WO2006128289A1 - Use of brevinin-2r in the treatment of cancer - Google Patents

Abstract

Described herein is a novel member of the Brevinin family of peptides which has anti-cancer activities but is also non-hemolytic. As a consequence, the peptide can be utilized to induce apoptosis in cancerous cells.

Description

Use of Brevinin-2R PRIOR APPLICATION INFORMATION

The instant application claims the benefit of US Provisional Application 60/686,414, filed June 2, 2005. BACKGROUND OF THE INVENTION

Antimicrobial peptides (AMPs) are widely distributed as an essential defense component of both invertebrates and vertebrates. Amphibians have a rich chemical arsenal as an integral part of their defense system and regulation of their dermal physiological actions. Antimicrobial peptides are produced and stored in specialized glands, which release their contents onto the dorsal surface or on the gut of the frog upon in response to a variety of stimuli (Bevins and Zasloff, 1990, Ann Rev Biochem 59: 395- 414; Barra and Simmaco, 1995, Trends Biotech 13: 205-209; Hancock and Scott, 2000, PNAS USA 97: 8856-8861 ). So far more than two dozen peptides have been reported in a single host (Hancock and Rozek, 2002, FEMS Microbiology Lett 206: 143-149). These peptides display a large amount of heterogeneity in primary and secondary structures but share common features that seem to underlie their cytotoxic function, such as amphipathy and net positive charge. Specifically, many of these peptides are believed to form ion channels in membranes. Therefore, the primary structure includes highly positive residues like lysine and arginine to form the internal part of ion channel; hydrophobic residues like leucine, isoleucine, and valine; and a region which can form a hinge, usually containing glycine or alanine.

Following well-advertised magainins from the skin of Xenopus laevis, a number of cationic peptides from various amphibians have been isolated and found to have a broad- spectrum of antimicrobial activity (Barra and Simmaco, 1995; Papagianni, 2003, Biotechnol Adv 21 : 465-499), Gaegurins (Park et al., 1994, Biochem Biophys Res Commun 205: 948-954) and Rugosins (Suzuki et al., 1995, Biochem Biophys Res Commun 212: 249-254) from Rana rugosa, Brevinins from Rana brevipoda porsa (Morikawa et al., 1992, Biochem Biophys Res Commun 189: 184-190), Rana esculenta (Simmaco et al., 1994, J Biol Chem 269: 11956-11961 ), Rana sphenocephala, (Conlon et al., 1999, J Peptide Res: 54: 522-527), Esculentins (Simmaco et al., 1993, FEBS Lett 324: 159-161) from Rana esculenta, Ranalexin (Clark et al., 1994, J Biol Chem 269: 10849- 10855), Ranatuerins (Goraya et al., 1998, Biochem Biophys Res Commun 250: 589-592) from Rana catesbeiana, Temporins (Simmaco et al., 1996, Eur J Biochem 242: 788-792) and Ranatuerin 1T from Rana temporaria (Goraya et al., 1999, Peptides 20: 159-163), Rana luteiventris, and Rana pipiens (Goraya et al., 2000, Eur J Biochem 267: 894-900). AMPs belonging to Brevinins; Esculentins and Ranatuerins families were also isolated from Rana luteiventris, Rana berlandieri and Rana pipiens (Goraya et al., 2000). Brevinin- 2, first isolated from an extract of the skin of the Japanese pond frog Rana brevipoda porsa (Morikawa et al., 1992), has a wide distribution in those species of Asian and European Ranid frogs examined to-date (Conlon et al., 2004, Biochim Biophys Acta 1696: 1-14). Furthermore, seven members of the brevinin-2 family were identified by analysis of skin secretions from a single specimen of Rana esculenta (Simmaco et al., 1994, J Biol Chem 269: 1 1956-11961 ) but a further three members of the family (Brevinin-2Ei, Brevinin-2Ej and Brevinin-2Ek) were identified in a pooled extract of skins from multiple specimens collected in the wild (Conlon et al., 2004; AIi et al., 2003, Peptides 24: 955-961) and two additional members (Brevinin-2Eg and Brevinin-2Eh) were isolated from a pooled extract of gastric tissue from R. esculenta (Conlon et al., 2004; Wang et al., 1998, Biochem Biophys Res Commun 253: 600-603).

So far nobody has reported any AMP from Rana ridibunda. This patent focuses on a new antimicrobial peptide belonging to Brevinin-2 family from the skin of the marsh frog, Rana ridibunda, a closely related species to European water frogs, Rana esculenta and Rana lessonae (Spolsky and Uzzell, 1986, MoI Biol Evol 3: 44-56). This peptide has been named Brevinin-2R (due to its similarity to the Brevinin 2 family of AMPs and R from genus Ridibunda). SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided an isolated peptide having an amino acid sequence substantially equivalent to SEQ ID NO. 1 , SEQ ID NO. 2 or SEQ ID NO. 3.

According to a second aspect of the invention, there is provided a method of generating a non-hemolytic, anti-cancer brevinin peptide comprising: decreasing the hydrophobicity of a brevinin peptide.

According to a third aspect of the invention, there is provided a method of treating cancer comprising administering to an individual in need of such treatment an effective amount of a peptide comprising an amino acid sequence as set forth SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3. According to a fourth aspect of the invention, there is provided a method of preparing a medicament for treating cancer comprising mixing an effective amount of a peptide comprising an amino acid sequence as set forth SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 with a suitable excipient. BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES Fig. 1. The cation exchange of skin extract on a SP-Sepharose FF column.

Conditions are as described in methods. Fractions indicated by horizontal bars were collected according to the absorbance at 280 nm and named from "I" to "IV". The inset was shown RDA different fractions of the chromatogram against K. pneumoniae. Of all fractions, three fractions "II", "III" and "IV" showed antibacterial activity against K. pneumoniae on trypticase soy broth (TSB). Letter "C" was negative control (PBS) and "AN" was 15 μg Amikacin antibiotic disk. Only fraction "IV" that had better antibacterial activity than the other fractions was applied to further purification using Reverse-Phase

HPLC.

Fig. 2. Reverse- Phase HPLC of active fraction "IV". (A) Fraction "IV" (40 mg) was separated in a C₈ reverse- phase semi-preparative column; the component labeled with a horizontal bar was subjected to further purification. (B) Horizontal bar labeled factions of

Fig.2A were separated in a C₁₈ reverse-phase semi-preparative column. The inset shows the HPLC profile of the pure component (fraction D of Fig.2B) using an analytical C₈ reverse-phase column.

Fig. 3. A comparison primary structure of Brevinin-2R from Rana ridibunda with Brevinin antimicrobial peptides isolated from the skin of Ranid frogs. With the exception of

N-terminal gap, Brevinin-2R is identical to both Brevinin-2Ee and Brevinin-2Ej. The family of Brevinin antimicrobial peptides was alphabetically arranged.

Fig. 4. Low hemolytic activity of Brevinin-2R against sheep erythrocytes.

Brevinin-2R up to 200 μg/ml has 2.5 % hemolytic activity. Positive control was 0.2 % triton X100 and negative control was PBS. Hemolysis assay were evaluated with triplicate assays in three independed experiments.

Fig. 5. CD spectra and Edmundson wheel projection of Brevinin-2R. (A)

CD spectra of Brevinin-2R in 50 mM NAPB (thin line) or the presence of 50 % TFE (thick line). (B) The Edmundson wheel projection shows the amphipathic structure of Brevinin- 2R. The N-terminal 1-18 region of peptide can be fitted to well-behaved amphipathic α- helical structure. Hydrophobic residues were marked by boldface.

Fig. 6. An unrooted neighbor-joining distance tree resulting from phylogenetic analysis of Brevinin-2 peptides isolated from seven Eurasian Ranid frogs. Numbers along branches are bootstrap values for 10000 replicate analyses; values < 50% are not indicated. Brevinin-2R from R. ridibunda was denoted by an arrow in clade "A".

Fig. 7. Effects of Brevinin 2R on the growth of HT29/219 (a) and SW742 (b) cell lines. The cells were treated with different concentrations of Brevinin 2R for 24 to 72 hrs and the viability was assessed by MTT assay. Results are expressed as percentage of corresponding control and represent the mean ± SD of 4 repeats. Fig. 8. Detection of apoptosis nuclear condensation upon stimulation of cells with Brevinin 2R by Hoechst 33258 staining. Fig. 9. Crude frog peptide mixture obtained from the skin secretions of

Rana Ridibunda kills L929 fibrosarcoma cells by a mechanism that partially depends on the pro-apoptotic Bcl-2 family member BNIP-3. L929 fibrosarcoma cells (A, B) and a clone that over-expresses the mutated (dominant negative) form of BNIP-3 L929-ΔTM-BNIP3 transfected cell line (C, D) were treated with crude frog peptide mixture (20 μg/ml) (B, D) for 8 hours. Their DNA was stained with DAPI and analysed by fluorescent microscopy. The visible light channel (Nomarski contrast) and the blue fluorescence channel (DAPI) have been overlayed. Whereas the majority of the non-transfected L929 cells shrink upon 8 hour treatment with the peptide extract (B), the L929-ΔTM-BNIP3 cells (D) that express the mutated form of BNIP3 are partially protected from the peptide extract induced cell death.

