with high concentrations of denaturants or by incubating at low pH, the native α-LA is converted to the partially unfolded protein form, so called the intermediate state or the molten globule state (Casbarra et al., 2004; Lassalle et al., 2003; Mok et al., 2005; Spolaore et al., 2010). After the addition of oleic acid to apo-state of α-LA, the formed complex can induce cell death in cancer cells (Svensson et al., 2003). α-LA contains conserved amino acid side-chains that can make salt bridges with the carboxylate group of oleic acid. Due to multiple sites with high or low binding affinities in α-LA, the binding of oleic acid to these sites becomes highly complicated (Petitpas et al., 2001). Moreover, oligomerization reaction rates are depended on α-LA concentration (Baumann et al., 2012).

For the production of this protein-fatty acid complex, human milk was used as a first source of α-LA, and the product was named as HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), (Svensson et al., 2000). While this complex induces an apoptotic mechanism in tumour cells, the healthy cells are resistant to this effect (Delgado et al., Håkansson et al., 1995; 2015; Jung et al., 2016; Svensson et al., 2003; Svensson et al., 2000;). Its anti-tumoural activity was shown on more than 40 different lymphoma and cancer cell lines (Hoque et al., 2015). It has been found that α-LA from different sources (human, bovine, goat, swine, sheep, equine and camelid) can form OA-complexes that can exhibit biological properties similar to HAMLET (Fontana et al., 2013; Pettersson et al., 2006; Spolaore et al., 2010). In other words, it has been proven that HAMLET-like complexes can also form with other α-LA varieties (Fang et al., 2014). For this purpose, bovine milk (BAMLET: Bovine Alpha lactalbumin Made LEthal to Tumor cells), which is more accessible source of milk, was used within the scope of the project.

In order to understand the tumoricidal activity of HAMLET, its molecular structure has been studied by researchers (Ho et al., 2012; Pettersson et al., 2006). Although the sequence variations in α-LA from animal sources exist, other α-LA variants can also bind oleic acid to form complex, as like in human α-LA. BAMLET, on the other hand, can bind high rates of oleic acid residues (5–8 oleic acids per protein molecule) than other protein-lipid complexes. It, therefore, has higher lipid-to-protein ratio than the HAMLET complex, and it might show different properties (Ho et al., 2012). In this study, bovine α-LA was fragmented into short peptides in various length with the help of hydrolysing enzymes, and hydrolysed BAMLET was produced subsequently. For this purpose, pepsin and trypsin peptidases were used. The activity of the formed complex was proven on DU145 and MCF7 cancer cell lines.

2.1 Purification of Bovine α-LA

α-LA was purified from skim bovine milk from commercial sources according to the method of Pettersson et al., (2006) with some modifications. Briefly, casein was removed by acid treatment adjusting the milk to pH 4.0 with HCI, and centrifuging at 4000g for 20 min at 4°C. α-LA was separated in fractional precipitation at the 55% ammonium sulfate saturation, and the precipitate was collected by centrifugation at 8000g for 20 min at 4°C. After dissolving, sample was desalted by dialysis in 0.1 M Tris-HCl buffer at pH 7.5 at 4°C, and ultrafiltrated using Amicon-Ultra 15 Centrifugal Filter Units (Merck-Millipore, Tullagreen, Cork, Ireland) with a molecular weight cut-off of 10 kDa. α-LA was purified by FPLC using size-exclusion (SEC) column (HiPrep Sephacryl S-200 HR) on ÄKTA Prime Plus FPLC (GE Healthcare) by eluting in 10 mM Tris-HCl buffer, pH 7.5 with 10 mM EDTA. Samples were concentrated by the ultrafiltration as described above. The purity was confirmed by SDS-PAGE and the molecular weight was estimated by using the gel filtration chromatogram (HiPrep Sephacryl S-200 HR) with low molecular weight markers (Gel Filtration Cal. Kit Low Molecular Weight, GE Healthcare). The total protein content was measured by SMART bicinchoninic acid assay (BCA) Protein Assay Kit (iNtRON Biotechnology, Sungnam, Korea). After mixing with loading buffer solution (Tris-HCl 0.5 M pH 6.8, glycerol, sodium dodecyl sulfate (SDS), β-mercapto ethanol and Bromophenol Blue), samples (5 µg) were denatured in boiling water for 3 min. The denatured protein was loaded and separated by 12% SDS-polyacrylamide gel (TGX FastCast acrylamide gels; BioRad, Hercules, CA, USA) and run at 120 V for 50 min. Bands were visualized in DNR Bio-Imagining Systems (MF-ChemiBIS 3.2, Jerusalem, Israel).

