NAKAHATA, Adriana Miti (Universidade Federal De São Paulo, Rua Três De Maio 10, Vila Clementino -020 São Paulo, 04044, BR)
SAMPAIO, Misako Uemura (Universidade Federal De São Paulo Rua Três De Maio, 100 Vila Clementino,Rua Três De Maio, 10, Vila Clementino -020 São Paulo, 04044, BR)
JASIULIONIS, Miriam Galvonas (Universidade Federal De São Paulo, Rua Botucatu 862 4º Anda, Vila Clementino -900 São Paulo, 04023, BR)
OLIVA, Maria Luiza Vilela (Universidade Federal De São Paulo, Rua Três De Maio 10, Vila Clementino -020 São Paulo, 04044, BR)
NAKAHATA, Adriana Miti (Universidade Federal De São Paulo, Rua Três De Maio 10, Vila Clementino -020 São Paulo, 04044, BR)
SAMPAIO, Misako Uemura (Universidade Federal De São Paulo Rua Três De Maio, 100 Vila Clementino,Rua Três De Maio, 10, Vila Clementino -020 São Paulo, 04044, BR)
JASIULIONIS, Miriam Galvonas (Universidade Federal De São Paulo, Rua Botucatu 862 4º Anda, Vila Clementino -900 São Paulo, 04023, BR)
WHAT IS CLAIMED IS:
1. The use of protease inhibitors isolated from Ba.uh.inia bauhinioides and/or Enterolobium contortisiliquum characterizing in that the use is in the preparation of a pharmaceutical composition comprising at least one of said inhibitor for cancer treatment .
2. The use, according to claim 1, characterizing in that the previously referred pharmaceutical composition additionally comprises a chemotherapic agent. 3. The use, according to claim 2, characterizing in that the said chemotherapic agent is 5-Fluorouracil.
4. The use, according to claim 2, characterizing in that the said chemotherapic agent is the carboplatinum.
5. A pharmaceutical composition comprising proteases inhibitor isolated from Bauhinia bauhinioides and/or
Enterolobium contortisiliquum characterizing in that it comprises an effective amount of at least one of said inhibitor and at least one pharmaceutically acceptable carrier. 6. A composition, according to claim 5, characterizing in that it also comprises at least one chemotherapic agent.
7. A Composition, according to claim 6, characterizing in that the said chemotherapic agent is 5-Fluorouracil .
8. A Composition, according to claim 6, characterizing in that the said chemotherapic agent is the carboplatinum. |
"USE OF PROTEASE INHIBITORS FROM BAUHINIA BAUHINIOIDES AND/OR ENTEROLOBIUM CONTORTISILIQUUM" .
FIELD OF THE INVENTION The present invention relates to the use of protease inhibitors isolated from the Bauhinia bauhinioides and/or Enterolobium contortisiliquum for cancer treatment, as well as a pharmaceutical composition comprising thereof.
BACKGROUND OF THE INVENTION
In the first half of XX century, infectious diseases became the biggest cause of human suffering and deaths,- however, due to research advances for vaccines and antibiotics, the amount of infectious diseases has been reducing. According, researcher's attention has been directed to immune system diseases and cancer (Fuchs & Matzinger, 1996) .
Cancer is not a disease itself, but a name employed to identify a plurality of malignant tumors. We may say cancer rises when some cells escape from intra and extra cellular vigilance mechanisms. These cells acquire the ability to invade tissues and colonize remote organs, characterizing invasion and metastatic processes (Chammas, 1998) , which is the major contributing factor for cancer morbidity and mortality (Stetler-Stevenson & Yu, 2001, Wollina et. al . , 2001) . The knowledge of molecular, genetic and biological bases for tumor dissemination and tumor angiogenesis has been dramatically increased in the last two decades (Stetler- Stevenson & Yu, 2001) . Tumor progression depends on a number of important processes. These include the continuous growth of tumor cells (autonomous growth) , its ability to avoid cellular programmed death (apoptosis) and inhibitory growth flagging, as well as
to develop a continuous angiogenic answers, to invade neighboring tissues and remote organs (metastasis) (Hanahan, 2000) .
Tumor invasion and metastasis are considered a multiple step phenomena, comprising proteolytic degradation of basal membrane and extra cellular matrix (ECM) , changing on cellular adhesion and on tumor cells physical movements
(Curran & Murray, 2000) . During invasion, tumor cells disengage from primary tumor, migrate and cross structural barriers, including basal membrane and surrounding stromal cells of the extra cellular matrix mainly consisting of collagen fibrin, and also fibronectin, laminin and several proteoglycan. Also, proteolytic degradation of extra cellular matrix is meaning as essential for tumor angiogenesis induction (Ka.ha.ri & Saarialho-Kere, 1999) .
Basal membrane and extra cellular matrix components degradation by proteolytic enzymes are essential for tumor progress (Wollina et al . , 2001) . Local proteolysis is turned easy for proteases outside the tumor cell, maybe linked to and/or secreted from tumor cell. More recent data suggest that proteases from tumor cell also participate of proteolysis by phagocytosis of the extra cellular matrix (Koblinski et al . , 2000; Wolf et al . , 2001).
Proteases are normally synthetized as latent forms, known as zymogene forms, which can be converted on its active forms. Interaction of several proteases leads to degradation of extra cellular matrix components. Therefore, a protease superexpression or activation results on a proteolytically activated environment surrounding tumor. Presently, it is already established that there is a straight correlation regarding increasing on expression, on activity and localization changing for many proteases and tumor progress (Koblinski et al., 2000).
Proteases can produce matrix proteins fragments, influencing liberation, activation and bio-availability of growth factors, and accordingly, modulating cellular growth, invasion, apoptosis and angiogenesis . Additionally, proteases, its receptors and inhibitors can be directly involved in the cellular migration and processing, or shedding, of cellular surface proteins (Noel et al . , 1997) . Many proteases have an important action in this degradation process, including serine-protease, cisteine-proteases, aspartic protease and metalo-proteases (Polette & Birembaut, 1998; Bode et al . , 2000).
It is good to evidence that tumor cells over express proteases to influence local blood supplement, leakage through vessels and migration through extra cellular matrix during metastasis (Wolf et al . , 2001), and that is the reason proteases inhibitors are introduced as strong candidates for controlling these events.
Inhibitors isolated from leguminous plants B. bauh±nioides, and Enterolobium contortϊsiliquum was isolated and was under chemical and biochemical characterization. Particularly, the influence of inhibitors activity was studied over a plurality of blood coagulation enzymes as calicrein, XIIa and Xa factors and thrombin, including proteases which play an important role during tumor development like plasmin and metalo-protease. All our references sowed these inhibitors are, all of them, highly homologues to soybean trypsin inhibitor. They showed too small structural differences possibly responsible for this choosing ability for different enzymes bonding (Olive et al., 2001 a, b) .
The most important role of proteases in metastatic process is regarding extra cellular matrix proteolytic degradation, which starts with the action of specific
proteases, secreted by different cell types, participating on tumor cells invasion activity and the increasing on several other enzymes expression or activity also involved on tumor cell malignity and invasion process . The action of proteolytic enzymes inhibitors, which showed differences for specificity action, allows the utilization of these effects for blocking proteases activity during cancer. For this reason, specific inhibitor for protease shows potential value for therapeutic activity for cancer.
Specifically in oncology area, protease inhibitors have been studied mainly for templates involving metastatic process, such as adhesion, migration, cycle, metabolism, cell proliferation and angiogenesis by using in vitro and in vivo model analysis for tumor development. Therefore, inhibitors, as well as peptides, isolated from plants and derived from their structures could have important participation over tumor process control.
BRIEF DESCRIPTION OF DRAWINGS
The following figures are part of the instant patent descriptive report and are included with illustrative purpose only for better understanding some specific aspects of the present invention. The object of the present invention will be better understood by reference to one or more of these figures associated with the detailed description of preferred embodiment showed herein.
FIGURE 1 shows the inhibition of HNE by EcTI. HNE (17.0 nM) and raising doses of inhibitor were preincubated over 10 minutes, at 37 0 C. The inhibitory activity was determined by the reduction enzyme activity, when using the substrate MeO-
Suc-Ala-Ala-Pro-Val-pNan (11.0 mM) .
FIGURE 2 shows the activation analysis of pro-MMP-9 in
MMP-9 and pro-MMP-2 in MMP-2 induced by plasminogen and/or PMA in HT1080 cells, treated or not treated with BbCI, BbKI, EcTI and aprotinin.
FIGURE 3 shows the action of (a) BbCI and (b) rBbCI in the adhesion of melan-a cells to IV collagen, fibronectin and vitronectin. Melan-a cells and raising concentration of BbCI and rBbCI were preincubated at room temperature during 15 minutes and placed in plates previously adsorbed with IV collagen or fibronectin or vitronectin (5 μg/ 100 μl/ well) or BSA, according to described herein.
FIGURE 4 shows the action of (a) BbCI and (b) rBbCI in the adhesion of Tm5 cells to IV collagen, fibronectin and vitronectin. Tm5 cells and raising concentration of BbCI and rBbCI were preincubated at room temperature during 15 minutes and placed in plates previously adsorbed with IV collagen or fibronectin or vitronectin (5 μg/ 100 μl/ well) or BSA, according to described herein.
FIGURE 5 shows the action of (a) BbKI and (b) rBbKI in the adhesion of melan-a cells to IV collagen, fibronectin and vitronectin. Melan-a cells and raising concentration of BbKI and rBbKI were preincubated at room temperature diuring 15 minutes and placed in plates previously adsorbed with IV collagen or fibronectin or vitronectin (5 μg/ 100 μl/ well) or BSA, according to described herein. FIGURE 6 shows the action of (a) BbKI and (b) rBbKI in the adhesion of Tm5 cells to IV collagen, fibronectin and vitronectin. Tm5 cells and raising concentration of BbKI and rBbKI were preincubated at room temperature during 15 minutes and placed in plates previously adsorbed with IV collagen or fibronectin or vitronectin (5 μg/ 100 μl/ well) or BSA, according to described herein.