Fig. 10 Enzymatic measurement of activity of caspase family of proteases, (a) Activity of caspase-3; (b) (DEVDase activity) caspase-9 (LEHDase activity) and (c) caspase-8 (lETDase activity) in HT29/219 and SW742 cells following treatment with Brevinin 2R for 24 hrs. Results are expressed as activity of the enzyme and represent the mean ± SD of 4 repeats.

Fig. 11. The effect of N-acetyl-L-cysteine on the cytotoxic effect of human

Brevinin 2R in (a) HT29/219 and (b) SW742 colon cell lines. The cells were treated with the indicated stimuli for 48 hrs; the cell death was detected by MTT assay. Results are expressed as activity of the enzyme and represent the mean ± SD of 4 repeats.

Fig 12. Effect of frog crude peptides on the rodent fibrosarcoma cell line L929. L929 and L929 ΔTM BNIP3 (stably transfected/overexpressing a BNIP-3 molecule that lacks its transmembrane domain, thus it behaves like BNIP-3 inhibitor) were treated with increasing concentrations of frog crude peptides for 4 hours (upper panel), or they were treated with fixed concentrations of the peptide extracts (15 μg/ml, 20 μg/ml), but for different time points (lower panels). The cells viability was then assessed by MTT assay. Results expressed as percentage of corresponding control and represent the mean SD of four repeats. Figure 13. Toxic effect of crude peptide extracts on cancer cell lines. Cells (Jurkat,

BJAB, MCF-7, and L929) were treated with indicated concentrations of crude peptide, for 4 h, and their viability was assessed by MTT assay. Results are expressed as percentage of corresponding control and represent the mean± SD of 4 independent experiments.

Figure 14: Brevinin-2R rapidly kills cancer cells from different histologic origins. Jurkat, BJAB, MCF-7 and L929 cells were treated with the indicated concentrations of Brevinin-2R for 4 h. Cell viability was then assessed by MTT-assay. The experiment was repeated 4 times and the average viability values are indicated.

Figure 15: Brevinin kills cancer cells by a mechanism resembling apoptosis, but insensitive to caspase inhibitors. (A) BJAB and MCF-7 cells were treated with Brevinin-2R (10μg/ml) for indicated time. Some samples were co-treated with zVADfmk (60μM) broad spectrum caspase inhibitor. Cell viability was assessed by MTT-assay. Data represent an average values obtained by 3 independent experiments. (B) Jurkat cells were treated with Brevinin-2R (10 μg/ml) and with anti-CD95 (0.5 μg/ml) for indicated time periods chosen based on an assumption that caspase activation should be measured about 1-2 h prior to morphologic manifestation of cell death. Total cell extracts were then harvested, resolved on SDS-PAGE and active subunits of caspase-3, -8, and -9 were detected by Western blot. (C) In an experiment parallel to the one depicted in (B), caspase activity in Jurkat cells was measured by a Caspase-Glo^® luminometric assay. The caspase activity is represent as a "-fold increase" in comparison to the control. The data represents triplicates of 3 independent experiments. (D) MCF-7 cells treated with Brevinin-2R for 6 h have been photographed to indicate the morphology of dying cells.

Figure 16: Brevinin-2R kills cancer cells by a novel pathway that involves BNIP3 and it is sensitive to inhibition by Bcl-2. (A) Cytofluorimetric analysis of mitochondrial transmembrane potential (ΔΨm) in Jurkat (left panel) and a clone that over-express Bcl-2 (Jurkat-Bcl-2, right panel). The cells were treated for 30 min. Cells were treated with medium alone (upper diagrams), or with Brevinin-2R (10 μg/ml) for 30 min. Brevinin-2R treatment showed obvious changes in mitochondrial membrane potential but Bcl2 over- expressed cell line was significantly resistance toward ΔΨm. (B) Bcl-2 over-expression significantly protects from Brevinin-2R induced cell death. Jurkat and MCF-7, and clones stably over-expressing Bcl-2 (Jurkat-Bcl-2, MCF-7-Bcl-2) were treated with Brevinin-2R (10μg/ml) for indicated period of time. Cell viability was then assessed by the MTT-assay. (C) Effect of Brevinin-2R on the growth of MCF-7, L929, and clones stably transfected with dominant negative mutant of BNIP3 (MCF-7-ΔTM-BNIP3, L929-ΔTM-BNIP3. Cells were treated with Brevinin-2R (10μg/ml) for the indicated time and then cell viability was assessed by the MTT-assay. Data represents the average values from triplicates from 3 independent experiments (9 measurement values in total).

Figure 17: Brevinin-2R shows higher toxicity toward cancer cells as compared to normal cells. PBMS and Jurkat were cells were treated either with 2.5μg/ml or 10μg/ml of Brevinin-2R and then after the indicated time, their viability was assessed by the MTT- assay. The respective Brevinin-2R concentrations are indicated in the figure legend as numbers standing next to names of used cells. The data represents the average values of triplicates from here independent experiments.

Table 1. Minimal inhibitory concentrations (MICs) of Brevinin 2R isolated from the skin of Rana ridibunda. MICs were determined with triplicate assays in three independent experiments.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

As used herein, "effective amount" refers to the administration of an amount of a given compound that achieves the desired effect. As used herein, "purified" does not require absolute purity but is instead intended as a relative definition. For example, purification of starting material or natural material to at least one order of magnitude, preferably two or three orders of magnitude is expressly contemplated as falling within the definition of "purified".

As used herein, the term "isolated" requires that the material be removed from its original environment.

As used herein, the term "treating" in its various grammatical forms refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causitive agent other abnormal condition. Described herein is a novel member of the Brevinin family of peptides which has anti-cancer activities but is also non-hemolytic. As a consequence, the peptide can be utilized to induce apoptosis in cancerous cells, as discussed below.

In a preferred embodiment, the peptide comprises

KLKNFAKGVAQSLLNKASCKLSGQC (SEQ ID NO: 1 ). As will be appreciated by one of skill in the art, the peptide may be synthesized de novo, isolated or purified from suitable extracts as discussed below or recovered from an expression system, as discussed below.

A comparison primary structure of Brevinin-2R from Rana ridibunda with Brevinin antimicrobial peptides isolated from the skin of Ranid frogs shows that with the exception of N-terminal gap, Brevinin-2R is identical to both Brevinin-2Ee and Brevinin-2Ej.

However, the low level hemolytic activity of Brevinin-2R supports the fact that it has a lower level of hydrophobicity than Brevinins family members especially such as: Brevinin- 2Ee and Brevinin-2Ej.

In a further aspect of the invention, there is provided a minimal "degenerated" brevinin sequence : KLKNXXKGVAQXLLXKASCKLSGQC (SEQ ID NO: 2) wherein X can be any amino acid. If X is a basic amino acid, the peptide may be more cytotoxic.

In other embodiments, there is provided a "degenerated" brevinin sequence: KLKNXXKGVAQXLLXKASCKLSGQC (SEQ ID NO: 3) wherein X is selected from the group consisting of alanine, leucine, isoleucine, phenylalanine, tryptophan, methionine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine. As will be apparent to one of skill in the art, in these embodiments, X is any amino acid except glycine, valine or proline, because these amino acids would cause an unwanted hinge or turn in the structure of the peptide which would certainly change the cytoxic effect of the peptide.

As will be appreciated by one of skill in the art, the anti-tumor activity of this peptide depends on amphipatic structure of this peptide. The most important residues for this property are basic (lysine, aspargine) and non polar (leucine) ones. The other important part of this peptide is hinge part. The tripeptide residues (GIy-VaI-AIa) can form a hinge in the BR2 structure. And finally the two c-terminal cysteine can form a disulfide bond. This part of the peptide is also important for binding to cell membranes and for cytotixic activity. By considering these factors, novel peptide variants can be designed and tested. Specifically, the above residues are likely to tolerate only highly conservative changes whereas other residues are likely to be more tolerant of less conserved substitutions.

It is of note that it is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. In one aspect of the invention, the above-described peptides may include peptides that differ by conservative amino acid substitutions. The peptides of the present invention also extend to biologically equivalent peptides that differ by conservative amino acid substitutions. As used herein, the term "conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar:

Ala, VaI, Len, lie, Phe, Trp, Pro, Met; acidic: Asp, GIu; basic: Lys, Arg, His; neutral: GIy,

Ser, Thr, Cys, Asn, GIn, Tyr.

In other embodiments of the invention, there is provided a nucleic acid molecule deduced from any one of the above peptides. The nucleic acid molecule may be operably linked to a suitable promoter for constructing an expression vector for expressing the peptide in a suitable expression system.

In a further embodiment of the invention, there is provided a method of generating a non-hemolytic, anti-cancer brevinin peptide comprising: decreasing the hydrophobicity of a brevinin peptide. In a preferred embodiment, the peptide is a purified, isolated or synthetic peptide having an amino acid sequence of SEQ ID NO.1 , SEQ ID NO. 2 or a variant thereof, as discussed above.