2.2. Enzymatic Hydrolysis of α-LA

For the hydrolysis of α-LA, the method described by Hernández-ledesma & Dávalos (2005) was used. α-LA (1 mg/mL) was first hydrolysed with trypsin (EC 3.4.21.4.; Type I; 10 900 U/mg protein; Sigma; St. Louis, MO, U.S.A.) in 0.1 M Tris-HCl buffer (pH 7.4) for 24 h at 37°C. Later, the pH was adjusted to pH 2.5 by HCl (1M), and pepsin (EC 3.4.23.1.; 1:60 000, 3400 U/mg protein; Sigma; St. Louis, MO, U.S.A.) was added at the ratio of enzyme-to-substrate by 1:20 (w/w). After the incubation for 2h at 37°C, enzymatic activity was stopped by heating at 95°C for 15 min.

2.3. Preparation of α-LA – oleic acid complexes

α-LA at 210 µM in phosphate-buffered saline (PBS) was mixed with oleic acid (1:2, v/v). The mixture was heated to 55°C, and incubated for 15 min at this temperature. Later, the mixture was allowed for 10 min to cool to room temperature. The sample was centrifuged (12.000g, 25°C, 15 min.). Excess oleic acids were removed (Kamijima et al., 2008).

2.4. Morphology study

For the ultrastructural analysis, scanning electron microscopy (SEM) was used. The samples were sprinkled onto a two-sided adhesive tape and then coated with a thin layer of gold. Morphologic features of samples observed by a scanning electron microscope (Quanta FEG 250) in high vacuum with 5 kV accelerating voltage.

2.5. FTIR study

Functional groups in α-LA, hydrolysed α-LA, BAMLET and hydrolysed BAMLET examined by Fourier transform infrared spectroscopy (FTIR) using the attenuated total reflection technique. After freeze-drying the samples were analyzed in the FTIR GX (Perkin-Elmer, Shelton, CT, USA) instrument. All spectra ranged from 200ˉ ˡ cm to 4000ˉ ˡ cm. The spectra were obtained by averaging 16 scans at 4 cmˉ ˡ resolution.

2.6. DSC study

Thermal behavior of α-LA hydrolysed α-LA, BAMLET and hydrolysed BAMLET were investigated by a differential scanning calorimeter (DSC-028 Mettler Toledo Inc., Columbus OH). The denaturation temperature range (ΔTd), peak denaturation temperature (Td) and the enthalpy of denaturation (ΔHd) of lyophilised samples were determined. About 5 mg of each sample was weighed in an aluminum pan and hermetically sealed. An empty pan was used as a reference. Samples were scanned at the temperature range of 40–300°C with the rate of 5°C/min.

2.7. Anticancer activity of BAMLET and BAMLET hydrolysates

2.7.1. Cell lines and Cell Culture

The analyses of the cancericidal effect of BAMLETs were performed using two cancer cell lines, MCF7 (human breast adenocarcinoma cell line) and DU145 (androgen-independent human prostate cancer cell line). To determine the cytotoxic effect of BAMLETs on normal somatic cells, fibroblast cells derived from foreskin tissue were used (Acemi et al., 2017). All cell lines were cultured in the complete RPMI 1640 medium [RPMI 1640 basal medium (Gibco, Paisley, UK) supplemented with 2.0 mM L-glutamine, 0.5 mM sodium pyruvate, 10% fetal bovine serum (Gibco) and 1% Penicillin-Streptomycin solution (Gibco). Cells were cultured under standard culture conditions in a humidified environment of 5% CO₂ at 37°C. Before the cell seeding into the culture plates, cells were detached by 0.25% trypsin-EDTA (Gibco).