FIGURE 7 shows the action of EcTI in the adhesion of (a) melan-a and (b) Tm5 cells to iv collagen, fibronectin and
vitronectin. Melan-a or Tm5 cells and raising concentration of EcTI were preincubated at room temperature during 15 minutes and placed in plates previously adsorbed with IV collagen or fibronectin or vitronectin (5 μg/ 100 μl/ well) or BSA, according to described herein.
FIGURE 8 shows the action of (a) BbCI and (b) rBbCI in the proliferation of melan-a cells. Melan-a cells and raising concentration of BbCI and rBbCI were preincubated for 15 minutes at room temperature, added to 96 well plates and analyzed in different incubation times (2h, 24h, 48h and 72h) .
FIGURE 9 shows the action of (a) BbCI and (b) rBbCI in the proliferation of Tm5 cells. Tm5 cells and raising concentration of BbCI and rBbCI were preincubated for 15 minutes at room temperature, added to 96 well plates and analyzed in different incubation times (2h, 24h, 48h and 72h) .
FIGURE 10 shows the action of (a) BbKI and (b) rBbKI in the proliferation of melan-a cells. Melan-a cells and raising concentration of BbKI and rBbKI were preincubated for 15 minutes at room temperature, added to 96 well plates and analyzed in different incubation times (2h, 24h, 48h and 72h) .
FIGURE 11 shows the action of (a) BbKI and (b) rBbKI in the proliferation of Tm5 cells. Tm5 cells and raising concentration of BbKI and rBbKI were preincubated for 15 minutes at room temperature, added to 96 well plates and analyzed in different incubation times (2h, 24h, 48h and 72h) . FIGURE 12 shows the action of EcTI in the proliferation of cells (a) melan-a and (b) Tm5. Cells and raising concentration of EcTI were preincubated for 15 minutes at room temperature, added to 96 well plates and analyzed in
different incubation times (2h, 24h, 48h and 72h) .
FIGURE 13 shows the cellular metabolism of Tm5 treated with 5-FU, rBbCI, rBbKI and EcTI. Tm5 cells (8xlO 3 / lOOμl/ well) were incubated for 24 hours and after that raising concentrations of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept incubated for (a) 24 hours and (b) 48 hours periods .
FIGURE 14 shows cellular cycle of melan-a with inhibitors rBbCI, rBbKI and EcTI. (a) melan-a, (b) melan-a treated with rBbCI, (c) melan-a treated with rBbKI, (d) melan-a treated with EcTI, after 24 hours incubation time as described hereinbefore. Steps from Ml to M4 indicate cellular cycle steps, Ml = fragmented cells, M2 = GO 4 Gl, M3 = S, M4 = G2 -» M. FIGURE 15 shows cellular cycle of Tm5 with rBbCI, rBbKI and EcTI inhibitors, (a) Tm5 , (b) Tm5 treated with rBbCI, (c) Tm5 treated with rBbKI, (d) Tm5 treated with EcTI, after 24 hours incubation time as described hereinbefore. Steps from Ml to M4 indicate cellular cycle steps, Ml = fragmented cells, M2 = GO → Gl, M3 = S, M4 = G2 -> M.
FIGURE 16 shows migration of melan-a and Tm5 cells. Melan-a and Tm5 cells were added to the plates previously adsorbed with fibronectin and kept under incubation for 1, 2 and 3 hours . FIGURE 17 shows the action of rBbCI in the migration of melan-a and Tm5 cells. Melan-a or Tm5 cells and raising concentration of rBbCI were preincubated for 15 minutes at room temperature, and subsequently added to plates previously adsorbed with fibronectin. FIGURE 18 shows the action of rBbKI in the migration of melan-a and Tm5 cells. Melan-a or Tm5 cells and raising concentration of rBbKI were preincubated for 15 minutes at
room temperature, and subsequently added to plates previously adsorbed with fibronectin.
FIGURE 19 shows the action of EcTI in the migration of melan-a and Tm5 cells. Melan-a or Tm5 cells and raising concentration of EcTI were preincubated for 15 minutes at room temperature, and subsequently added to plates previously adsorbed with fibronectin.
FIGURE 20 shows the effect of BbCI and rBbCI in Tm5- induced tumorgenesis . Tumorgenesis was screened depending on tumor volume (mM 3 ) . Mice C57B1/6 (n=5) received subcutaneous injection with Tm5 and daily treatment with inhibitor, (a) BbCI (2mg/ kg/ day/ animal) , in detailed tumor photos of control groups and trial groups receiving BbCI; (b) rBbCI (2mg/ kg/ day/ animal) . FIGURE 21 shows the effect of BbCI and rBbCI in Tm5- induced tumorgenesis. Tumorgenesis was screened depending on tumor volume (mM 3 ) . Mice C57B1/6 (n=5) received subcutaneous injection with Tm5 and daily treatment with inhibitor, (a) BbKI (2mg/ kg/ day/ animal) , in detailed tumor photos of control groups and trial groups receiving BbKI; (b) rBbKI
(2mg/ kg/ day/ animal) .
FIGURE 22 shows the effect of EcTI in Tm5-induced tumorgenesis. Tumorgenesis was screened depending on tumor volume (mM 3 ) . Mice C57B1/6 (n=5) received subcutaneous injection with Tm5 and daily treatment with inhibitor, (a) EcTI (2mg/ kg/ day/ animal) , in detailed tumor photos of control groups and trial groups receiving EcTI; (b) EcTI (2mg/ kg/ day/ animal) .
FIGURE 23 shows the material with affinity for rBbCI- Sepharose reverse-phase chromatography (RPC) . Sample: 20 μg
(A 2 15) from affinity chromatography rBbCI-Sepharose (fraction c) was applied at the top of a C4 column (4.6 mM x 15 cm) balanced with 0.1 % TFA in water. Elution through TFA 0.1 %
acetonitrile linear gradient in water, from 0 to 100 %, in 60 minutes. Arrows indicate materials submitted to consecutive sequencing. At detail, Affinity Chromatography rBbCI- Sepharose. Affinity Chromatography rBbCI-Sepharose column (2.0 ml) balanced with Tris/HCl 0.1 M buffer, pH 8.0. (a) non-adsorbed material; (b) material eluted with Tris/HCl pH 8.0, with NaCl 0.15 M; (c) elution with KCl/HCl 0.5 M, pH 2.0, followed by immediate neutralization with Tris/HCl 1.0 M; (d) material eluted with glucose 0.5 M; (e) material eluted with Tris/HCl pH 8.0, containing urea 1 M.
FIGURE 24 shows the material with affinity for rBbKI- Sepharose reverse-phase chromatography (RPC) . Sample: 20 μg (A215) from affinity chromatography rBbKI-Sepharose (fraction c) was applied at the top of a C4 column (4.6 mM x 15 cm) balanced with 0.1 % TFA in water. Elution through TFA 0.1 % acetonitrile linear gradient in water, from 0 to 100 %, in 60 minutes. Arrows indicate materials submitted to consecutive sequencing. At detail, Affinity Chromatography rBbKI- Sepharose. Affinity Chromatography rBbKI-Sepharose column (2.0 ml) balanced with Tris/HCl 0.1 M buffer, pH 8.0. (a) non-adsorbed material; (b) material eluted with Tris/HCl pH 8.0, with NaCl 0.15 M; (c) elution with KCl/HCl 0.5 M, pH 2.0, followed by immediate neutralization with Tris/HCl 1.0 M; (d) material eluted with glucose 0.5 M; (e) material eluted with Tris/HCl pH 8.0, containing urea 1 M. The arrow indicates the sequencing fraction analyzed.
FIGURE 25 shows the material with affinity for EcTI- Sepharose reverse-phase chromatography (RPC) . Sample: 20 μg (A 2 15) from affinity chromatography EcTI-Sepharose (fraction c) was applied at the top of a C4 column (4.6 mM x 15 cm) balanced with TFA 0.1 % in water. Elution through trifluoroacetic acid (TFA) 0.1 % acetonitrile linear gradient in water, from 0 to 100 %, in 60 minutes. Arrows indicate
materials submitted to consecutive sequencing. At detail, Affinity Chromatography EcTT-Sepharose. Affinity Chromatography EcTI-Sepharose column (2.0 ml) balanced with Tris/HCl 0.1 M buffer, pH 8.0. (a) non-adsorbed material; (b) material eluted with Tris/HCl pH 8.0, with NaCl 0.15 M; (c) elution with KC1/HC1 0.5 M, pH 2.0, followed by immediate neutralization with Tris/HCl 1.0 M; (d) material eluted with glucose 0.5 M; (e) material eluted with Tris/HCl pH 8.0, containing urea 1 M. FIGURE 26 shows the sequencing of the amino-terminal region of the material eluted within 38 minutes, in the reverse phase chromatography (RPC) , referring to the material showing affinity and similarity to rBbCI-Sepharose. Indication shows the sequencing of the amino-terminal region. Following, there are other proteins showing similarity to this sequence, obtained through analysis in database of primary structure NCBI-Blast2 (www. ebi . ac .uk/blastall) . In evidence is the name of the protein showing identity similarity and residues. FIGURE 27 shows the sequencing of the amino-terminal region of the material eluted within 40 minutes, in the reverse phase chromatography (RPC) , referring to the material showing affinity and similarity to rBbCI-Sepharose. Indication shows the sequencing of the amino-terminal region. Following, there are other proteins showing similarity to this sequence, obtained through analysis in database of primary structure NCBI-Biast2 (www. ebi . ac . uk/blastall) . In evidence is the name of the protein showing identity similarity and residues. FIGURE 28 shows the sequencing of the amino-terminal region of the material eluted within 35 minutes, in the reverse phase chromatography (RPC) , referring to the material showing affinity and similarity to rBbKI-Sepharose .
Indication shows the sequencing of the amino-terminal region. Following, there are other proteins showing similarity to this sequence, obtained through analysis in database of primary structure NCBI-Blast2 (www. ebi . ac . uk/blastall) . In evidence is the name of the protein showing identity similarity and residues.
FIGURE 29 shows the sequencing of the amino-terminal region of the material eluted within 37 minutes, in the reverse phase chromatography (RPC) , referring to the material showing affinity and similarity to rBbKI-Sepharose. Indication shows the sequencing of the amino-terminal region. Following, there are other proteins showing similarity to this sequence, obtained through analysis in database of primary structure NCBI-Biast2 (www. ebi . ac .uk/blastall) . In evidence is the name of the protein showing identity similarity and residues.