The hydrophobicity may be decreased by several means known in the art, for example by substituting one or more of the hydrophobic residues of the native amino acid sequence of said brevinin peptide with non-hydrophobic or less hydrophobic residues as described above, or by inserting or adding non-hydrophobic or hydrophilic residues to the native amino acid sequence. As will be apparent to one of skill in the art, these insertions may be made at the C-terminus or N-terminus of the peptide or may be made at locations within the peptide where insertions are likely to be tolerated, for example, locations within the peptide where insertions/deletions have been observed in other brevinin family peptides.

In a yet further embodiment of the invention, there is provided a method of generating a non-hemolytic, anti-cancer brevinin peptide comprising: increasing the positive charge of a brevinin peptide. In a preferred embodiment, the peptide is a purified, isolated or synthetic peptide having an amino acid sequence of SEQ ID NO.1 , SEQ ID NO. 2, SEQ ID NO. 3 or a variant thereof, as discussed above.

As will be appreciated by one of skill in the art, the positive charge of the brevinin family peptide may be increased by carrying out at least one of the following: substituting one or more negatively charged amino acid residues in the native brevinin family peptide amino acid sequence with a non-charged or positively charged residue; by substituting a non-charged amino acid residue with a charged amino acid residue; by deleting negatively charged amino acid residues; or by adding or inserting positively charged amino acid residues to the native amino acid sequence of the brevinin family peptide. As will be appreciated by one of skill in the art, in some embodiments, the brevinin family peptide may be an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3. In some embodiments discussed below, the non-hemolytic anti-cancer brevinin peptide may be administered to an individual in need of such treatment at a localized dosage or concentration of 2.5-10 μg/ml. As will be appreciated by one of skill in the art, the actual dosage will of course depend on the age, weight and condition of the patient and other similar factors, in addition to other factors such as the location and size of the tumor to be treated and the mode of delivery.

While not wishing to be bound to a particular theory, the inventors have the following evidence for frog peptide and BR2 cytotoxic effect:

1- We did not observe any significant difference in toxicity of crude peptides between BJAB Wt, and BJAB FADD-DN cell lines (FADD is a key component of the death-receptor-dependent apoptotic pathway). In addition we did not observe any significant difference in toxicity of crude peptides between J-16 and J-16 FADD-DN cells. Therefore cytotoxic activity of crude frog peptides is not dependent on a cell surface receptor pathway.

2- The toxicity of crude peptides is significantly lower in J16-Bcl2 over expressed cell line than J16 wild type cell. This indicates that the apoptoic pathway triggered by this peptide relies on the mitochondrial death pathway.

3- In J16 and BJAB wild type cells the toxicity of frog crude peptides and BR2 is accomplished with loss of mitochondria membrane potential but in L929 cell this toxicity occurs independently of the loss of mitochondria membrane potential. These results demonstrate that different apoptotic mechanisms may be triggered by these peptides in different cell types.

Thus, we can conclude that the toxic effect of this peptide is independent of the extrinsic/death-receptor dependent pathway, it is mediated over the mitochondria apoptotic pathway, but it may also involve other pathways. The mitochondria pathway is activated under conditions that induce "cellular metabolic stress" by disrupting important physiologic processes, for example, disruption of cytoplasmic membranes by these peptides which causes a influx of calcium ion in the intracellular medium which then causes mitochondria pathway activation.

As will be apparent to one skilled in the art, a therapeutically effective amount of the non-hemolytic, anti-cancer brevinin peptide is the amount sufficient to achieve the desired result. For example, for treating cancer, for example, prostate cancer, the therapeutically effective amount is the amount sufficient to accomplish at least one or more of the following: reduce tumor size, induce apoptosis in cancerous cells, and inhibit tumor growth. The amount administered will vary according to the concentration of the active agent and the body weight of the patient. Other factors include the degree of tumor progression, the body weight and the age of the patient.

In some embodiments, the non-hemolytic, anti-cancer brevinin peptide as described above, that is, substantially as set forth in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3, or consisting essentially of an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or consisting of an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 at concentrations or dosages discussed above may be combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non-biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, mathacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethycellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars and starches. See, for example, Remington: The Science and Practice of Pharmacy, 2000, Gennaro, AR ed., Eaton, PA: Mack Publishing Co.

As will be apparent to one knowledgeable in the art, specific carriers and carrier combinations known in the art may be selected based on their properties and release characteristics in view of the intended use. Specifically, the carrier may be pH-sensitive, thermo-sensitive, thermo-gelling, arranged for sustained release or a quick burst. In some embodiments, carriers of different classes may be used in combination for multiple effects, for example, a quick burst followed by sustained release. In other embodiments, a non-hemolytic, anti-cancer brevinin peptide at concentrations or dosages described above may be encapsulated for delivery. Specifically, the non-hemolytic, anti-cancer brevinin peptide may be encapsulated in biodegradable microspheres, microcapsules, microparticles, or nanospheres. The delivery vehicles may be composed of, for example, hyaluronic acid, polyethylene glycol, poly(lactic acid), gelatin, poly(E-caprolactone), or a poly(lactic-glycolic) acid polymer. Combinations may also be used, as, for example, gelatin nanospheres may be coated with a polymer of poly(lactic-glycolic) acid. As will be apparent to one knowledgeable in the art, these and other suitable delivery vehicles may be prepared according to protocols known in the art and utilized for delivery of the non-hemolytic, anti-cancer brevinin peptide. Furthermore, the delivery vehicle may be dispersed in a gel or paste, thereby forming a nanopaste for coating a tissue or tissue portion.

It is of note that in other embodiments, the non-hemolytic, anti-cancer brevinin peptide described above may be combined with permeation enhancers known in the art for improving delivery. Examples of permeation enhancers include, but are by no means limited to those compounds described in U.S. Pat. Nos. 3,472,931 ; 3,527,864; 3,896,238; 3,903,256; 3,952,099; 4,046,886; 4,130,643; 4, 130,667; 4,299,826; 4,335,115; 4,343,798; 4,379,454; 4,405,616; 4,746,515; 4,788,062; 4,820,720; 4,863,738; 4,863,970; and 5,378,730; British Pat. No. 1 ,011 ,949; and Idson, 1975, J. Pharm. Sci. 64:901-924.

In some embodiments, the non-hemolytic, anti-cancer brevinin peptide in any suitable form as described above, may be combined with biological or synthetic targetting molecules, for example, site-specific binding proteins, antibodies, lectins or ligands, for targetting the non-hemolytic, anti-cancer brevinin peptide to a specific region or location.

In some embodiments, the non-hemolytic, anti-cancer brevinin peptide, with or without specific targeting sequences, as discussed above may be delivered in a form of a coding cDNA or mRNA and produced by cell's own machinery upon the delivery to the cell of nucleic acid sequences and regulatory nucleic acid sequences that will cause intracellular production of the non-hemolytic, anti-cancer brevinin peptide, and also its organelle-targetting sequence(s). That is, a nucleic acid molecule deduced from the amino acid sequence of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or variants thereof prepared as described above may be operably linked to a suitable promoter active in the intended host as well as other control sequences necessary for expression in said host. As will be appreciated by one of skill in the art, the promoter may be a constitutive promoter, an inducible promoter, a cell-type specific promoter or other suitable promoter known in the art. As discussed above, these constructs may also include targeting or other similar sequences as known in the art. Purification of the small cationic antimicrobial peptides

The SP-Sepharose FF as cation exchange chromatography was applied to separate the small cationic peptides from other small non-cationic compounds (Fig.1). About 500 mg of the crude extract was dissolved in 10 ml of start buffer A (50 mM acetate buffer pH 4.8) and loaded on the column (1 x 10 cm). Elution of the peptides were accomplished with a linear gradient of 0-50% buffer B (50 mM acetate buffer, pH 4.8, 1 M NaCI) at a flow rate of 1 ml/min. Tubes with a volume of 2 ml were collected and grouped according to the absorbance at 280 nm as shown (horizontal bar). The total extract, approximately 30%, corresponds to fraction "I", 5.5% to fraction "II", 44% to fraction "III", and 20.5% left to fraction "IV". Different fractions were lyophilized and desalted for antibacterial activity. Fractions "II", "III" and "IV" showed antibacterial activity against K. pneumoniae (inset of Fig.1 ), but Fraction "II" due to its low antibacterial activity was not analyzed further. Fraction "IM" also had moderate antibacterial activity. The strong cationic antibacterial compounds presented in peak "IV" (Fig.1 ) were purified to homogeneity by the following multisteps procedure.

Fraction "IV" was subjected to Vydac semi-preparative C₈ reversed-phase column (Fig. 2A). Fraction labeled by the horizontal bar was injected to Macherey -Nagel semi- preparative C₁₈ reversed-phase column (Fig. 2B). Only peak "d" from a series of peaks named "a" to "e" in Fig. 2B was again injected to the semi-preparative Ci₈ reversed-phase column and eluted by the same conditions. A pure antibacterial peptide "d" was obtained as judged by analytical C₈ reversed phase chromatography as shown the inset of Fig.2B. Mass spectrometry and structural characterization

Based on the mass spectrometry result our peptide sequence is: KLKNFAKGVAQSLLNKASCKLSGQC. Brevinin-2R (MH⁺=2636.28) like other AMPs of Rana contains a C-terminal hepta-membered ring stabilized by a disulfide bridge with 6 amino acids upstream. Brevinin-2R from Rana ridibunda is 25 amino acids long, and similar to Brevinin-2Ee and Brevinin-2Ej from R. esculenta except for a four and a five N- terminal amino acid gap respectively (Fig. 3). The amino acid sequence of Brevinin-2Ee, having 29 residues, was deduced from its cloned cDNA (Simmaco et al., 1994) whereas isolates from skin extractions of multiple Rana esculenta typically have 30 residues (AIi et al., 2003). In fact, 25 of the residues of Brevinin-2Ee and Brevinin-2Ej were identical to Brevinin 2R. Rana ridibunda's Brevinin also shared 79% homology to the Brevinin-2Ec and Brevinin-2Ed and 75% homology to Brevinin-2E which were previously isolated from Ranid frogs (Conlon et al., 2004; Simmaco et al., 1994; AIi et al., 2003).