2.7.2. Determination of cell viability by WST-1

To determine the effect of BAMLETs on fibroblast, MCF7 and DU145, cells were incubated with different concentrations of BAMLET and hydrolysed-BAMLET in RPMI 1640 complete medium for 24 h. The medium was replaced with RPMI 1640 basal medium supplemented with 10% WST-1 (Roche, Mannheim, Germany), and further incubated for 30 min at 37 °C. The absorbance at 450 nm was measured. The changes in cell numbers were expressed in percentage with respect to control group (medium without BAMLET).

2.8. Lactate dehydrogenase (LDH) activity assay

Cytotoxicity was determined with Cytotoxicity Detection Kit LDH (Roche) according to the manufacturer’s instructions (Duruksu & Aciksari, 2018). MCF7 and DU145, cells were incubated with different concentrations of BAMLET and hydrolysed-BAMLET in RPMI 1640 complete medium for 24 h. After performing the assay, the absorbance of the collected supernatant was measured at 490 nm and a reference wavelength of 620nm with controls including medium alone. The cell culture without BAMLET or hydrolised BAMLET was used as control.

2.9. Statistical analysis

Data were measured in triplicate. All statistical analyses were performed with the use of SPSS 10.0 (SPSS Inc, Chicago, IL, USA). The significance of results was analyzed by means of paired t test. In all analyzed cases, the results were regarded as statistically significant at p < 0.05.

3.1. Identification of purified α-LA and hydrolysed α-LA

α-LA was purified by FPLC method. A protein peak of 14.2 kDa was obtained according to the calibration curve of standard proteins with known molecular mass (Fig. 1). In agreement with the results by Svensson et al., (2003), α-LA was observed as an intense, single band around 14 kDa as shown on SDS-PAGE (Fig. 2). Hydrolysis of α-LA with pepsin and trypsin generated peptide fragments with small molecular weight (Table 1). Hydrolysed α-LA on the SDS-PAGE showed a faint band around 15 kDa, indicating that the majority of α-LA is hydrolysed by pepsin and trypsin and minor amount of unhydrolysed α-LA still existed as a peptide band of 15 kDa. The digestion of α-LA protein into smaller fragments by the hydrolysing enzymes provided to separate into functional peptides, which was predicted putatively on Table 1. Many functional groups on α-LA was expected to become available to interact with other molecules by this process (Gómez-Mascaraque et al., 2016; Jia et al., 2020; Lacroix & Li-Chan, 2014, Url-1).

Table 1

Putative digestion products of α-LA by pepsin or trypsin (Lacroix & Li-Chan, 2014; Jia et al., 2020; Gómez-Mascaraque, Miralles, Recio & López-Rubio, 2016, Url-1)
Fragment	Sequence	Pepsin / Trypsin
1–5	EQLTK	Trypsin
1–10	EQLTKCEVFR	Trypsin
10–17	RELKDLKG	Pepsin
12–16	LKDLK	Trypsin
16–25	KGYGGVSLPE	Pepsin
32–36	HTSGY	Pepsin
37–43	DTQAIVQ	Pepsin
37–44	DTQAIVQN	Pepsin
41–53	IVQNNDSTEYGLF	Pepsin
45–50	NDSTEY	Trypsin
51–58	GLFQINNK	Trypsin
60–79	WCKDDQNPHSSNICNISCDK	Pepsin
63–68	DDQNPH	Trypsin
90–95	CVKKIL	Pepsin
95–104	ILDKVGINY	Pepsin
94–98	KILDK	Trypsin
94–108	KILDKVGINYWLAHK	Trypsin
95–108	ILDKVGINYWLAHK	Trypsin
99–104	VGINYW	Trypsin
104–117	WLAHKALCSEKLDQ	Pepsin
105–111	WLAHKAL	Pepsin
105–115	LAHKALCSEKL	Pepsin
110–117	LCSEKLDQ	Pepsin
115–123	LDQWLCEKL	Trypsin
115–122	LDQWLCEK	Trypsin