FIGURE 30 shows the sequencing of the amino-terminal region of the material eluted within 31 minutes, in the reverse phase chromatography (RPC) , referring o the material showing affinity and similarity to EcTI-Sepharose. Indication shows the sequencing of the amino-terminal region. Following, there are other proteins showing similarity to this sequence, obtained through analysis in database of primary structure NCBI-Blast2 (www. ebi . ac . uk/blastall) . In evidence is the name of the protein showing identity similarity and residues.
FIGURE 31 shows the sequencing of the amino-terminal region of the material eluted within 35 minutes, in the reverse phase chromatography (RPC) , referring to the material showing affinity and similarity to EcTI-Sepharose. Indication shows the sequencing of the amino-terminal region. Following, there are other proteins showing similarity to this sequence, obtained through analysis in database of primary structure NCBI-Blast2 (www. ebi . ac . uk/blastall) . In evidence is the name
of the protein showing identity similarity and residues.
FIGURE 32 shows cell metabolism of MKN28 (gastric cancer - less invasive strain) treated with 5-FU, rBbCI, rBbKI and EcTI. Cells MKN28 (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours.
FIGURE 33 shows cell metabolism of Hs746T (gastric cancer - more invasive strain) treated with 5-FU, rBbCI, rBbKI and EcTI. Cells Hs746T (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours . FIGURE 34 shows cell metabolism of HT-29 (colon cancer - less invasive strain) treated with 5-FU, rBbCI, rBbKI and EcTI. Cells HT-29 (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours.
FIGURE 35 shows the cellular metabolism of HCT 116
(cancer of colon - more invasive strain) treated with 5-FU, rBbCI, rBbKI and EcTI. Cells HCT 116 (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours .
FIGURE 36 shows the cellular metabolism of MCF-7 (breast cancer - less invasive strain) treated with 5-FU, rBbCI, rBbKI and EcTI. Cells MCF-7 (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48
hours .
FIGURE 37 shows the cellular metabolism of SKBR-3
(breast cancer - more invasive strain) treated with 5-FU, rBbCI, rBbKI and EcTI. Cells SKBR-3 (4 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours .
FIGURE 38 shows the cellular metabolism of K562 (leukemia cells strain) treated with 5-FU, rBbCI, rBbKI and EcTI. Cells K562 (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours. FIGURE 39 shows the cellular metabolism of THP-I (monocyte leukemic cells strain) treated with 5-FU, rBbCI, rBbKI and EcTI. Cells THP-I (2 x 10 4 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, rBbCI, rBbKI and EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours .
FIGURE 40 shows the cellular metabolism of MKN28 (gastric cancer - less invasive strain) treatment with 5-FU, EcTI and 5-FU+EcTI. Cells MKN28 (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, EcTI and 5-FU EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours .
FIGURE 41 shows the cellular metabolism of Hs746T (gastric cancer - more invasive strain) treatment with 5-FU, EcTI and 5-FU EcTI. Cells Hs746T (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of 5-FU, EcTI and 5-FU+EcTI were added to
plates and kept under incubation for (a) 24 hours and (b) 48 hours .
FIGURE 42 shows the cellular metabolism of MKN28 (gastric cancer - less invasive strain) treatment with carboplatinum, EcTI and carboplatinum+EcTI . Cells MKN28 (8 x 10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of carboplatinum, EcTI and carboplatinum+EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours. FIGURE 43 shows the cellular metabolism of Hs746T
(gastric cancer - more invasive strain) treatment with carboplatinum, EcTI and carboplatinum+EcTI . Cells Hs746T (8 x
10 3 / 100 μl/ well) were incubated for 24 hours and after this period raising concentration of carboplatinum, EcTI and carboplatinum+EcTI were added to plates and kept under incubation for (a) 24 hours and (b) 48 hours.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the use of protease inhibitors isolated, mainly, from Bauhinia bauhinioides and
Enterolobium contortisiliquum seeds for cancer treatment.
Inhibition specificity over different proteases, showed by isolated compounds from Bauhinia and Enterolobium seeds leads to a strong activity over cancer, which metastatic process involves the action of different proteases. These products were able to act on blocking of "in vivo" model of tumor development without any apparent toxicity. In cellular strain of human cancer, takes place important inhibition of the cellular proliferation. The combined therapy of a protease inhibitor in accordance with the present invention and a chemotherapic agent reduces by an expressive amount the amount of the necessary chemotherapic drugs for inhibition of tumor cell proliferation. In this aspect, inhibitors can
reduce the side effects showed by these chemotherapic agents.
The present invention also includes pharmaceutical compositions comprising an effective amount of at least one of inhibitors of the invention and at least one pharmaceutically acceptable carrier.
In accordance with another embodiment, the compositions of the present invention may still comprise at least one chemotherapic agent. Such chemotherapic agent includes, for example, the 5-Fluorouracil (5-FU) and carboplatinum. When a second chemotherapic drug is used, the second chemotherapic drug can be administered in much lower amount, while increasing composition effectiveness.
Typically, the pharmaceutical compositions of the present invention comprise at least one inhibitor in accordance with the present invention which can be administered in combination with at least one chemotherapic agent and at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers include, but are not limited by aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and other additives known in the art. pH and exact concentration of several components of composition can be adjusted according to current practice.
Also it will be understood that a specific dosage and a treatment regime for any particular patient will depend on a plurality of factors, including the activity of the specific used compound, the age, physical weight, general health, sex, diet, time of administration, rate of excretion, drugs combination and doctor's judgement about disease seriousness which is specifically being treated. The amount of active ingredients also will depend on the specific compound and of the other therapeutic agent, if present, in composition.
Following, the present invention will be more detailed explained with the help of a preferred embodiment
description.
Inhibitors were purified through usual chromatographic techniques which consist of ion exchange chromatography, affinity chromatography, gel filtration chromatography, RP- chromatography, and in accordance with methods described hereinbefore, for example, for Oliva et al., 1999; Oliveira et al., 2001b, Batista et al . , 1996 and recombinant inhibitors obtained according to described method, for example, by Hansen, 2004.
Enzymatic Dosage and Determination of Inhibition Constants .
Chromogenic substrates, like peptides derived from p- nitroanilide, were used mainly because the high sensibility for photometric detection (at A 40 5) of the p-nitroaniline released after enzymatic hydrolysis (Erlanger et al . , 1961), in Packard spectrophotometer (model SpectraCount) .
Used substrates in each experiment were what which showed better specificity for assayed enzymes. Substrates derived from the p-nitroanilide were initially diluted in dimethyl sulfoxide (DMSO) and subsequent dilution was carried out in appropriated buffer for each test .
Fluorogenic substrates, amino methyl coumarin (AMC) derived peptides, also were used in some dosages, because they are more sensitive than chromogenic substrates derived from p-nitroanilide. The hydrolysis was monitored in wavelengths 380 nm (excitement) and 460nm (emission) , using a Spectrofluorometer Packard (model FluoroCount) . These substrates were diluted in dimethylformamide (DMF) .
Tests using substrates of out-fluorescence (ortho aminobenzoic acid [Abz] -X- 2,4 - ethylene diamine dinitrophenyl [EDDnp] ) were carried out in Hitachi's Spectrofluorometer (model F-2000) , and hydrolysis detection was carried at wavelengths 320nm (excitement) and 420 nm
(emission) and diluted in dimethyl formamide and water 1:1 (v/v) .
Inhibition Constants were determined ' by the value of the
Dissociation Constant of the inhibitor-enzyme complex (Ki) . Determinations were carried out following Morrison model
(1989), whose final equation was defined by Knight, 1986, adapted to an enzymatic kinetic plotting computer program, the value being calculated by GraFit ® program (version 3.0) .
Proteases substrate hydrolysis for determination of inhibitory activities .
Proteases enzymatic activities were carried out on specific substrates, using enzyme [M] that was preincubated at 37°C, with different inhibitor concentrations, in appropriate buffer, and after 10 minutes substrate [M] was added (Table I) , in final volume of 250μl, incubation continuing by 20-30 minutes (specific time for each enzyme) at 37°C, stopping the reaction with 40μl of acetic acid 40 %.
Substrate hydrolysis by enzyme was followed by photometric readings (at A 405 ) of the released p-nitroaniline or substrate hydrolysis (AMC) was monitored by fluorescence detection in wavelengths of 380nm (excitement) and 460nm (emission) . The inhibitory activity was calculated by the determination of the residual activity of enzyme in assays . Concentration of inhibitors was calculated considering stoichiometry ratio of 1:1.
This method was also used to localize inhibitory activity during inhibitors purification process. Experiments were carried out in triplicate. Table I shows the conditions used for determination of studied inhibitor activity over proteases which are involved in cell activity.
Table I: Enzyme, substrate and buffer used to verify
proteases substrates hydrolysis and inhibitory activities determination .
* inhibitor used for enzyme titration.
Hydrolysis of Z-Phe-Arg-AMC for cisteine-proteases and inhibitory activities determination
The cisteine-proteases concentration was obtained through titration with egg cistatine. The catepsina L or cruzipain or cruzain inhibitory activity was determined by residual activity of the enzyme over substrate Z- phenylalanyl-arginine-aminomethylcoumarinic (z-Phe-Arg-AMC) . Enzymes were activated in Na 2 HPO 4 0.1 M buffer, pH 6.3, 1OmM EDTA, NaCl 40OmM and DTT 2mM, stored at 37 0 C during 10 min. In typical experiments carried out in the same activation
buffer, catepsina L (18nM) or cruzipain (18nM) or cruzain (3.2nM), was preincubated with raising concentrations of purified inhibitor preparations, during 10 minutes at 37°C, and than added Z-Phe-Arg-AMC (0,3mM) . Substrate hydrolysis was monitored by fluorescence detection in the wavelengths 380nm (excitement) and 460nm
(emission) , using a Spectrofluorometer Hitachi (model F-
2000) . The fluorescence increasing was continuously recorded during 10 minutes. The experiments were carried out in triplicate.
The residual activity was determined by comparing enzymatic hydrolysis curves, in the presence and absence of the inhibitor.