It is of note that Brevinin-1 Ed and Brevinin-1 Eb had very limited antimicrobial activity. In addition these two brevinin had very low hemolytic activity too. Brevinin-1 E on the other hand is more potent antimicrobial agent and has hemolytic activity as well. Antibacterial, antifungal and hemolysis assay Antibacterial activity was examined during each purification step by the radial diffusion assay on K. pneumoniae (the inset of Fig. 1 ) but the antimicrobial spectrum of Brevinin-2R was determined by measuring the minimal inhibitory concentrations. As shown in Table 1 , Brevinin-2R displayed antimicrobial activities against bacteria including but by no means limited to: S. aureus, M. luteus, Bacillus spKR-8104 , E. coli, S. typhimurium, P. aeruginosa, K. pneumoniae, and fungi including: C. albicans and C. tropicalis. Hemolysis assay of the isolated peptide Brevinin-2R was tested against sheep erythrocytes based on release of hemoglobin assay shown in Fig.4. This peptide had no appreciable (2.5%) hemolytic activity up to 200 μg/ml of the peptide. CD spectroscopy

The secondary structure content of Brevinin-2R was calculated by circular dichroism (CD) in the absence and presence of TFE (Fig. 5A). The CD spectrum of Brevinin-2R in 50 mM NAPB indicated that the content of α-helix, β-sheet, turn and random coils were 9.3%, 19.9%, 38.2% and 32.6% respectively. Under the hydrophobic condition of 50% TFE solution, the contents of α-helix, β-sheet, turn and random coils changed to 43%, 18.2%, 27% and 11.8% respectively. The Edmunson wheel projection was shown that Brevinin-2R can potentially form an amphiphatic structure. The N-terminal 1-18 residues of Brevinin-2R can be fitted to an α-helix. A polar and positively charged hydrophilic side and hydrophobic side are clearly distinguishable on each side of the cylindrical surface (Fig. 5B). Like other antimicrobial peptides isolated from the family of Ranidae, the polar residues in Brevinin-2R are well interspersed among the hydrophobic residues, interrupting the contiguity of hydrophobicity, which gives the potential to form an amphipathic helix. Phylogeny Phylogenetic analysis shows that Brevinin-2 peptides from seven Eurasian Rana species were segregated into three major clades, "A", "B" and "C" (Fig. 6). The results revealed that Brevinin-2R from Rana ridibunda is nested in clade "A" comprising the Brevinins from European R. esculenta. In this clade, Brevinin-2R along with Brevinin-2Ee are sister sequences to R. esculenta-2Ej. But, both Brevinin-2Ei and Brevinin-2Ek from R. esculenta are members of clade "C" in which the former are sister to the remainder of the clade and Brevinin-2Ek is allied with a subclade of Brevinin-2Tc and Brevinin-2Td from R. temporaria. Rugosa-C and Gaegurin-4 (from R. rugosa), two Brevinin-2 like peptides, are sister sequences and assembled with R. brevipoda-2 and R. nigromaculata-2 (Nigrocin-1) in clade "B". However, the remaining Brevinin-2 peptides from R. rugosa are nested in clade "C" which also contained R. ornativentris-20a and -20b. Hemolysis assay Hemolysis assay of the isolated peptide Brevinin 2R was tested against sheep erythrocytes. This peptide had no appreciable (2.5%) hemolytic activity up to 200 μg/ml peptide (Fig. 4).

Cytotoxicity assay and detection of apoptosis using Hoechst 33258 Viability tests were applied using MTT assay to determine cytotoxicity of Brevinin

2R. As shown in Fig. 8a, treatment of HT29/219 cells with Brevinin 2R resulted in significant cell death in all considered concentrations and times. The 50% viability at 24 hrs was at concentrations higher than 40 μg/ml in HT29/219 cell line (Fig. 8a). The Brevinin 2R treated SW742 cells showed a significant cell death at all examined concentrations during studied time periods (Fig. 8b). In this case 50% viability at 24 hrs was determined at concentrations higher than 25 μg/ml (Fig. 8b) in SW742 cell line. In order to confirm the apoptotic cell death, cell nuclei were stained with Hoechst 33258. Brevinin 2R peptide caused typical apoptotic changes in the nuclear morphology, with pronounced condensation of cell nuclei and nuclear fragmentation. DNA fragmentation

Treatment of HT29/219 and SW742 cells with 20 and 40 μg/ml of Brevinin 2R for 24 hrs resulted in formation of DNA fragments of oligonucleosomal size (180-200bp), a hallmark of apoptosis, as shown in Fig. 10. Caspase -3, -8 and -9 activation assays To explore the possible biochemical mechanisms underlying Brevinin 2R-induced apoptosis, the activation of caspase-3, caspase-8 and caspase-9 were assayed. The results demonstrated that the activity of caspase-3 and -9 were significantly (p<0.05) increased in both cell lines treated with Brevinin 2R (Fig. 11a, b). But, the increases in caspase-8 activity in both cell lines were not significant (Fig 11c). The effect of N-Acetyl-L-cysteine on the Brevinin 2R-induced cytotoxicity

The activation of caspase-9 is a classic characteristic of the mitochondrial cytochrome c-dependent pathway. In addition, death induced by some stimuli, including tumor necrosis factor-α significantly relies on ROS production by mitochondria. In order to get further insights into Brevinin 2R toxicity pathways we examined the effects of NAC, a broadly used clinical antioxidant. As shown in Figure 12, NAC has the potential to protect from Brevinin 2R toxicity in a dose-dependent manner. NAC showed typical linear dose- dependent activity on Brevinin 2R treatment and up to 10 mM concentration is required to fully counteract the stimulus.

We purified a cationic antimicrobial peptide, called Brevinin-2R, from skin of Rana ridibunda. The circular dichroism studies and Edmunson wheel projection showed that Brevinin-2R has a random coil conformation and changes to an amphipathic α-helical structure at 50% TFE (Fig. 5A, B). Our results showed that Brevinin-2R exhibits a broad spectrum of antimicrobial activity against gram-negative and gram-positive bacteria and fungi at various concentrations ranging from 2.5 to 30 μg/ml (Table 1).

The antimicrobial and low level hemolytic activities of Brevinin-2R on the sheep erythrocytes could be due to the differences in the membrane compositions of its targets. It was suggested that the difference between bacteria and eukaryotic cells exists in the electrostatic properties of their cell surfaces (Oren et al., 1997, J Biol Chem 272: 14643- 14649). Bacteria have negatively charged surface structures, such as lipopolysaccharides or lipoteichoic acids, and their membranes contain negatively charged phospholipid, such as phosphatidylglycerol. On the other hand, eukaryotic cells such as erythrocytes contain zwitterionic phosphatidylcholine. Therefore, the antimicrobial peptides with positively charged amino acid residues might selectively bind to the outer leaflet of the bacterial membranes via electrostatic interaction rather than to the eukaryotic membranes (Kim et al., 2003, Peptides 24: 945-953; Hong et al., 1998, Antimicrob Agents Chemother 42: 2534-2541 ).