3.2. Characterization of BAMLET and Hydrolysed BAMLET complexes

3.2.1. Molecular weight characterisation

In the current study, the heating method was preferred to generate BAMLET because of the high efficient production of BAMLET. The generation of BAMLET by an anion exchange column, which was loaded with oleic acid, has been described previously, but the method by heating a mixture of α-LA and oleic acid generated with higher efficiency (Håkansson et al., 1995; Svensson et al., 2003; Svensson et al., 2000). By heating the α-LA protein, partial denaturation was induced, and the complex formation took place efficiently (Kamijima et al., 2008).

After the hydrolysis of α-LA, the same procedure for BAMLET was applied to the smaller fragments. SDS-PAGE was insufficient to separate these products, but the single band of BAMLET produced from the α-LA could be detected on the gel (Fig. 2). Peptides in hydrolysed BAMLET are mostly smaller than 10 kDa. While hydrolysis-derived peptides formed small aggregates with the effect of low pH (~ 2.5), the cross-links they formed between them consist of disulfide bonds. In addition to disulfide bonds, other intermolecular cross-links were also formed between protein aggregates formed at higher pH values (above 4.5), respectively (Gulzar et al., 2011). Observation of a faint band around 15 kDa indicates that new and strong covalent bonds were not formed when hydrolysed BAMLET was formed from hydrolysed α-LA (Gulzar et al., 2011), but newly formed disulfide bonds between peptides (Maux et al., 2013). During the production of BAMLET, in which the mixture of molecules were heat treated for 15 min., two main forms of BAMLETs were mainly generated with molecular weights of about 25.5 kDa and 51 kDa, which was predicted as the monomeric and dimeric forms of complexes, respectively (Fig. 3). The intense bands on SDS-PAGE and the data of SEC analysis of BAMLET indicate that BAMLETs interconnected by disulfide bonds and by intermolecular cross-linkages to form larger insoluble and soluble aggregates. The molecular mass of the monomeric BAMLET was estimated to be approximately 25.5 kDa (Fig. 3). In a complementary approach, it confirmed that the SDS-PAGE showed another peak around 51 kDa, a dimeric form of BAMLET. Remarkably, another peak at 14.2 kDa, which refers to α-LA, could be observed to remain as residue in the medium left without interacting with oleic acid (Fig. 3). This α-LA residue of BAMLET reaction was left to interact with oleic acid overnight at 55°C. It was observed that BAMLET complexes were formed at various molecular weights (Fig. 4). During the synthesis process, high temperature and high pH led some modifications to form high or low binding affinity sites on α-LA. These changes further supported the production of new oligomers after the process. Therefore, oleic acid:α-LA complexes could be formed at different ratios. A continuous complex formation took place between α-LA and excess oleic acid, which cannot be eliminated. The various form of BAMLET complexes formed after processing could be explained by either the formation of peptide aggregates that cannot be dissociated by the SEC eluent or newly formed disulphide bonds between peptides and proteins interconnected by intermolecular cross-links (Maux et al., 2013).

3.2.2. Morphological characterisation

The SEM micrographs of BAMLET and hydrolysed BAMLET showed that the BAMLET and BAMLET-like complexes have a globular conformation due to the globular structure of α-LA (Fig. 5), as stated before (Delgado et al., 2015; Mossberg et al., 2010). Similarly, the peptide fragments of hydrolysed BAMLET appeared as spherical structures in the SEM image. In general, smaller microparticles were obtained when pepsin and trypsin enzymes were used as hydrolysing substance (mean diameter: 0.192 µm). Particle sizes and polydispersity index (PDI) are extremely important for drug formulations. It has been reported that particles with a diameter of 50–500 nm are known to be suitable for accumulate in tumor cells (Delgado et al., 2015). The relatively low polydispersity index (PDI) value of hydrolysed BAMLET indicated that it has a more regular particle size distribution than BAMLET.