Determination of BbKI, rBbKI and EcTI concentration Different concentrations of humane plasmatic calicrein inhibitor from Bauhinia bauhinioides (BbKI) , humane plasmatic calicrein recombinant inhibitor from Bauhinia bauhinioides (rBbKI) and trypsin inhibitor from Enterolobium contortisiliquum (EcTI) were preincubated at 37°C for 10 minutes in Tris/HCI 0.05M buffer, containing CaCl 2 0.02 %
(v/v) , pH 8.0, with known trypsin concentration. To the preincubated were added 25μl of H-D-Benzoyl-L-arginine-p- nitroanilide (Bz-Arg-pNan) 10.OmM sustrate till a 250μl final volume. Substrate enzymatic hydrolysis was followed by photometric reading (at A405) of released p-nitroaniline, according to previously described.
Concentration of inhibitors was calculated considering stoichiometry ratio of 1:1.
Determination of BbCI and rBbCI concentration Different inhibitor concentrations of cruzipain from Bauhinia bauhinioides (BbCI) and recombinant inhibitor of cruzipain from Bauhinia bauhinioides (rBbCI) were preincubated at 37 0 C for 10 minutes, in Tris/HCI 0. IM buffer
pH 7.0, containing NaCl 0.5M with human neutrophils elastasis (HNE) (17nM) , and determined according to described previously. To the preincubated 25μl of methyl ester-L- succinyl-L-alanyl-L-alanyl-L-proIyI-L-valine-p-nitroanilide (MeO-Suc-Ala-Ala-Pro-Val-pNan) substrate (11. OmM) were added, in a final volume of 250μl. Substrate enzymatic hydrolysis was followed by photometric readings (at A 405 ) of the released p-nitroaniline .
Concentration of inhibitors was calculated considering stoichiometry ratio of 1:1.
Poliacrylamide gel electrophoresis
The protein electrophoresis analysis was carried out according to Laemmli, 1970. Glass plates (8.0 x 10.0 cm) were previously washed and rinsed with distilled water and cleaned with ketone, to be used in gel polymerization process using a
15 % acrylamide solution.
For the analyses 15μg of lyophilized resuspended proteins in appropriate distilled water volume and an equal volume of buffer Tris/HCl 0.12 M containing SDS 4 % (p/v) and glycerol 20 % (v/v) were used; when indicated, proteins were reduced, by addition of a buffer volume equal to dithiothreitrol (DTT) sample volume (200 mg/ml) . All the samples were heated to 100 0 C during 7min, centrifuged, and was added 1 μl of solution of blue of bromophenol 0.02 % (p/v), containing SDS 0.1 % and glicerol 10 % in phosphate buffer 0.1 M, pH 6,8.
The proteins used as molecular weight standards were fosforilase b (94 kDa) , bovine albumin (66 kDa) , egg albumin (45 kDa) , anidrase carbon (31 kDa) , trypsin inhibitor of soy (20 kDa) and α-lactalbumina (14 kDa) .
The eletroforese was carried out in buffer Tris 0.025 M and glicina 0.18 M, when SDS is containing 0.1 %. After the race eletroforetica, the gel was immersed in solution of acid
tricloroacetico (TCA) 10 % (v/v) during 30 min for haste of the proteins. Subsequently washed with water, the gel was colored by immersion, by the period of 1 hour in the solution of Coomassie Brilhant Blue R 250 0.5 % (p/v) in solution of methanol/water/acetic acid (4,35:4,65:1, v/v/v) , in which the gel was immersed by 30 min, the room temperature. The excess of coloring was moved by washing in solution of ethanol, water and acetic acid (4,35:4,65:1, v/v/v) up to the appearance of the directions of proteins . The gels were maintained in acetic acid 1,0 %. Zimography
Fibrosarcom humanize cells (HT1080) (2 x 10 5 cells / wells) were cultivated in plates of 24 wells for 48 hours and next cells were treated by inhibitors (BbKI, BbCI, EcTI and aprotinin) , plasminogen and or PMA. In the samples none of inhibitors controls it was added, while plasminogen and PMA were added in the absence of inhibitors, and when were maintained for 48 hours 37°C under tension of 5 % of CO 2 . After this period, the conditioned way of cells, rascalities or not, was collected, centrifuged and subjected to the electrophoresis analysis in gel of 10% of acrylamide containing 1 mg/ml of jelly, according to described by Baramova et al., 1997. After the race, the gel was washed in 2 % of Triton X-100 for 1 hour and next, when Tris/HCl 0.05 M incubated for 16 hours in the activation buffer, pH 7.4 when NaCl 0.2 M, CaCl2 is containing 5 mM and 0.02 % NaN3. The gel was colored by immersion, by the period of 1 hour in the solution of Coomassie Brilhant Blue R 250 0.5 % (p/v) in solution of methanol/ water/ acetic acid (4.35:4.65:1, v/v/v) , in which the gel was immersed by 30 min, the room temperature. The excess of coloring was moved by washing in solution of ethanol, water and acetic acid (4,35:4,65:1, v/v/v) up to the appearance of the directions of proteins .
The gel was maintained in acetic acid 1,0 %.
This test was carried out in collaboration with Dr. Christian Ries of the University Ludwing Maximilians Munich, Germany. Cellular culture
Conditions of cultivation of the cellular strain Tm5 and meIan-a
The cellular strain mice's murine melanocytes (melan-a) and murine melanoma (Tm5) (Correa et al . , 2005) were maintained in environment of culture of cells (RPMI) pH 6,9 made rich with 5 % of serum fetal bovine (SFB) and peniciline/estreptomicine. The strain melan-a needs it of phorbol esther (PMA) for growth. PMA maintains all the tested characteristics of normal melanocytes except the senescence (Bennet et al . , 1987).
Cells were maintained to 37°C under tension of 5 % of C02. The subcultivation was carried out weekly, how described following. For maintenance of the cellular stocks, initially the way was moved of the plate of cells tributaries that were subsequently washed by solution of PBS pH 7.4. Next, cells were incubated by a solution of trypsin (2,5 %) (ImI in each plate of 60 x 10 mM) when the solution is maintained for approximately 1 minute in the plate, necessary time so that cells come off of the plate. Then, they were resuspended, carefully, several times with a Pasteur pipette and
transferred (approximately 1 x 10 5 cells) to a new plate with RPMI mean enriched with 5 % SFB, previously balanced at 37 0 C. The mean was substituted in the next day to subcultive, and for each 3 days. To the melan-a strain was added PMA 200 nM. At the cells observation at microscope, when the confluence was achieved of approximately 80-90 %, the assays were begun.
Cultive conditions to human strain cells
The cellular strain cells of gastric human cancer (MKN28) and cells of gastric human cancer (Hs746T) , cells of cancer of human colon (HT-29) and cells of cancer of human colon (HCT 116) , cells of human breast cancer (SKBR-3) and cells of human breast cancer (MCF-7) , leukemic cells (K562) and monocyte leukemic cells (THP-I) were maintained in environment RPMI pH 7.4 made rich of 7,5 % of serum fetal bovine (SFB) and peniciline/estreptomicine.
The strain HT1080 (fibrosarcom humanize) was maintained in environment DMEM pH 7.4 made rich of 10 % of serum fetal bovine (SFB) and peniciline/estreptomicine. Cells were maintained to 37 0 C under tension of 5 % of C02. The subcultivation was carried out how described in the previous item.
Tests of cellular adhesion
The tests of cellular adhesion were carried out for melan-a and Tm5, through modifications of the method described by Solimene et al . , 2001.
Plates with 96 wells were previously adsorbed with proteins of the extra cellular matrix (fibronectin/vitronectin/IV collagen) when 7.4 diluted in PBS (137 mM NaCl, 2 , 7 mM KCl, 8,1 UiM Na2HPO4 and 1,5 mM KH2PO4) pH, sterile, in the concentration of 5 μg/100 μl/well, when were incubated 37 0 C under tension of 5 % of CO 2 , for two hours or " overnight market " to 4 0 C.
The wells of the plates were blockaded by bovine albumin
(BSA) 1 % when was diluted in sterile PBS (100 μl/well) for 1 hour 37°C. After the incubation, the excess of BSA was discarded and the plates were washed three times with PBS pH 7.4.
Cells were moved of the bottle of cultivation using solution of trypsin (2,5 %) , washed with PBS pH 7.4 sterile, centrifuged and lifted in RPMI pH 6,9, next same were counted in camera of Neubauer. We use the concentration of 5 x 104 celulas/50 μl/well.
Cells and inhibitors (1,00-25,00 μM) diluted in RPMI pH 6,9 sterile (100 μl/well) were preincubated for 15 minutes at room temperature and, next added simultaneously in the plates
and when were incubated 37° C under tension of 5 % of C02, for period of 2 hours .
After the incubation not stuck cells were moved through three washings with PBS, pH 7.4. In the wells 100 % was added methanol for 5 minutes, to fix the stuck cells. Subsequently, the plates were washed three times with PBS, pH 7.4 and when 1 % was colored with blue solution of toluidine in borax 1 % for 5 minutes. The wells were washed four times with PBS, pH 7.4 and the pigment kept by the cell was released by a solution of SDS 1 %. The absorbance is detected in A570 in the Packard spectrophotometer (model SpectraCount) .
The tests were carried out in triplicate for each concentration of inhibitors and the experiments were carried out at least two times. Tests of cellular proliferation
Cells were moved of the bottle of cultivation using solution of trypsin (2,5 %) , washed with PBS pH 7.4 sterile, centrifuged and lifted in RPMI pH 6,9 added of 5 % of SFB, next same were counted in camera of Neubauer. We use the concentration of 5 x 10 3 celulas/100 μl/well.
Inhibitors (1,00-12,50 μM) diluted in RPMI pH 6,9 added of 5 % of SFB and previously filtered in Millipore (0.22 μm) were preincubated for 15 minutes at room temperature was incubated with cells for 15 minutes and added simultaneously
in plates of 96 wells, when were incubated 37 0 C under tension of 5 % of CO 2 for period of 2, 24, 48 and 72 hours.