In a conventional hemolytic assay, Brevinin-2R had no or low hemolytic activity at the concentrations at or near to MICs (Fig. 4 and Table 1 ) whereas Brevinin-2 family members were shown previously to have hemolytic activity (Simmaco et al., 1994; Simmaco et al., 1993; Conlon et al., 2004). For example, Brevinin 1 E had haemolytic activity in the range of antimicrobial activity concentration. The low level hemolytic activity of Brevinin-2R supports the fact that it has a lower level of hydrophobicity than other Brevinins family members especially such as: Brevinin-2Ee and Brevinin-2Ej. As will be apparent to one of skill in the art, it is likely that changing the hydrophobicity too much will affect cytoxicity. It has been demonstrated that the hemolytic activity of the majority of antimicrobial peptides increases with an increase in their hydrophobicity and a decrease in their net positive charge (Mangoni et al., 2003, Peptides 24: 1771-1777; Thennarasu and Nagaraj, 1995, lnt J Pept Protein Res 46: 480-486). The N-terminal sequence of Brevinin-2Ee is GIy-I le-Phe-Asp and Brevinin-2Ej is Gly-lle-Phe-lle-Asp. The presence of these gaps from the N-terminal of Brevinin-2R in comparison with the other Brevinin-2 peptides could contribute to low hemolytic activity of this peptide (Fig. 3). Hydrophobic residues such as lie, Leu and Phe at the N-terminal of Brevinin-2 family (in the case of Brevinin 2Ee and Brevinin 2Ej) could increase their hydrophobicity, and therefore influence their hemolytic activity. This is supported by reported data from Kwon et al that a deletion of three amino acids (FLP) from the N-terminal region of Brevinin-1 E did not greatly affect antimicrobial activity but dramatically reduced hemolytic activity (Kwon et al., 1998, Biochim Biophys Acta 1387: 239-248). It has been shown that deletion of a few amino acids from either the human LL-37 (Oren et al., 1999, Biochem J 341 : 501-513) or dermaseptins (Strahilevitz et al., 1994, Biochemistry 33: 10951-10960; Kustanovich et al., 2002, J Biol Chem 277: 16941-16951) preserved their antimicrobial activity but decreased their hemolytic activity. In addition to Brevinin-2R, other AMPs belonging to the Brevinin-2 family, such as: Gaegurin-2, Gaegurin-3 from R. rugosa (Park et al., 1994; Suzuki et al., 1995) and Nigrocin-1 from R. nigromaculata (Park et al., 2001 , FEBS Letters 507: 95-100) were reported to show non hemolytic activity. Therefore, Brevinin-2 antimicrobial peptides apparently are a superfamily of several hemolytic and non-hemolytic AMPs which were isolated from different Rana species. As discussed above, Brevinin-1 Ed and Brevinin-1 Eb have very low haemolytic activity but brevinin-1 E has very high haemolytic activity at its antibmicrobial concentration.

Diversity in primary structure and function of Brevinins suggests that they may be useful as molecular markers to infer phylogenetic relationships among different species of the genus Rana. Therefore, the existence of Brevinin-2R in skin secretion of R. ridibunda and its similarity to both Brevinin-2Ee and Brevinin- 2Ej from R. esculenta clearly indicates a close relationship between R. ridibunda and the latter species (Fig. 6).

We have examined the effect of Brevinin 2R peptide on colon cancer cell lines viability and their mechanism of action. This peptide has a significant cytotoxic apoptotic activity toward HT29/219 and SW742 cell lines (Fig. 8a, b).

Upon treatment of colon cancer cell lines with varying times and doses of Brevinin 2R, cytotoxic effects were observed (Fig. 8). In addition, incubation of the cells with Brevinin 2R for 24 hrs results in a nuclear morphology having pronounced condensation of cell nuclei and nuclear fragmentation, typical of cells undergoing apoptosis (Fig. 8 and 9). This indicates that Brevinin 2R can induce apoptosis.

The Brevinin 2R-induced apoptotic activity was induced through the mitochondrial cytochrome c-dependent (intrinsic) pathway as verified by the activation of caspase-9 (and caspase-3) but not caspase-8. The finding that caspase-8 activity was only slightly increased after Brevinin 2R treatment clearly indicates that the caspase-8/death receptor pathway was not involved in the Brevinin 2R-induced apoptosis. Release of cytochrome c upon mitochondrial damage facilitates mediated caspase activation through caspase-9 (Risso et al., 2002, Molecular and Cellular Biology 22:1926-1935). Decreased cellular ATP levels after treatment of cancer cells with increased concentrations of Brevinin 2R clearly showed malfunctioning mitochondria in cancer cells. It has been reported that tumoricidal peptides originating from amphipathic antimicrobial peptides could induce apoptosis by being internalized and interacting with mitochondrial membranes (which are similar to the membranes of prokaryotic cells) and inducing apoptosis via the mitochondrial pathway (Risso et al., 1998, Cell Immunology 189: 107-115). Another investigation has also shown that BMAP-28 induces the opening of the mitochondrial permeability transition pore and that cytotoxic potential depends on alteration of mitochondrial permeability (Risso et al., 1998). Perturbation of mitochondrial function and cytochrome c release could be key events in the cytotoxicity of Brevinin 2R similar to other reports (Risso et al., 1998). Reactive Oxygen Species (ROS) which are the by-products of normal cellular oxidative processes have been suggested as regulatory molecules for the initiation of apoptotic signalling (Mayer and Noble, 1994, PNAS USA 91 : 7496-7500; Ghavami et al., 2004; Monitoring of programmed cell death in vivo - new methods of cancer therapy monitoring in Apoptotic pathways as target for novel therapies in cancer and other diseases, edited by M. Los and S. B. Gibson, Kluwer Academic Press, ISBN: 0-387-23384-9). Finally, we showed that apoptosis induced by Brevinin 2R is prevented by pre-treatment of the cells with the antioxidant NAC which inhibits ROS formation. Therefore, it is suggested that Brevinin 2R facilitates a pro-oxidant state that likely contributes to the molecular mechanism of its apoptotic effect. To conclude, it is possible to exploit this biochemical feature and develop therapeutic strategies to preferentially kill cancer cells through ROS- mediated-, or other mitochondria-dependent mechanisms.

Fig 12 shows that there was significant difference between cytotoxic effect of crude peptide on wild type L929 and ΔTM BNIP3 L929 transfected cells (p<0.05). It means that cytotoxic effect of crude peptides is BNIP3 dependent. BNIP3 (Bcl-2/E1 B 19kDa interacting protein) was discovered in a yeast two-hybrid screen for proteins that interact with adenovirus E1 B 19K, which is homologous to Bcl-2. BNIP3 belongs to the BH3-only subfamily and has a C-terminal transmembrane (TM) domain. The TM domain of BNIP3 is required for dimerization, pro-apoptotic function, and mitochondrial targeting. Over- expression of BNIP3 opens the mitochondrial permeability transition pore (PTP) thereby suppressing the proton electrochemical gradient (ΔΨ_m), and this is followed by chromatin condensation and DNA fragmentation. BNIP3-mediated cell death is independent of the release of cytochrome c from mitochondria and the activity of the caspase family of cell death proteases. BNIP3 is implicated in the killing of tumor cells under hypoxic conditions, therefore it can be considered anti-oncogenic. On the other hand, it has been reported that there might be nuclear localization for BNIP3. Therefore our findings show that in crude peptides, the cytotoxic effect may be located at the mitochondria or nucleus. Anticancer activity of purified Brevinin-2R Brevinin-2R was the main component of fraction IV of the crude peptide extract (Fig. 1). Thus, we have tested the cytotoxic effect of Brevinin-2R on various cancer cells. As shown in figure 14, Brevinin-2R efficiently killed our model cell lines (Jurkat, BJAM, MCF-7, L929) at concentrations 2-4 times lower than the concentrations of crude peptide used in earlier experiments (Fig. 13). Again, the breast cancer adenocarcinoma cell line, MCF-7, was most sensitive. It is worth noticing that Brevinin-2R at concentrations that were toxic for our model cell lines (7.5-10 μg/ml) had absolutely no hemolytic activity (less than 0.5%, Fig. 4). Thus, the simple cell lysis can be excluded as the anticancer mechanism of action of this defensin. Four hours of incubation was sufficient for Brevinin-2R to induce its toxic effect. Longer exposure times (e.g. 6 or 8 h) killed cancer cells with even lower concentrations of Brevinin-2R. Brevinin-2R kills cancer cells by a distinct mechanism that only partly relies on classical apoptotic pathways.

In order to gain insight into the mechanism of cell death triggered by Brevinin-2R, we have examined the time kinetics of Brevinin-2R induced cell death and its dependence on the caspase family of proteases. As shown in figure 14A, Brevinin-2R that was applied at a concentration of 10 μg/ml, induced cell death quickly. Similarly with the experiments involving 'crude extract', the toxic effect of Brevinin-2R was already pronounced within the first 2-4 h. Brevinin-2R triggered toxicity could not be efficiently blocked by the broad- spectrum caspase inhibitor zVADfmk. Figure 15A shows data obtained on MCF-7 and BJAB cells, but similar results were also obtained with Jurkat and L929 cell lines. Concomitantly, processing and activation of caspase-3 -9, and -8, could not be detected in Brevinin-2R treated Jurkat cells (Fig. 15B). This was further confirmed by the lack of detection of the respective enzymatic activities (DEVD-ase, LEHD-ase, IETD-ase), (Fig. 15C). Only some DEVD-ase (Caspase-3/7) activity (around 1 fold increase) could be detected. These traces of DEVD-ase activity may explain some protection from Brevinin- 2R -induced cell death, observed upon the treatment with zVADfmk pan-caspase inhibitor. The morphology of dying cells resembled apoptosis to some degree. The cells were rapidly shrinking, but they rarely exhibited some other apoptosis-typical features like for example membrane blebbing or cell detachment from their growth support (Fig. 15D).

We then performed a series of tests that allowed for better characterization of Brevinin-2R induced cell death. Brevinin-2R killed cancer cells by a non-classical apoptotic mechanism that is insensitive to the broad-spectrum caspase inhibitor zVADfmk. BNIP3, a pro-apoptotic Bcl-2 family member that mediates cell death involving ΔΨm, is sensitive to inhibition by Bcl-2, but it is caspase-independent and it does not rely on mitochondrial release of cytochrome c. We thus tested the involvement of mitochondria in Brevinin-2R induced death and also the role of BNIP3 and related cell death mechanisms. Our experiments revealed that Brevinin-2R triggers ΔΨm that could be significantly counteracted by the over-expression of Bcl-2 (Fig. 16A). Bcl-2 over-expression not only protected from ΔΨm, but it also significantly abolished Brevinin-2R induced toxicity in MCF-7, Jurkat (Fig. 6B), BJAB, and L929 cells. Finally, stable over-expression of DTM- BNIP3, a dominant-negative mutant of BNIP3, lacks the trans-membrane domain and thus it cannot insert into the mitochondrial membrane, and is strongly protected from Brevinin- 2R induced cell death (Fig. 16C).