3.2.3. FTIR characteristics

The FTIR spectrum of α-LA (Fig. 6a) showed a broad band at around 3000 ~ 3700 cm ˉ ˡ, indicating enhanced hydrogen bonding compared to that of BAMLET seen in Fig. 6a. This suggests that hydrogen bonding is also involved in the interaction between α-LA and oleic acid. The lipid interaction effect formed by the binding of oleic acid to α-LA is clearly visible in the stretching vibration in the range of 2800 ~ 3000 cm ˉ ˡ (yellow circle). Characteristic bands were observed for both molecules (α-LA and BAMLET) at 1642 cmˉ ˡ (yellow circle) in a characteristic amide I band attributed to the C = O vibration (C = O stretching) of the acetylated units. The decrease in 1408 cm ̄ ˡ is due to the vibration of -OH and -CH (Chang et al., 2016). However, although there are many higher or lower wave numbers between the 1000 ~ 1500 cm ˉ ˡ range in both groups, due to the overlap of the amide I region and most spectral bands, these different fluctuations can be attributed to oleic acid binding to α-LA and to the application of heating treatment while BAMLET was formed. The broad band seen in the FTIR spectrum of BAMLET and hydrolysed α-LA in the range 3000 ~ 37000 cm ˉ ˡ was not seen for hydrolysed BAMLET (Fig. 6b). However, stronger stretch vibration and a wider band are observed in the range of 2800 ~ 3000 cm ˉ ˡ (yellow circle). This situation draws attention to the lipid interaction density between hydrolysed α-LA and oleic acid, which creates hydrolysed BAMLET. The FTIR spectrum of hydrolysed α-LA showed absorption bands at 1615 cm ˉ ˡ (amide I, C ═ O and C ― N stretching), 1410 cm ˉ ˡ and 1320 cm ˉ ˡ (amide II, N ― H deformation and C ― N stretching), (Huang et al., 2012). For Hydrolysed BAMLET, while none of the amide groups were observed, a single distinct peak was detected. The peak at 1747.33 cmˉ ˡ corresponds to the esterified group of hydrolysed BAMLET (yellow circle), (Raei et al., 2017). BAMLET, on the other hand, has only amide I bonds in this domain (yellow circle, Fig. 6c).

3.2.4. Thermal characteristics (DSC)

The DSC thermograms of BAMLET, hydrolysed BAMLET, α-LA and hydrolysed α-LA were shown in Fig. 7A and Fig. 7B respectively. The thermograms of both α-LA and hydrolysed α-LA exhibited a weak melting behavior at around 160°C, which associated with the melting of the network structures (Huang et al., 2012). The melting behavior in the network structures continued for the enzyme-treated α-LA, which is corresponding to the thermal denaturations of less stable peptide fragments have been observed at temperatures lower than 200°C (Table 2). However, α-LA exhibited a sharp peak. A higher denaturation temperature and denaturation enthalpy of α-LA shows a network structure with improved thermal stability compared to hydrolysed α-LA. It has been announced that, complex proteins are more stable against thermal denaturation (Capitani et al., 2007; Chang et al., 2016; Huang et al., 2012; Timilsena et al., 2015). Similar to α-LA and hydrolysed α-LA, both thermograms of BAMLET and hydrolysed BAMLET begin with melting of the network structures; hydrolysed BAMLET exhibited a very different diagram. Hydrolysed BAMLET composed of very small peptides showed only slight crystallinity, attributed to monomerization causing weaker network structure. BAMLET, on the other hand, showed an endothermic peak at higher temperature (Td = 231.44°C). This denaturation temperature could be ascribed to a tendency to dimerisation. The same situation was observed for α-LA at Td = 236.8°C.