After each period of incubation there was added 5 μl of bromide of (3 - (dimethyltiazole 2-il 4, 5-dimethyltiazole~2- il) - 2,5-diphenil tetrazolio (MTT) (5 mg/ml) in each well and cells when were incubated for 2 hours 37 0 C under tension of 5 % of CO 2 . After this period the way was moved and added isopropanol and when was maintained for 20 minutes 37 0 C.
Finally, the absorbance was detected in A620 in the Packard spectrophotometer (model SpectraCount) .
The tests were carried out in triplicate for each concentration of inhibitors and the experiments were carried out at least two times.
Tests of cellular metabolism The metabolism of the cellular strain was analyzed when MTS is used, a salt (tetrazolium) which exchange to the color caused it shears bioreduction of the MTS in a soluble form in water called formazan, what accompanies the active enzymes desidrogenases found in the mitochondria and the reaction takes place only in vivo cells.
The tests of cellular metabolism were carried out for Strain Tm5, MKN28, Hs746T, HT-29, HCT116, SKBR-3, MCF-7, K562 and THP-I, through modifications of the method described by Mayer et al. (2001) .
Cells 4 x loV-LOO μl/well (SKBR-3) , 2 x 10 4 /l00 μl/well
(THP-I) and 8 x 10 3 celulas/lOO μl/well (for too many strain used in this study) were incubated to 37° C under tension of 5
% of CO 2 , for period of 24 hours for strain metastatic and 48 hours for strain not - metastatics in appropriate added environment of SFB, to allow the adhesion.
Inhibitors (1,00-25,00 μM) diluted in way adapted for each strain, added of SFB previously filtered in Millipore (0.22 μm) were added to plates with volume of 100 μl, when were incubated 37 0 C under tension of 5 % of CO2, for period of 24 and 48 hours. Additionally, there were carried out tests of cellular metabolism, of combined therapy of the protease inhibitors with 5-FU or carboplatinum, in the same concentrations carried out with the isolated therapy. After each period of incubation there was added 5 μl of
MTS (2 mg/ml; pH 6,5) and 3-mM PMS was dissolved in PBS pH
7.4, separately and sterilized in filter of 0.22 μm. These solutions were stored to-20°C protected of light. 1-metoxi
(PMS) was added to 3- (dimethyltiazole 2-il 4,5- dimethyltiazole-2-il) -5- (3-carboximetoxiphenil) -2- (4- sulfofenil) -2H-tetrazolium] (MTS) immediately before the use (MTS: PMS, 1:20), 20 μl was added in each well and when was incubated 37 °C under tension of 5 % of CO 2 , for period of 2 hours. The absorbance was detected in A490.
The tests were carried out in triplicate for each concentration of inhibitors, the experiments were carried out at least two times and the cellular viability was controlled by coloration of blue of Trypan in the beginning of all the tests, and 0.1 % of non-viable cells was found. Tests of Cellular Migration
Chambers of modified Boyden Transwellό (Costar,
Cambridge, USA) with membranes of polycarbonate containing pores of 8 μm of diameter were moved in his inferior faces with 10 μg of fibronectin / IV collagen, for 2 h to 37°C.
Melan-a or Tm5 cells 2 x 105/100 μl they were pre incubated with 100 μl of rBbCI or rBbKI (1,00-12,50 μM) or EcTI (1,00 μM) in environment RPMI pH 6,9, and finally the inner face of the insert one was filled out by 200 μL of the suspension of cells in the presence of the inhibitor one, when was incubated for 2 hours 37 0 C in wet stove containing 5 % of
CO 2 . Next, the membranes were washed carefully with PBS fixed by 5 min in methanol 100 %, washed with water, and when 1 % was colored with blue solution of toluidine in borax 1 % for 15 minutes.
Cells that remained in the superior face were carefully moved by cotton, remaining you punish that what migrated for inferior face of the membrane . The elution of the coloring was carried out by 1 % (p/v) of SDS in water, when there are
applied 200 μL by camera and incubating for 30 'a 37°C. The absorbance was detected in A570 in the Packard spectrophotometer (model SpectraCoimt) . The controls of the migration unspecific towards 1 % (p/v) of BSA in PBS were deducted. All the experiments were carried out in duplicate.
Tests of Cellular Cycle
Melan-a cells and Tm5 (2 x 10 5 cells/ml) were diluted in RPMI pH 6,9 added of 5 % of SFB and cultivated in plates (60 x 10 mM) for 24 hours. After this period cells were washed by PBS pH 7.4 and maintained in RPMI pH 6,9.
After 24 hours inhibitors (2,50 μM) previously diluted in environment RPMI pH 6,9 were added to plates, they were previously washed with PBS pH 7.4 and incubated for 24 hours . Next the way was transferred to a tube of 15 ml and cells were washed by PBS moved of the bottle, and joined in the same tube in which the way was transferred and centrifuged to 2.000 rpm for 5 min, to 2O 0 C.
The supernatant was despised, to a pellet 5 was added ml of PBS pH 7.4 and again centrifuged. The PBS pH 7.4 was despised, cells resuspended in 0.5 ml of PBS transferred to an eppendorf and 1 ml of ethanol 100 % was added slowly under agitation constant and when were stored 4°C for 16 hours.
After this period the materials were centrifuged to 2.000 rpm for 5 min, to 20 0 C, the ethanol was moved
carefully, cells were washed by 1 ml of PBS pH 7.4 and again centrifuged to 2.000 rpm for 5 minutes.
The PBS pH 7.4 was moved, cells resuspended in 100 μl of PBS pH 7.4 and 100 μl of RNase 100 μg/ml when the room temperature is remaining for 5 minutes.
After this period was added 400μl of propidium iodide 50 μg/ml to 4 0 C for 30 minutes without the presence of the light . Next the samples were analyzed in citomere of flow (FACS; Becton-Dickinson, San Diego, HERE) . Tumorgenesis in vivo
To study the effect of inhibitors in the growth tumoral in I live, mice singenic C57BL/6 were inoculated subcutaneousIy in the left flank with 2 x 10 s cells of melanoma (Tm5)/l00 μl. Inhibitors were injected subcutaneousIy near to the region where cells were inoculated. The animals were treated, in the concentration (2 mg/lOO μl/kg/mice/day) .
The tumors were measured daily with the help of paquimeter. After the tumors reached a volume of approximately 1500 mM3 or after 20 days of experiment, the animals were sacrificed and examined so much regarding the local invasion like spontaneous metastasis in different organs .
The volume was calculated by the formula described following:
Volume = (less diameter) 2 x bigger diameter
2 Affinity chromatography EcTI-Sepharose, rBbCI-Sepharose and rBbKI-Sepharose
Control the tumors of the group that they were excised and homogenized in Tris/HCl 1 tnM pH 8.0 containing 0.02 % Triton X-100 maintained by 20 minutes in bath of ice, next cells were used with sonication for 2 wrists of 30 seconds each, in bath of ice with intervals of 30 seconds. Next, there went chromatographied in column of affinity where inhibitors (rBbCI, rBbKI and EcTI) coupled to the resin Sepharose according to methodology described by Olive, 1988. In the columns (2,0 ml of resin) rBbCI-Sepharose, rBbKI- Sepharose and EcTI-Sepharose previously balanced in buffer Tris/HCl 0.1 M, pH 8.0. The not kept material was collected and the resin was washed by the buffer of balance up to the fall of the absorbance in 280 of the effluent one the values less than 0,03. The resin was subsequently washed by buffer Tris/HCl 0.1 M, pH 8.0, containing NaCl 0.15 M. The kept inhibitor thing was eluted of the column for acidification with KC1/HC1 0.5 M, pH 2,0 were neutralized immediately by the addition of buffer Tris/HCl 1,0 M, pH 8.0. The materials were eluted subsequently with glucose 0.5 M, next with
Tris/HCl 0.1 M pH 8.0 containing urea 1 M. The elution of the proteins was followed by photometric reading in 215 nm. The fractions were eluted with KC1/HC1 0.5 M, pH 2,0 were brought together, liofilized and chromatographied in phase it re-is (system HPLC) . The fractions presented higher absorbance of the chromatography of phase it re-is that they were sequenced, analyzed in database of primary structure HCBI- Blast2 (www.ebi.ac.uk/blastall) and aligned. Statistical treatment of the data Given for curves of survival Kaplan-Meyer, analyzed by Mantel-Haenszel of the experiments in vivo they were carried out that they were determined when GraphPadPrism version 3.03 is used for Windows (GraphPad, San Diego, HERE; www. graphpad.com) . The data of the tests of cellular adhesion, cellular proliferation, cellular migration and cellular metabolism were analyzed using Excell (for Windows XP) , for determination of SD and statistical parameters (paired t test) . RESULTS
Characterization of the inhibitory activity of BbCI, rBbCI, BbKI, rBbKI and EcTI
The inhibitory activities of BbCI, BbKI, rBbCI and rBbKI on the proteases they were confirmed using the methodology described previously, when as al was described by Batista
et., 1996; Olive et al . , 1999; Oliveira et al . , 2001; Araύjo et al., 2005 (II Fixes). EcTI inhibited the HNE and the constant one of apparent inhibition was determined like 55,00 nM. Through the curve of inhibition in which growing concentration inhibitor was used it was possible to define the estequiometria of reaction like of 1:1 it shears
- extrapolaςao linearly for 100 % of inhibition enzymatic (Figure 1) .
II fixes: Determination of θI --nhibition Constants of EcTI, BbCI, rBbCI, BbKI and rBbKI.