Brevinin-2R is significantly more toxic towards transformed cells as compare to primary cells.

The data presented above indicates that Brevinin-2R applied at clinically- achievable low-micromolar concentrations rapidly kills cancer cells of different histologic origin and from different species by a distinct, apoptosis-like mechanism. Thus, we have tested how normal, PBMC react to Brevinin-2R. Brevinin-2R at a concentration of 2.5 μg/ml was virtually non-toxic for PBMC (toxicity: ≤5%) even after 4 h, whereas at the same concentration about 25% of Jurkat T-cell leukemia cells were killed (Fig. 17). Brevinin-2R at this concentration also killed 30-40% of L929, BJAB, and MCF-7 cells (Fig. 14). At higher concentrations (10 μg/ml) over 70% of Jurkat cells were killed after 4 h, whereas the toxicity towards PBMC was only -30%. Thus, the above experiments show that Brevinin-2R (/) kills cancer cells by a distinct, apoptosis-like mechanism that is insensitive to caspase inhibition, but (H) is modulated by Bcl-2 family members and (Hi) Brevinin-2R toxicity is semi-selective towards cancer cells.

This study describes the isolation and initial characterization of a new defensin, Brevinin 2R, an antibacterial peptide with strong and semi-selective anticancer properties. Anticancer defensins, which are mostly cationic and adopt an amphipathic structure (Steiner et al., 1988, Biochim Biophys Acta 939: 260-266; Matsuzaki et al., 1989, Biochim Biophys Acta 981 : 130-134; Pouny et al., 1992, Biochemistry 31 : 12416-12423; Gazit et al., 1994, Biochemistry 33: 10681-10692; Boman, 1995, Annu Rev Immunol 13: 61-92; Dimarcq etal., 1998, Biopolymers 47: 465-477; Shai, 1999, Biochim Biophys Acta 1462: 55-70; Tossi et al., 2000, Biopolymers 55: 4-30; Zelezetsky et al., 2005, Peptides 26: 2368-2376), were initially discovered due to their role in antibacterial defense (Ganz and Lehrer, 1998, Curr Opin Immunol 10: 41-44; Zasloff, 2002, N Engl J Med 347: 1199-1200; Ganz, 2004, C R Biol 327: 539-549). They are found in most living species, and they are released to the epithelium either continuously or in response to bacterial infection (Brotz and Sahl, 2000, J Antimicrob Chemother 46: 1-6; Hancock and Diamond, 2000, Trends Microbiol 8: 402-410; Hancock and Rozek, 2002, FEMS Microbiol Lett 206: 143-149). Based on their spectrum of activity, these peptides can be divided into two major groups. The first group includes peptides that are highly potent against both bacteria and cancer cells, but not against normal mammalian cells, e.g. some insect cecropins (Chen et al., 1997, Biochim Biophys Acta 1336: 171-179) and magainins (Cruciani et al., 1991 , Proc Natl Acad Sci USA 88: 3792-3796; Baker et al., 1993, Cancer Res 53: 3052-3057). The second group includes peptides that are toxic to bacteria and both mammalian cancer and non-cancer cells; some examples include the bee venom melittin (Mai et al, 2001 , Cancer Res 61 : 7709-7712), tachyplesin Il isolated from the horseshoe crab (Mai et al., 2001 ), human neutrophil defensins (Lichtenstein et al., 1986, Blood 68: 1407-1410), insect defensins (Papo and Shai, 2005, Cell MoI Life Sci 62: 784-790) and the human LL-37 (Johansson et al., 1998, J Biol Chem 273: 3718-3724). Thus, Brevinin-2R can be classified in the first group since it shows preferential toxicity towards cancer cells.

The anti-cancer effect of the Brevinin-2R peptide could be observed upon treatment of several cell lines. This peptide exhibited significant cytotoxic activity toward, MCF-7, L929, BJAB, and Jurkat cell lines (Fig. 14) (and also against HT29/219 and SW742 colon cancer cell lines). Interestingly, Brevinin-2R possesses virtually no hemolytic activity (Fig. 4). In contrast, frog antibacterial peptides called magainins are toxic for various tumor cells (Cruciani et al., 1991), but they also show significant hemolytic activity that precludes their internal use in vivo (Dathe et al., 2001 , FEBS Lett 501 : 146-150). The hemolytic activity of the majority of antimicrobial peptides become more potent with the increase of their hydrophobicity and the decrease in their net positive charge (Thennarasu and Nagaraj, 1995, lnt J Pept Protein Res 46: 480-486; Mangoni et al., 2003, Peptides 24: 1771-1777). Hydrophobic residues such as lie, Leu and Phe at the N-terminal of the Brevinin-2 family (found for example in Brevinin-2Ee and Brevinin-2Ej) could increase their hydrophobicity, and therefore influence their hemolytic activity (Fig. 1 D).

The cytotoxic effect of Brevinin-2R was dose- and time dependent (Fig. 14-15A). Surprisingly, Brevinin-2R induced cell death was almost completely insensitive towards the co-treatment with the broad-spectrum caspase inhibitor zVADfmk (Fig. 15A). Also, no significant proteolytic activation (Fig. 15B) or activity of caspase-3, -9, and -8 (Fig. 15C) have been detected in Brevinin-2R treated cells. Furthermore, Brevinin-2R treatment caused ΔΨm that was counteracted by the over-expression of Bcl-2. The above findings indicate that Brevinin-2R activates the intrinsic mitochondrial death pathway, but the cell death execution, although resembling apoptosis does not absolutely rely on the caspase- dependent proteolytic cascade. This is in agreement with earlier publications that describe caspase-independent, apoptosis-like cell death (reviewed by: Borner and Monney, 1999, Cell Death Differ 6: 497-507; Leist and Jaattela, 2001 , Nat Rev MoI Cell Biol 2: 589-598; Kogel and Prehn, 2003, Caspase-independent Cell Death Mechanisms. In: Caspases - their Role in Cell Death and Cell Survival, eds. Los and Walczak, New York: Kluwer Academic Press, 237-248). Thus, in addition to the "classical apoptosis" that can be inhibited to various degree by caspase inhibitors, an "apoptosis-like" cell death has been described that may involve activation of the caspase-family members, but other proteases, like calpains and cathepsin may functionally replace caspases if they become inhibited (Sarin et al., 1997, Immunity 6: 209-215). Such a form of cell death is still partly sensitive to the inhibitory action of Bcl-2 (Denecker et al., 2001 , Cell Death Differ 8: 829-840). Furthermore, caspase inhibition in some experimental systems leads to the change of cell death morphology (from necrotic to apoptotic), rather than preventing cell demise or it can even be accelerated by caspase inhibitors (Vercammen et al., 1998, J Exp Med 187: 1477-1485; Los et al., 2002, MoI Biol Cell 13: 978-988).

The data summarized above point towards BNIP3 as a possible mediator of Brevinin-2R triggered apoptosis. Indeed, BNIP3 kills cells in a caspase-independent manner, it targets mitochondria, and this type of cell death is Bcl-2 sensitive (Vande Velde et al., 2000, MoI Cell Biol 20: 5454-5468). The transmembrane domain of BNIP3 is required for dimerization, pro-apoptotic function, and mitochondrial targeting (Yasuda et al., 1998, J Biol Chem 273: 12415-12421 ; Ray et al., 2000, J Biol Chem 275: 1439-1448). Over-expression of BNIP3 opens the mitochondrial permeability transition pore (PTP), thereby suppressing the proton electrochemical gradient (Ψm), and this is followed by chromatin condensation. L929 and MCF-7 cells which were stably transfected with ΔTM BNIP3, (a BNIP3 that lacks the transmembrane domain) were treated with Brevinin-2R. Cells expressing dominant-negative BNIP3 were significantly protected from Brevinin-2R induced cell death. Furthermore, the morphology of MCF-7 cells incubated with Brevinin- 2R resulted in the condensation of cell nuclei typical of cells executing the apoptotic program, but the cells tended to remain attached and cell membrane blebbing was in general not observed, (Fig. 15D) once again pointing to 'apoptosis-like' rather than classical apoptotic cell death.