Table 2

Denaturation temperature range (ΔTd), peak denaturation temperature (Td) and denaturation enthalpy (ΔHd) of CPI and α-LA, hydrolysed α-LA, BAMLET and hydrolysed BAMLET.
Materials		ΔTd (°C)	Td (°C)	ΔHd (j/g)
α-LA	First peak	203.67-219.12	199.33	+ 270.7
α-LA	Secound peak	244.25-235.12	236.8	+ 5.16
Hydrolysed α-LA	First peak	190.96-183.63	185.22	+ 38.96
	Secound peak	201.87-190.96	192.15	+ 59.28
	Third peak	231.78-201.87	204.94	+ 101.2
BAMLET	First peak	129.68–114.60	116.73	+ 50.54
	Secound peak	153.65-129.68	142.91	+ 32.69
	Third peak	183.56-153.65	231.44	+ 122.5
Hydrolysed BAMLET	First peak	190.8–82.7	143.7	-100.9

3.3. Toxicity of BAMLET products on fibroblast cells

LDH activity of BAMLET and hydrolysed BAMLET was estimated for both MCF7 and DU145 cell lines after incubation for 24 h (Fig. 8). The toxicity of BAMLET was rather limited for MCF7 cells. At the highest BAMLET concentration (10 µg/µl), less than 6.6% ±1.3 of LDH activity level was observed compared to the control (without BAMLET). On the other hand, the highest toxicity level with 25.6% ±2.2 toxicity was achieved in DU145 cell lines at the BAMLET concentration of 2.14 µg/µl. α-LA has a toxicity effect with 0.83% ±2.2 and 8.8% ±0.9 for MCF7 and DU145, respectively. Compared to α-LA, the BAMLET showed improved toxic effect, but the toxicity level for BAMLET could not be detected higher than 8% and 28% for MCF7 and DU145 over the BAMLET concentration ranging from 1.07 to 10.00 µg/µl.

The hydrolysed BAMLET possessed significantly improved toxic effect for both cancer cells. The toxicity levels of hydrolysed BAMLET for MCF7 and DU145 were estimated as 49.2% and 55.2% for the concentration at 6.38 µg/µl of hydrolysed BAMLET compared to the control. The effectivity was improved by almost 6.2-fold and 2-fold against MCF7 and DU145, respectively.

In the previous studies with BAMLET, it was shown that two types of cytotoxicity effect of BAMLET could be observed depending on its concentration. Cell apoptosis could be observed at low concentrations, but at high concentrations cell lysis could be detected (Brinkmann et al., 2011). In this study, higher BAMLET concentrations were required to observe the cytotoxic effect after 1 hour of incubation.

3.4. Anti-cancer effect of BAMLET and hydrolysed BAMLET products

The viability analyses of MCF7 and DU145 cells treated with BAMLET / hydrolysed BAMLET complexes was performed by WST-1. We found that the synthesized complexes showed varying toxicities (Fig. 9). Although α-LA demonstrates relatively better cytotoxic effect for the prostate cancer cells (DU145), it also caused to decrease the cell viability of breast cancer cells, MCF7 (Fig. 9B and Fig. 9A). After the treatment with BAMLET at the concentration of 2.14 µg/µl for 24 hrs, the viability of DU145 cells were decreased more than 50%. (Fig. 9B). However, the same BAMLET concentration did not have the same cell viability decreasing effect on MCF7 (Fig. 9A). The MCF7 viability was slightly decreased to 89.16% at the concentration of 10 µg/µl (Fig. 9A).

After the treatment with the hydrolysing enzymes, tumoricidal effect of BAMLET was significantly improved. It was observed that the formed peptide fragments retained their ability to form a complex with oleic acid possessing a tumoricidal activity, similar to BAMLET. Hydrolysed BAMLET was observed to have improved cytotoxicity at the dose of 6.38 µg/µl for both cancer cells (Fig. 9C and Fig. 9D). The effect of BAMLET and hydrolysed BAMLET on somatic cells (fibroblasts) at the specified dosage ranges is quite important for its safely usage. BAMLET supported the cell viability of fibroblast cells until the concentration of 2.14 µg/µl, but the viability suddenly dropped at the concentrations higher than 4.25 µg/µl of BAMLET (Fig. 9E). Interestingly, the hydrolysed BAMLET did not showed any detrimental effect on the fibroblast cell viability (Fig. 9F). On the contrary, it supported the cell proliferation. The cell population was enhanced by 2.71-fold at the concentration of 6.38 µg/µl, at which the prostate and breast cancer showed %32.2 and %22 cell viability. BAMLET had a tumoricidal effect of 2.89% (Fig. 9A) on MCF7, while on DU145 the level of this effect was 51.9% (Fig. 9B). On the other hand, the hydrolysed BAMLET had a tumoricidal effect of 78.04% and 67.83% on MF7 and DU145 cells, respectively (Fig. 9C and Fig. 9D).