Enzyme EcTI BbCI rBbCI BbKI rBbKI (Kiapp (Kiapp (Kiapp (Kiapp (Kiapp nM) nM) nM) nM) nM)
Trypsin 0.88 φ 20.00 28.00
Chimotrypsin 1.11 φ φ 26.00 ND
Plasmina 9.36 φ φ 330.00 ND
HuPK 6.15 φ φ 2.40 2.00
PoPK φ φ φ 200.00 900.00
Catepsina G ND 160.00 ND φ φ Catepsina L ND 0.22 ND φ ND
Enzyme EcTI BbCI rBbCI BbKI rBbKI
(Kiapp (Kiapp (Kiapp (Kiapp (Kiapp πM) roM) nM) nM) nM)
Cruzain ND O .30 O .30 φ φ
Cruzipain ND 1 .30 1 .20 φ φ = it does not inhibit; ND = done not not determined
Zimography
Studies driven by Baramova and collaborators (1997) showed that the addition of plasminogenio and or the combination with PMA induces the activation of pro-one metaloprotease-2 (pro-MMP-2) in cells HT1080 and what, in the presence of plasminogen, takes place a weak activation of the active form of MMP-2 (Mr~62 kDa) . The authors propose that the activation of the pro-one - MMP-2 takes plaθc- -e in two phases. In the first phase, matrix type 1 metaloprotease (MTl-MMP) when the cellular hydrosmooth pro-surface was tied - MMP-2, converting it in the intermediary form of 64 kDa. Next, in the second phase the plasmin mediates the processing in an active form of metaloprotease-2 (MMP-2) of 62 kDa. The EcTI inhibits the generation of the two active forms of pro-one - MMP-2, blockading completely the generation of MMP-2. While the inhibitor aprotinin of serinoprotease, what it inhibits plasmin, is able to interfere only in the action proteolitic of this enzyme under the form of 64 kDa,
blockading the generation of the ripe form of MMP-2 (62-kDa) , while the intermediary active form (64 kDa) is still produced (Figure 2) .
On the other side, the addition of plasminogen in cells HT1080 turns in the activation of pro-one - metaloprotease-9
(pro--MMP-9) in his form of 72 kDa (Ramos-DeSimone, 1999) .
EcTI reduces the activation of pro-one - MMP- 9 induced for plasmin, indicating that EcTI shows a straight influence on the activation of pro-one - MMP-9 because of inhibitor the activity of plasmin, since II was characterized and described previously in the Table. Aprotinin used like control inhibits clearly the activation of pro-one - MMP-9. The results demonstrate that EcTI regulates the activity of MMP-2 and
MMP-9 in the strain HT1080 ' and his effect is not owed only to the inhibitory activity of plasmin, but also to the blockade of the first phase of the activation of pro-one - MMP-2 mediated for MTl-MMP through a process independent of the action plasmin (Figure 2) .
Action of the inhibitor BbCI, rBbCI, BbKI, rBbKI and EcTI in the strain Tm5 and melan-a
Action of inhibitors in the cellular adhesion The results of the action of inhibitors in the cellular adhesion of the strain melan-a and the TM5 to fibronectin, vitronectin and IV collagen showed that the BbCI and his form recombinant (1,00-25,00 μM) showed similar activity and did
not interfere significantly in the adhesion of melan-a and TM5 on fibronectin, vitronectin and IV collagen (It imagines 3 and 4, respectively). BbKI and rBbKI (1,00-25,00 μM) also they did not inhibit the adhesion of the significant form of • this strain on the used glicoproteins (Figure 5 and 6, respectively) .
On the other side, EcTI inhibited the adhesion of melan- a and Tm5 on IV collagen, vitronectin and fibronectin. The results showed action dependent dose caused by EcTI, and inhibitor East was more efficient in the inhibition of the adhesion of Tm5 on the proteins of used MEC (Figure 7) . Action of inhibitors in the cellular proliferation The cellular proliferation is one of the principal factors for tumor development. Therefore, the action of BbCI, rBbCI, BbKI, rBbKI and EcTI (1,00-12,50 μM) were analyzed in the cellular proliferation of melan-a and Tm5, in different incubation times (2h, 24h, 48h and 72h) , according to described previously.
The results demonstrate that BbCI and rBbCI when incubated by 24 h-72 h they interfere in the proliferation of melan-a (the very inhibition of @ 30 %) . After 24 hours the inhibition was more efficient for transformed strain, Tm5 (the very activity of @ 50 %) (Figures 8 and 9, respectively) .
Analyzing the effect of BbKI and rBbKI in the proliferation of melan-a and TM5 there happened an inhibitory dependent effect of the time of incubation and of the concentration, being more efficient on TM5, mainly after 24 and 48 hours of incubation (Figures 10 and 11, respectively) .
EcTI inhibited the proliferation of two cellular strain of form dose and dependent time, reaching 80-900 % of inhibition when used in the concentrations of 6,25 and 12,50 μM, after 48 hours of incubation (Figure 12) . Action of inhibitors in the metabolism of Tm5 and comparison with 5-FU
The cellular metabolism of Tm5 was analyzed after treatment with the inhibitor rBbCI, rBbKI and EcTI (1,00-
12,50 μM) , and compared with the chemotherapic drugs 5-FU, widely administered clinically for treatments of different types of tumors
The rBbCI inhibited in the form dependent dose being more efficient than rBbKI, and both more efficient than 5-FU (Figure 13) . The rBbKI (12,50 μM, 24 hours) inhibited approximately 30 % the metabolism of Tm5. And after 48 hours rBbKI caused decrease of the inhibitory activity (Figure 13) .
The EcTI inhibited in the form dependent dose, in 24 and 48 hours, EcTI still demonstrated to be more efficient than 5FU in concentrations above 6,25 μM (Figure 13) .
The chemotherapic drugs 5-FU inhibits the cellular proliferation independent of the concentration and of the time.
Action of inhibitors in cellular cycle steps Cellular cycle steps of melan-a and Tm5 were analyzed by them after treatment with the inhibitor rBbCI, rBbKI and EcTI
The rBbCI caused significant increase in the number of melan-a cells in the phase of fragmentation of the DNA
(Figure 14b) , when cells that did not receive treatment (Figurel4) were compared. And the rBbCI does not interfere in the significant form in steps of the cycle of Tm5 (Figure
15b) , when Tm5 cells that did not receive treatment (15th
Figure) were compared.
The rBbKI caused significant increase in the percentage of cells in the phase G0-> Gl of both studied strain (Figure 14c and 15c) .
The treatment with EcTI caused increase in the number of cells in phase Ml, corresponding the fragmentation of the DNA, when compared to the control (Figure 14d and 15d) . Action of inhibitors in the cellular migration
According to showed in the Figure 16, Tm5 it migrated with speed superior to melan-a on fibronectin, in all the incubation times. Between 1 and 2 hours of incubation, the numbers of cells Tm5 migrated doubled, and between 2 and 3 hours it did not take place significant differences. While,
for melan - there her took place a gradual increase of the number of migrated cells. Therefore, the interference of inhibitors on the migration of the strain melan-a and TM5 using fibronectin, as chemoatractive, they were valued after two hours of incubation.
The migration of melan-a and Tm5, treated with rBbCI was inhibited in the form dependent-dose. And in the concentrations of 6,25 and 12,50 μM it was twice more efficient for Tm5 (Figure 17) . The treatment of rBbKI (1,00 and 2,50 μM) in Tm5 caused increase of the number of cells migrated in relation cells that did not receive inhibitor any, and only in the concentrations of 6,25 and 12,50 μM inhibition of the migration happened. , the effect rBbKI in melan-a was still similar in all the concentrations (Figure 18) .
The EcTI (1,00 μM) did not interfere significantly in the cellular migration (Figure 19) . In all the tests, BSA was used like control, for analysis of the migration of the strain Tm5 and melan-a in a unspecific form. Tumorgenesis in vivo.
The effect of inhibitors at the growth of tumors in vivo was valued according to described previously. The volume tumoral was measured daily with the help of a paquimeter, and the results demonstrated that the BbCI and the rBbCI delayed the growth of the tumor (Figure 20) . The EcTI delayed the
growth of the tumor of form dependent-dose, (Figure 22) . On the other side, BbKI and rBbKI (Figure 21) they stimulated tumor development.
Analysis of the proteins from tumors and when they took notice of the resin inhibitor-Sepharose
Affinity chromatography inhibitor-Sepharose and chromatography in phase it re-is
The tumor homogenized was submitted to affinity chromatography rBbCI-Sepharose ou rBbKI-Sepharose ou EcTI- Sepharose, as described above. The materials retained to the support and eluted with a solution of KC1/HC1 0.5 M, pH 2,0 were submitted to chromatographic analysis in reverse phase
(Figures 23-25) .
Determination of amino-terminal region The material sequences of amino-terminal region obtained with reverse phase chromatography were determined by the automatized process of Edman's degradation reaction (1956).
The sequence of the amino-terminal region of the obtained material of the affinity rBbCI-Sepharose and eluted with 38 minutes it demonstrated similarity with isolated regulating proteins of mice of the signalling of the protein G. And the material eluted with 40 minutes demonstrated similarity with proteins associated to metastasis (Figures 26 and 27, respectively) .
The sequence of the obtained material of the affinity chromatography rBbKI-Sepharose, . and subsequently eluted with 35 minutes it demonstrated similarity with P-selectin and with her subunit-α of the factor 3 induced shears hipoxia (Figure 28) . The amino-terminal region of the material eluted with 37 minutes showed similarity with receiver containing power type imunoglobulin (Figure 29) .
The amino-terminal region of the material eluted with 31 minutes, referent to the fraction tied in the affinity chromatography EcTI-Sepharose, demonstrated similarity with inositol 1,4, 5-trifosfato kinase (Figure 30). And the material eluted with 35 minutes type - lectina showed similarity with receivers of natural cell " killer " and with protein associated with neoplasic progression (Figure 31) . Studies of the metabolism in tumorogenic human cells treated with rBbCI, rBbKI and EcTI
The effects of the inhibitor rBbCI, rBbKI and EcTI in the cellular metabolism was compared with the chemotherapic drugs 5-FU. The cellular strain been derived from different types of tumor was: MKN28, Hs746T, HT-29, HCT 116, MCF-7, SKBR-3, K562 and THP-I. The results demonstrated what the effect of 5-FU does not modify with the increase of the concentration.
The effect of rBbCI (24, 48 hours), in the strain MKN28 (gastric cancer - less invasive strain) it inhibited the
metabolism of a form dose and dependent time, being more efficient than 5-FU, in concentrations superior to 6,25 μM. The inhibitor rBbKI in 24 hours inhibited in the form dependent-dose. After 48 hours and in the concentration of 12,50 μM 20 superior % to inhibition showed effect caused for 5-FU. The EcTI (6,25 μM and 12,5 μM) was approximately a 50 more efficient % than it 5-FU in two incubation times (Figure 32) .