Finally we have shown that Brevinin-2R is much less toxic towards PBMC than towards Jurkat and other cell lines. The differential toxicity could be explained by several mechanisms: (/) the outer membrane of cancer cells contain higher amounts of negatively charged phosphatidylserin (PS) (3-9% of the total membrane phospholipids) as compared to normal cells (Connor et al., 1989, Proc Natl Acad Sci USA 86: 3184-3188; Utsugi et al., 1991 , Cancer Res 51 : 3062-3066). Thus, positively charged defensins, like Brevinin-2R will have higher affinity to cancer cell membranes than to normal cells. Such interaction would lead to direct depolarization of the cell membrane in cancer cells and to interference with membrane metabolism (Papo et al., 2003, J Biol Chem 278: 21018-21023; Papo and Shai, 2003, Peptides 24: 1693-1703). For example, a short cationic diastereomeric peptide composed of D- and L-leucines, lysines, and arginines were selectively toxic toward cancer cells and significantly (86%) inhibited lung metastasis formation in a mice model with no detectable side effects (Papo et a/., 2003). Their cytotoxic action was attributed to their ability to depolarize the transmembrane potential of cancer cells at low micromolar concentrations (3 μM) that were comparable to concentrations of Brevinin-2R used in our study. (H) The membranes of many cancer cells contain higher levels of O- glycosylated mucines (high molecular weight glycoproteins consisting of a backbone protein to which oligosaccharides are attached via the hydroxyl groups of serine or threonine) (Papo and Shai, 2005, Cell MoI Life Sci 62: 784-790). These glycoproteins create an additional negative charge on the cancer cell's surface that may facilitate more efficient interaction with positively charged defensins like Brevinin-2R. Furthermore, (Hi) we cannot rule out the possibility that the higher negative potential within cancer cells, compared with that of non-cancer cells, may also contribute to the selective lytic activity of antimicrobial peptides (Cruciani et a/., 1991), since pharmacologic cell membrane depolarization prevented cytotoxicity caused by defensisns from the magainin family. (/V) Another plausible explanation for the different susceptibilities of normal and cancer cells to cytolytic peptides is based on the relatively higher number of microvilli on tumorigenic cells compared with normal cells (Zwaal and Schroit, 1997, Blood 89: 1121-1132). This consequently increases the surface area of the tumorigenic cell membrane and enables binding of a larger amount of peptides (Chan et al., 1998, Anticancer Res 18: 4467-4474). Finally, (v) cell membranes of cancer cells may be more permeable towards Brevinin-2R, thus it may achieve a higher concentration in malignant cells. Since mitochondrial membranes resemble bacterial cell membranes more closely, this defensin may "attack" mitochondria within cancer cells, thus inducing cell death. In conclusion, we present the purification and the initial characterization of a novel defensin, called Brevinin-2R, with semi-selective anti-cancer activity. Brevinin-2R kills cancer cells by a mechanism that has some morphologic resemblance to apoptosis, its action is death receptor-independent, it can be modulated by Bcl-2, Brevinin-2R triggered cell death presumably involves BNIP3, it is insensitive to caspase inhibition, and it is associated with ΔΨm drop. We also extensively discuss possible mechanisms that are responsible for its semi-selective anti-cancer activity. As Brevinin-2R appears to be a very promising new anticancer drug, our attention is currently focused on the interaction of Brevinin-2R with the cell membrane, and with the isolated mitochondria in the quest to better characterize its molecular mechanism of toxicity. Our results also show that Brevinin-2R has a potent antimicrobial activity but no hemolytic activity around MICs. It may be utilized as a useful topical antimicrobial agent and a model peptide for studying the relationships between structure and antimicrobial activity.

Materials and Methods Materials Chemicals, culture media and related compounds were purchased from Sigma Co.

(USA) and Pharmacia Biotech. Uppsala (Sweden). All additives have been tested for endotoxin contaminations. Cell culture plastic wares were obtained from Nunc Co. (Denmark), caspase-3 colorimetric assay kit and caspase-9 colorimetric assay kit and annexin V-FITC apoptosis detection kit were purchased from R&D Systems Co. (USA). HPLC grade solvents, TFA and TFE were obtained from Merck Co. (Germany).

Chemicals, culture media and related compounds were purchased from Sigma Co. (USA) and Pharmacia Biotech. Uppsala (Sweden). The strains used for determining antimicrobial activity included: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Klebseilla pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028, Micrococcos luteus and Bacillus spKR-8104 strain are from our strain bank (Tarbiat Modares University, Iran), Candida albicans and Candida tropicalis were clinical isolates. Preparation of skin secretions

150 specimens of both adult males and females of Rana ridibunda were collected from Gonbad region of Golestan province in Northeast Iran and maintained in captivity at the Tarbiat Modarres University. Secretions were obtained from parotoid and dorsal glands by the non-invasive electrical stimulation method (Tyler et al., 1992, J Pharm & Toxicol Methods 28: 199-200). The skin secretions were washed from the animals with 5 ml of 0.01 % acetic acid solution. The mixture was centrifuged and supernatant was passed through double stack Amicon filter (cut-off 10 kDa) and the utterance solution was collected and finally lyophilized. Peptide purification

The skin extract was dissolved in 50 mM acetate buffer pH 4.8 and chromatographed on a (1 *10 cm) SP-Sepharose FF (Pharmacia Biotech. Uppsala Sweden) equilibrated with buffer A (50 mM acetate buffer pH 4.8). The column was eluted with a 10 column volume (CV) gradient of 50% buffer B (50 mM acetate buffer pH 4.8, 1 M NaCI) at a flow rate of 1 ml/min and fractions (2 ml) were collected. Absorbance was measured at 280 nm with a Pharmacia FPLC detector. Fractions designated "IV" which had higher antibacterial activity compared to other fractions, were lyophilized and redissolved in 0.1% (V/V) trifluoroacetic acid (TFA)/water and injected onto a (1 * 25 cm) Vydac 208TP710 C₈ reverse- phase HPLC column (Separation group, Hesperia, CA, USA) equilibrated with 20% acetonitrile/water and 0.1% TFA. The elution was achieved with an initial 10-min wash in the equilibrated solution and then 20-60% linear gradient of acetonitrile containing 0.085% TFA over 60 min at a flow rate of 2 ml/min. The UV absorbance was monitored at 214 nm. Selected fractions were further purified by reverse- phase HPLC on a (1 * 25 cm) VP Nucleosil 300, Ci₈ reverse-phase HPLC column (Macherey-Nagel, GmbH, Co. Germany) with the same solvent system described above with successively lower gradient of acetonitrile at the flow rate of 1.5 ml/min. Finally, the purity of peptides was checked with an Aquapore RP-300 7micron (0.46 * 25 cm) equilibrated with 20% acetonitrile /water, 0.1% TFA and then elution was done with 20- 60% linear gradient of acetonitrile over 70 min at a flow rate of 1 ml/min. Nanocapillary HPLC - MS/ MS analysis

Nanocapillary reversed-phase liquid chromatography (LC) was performed using a capillary LC system (LC Packing, Netherlands) coupled online to an ion-trap (IT) mass spectrometer (LCQ Deca XP; Thermo-Finnigan, San Jose, CA, USA). Reverse-phase separations were performed using 75 nm ID x 360 mm x 15 cm long capillary columns (Dionex, Netherlands). After injecting 5 microlitres of the sample onto the column, a 20- min wash with 95% buffer A (0.1% v/v formic acid in water) was applied, and peptides were eluted using a linear gradient of 5% solvent B (0.1 % v/v formic acid in acetonitrile) to 85% solvent B in 60 min with a constant flow rate of 0.2 microlitres/min. For the analysis, 2 microgrammes of total sample was loaded onto the column. The IT mass spectrometer was operated in a data-dependent mode where each full MS scan was followed by three MS/MS scans in which the 3 most abundant peptide molecular ions were dynamically selected for collision-induced dissociation (CID) using a normalized collision energy of 35%. The temperature of the heated capillary was 180°C, and the electrospray voltage was 1.8 kV. CID spectra from the nanoLC-MS/MS analysis were searched against the frog FASTA database using SeQuest software (Thermo-Finnigan, San Jose, CA, USA). Only those peptides identified as possessing cross-correlation scores (Xcorr) greater than 1.9 for singly charged peptides, 2.2 for doubly charged peptides, and 2.9 for triply charged peptides (each with delta correlation scores (DelCorr) greater than 0.1) were considered as legitimate identification.