In the current study, the BAMLET was produced from the hydrolysed α-LA fragments, so-called “hydrolysed BAMLET”, with improved cytotoxicity against MCF7 and DU145 cells. Moreover, this hydrolysed BAMLET supported the cell population of somatic cells while decreasing the number of cancer cells. From this aspect, the hydrolysed BAMLET might be an ideal anti-cancer agent to be used in the cancer treatment. The hydrolysis process released the functional groups of α-LA, which were more effective in cellular toxicity against MCF7 and DU145 cells. It is still unclear which released functional peptide fragments of α-LA possessed the toxic effect or which fragments involved in the complex formations with oleic acid. Long-term incubation of α-LA and oleic acid led the formation of complexes with various dimerization rates. The dimerization trend in BAMLET is consistent with the DSC thermograms. Although hydrolysed BAMLET yields a less stable thermogram, an intense lipid interaction between α-LA and oleic acid according to the FTIR spectrum. Moreover, the collection of functional groups in a single peak at 1615 cm ˉ ˡ indicates the presence of structurally esterified groups.

BAMLET and hydrolysed BAMLET complexes were identified as significant growth inhibitors on the MCF7 and DU145 cell lines. However, hydrolysed BAMLET is more effective than BAMLET from the point of toxicity. Hydrolysed BAMLET, at the concentration of 6.38 µg/µl where it has a strongest tumoricidal effect on MCF7 and DU145 cancer cells, showed a supportive effect on the viability of fibroblast cells. In the future, BAMLET and hydrolysed BAMLET could be considered for using in drug formulations with the appropriate method.

α-LA, alpha lactalbumin; BAMLET, Bovine α-Lactalbumin Made LEthal to Tumors; BCA, bicinchoninic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; FTIR, Fourier transform infrared spectroscopy; DSC, Differential scanning calorimetry; SEM, scanning electron microscopy; LDH, Lactate Dehydrogenase; WST, Water-soluble tetrazolium; PDI, polydispersity index.

Acknowledgments

We would like to thank to Evren DEMİRCAN from the Department of Food Engineering at Istanbul Technical University for his technical assistance in DSC and FTIR analyses.

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Declaration of competing interest

No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.

Credit authorship contribution statement

Reyhan Koyuncu: Methodology, Formal analysis, Resources, Investigation, Writing-original draft, Revision, Conceptualization, Project administration. Gokhan Duruksu: Conceptualization, Validation, Reviewing & editing, Supplementary data, Data curation. Beraat Ozcelik: Review & editing, Supervision. Yusufhan Yazır: Investigation, Supervision.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Enhancement of Cancericidal Activity of Bamlet Complexes by Enzymatic Treatment

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Purification of Bovine α-LA

2.2. Enzymatic Hydrolysis of α-LA

2.3. Preparation of α-LA – oleic acid complexes

2.4. Morphology study

2.5. FTIR study

2.6. DSC study

2.7. Anticancer activity of BAMLET and BAMLET hydrolysates

2.7.1. Cell lines and Cell Culture

2.7.2. Determination of cell viability by WST-1

2.8. Lactate dehydrogenase (LDH) activity assay

2.9. Statistical analysis

3. Results And Discussions

3.1. Identification of purified α-LA and hydrolysed α-LA

3.2. Characterization of BAMLET and Hydrolysed BAMLET complexes

3.2.1. Molecular weight characterisation

3.2.2. Morphological characterisation

3.2.3. FTIR characteristics

3.2.4. Thermal characteristics (DSC)

3.3. Toxicity of BAMLET products on fibroblast cells

3.4. Anti-cancer effect of BAMLET and hydrolysed BAMLET products

4. Conclusion

Abbreviations

Declarations

References

Supplementary Files

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