In the strain Hs746T (gastric cancer - more invasive strain) rBbCI and rBbKI, in concentrations superior to 6,25 μM they showed similar effect to an obtained one in the strain of gastric cancer - the least invasive strain. Meantime, they were more efficient in 24 hours of incubation and with 12,5 μM. The EcTI (6,25-12,50 μM, 24 hours) inhibited above 60 %, while 5-FU it inhibited 15 %. After 48 hours EcTI caused inhibition of the metabolism above 80 %, t while 5-FU it inhibited only 25 % (Figure 33) .
In the strain HT-29 (cancer of colon - less invasive strain) we notice that the inhibitor rBbCI and rBbKI (6,25 and 12,50 μM, 24 hours) were more efficient than 5-FU. After 48 hours with 12,50 μM were approximately a 20 % more efficient than the chemotherapic drugs. EcTI (6,25 and 12,50 μM) was more efficient than 5-FU presenting action it dependent-doses (Figure 34) .
In contrast, in the strain of cancer of colon - strain the least invasive HCT 116, the inhibitor rBbCI and rBbKI
(1,00-6,25 μM, 24 and 48 hours) are less efficient than 5-FU.
Meantime, EcTI (6,25 and 12,50 μM, 24 hours) presents superior effect to of 5-FU (It imagines 35) .
In the strain of breast cancer MCF-7 (less invasive strain) and SKBR-3 (more invasive strain) , the inhibitor rBbCI and rBbKI (6,25 and 12,50 μM, 24 hours) it were more efficient than 5-FU and, after 48 hours with 12,50 μM was observed, what both were more efficient than 5-FU. EcTI (24 hours) was more efficient than 5-FU in all the concentrations used in the strain of breast cancer. , after 48 hours EcTI was still approximately twice more efficient than 5-FU when used with 12,50 μM (Figures 36 and 37) . In the strain K562, rBbCI (12,50 μM, 24 hours) it inhibited approximately 50 % while 5-FU you punish 30 %. On the other side, rBbKI was not efficient. The result demonstrated that EcTI (2,5-12,50 μM, 24 hours) is more efficient than 5-FU (Figure 38) . The rBbCI (6,25 and 12,50 μM, 24 hours) caused inhibition in the metabolism of THP-I and was more efficient than it 5-FU approximately 2 times. On the other side, rBbKI was not effective for this strain. The EcTI was more efficient than 5-FU in all the conditions analyzed in the concentrations of 6,25 and 12,50 μM, (Figure 39).
Studies of the cellular metabolism using combined therapy: EcTI with 5-FU and EcTI with carboplatinum.
The combined therapy is used to increase the activity of anti-neoplasic agents, as well as, to minimize his toxicological effects. Therefore, we analyze the effect of the combined therapy of EcTI with chemotherapic agents what act on the DNA.
The metabolism of the strain of gastric cancer MKN28 and Hs746T used previously in the individual therapy, was analyzed by EcTI (1,00-6,25 μM) combined with 5-FU or with carboplatinum, both in the same concentrations of EcTI.
The combined therapy (5-FU and EcTI) showed himself more efficient for inhibition of the metabolism of MKN28, so took place a significant decrease in the necessary concentration to cause the same effect regarding individual therapy. This effect is observed more well in 48 hours with 1,00 μM (Figure 45) .
In the strain Hs746T (gastric cancer - more invasive strain) the positive effect of the therapy combines that it was observed after 48 hours and in the concentration of 2,50 μM, doubling the effect caused by the individual therapy
(Figure 41) .
The combined therapy of carboplatinum with EcTI showed efficiency on the individual therapy in MKN28, when one used low concentrations (1,00 μM and 2,50 μM) , in 24 hours (It
imagines 42) . Similar effect was obtained by the invasive strain, Hs746T, of gastric cancer (Figure 43) . EFFECT OF BbCI IN MODELS TUMORAIS
In the first stage, in models of the dissemination of the tumor, we use inhibitors in the strain of mice melanocytes, when was called melan-a and of melanoma Tm5, when it was derived from the strain melan-a through the forced impediment of the adhesion cell-substrate (Correa et al., 2005) Studies effectuated in melanoma showed a straight correlation between alteration in molecules of cellular adhesion, like the integrin αvβ3 and the tumoral progression. , the presence of this integrin that was not detected in normal melanocytes, was still connected to the least over life of the patients with tumor (Edward, 1995; Natali et al . , 1997; Ebrahimnejad et al . , 2004). Therefore we use inhibitors in order that it checks his action in the process of adhesion of the strain melan-a and Tm5. BbCI and rBbCI inhibitor with specificity for HNE, did not interfere significantly in the adhesion of the strain of mice melanocytes (melan-a) and melanoma (Tm5) to IV collagen, fibronectin and vitronectin (Figures 3 and 4, respectively)
Additionally, Champagne and collaborators (1998) demonstrated that HNE cliva to intercellular connection of ICAM-I, promoting the migration of neutrophils for sieges of
adhesion. The action of BbCI and rBbCI on elastase can have interfered in the action hydrolytic of this enzyme on ICAM-I interfering in the process of migration.
Besides the inhibitory activity BbCI and rBbCI might be if if they took notice of the family of molecules of adhesion definite in the strain melan-a and Tm5, if they not contributed with the mobility of Tm5. Nevertheless , no structural similarity was observed by molecules of adhesion that were measuring this process, between the extracted proteins of the tumors kept in the resin of affinity (rBbCI- Sepharose) (Figures 26 and 27) .
The results of the proliferation of the analyzed strain demonstrated that as many BbCI as rBbCI caused a more efficient inhibition from 24 hours in the proliferation of the strain of melanoma (Tm5) that in the strain not tumorogenic of melanocytes (melan-a) . The presence of a protease can induce a mistake in the system of repair to the DNA through the hydrolysis of repressing proteins, turning in the cellular transformation (Borek et al . , 1977). Studies with defective mice in elastase induced with UVB demonstrate that these animals are more resistant to the development of tumors from skin (Starcher et al., 1996). So, the inhibition of the proliferation of Tm5 caused by BbCI, his activity can be made a list of HNE inactivation and catepsina G, proteases connected with the progression of the tumor.
As well as in the proliferation, the rBbCI inhibits the cellular metabolism of Tm5, fact observed through the activity of mitochondrial enzymes so-called deshydrogenases
(Figure 13) . In spite of two tests to use markers of mitochondrial enzymes, in the test of proliferation inhibitors were added simultaneously to cells on the plate, whereas in the test of metabolism cells were stuck to the plate and after 24 hours the added inhibitor ones. The obtained results demonstrate that inhibitor did not interfere in the adhesion of the cells to the plastic.
The rBbCI causes increase of the number of melan-a cells in the phase of fragmentation of DNA, this fact not observed in cells Tm5 in the analyzed conditions. Sun and Yang (2004) suggest what the imbalance between elastase and αl- antitrypsin the carcinogenesis can promote. The tall concentration of HNE can turn in the inhibition of the apoptosis of tumor cells through his contribution in the degradation of the MEC. In spite of the low interference when was watched in melan-a the results obtained on Tm5 can be explained as a result of the insufficient amount inhibitor
(6,25 μM) , used to provoke the same effect when was caused in melan-a. As soon as, in tumor cells the imbalance of proteases-inhibitor ones takes place and since Tm5 is a strain of melanoma, it can need a bigger concentration of the
inhibitor one to produce the effect caused in the strain not - tumorogenic of melanocytes
On the other side, Porteu and collaborators (1991) demonstrated what elastase is able to move fragment of the receiver 2 of TNF (factor of tumoral necrosis) of the cellular surface, reducing his affinity for receiver, so, elastase can be interfering in the answer of TNF, preventing from practicing his function. Still, van Kessel and collaborators (1991) showed that catepsina G inhibits the activity of TNF-α . Besides, the secreted proteases for neutrophils what are recruited in the inflammation, participate in tumor development (Iwatsuki et al . , 2000). BbCI inhibitor activity of the elastase and of the catepsin G, can prevent these proteases from interfering in the apoptotic process through TNF, that this one involved directly with the road of signaling of apoptosis.
The migration of the strain Tm5 of melanoma of mice (when the mice's melanocytes) was derived of melan-a it demonstrated to be much quicker on fibronectin, in comparison to the not transformed strain melan-a. The inhibition of the migration of Tm5 caused for rBbCI was twice more efficient when was when compared with the inhibition of the migration of melan-a. In the process of cellular migration elastase connected with membrane is mainly located in the direction of the migration and can make easy the passage trans endothelial
of the leucocytes making easy tumor development (Cepinskas et al. , 1999) .
Besides the elastase, studies carried out with the strain B16 (murine melanoma) in test of invasion in vitro, showed inhibitor synthetic what of catepsin B and L caused a decrease of the invasion (Sever et al . , 2002). The inhibitory activity of BbCI is left catepsines L, G and of elastase, it indicates that this can be the road of inhibition of the migration of Tm5. In contrast with the results obtained with BbKI and rBbKI, the studies with model of tumorogenesis in mice treated with BbCI and his form recombinant demonstrated a retardation in tumor development when compared to the group that received PBS (It imagines 20) . Yamashita and collaborators (1996, 1997) demonstrated that the concentration of elastase is connected with the progression of the tumor, being which high concentrations of elastase are present in patients with bigger tumors . These data suggest probably that the located production of HNE is connected with the invasion tumoral.
The superexpression of the pro-one - catepsin L in cells of melanoma increased his potential of invasion and turned the phenotype of the least invasive strain into more invasive strain (Friar et al., 1998). So, BbCI can be delaying the progression of the tumor through the inactivation of
proteases involved with the acquisition of an invasive phenotype .
A possible paper of BbCI in tumor development, is still through the indirect inactivation of other proteases, as soon as elastase can activate other proteases, like components of the uroquinase type pasminogen activator system (caprioles)
(Schmitt et al . , 1991), MMP-2 through MTl-MMP and metaloprotease-3 (MMP-3) (Okada et al., 1989; Shamamian et al . , 2001), as well as catepsin L can activate the simple chain of pro-one - uroquinase type plasminogen activator (Pro-It Pro-caprioles) (Goretzki et al . , 1992).