Antimicrobial assays and MIC determination

The antimicrobial activity against K. pneumoniae was examined during each purification step by radial diffusion assay (Lehrer et al., 1991 , J Immunol Methods 137: 167-173). The antimicrobial activity was evaluated by observing the suppression of the bacterial growth around the 3-mm diameter wells. The MIC of the peptide was determined using a broth dilution assay. Briefly, the serial dilution of the peptide was made in 0.2% BSA, and 0.01 % acetic acid solutions in 96-well polypropylene microtiter plates (Costar, Corning Incorporated, New York. N.Y.). Each well was inoculated with 100 μl of the test organism in MHB to a final concentration of 5-7 * 10⁵ CFU/ml. The MIC was taken as the lowest peptide concentration at which reduces growth by more than 50% after 18 hrs of incubation at 37⁰C. For the MIC of peptide against Candida albicans and Candida tropicalis, 2500 CFU/ml in the Sabouraud dextrose broth were tested with a serial dilution of the peptide and incubated for 18 hrs at 3O⁰C. Hemolytic assay

The hemolytic activity was assayed as described by Minn et al., 1998, with a slight modification (Minn et al., 1998, Biochim Biophys Acta 407: 31-39). 3 ml of freshly prepared sheep erythrocytes was washed with isotonic phosphate-buffered saline, pH 7.4 (PBS), until the color of the supernatant turned clear. The washed erythrocytes were then diluted to a final volume of 20 ml with the same buffer. Peptide sample (20 μl) serially diluted in PBS, were added to 180 μl of the cell suspension in microfuge tubes. Following gentle mixing, the tube were incubated at 37⁰C for 30 min and then centrifuged at 4000 * g for 5 min. 100 μl of supernatant was taken, diluted to 1 ml with PBS, and absorbance at 567 nm was determined. The relative optical density was compared with that of the cell suspension treated with 0.2% Triton X-100 as 100% hemolysis. Circular dichroism (CD) CD spectra were measured in either 20 mM NAPB, or 50% (v/v) trifluoroethanol

(TFE) in 20 mM NAPB, and recorded on a JASCO J-715 spectopolarimeter (Japan) using solutions with a peptide concentration of about 0.1 mg/ml. The CD results were expressed as molar ellipticity [θ] (deg.cm²d.mol^'1) based on a mean amino acid residue weight (MRW) assuming its average weight 110 (Protasevich et al., 1997, Biochemistry 36: 2017- 2024). The molar ellipticity was determined as [θ] = (θ. 100 MRW) / (cl), where c is the protein concentration in mg/ml, I is the light path length in cm, and θ is the measured ellipticity in degree at wavelength λ. Noise in the data was smoothed using the Jasco J- 715 software including the fast Fourier-transform noise reduction routine which allows enhancement of most noisy spectra without distorting their peak shape (Protasevich et al., 1997).

Phylogenetic analysis

Twenty-five Brevinin-2 amino acid sequences from six Rana species obtained from Genbank along with that of R. ridibunda were aligned using program Blast (National Center for Biotechnology Information, Bethesda, MD) and manually adjusted. A phylogenetic tree was obtained using the neighbor-joining method in PAUP^* ver. 4.0b4a (Swofford, 2000, PAUP, Version 4, Sinauer Associates, Sunderland Massachusetts) using distance option setting with mean character difference. To obtain confidence limits for various clades, bootstrap support values (Felsenstein, 1985, Evolution 39: 783-791) were calculated from 10000 replicates of neighbor-joining search. Cell Culture HT29/219 (NCBI C154) and SW742 (NCBI C146) colon carcinoma cells (obtained from National Cell Bank of Iran (NCBI)) were cultured in RPMI 1640 and were supplemented with 10% fetal calf serum, 100U/ml penicillin and 100μg/ml streptomycin. They were incubated at 37⁰C in a humidified CO₂ incubator with 5% CO₂ and 95% air. Cultures were regularly examined. Cytotoxicity Assay

To evaluate the cytotoxic effect of Brevinin 2R on these cell lines, MTT (3-(4, 5- Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) colorimetric assay was applied (Yui et al., 2002, Mediators of Inflammation 11 : 165-172). Briefly, asynchronously growing cells (1.5 X 10⁴ cells/ml) were transferred into 96-well culture plates containing 200 μl of medium and incubated for 24 hrs. Brevinin 2R at 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg was added and incubated for 24, 48 and 72 hrs after which MTT assay was performed. The percent of cell viability was calculated using the equation: (mean OD of treated cells/mean OD of control cells) X 100. Analysis of nuclear morphology Cells were plated in 8 well chamber slides and allowed to adhere. Brevinin 2R treated cells were fixed with methanol-acetic acid 3:1 (v/v) for 10 min after which staining was carried out with Hoechst 33258 (200 μg/ml). The slides were then washed with PBS (pH 7.4) and examined by an epifluorescence microscope (Micros, Austria). Apoptotic cells were defined on the basis of changes to nuclear morphology such as chromatin condensation and fragmentation, as well as overall cell shrinkage. Genomic DNA isolation

Cells were washed thoroughly in PBS and pelleted by centrifugation at 3000 rpm for 10 min at room temperature. The pellet was then resuspended in 0.5 ml of lysis buffer (150 mM NaCI, 10 mM EDTA, 0.1 M Tris HCI pH 7.75, 0.4% SDS, 200 μg/ml Proteinase K) vortexed hard and incubated at 37⁰C until the pellet dissolved completely. DNA was then extracted with phenol chloroform and precipitated overnight with 100% isopropanol at -2O⁰C. It was then spun down and resuspended in sterile water. Genomic DNA was fractionated on 0.8% agarose containing ethidium bromide (0.25 μg/ml). Caspase -3, -8 and -9 activation assays Caspase-3 (using DEVD-pNA as substrate), caspase-8 (using Ac-IETD-pNA as substrate) and caspase-9 (using LEHD-pNA as substrate) colorimetric assay kits were used to investigate the activation of these caspases in the treated HT29/219 and SW742 cells. To estimate caspase-3 and -8 activities, cells were lysed by incubation with cell lysis buffer on ice for 15 min and then centrifuged at 20,000 X g for 10 min (at 4⁰C). For caspase-9 activation assay, cells were lysed by incubation with cell lysis buffer on ice for 10 min and then centrifuged at 10,000 X g for 1 min (at 4⁰C). Enzymatic reactions were carried out in a 96 well flat bottom microplate. To each reaction sample 5, 25 and 50 μl of cell lysate (100-200 μg total protein) was added for caspase-3, -8 and -9, respectively. Additional controls, one free from cell lysate and the other lacking substrate as well as caspase-3 and 8 positive controls have been included. Protein content was estimated by the Bradford method (Bradford, 1976, Analytical Biochemistry 72: 248-254). These activities were expressed as nmole/min/mg protein. ATP assay

Intracellular ATP was measured by a bioluminescence assay using luciferin- luciferase. The assay is based on the requirement of ATP for producing luciferase- generated light (emission maximum λ 560 nm at pH 7.8). Cells (5 X 10⁶) either untreated or treated with 10 and 20 μg/ml of Brevinin 2R for 12 hrs were collected by centrifugation, resuspended in 250 μl of extraction solution (100 mM Tris Buffer, 4 mM EDTA, pH 7.75), heated at 98⁰C for 4 min, and placed in -2O⁰C. For ATP measurement, a 50 μl aliquot of a sample was added to 150 μl of reaction solution (50 mM Tris buffer, 20 mM magnesium acetate, 1 mM dithiothreitol pH 7.8) containing 0.5 mM luciferin, and 10 μg/ml luciferase. Light emission was quantified in a Turner Designs™ TD 20/20 luminometer (Stratec Biomedical Systems, Germany). For all experiments, ATP standard curves were run and were linear in the range of 5-500 nM. Concentrations of ATP stock solution were calculated from spectrophotometric absorbance at 259 nm using an extinction coefficient of 15,400 and calculations were made against the curve, and cellular ATP levels were expressed as nmol/10⁶ cells. Effect of N-Acetyl-L-Cysteine on Brevinin 2R cytotoxic effect

To study the involvement of reactive oxygen species (ROS) in the induction of apoptosis by calprotectin, the cell lines were pretreated with increasing concentration (0- 10 mM) of N-Acetyl-L-cysteine (NAC) for 24 hours. The cell lines were then treated with Brevinin 2R (30 μg/ml) for 48 hours. Statistical analysis

The results were expressed as the mean ± SD and statistical differences were evaluated by one-way ANOVA and P<0.05 was considered significant. While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

Table 1. Minimal inhibitory concentrations (MICs) Brevinin 2R isolated from the skin of Rana ridibunda.

Microorganism MIC (μg/ml) Staphilococcus aureus ATCC 25923 7.0

Micrococcus luteus 2.5

Bacillus spKR-8104 7.2

Escherichia coli ATCC 25922 3.0

Salmonella typhimorium ATCC 14028 4.4 Pseudomonas aeruginosa ATCC 27853 22.0

Klebseilla pneumoniae ATCC 13883 3.0

Candida albicans (clinical isolate) 4.6

Candida tropicalis (clinical isolate) 30.0

Claims

1. An isolated peptide having an amino acid sequence substantially equivalent to SEQ ID NO. 1 , SEQ ID NO. 2 or SEQ ID NO. 3.

2. The isolated peptide according to claim 1 having an amino acid sequence as set forth in SEQ ID NO. 1.

3. The isolated peptide according to claim 1 having an amino acid sequence as set forth in SEQ ID NO. 3.

4. A method of generating a non-hemolytic, anti-cancer brevinin peptide comprising: decreasing the hydrophobicity of a brevinin peptide.

5. The method according to claim 4 wherein the hydrophobicity of the brevinin peptide is increased by substituting one or more nonessential non-hydrophobic amino acids with one or more hydrophobic amino acids.

6. The method according to claim 4 wherein the hydrophobicity of the brevinin peptide is increased by adding one or more hydrophobic amino acids to the brevinin peptide.

7. The method according to claim 4 wherein the brevenin peptide has an initial amino acid sequence as set forth SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3.

8. A method of treating cancer comprising administering to an individual in need of such treatment an effective amount of a peptide comprising an amino acid sequence as set forth SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3.

9. The method according to claim 6 wherein the cancer is colon cancer.

10. The method according to claim 6 wherein the cancer is a cancer of mesodermal origin.

11. A method of preparing a medicament for treating cancer comprising mixing an effective amount of a peptide comprising an amino acid sequence as set forth SEQ ID

NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 with a suitable excipient.