The peptide INKKI was previously isolated from bovine β-casein by HPLC. The sequences were confirmed by amino acid analysis and also by mass spectrometry on a triple quadrupole Micromass mass spectrometer, model Quatro II (Micromass, Milford, MA, USA). After primary sequence determination, the peptides were synthesized by solid phase peptide synthesis and sequenced using Edman degradation. The natural and synthetic peptides were compared by MALDI-TOF. The synthetic peptides were chemically and biologically identical as described in previous works [17,18]. The hydrophobic/hydrophilic balance of INKKI was also calculated considering neutral and ionized forms. The n-octacnol/water partition coefficients, ClogP (neutral) and ClogD (ionization), which expressed this kind of nonpolar/polar equilibrium, were calculated employing the Marvin-Calculator Plugins software available from http://www.chemaxon.com [19-21].

The B16F10 (murine malignant melanoma ATCC® CRL- 6475) cells were obtained from the American Type Culture Collection (Mannasa, VA, USA). FN1 (normal human fibroblasts) cells were obtained from the laboratory of Biochemistry and Biophysics (Butantan Institute, SP, Brazil). All cells were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS), 100 µg/ml L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin in culture flasks at 37˚C in a humid atmosphere containing 5% CO₂.

The cytotoxicity study was performed when cell growth reached 80% - 90% confluence. The cells were then seeded into 96 well plates at a density of 1 × 10⁴ cells/well. Briefly, B16F10 and FN1 cells were plated in triplicate and incubated overnight at 37˚C in a humidified incubator containing 5% CO₂. After this, the cells were treated with concentrations of 20 µM to 0.03 µM INKKI. After 24 h of treatment, cells were exposed to 5 mg/ml MTT for 2 h and the precipitated formazan was dissolved in 0.1 N HCl in isopropanol. The IC50 values were determined and measured at 540 nm on a microplate reader Thermo Plate (Rayto Life and Analytival Sciences C. Ltd, Germany).

The proportion of apoptotic cells was estimated by double staining with Annexin V-fluorescein isothiocyanate (FITC) (Boehringer-Mannheim GmbH, Mannheim, Germany) and propidium iodide-(PI) (Sigma-Aldrich, St Louis, MO, USA). B16F10 cells at a concentration of 1 × 10⁶ cells were treated for 24 h with 1.7 µM INKKI and untreated cells were considered as control. After this period the cells were stained with 4 µg/ml Annexin V and 18 µg/ml PI for 1 h at 37˚C. Stained cells were analyzed by flow cytometry in a FACSCalibur (Becton Dickinson Immunocytometry, San Jose, CA, USA), and the data were analyzed with the software WinMDI 2.6 (Scripps Institute, La Jolla, CA, USA).

Aliquots of 100 μl (1 × 10⁶) of the B16F10 cells were treated with 1.7 µM INKKI and untreated cell were considered as control. Cells were washed 3 times with Krebs Ringer (124 mM NaCl, 4 mM KCl, MgSO₄ 1.2 mM NaHepes 25 mM glucose and 12 mM CaCl₂ 1 mM, pH 7.4). The samples were lysed in PBS (Na₂HPO₄ 0.0004 mg/ml, NaCl 0.008 mg/ml, KCl 0.0002 mg/ml, pH 7.4) with triton X-100 0.2% for the test with the fluorogenic substrate DEVD-MCA (Peptide Institute), specific for caspase-3. The kinetics of hydrolysis of the substrate was monitored in a microplate fluorometer, with excitation at 370 nm and emission at 460 nm. The results are expressed in v/max of substrate hydrolyzed per minute.

B16F10 cells previously treated with 1.7 µM INKKI and untreated cells, considered as control, were harvested, washed with PBS and resuspended in 300 µl of trypsin 0.03 g/l, 10 mM Tris (pH 8.0). After 15 min of incubation at room temperature, the neutralization solution (tripsin inhibitor 0.5 g/l, RNase A 0.1 g/l and spermine 1.2 g/l) was added and incubation continued for 15 min. Pelleted cells were resuspended in 0.3 ml PBS and fixed by addition of ice-cold ethanol (70%). Prior to analysis, cells were incubated with 18 mg/ml propidium iodide solution and incubated in the dark for 30 min. Flow cytometry analysis was performed on a FACScan flow cytometry system (Scalibur-Becton Dickinson, San Jose, CA). The DNA content in the cell cycle phases (G0/G1, S and G2/M) was analyzed by the Cell-Quest software and by the Modfit software cell (Becton, Dickinson, NJ, USA).

The B16F10 cells were maintained according to protocol of the cell culture. Adherent cells were detached from plates with 0.1% trypsin and 0.2% EDTA. After trypsin inactivation with 10% FBS, viable cells were counted based on trypan blue dye exclusion method. For the in vivo experiments 5 × 10⁴ cells suspended in 100 μl of PBS were injected subcutaneously (s.c) in the flanks of C57BL/6J mice.

Mice previously were inoculated with B16F10 melanoma cells as described above. 24 h after tumor implantation, the animals were randomized and divided into different groups (n = 5). Animals were treated with administration into the peritumoral region of 55 mg/kg INKKI and saline for the control group, the treatment continued for 21 days. The tumor sizes were measured three times a week using a caliper-like instrument during the experiment, and converted to tumor weight by the equation: tumor weight = (length2 × width)/2. We also evaluated the tumor growth delayed and tumor doubling time.

All experiments were performed according to institutional ethics commission guidelines concerning the protection of animals used for experimentation and based no the current international procedure rules in animal care and handling (protocol number 349/06 CEUAIBU).

Tumor tissue of the mice previously treated with 1.7 µM INKKI and saline solution mice, considered as control were fixed in 10% buffered formalin and routinely processed for paraffin embedding. Sections of 5 µm were obtained and stained with Hematoxylin-Eosin.

The results were expressed as the mean value ± standard deviation (SD). Statistics analysis was carried out with Student’s t test and analysis of variance (ANOVA) using the Instat, version 2.0, program, with the level of significance set at *p < 0.05, **p < 0.01 and ***p < 0.001.

INNKI corresponds to residues 41 - 45 of the bovine β-casein sequence, and presents a non-polar isoleucine residue at each extremity, which can be visualized by a green to blue color on the molecular surface. The two lysine residues confer positive net charge to the peptide. Asparagine and two lysine residues are responsible for the most hydrophilic feature of this peptide (intense blue regions on surface (Figure 1), and the negative values obtained for ClogP (–1.77) and ClogD (–4.36) corroborate this finding.

The first step in order to evaluate a potential antitumor activity of the peptide was to perform an MTT assay. INKKI had no effect on the viability of FN1 cells. On the other hand, after 24 h of treatment, INKKI exhibited a potent activity against B16F10 cells (IC50 1.7 µM). These results suggest a preferential activity of INKKI on tumor cells (Figures 2(a)-(b)).

The fact that most cells were in the sub-G1 peak indicated that they were dying. To verify if the cells were undergoing an apoptotic process, we used annexin V. The cells were stained with two cell markers, annexin V, to measure phosphatidylserine translocation to the extra-cellular leaflets (early apoptosis), and PI to measure the loss of phospholipid membrane integrity (late apoptosis/necrosis). The treatment of the B16F10 murine malignant melanoma cells with INKKI at 1.7 µM concentration resulted in an 82.82% ± 8.56 increase of apoptotic cells as compared with the control (3.99% ± 1.2 apoptotic cells) (Figure 3(a)). These data suggest that INKKI is highly effective in controlling tumor progression by apoptosis (Figure 3(b)). To confirm the annexin V assay, we performed a fluorometric analysis that showed that INKKI induces apoptosis probably mediated by the increase of caspase-3 activity (Figure 3(c)).

We then analyzed the effect of the peptides on the cell cycle. After 12 h of treatment with INKKI at 1.7 µM, showed a significant (***p < 0.001) increase in the median percentage of apoptotic cells as evidenced by the sub-G1 peak, as compared to the untreated cells. In addition, the proportion of cells in G2/M phases decreased. These data suggest that INKKI mediate antiproliferative and proapoptotic effects on B16F10 cells (Figure 3(d)).

All previous results were obtained in vitro. To evaluate the in vivo effect, the peptide was injected in the peritumoral region of mice bearing B16-F10 melanoma cells. INKKI reduced significantly (78.8%) the tumor volume as compared to the control animals (Figures 4(a)-(b)). Histological analysis revealed an increase of necrosis areas and reduction of tumor cells in tumor treated with INKKI, it was a many hallmark of its antitumor effects observed from in vivo experiments (Figure 4(c)). The mice treated with 55 mg/kg of INKKI presented inhibition of total metastasis (72.62% ± 3.7%) in comparison with the control group (Figure 5(a)). Mice treated only with saline showed increased metastasis (average of 84.5% ± 5.2%), located mainly in the lung, lymph node and spleen (Figure 5(b)). In addition, the tumor volume measurements allowed calculate the tumor growth delay and tumor doubling time. In agreement with the reduction of tumor volume, the treatment with INKKI caused an increase of the tumor doubling time and tumor growth delay, i.e., the time for the tumor to reach at 1 cm³ (Figures 5(c)-(d)).