In tumors, chimioatractive citocines are responsible for recruitment of leucocytes, which answer the organization and abolition of cellular answer for reorganization of the cloth. With the accumulation of changes in the process tumorogenic the imbalance of this organization takes place inducing aberrant proliferation, adhesion and cellular migration being able to promote a malignant behavior (Di Carlo, 2001; Cerwenka and Lanier, 2001; Coussens and Werb, 2002; Lin and Pollard, 2004) .
Data obtained with work developed in model of pleurisy in mice induced for carragenin, show that there takes place a reduction of 50 % of CINC-I (IL-8 in human) in the serum and in the exudate of the animals treated with BbCI, when when control (of Oliveira, 2004) was compared to the group.
Varney and collaborators (2003) demonstrated what the strain of melanoma, melanoma humanize (A375SM) , highly metastatic, it expresses elevated levels of mRNA and of the proteins, CXCRl and CXCR2 (receivers of chimocinas, like the IL-8) when compared with the strain not - metastatic of melanoma humanize SBC-2. Besides this, the treatment with IL- 8 recombinant increased the potential of proliferation and invasion of these cells. Antibodies special for these receivers inhibited the potential of proliferation and invasion of cells of melanoma I humanize A375P, when they were stimulated or not with IL-8. Besides, works show that IL-8 can contribute like a factor of growth autocrine to tumor cells (Schadendorf et al . , 1993). Therefore, BbCI can reduce the invasive potential and the proliferation of Tm5 through the decrease of the IL-8.
Another explanation of the effects of observed BbCI can be made a list his action on receivers transmembranic PARs, activated directly shears clivage of the amino-terminal region for serino-protease (Loew et al . , 2000; Sambrano et al., 2000; Cumashi et al . , 2001; Macfarlane et al . , 2001). There are known four members of this family (PAR-I the PAR- 4) , which they announce of the plaquetary aggregation, stimulus of inflammatory cells, cellular proliferation and mobility (revised by Dery et al., 1998) .
The protein eluted with 38 minutes of the chromatography of -reverse phase showed similarity with isolated regulating proteins of mice of the signaling of the protein G. (It imagines 27) (Sierra et al . , 2002). Therefore, besides the inhibitory effect on proteases, rBbCI can be interfering in the signaling through the interaction with this protein.
Still, the amino-terminal region of the material eluted with 40 minutes, he presents similarity with proteins associated to the metastasis, called (Mta) , the family of the genes of these proteins are called (MTA) (Kumar et al., 2003) . The expression of these genes is being associated by the invasive potential in cellular human strain and in tissues by tumor (Toh et al., 1997). Consequently, while interacting with this protein, inhibitor can be inhibitor the potential of invasion in vivo.
Effect of BbKI in models tumorais
Components of the system calicreins (plasmatica and/or tecidual) / cininas - KKS can be directly involved by tumors through the increase in the expression and of the deregulation of the activity proteolitic detected in tumor in the pancreas (Yousef et al., 2004), prostate (Rittenhouse et al., 1998), it suckles (Luo et al . , 2002), ovary (Obiezu et al., 2001), between others, or indirectly for generation of cininas and/or activation of other proteases (revised by Bhoola, 1992; Shariat-Madar et al, 2002) .
Meantime, the treatment of cells with the BbKI and the rBbKI did not alter the process of adhesion of melan-a and Tm5 to the glicoproteins: IV collagen, fibronectin and vitronectin (You imagine 4 and 5) BbKI and his form recombinant showed itself quite efficient in the inhibition of the proliferation of the cellular strain Tm5 regarding the strain of melanocytes, which can be explained by the effect of BbKI on the plasmatic and tissue's calicreins. When were turned out to be done not not showed they showed bigger concentration of bradicinine in the middle of culture of Tm5 regarding melan-a, indicating
• important paper of enzymes cinino liberator in this strain.
The superexpression of calicreins in strain tumorogenics is already reported by different groups of inquiries (Denmeade et al., 2001; Yousef et al . , 2004). The tissue calicrein, hK3
(PSA) , for example, is one of the newspaper commentaries human calicreins used like marker tumoral for diagnosis, prediction and monitoring of the cancer of the prostate
(Rittenhouse et al . , 1998; Diamandis and Yousef, 2002; Gray, 2005; Ryan and Small, 2005) .
The results of inhibitors in the proliferation were confirmed using the model of cellular metabolism (It imagines 13) and, though interference has taken place in the cellular metabolism the showed effect was not better to the obtained one with the chemotherapic drugs 5-FU. The differences showed
for rBbKI when employed in low concentrations (It appears 13b, 48 hours) can be explained by the methodologies employed in two experiments . The prior addition of cells to the plate modified the efficiency of inhibition, which can be explained by the interference of inhibitors in the adhesion of cells to the plastic. Therefore, a more elevated concentration would be necessary to interfere in the process of metabolism, if it indicated that the functionality of the inhibitor one is better when this one is incubated previously with cells before same they were added to the plate.
The analysis of the effect in the cellular cycle shows that rBbKI cause increase of the number of melan-a and Tm5 cells in steps GO ® G1 (You appear 14c and 15c, respectively) , regarding the control in the absence of the inhibitor one in the used conditions . The phase GO is the period in which the cell maintains his metabolic tax, but do not grow in size, unless it receives extra cellular signs and the phase Gl corresponds to phase of cells metabolically active with continuous growth (Jogging, 1996) . The results of the rBbKI in the cellular migration were surprising, as soon as due to his activity anti-proteolitic, it was of a reduction was waited in the invasive process. As soon as, since we mention, the proteases have paper of distinction, active process of the migration of neoplasic cells through the physical barrier formed by the MEC. The
proteases secretadas make easy the degradation of this barrier (Liotta and Clair, 2000; Nguyen, 2004) . However, in low concentrations, we observe a significant increase of the migration of cells of melanoma, fact not observed in the not transformed cells. On the other side, inhibition of the migration takes place when he is used in high concentrations, probably due to his effect on the proteases. Wolf and collaborators (2001) demonstrated the inhibition of the invasion of adenocarcinomas on the matrigel (mimetiza the MEC in vitro) for inhibitor synthetic of tissue's calicreins, indicating the participation of the calicreins in the invasion of the tumor.
The tissue's calicreins participate of the invasion through the activation of metalo-proteases like the MMP-2 and to metaloprotease-9 (MMP- 9) (Tschesche et al . , 1989; Desrivieres et al., 1993). We emphasize that, in 1997, Frenette and collaborators demonstrated what the calicrein hK2 activates whoops (ativador of plasminogenio type urokinase) , unleashing the activation of plasmin. So, since rBbKI inhibits the activity of the calicreins and of plasmin, consequently, inactive the participation of these enzymes in this process.
In contrast, the increase of the migration of Tm5 for action of rBbKI in less concentrations can have taken place due to insufficient amount inhibitor for inactivate the
calicreins definite in this strain, turning in an imbalance proteolitic or the possible activation of other proteases involved with the cellular migration induced for calicreins.
In model experimental vivo in, BbKI caused a significant increase of the volume tumoral in the group BbKI, being that some animals went to the death before the group control (not showed data) . Shin and collaborators (1996) demonstrated the SbTI (trypsin inhibitor of soy) whpresentlys a low middle- life due to action of proteases on his degradation. However, only the processing of the inhibitor one does not allow us to explain the effect of the increase tumoral .
As mentioned previously, the reactive siege of BbKI presents similarity with the bradicinina (Cagliari et al . , 2003) . Works developed by the present inventors (Santomauro- Vaz, 2005;) they demonstrated Andrade, personal communication what peptides synthetic with the sequence of the reactive siege act in the similar form to the bradicinina (BK) in models of muscular contraction, liberation of calcium and of NW. The BK is involved in processes of tumorogenesis when the angiogenesis (Bhoola et al was associated. , 2001) . The growth of the tumor is a dependant of the angiogenesis, being that in his absence the growth of the tumor is limited to a microscopic size and tumor cells do not go out from the circulation (Folkman, 2003) . BK presents angiogenic activity
for connection to the receivers Bl and B2 in endothelial cells. The antagonists' use of these receivers showed that the blockade of this connection reduces the vascular permeability, activation of the nitric oxide, growth and progression of the cancer (Hu and Fan, 1993; Maeda et al., 1999; Hayashi et al . , 2001).
Results obtained by Santomauro-Vaz (2005) , in model of isquemia and reperfusion in mice treated with BbKI, demonstrate clearly an increase of ONOO-, indicating that the protein also is tied to a receiver. Free roots, like NW can be induced by the connection of the BK to a receiver, for citocinas and growth factors (Knowles and Moncada, 1994; Witte and Barbul, 2002) . The NW reacts quickly with superoxides forming ONOO - (revised by Bongdan, 2001) . Okamoto and collaborators (1997) demonstrated that ONOO - pro-pro-active service - MMPs involved in the degradation of the MEC (Maeda et al . , 1999). Therefore, BbKI can be presenting pro-activity - angiogenic for property of connection to a receiver of BK and stimulate the production of NW.
The receiver B2 has been detected in different tissues and in tumorals strain of mice (Wu et al., 2002), being that migration does not take place of neutrophils for sieges of tissues inflamed in defective mice of the receiver Bl, which turns in the inhibition of the development of tumors after
CO-inoculation from apoptotic cells and cells Tm5 (Correa et al . , 2005). The results obtained with BbKI open perspectives to investigate the action of BbKI and the effects of peptides derived from BbKI, with sequence similar to BK on the receivers Bl and B2, in the models of progression of the vivo melanoma in.
Still, the works developed for of Oliveira (2004) , showed that the serum of animals treated with BbKI they present increase of the expression of IL-lα. Vidal-Vanaclocha and collaborators (2000) , demonstrated which defective mice in IL-lβ are more resistant to the development of metastasises. Additionally, IL-lβ increase the connection of linfόcitos and monόcitos to cells endoteliais, induce the neovascularizagao and it stimulates production of TNF-α (revised for Add et al . , 2003). The participation of TNF-α can take place through the induction of the production of proteases important for invasion of tumor cells and macrόfagos, like HNE, and can provoke an increase in the expression of molecules of adhesion in cells endoteliais (Jonjic et al., 1992; Balkwill and Mantovani, 2001). So, BbKI can be causing an increase of IL-lβ and TNF-α in the model used in accordance with the present invention, inducing the progression tumoral.
In the model studied for of Oliveira (2004) also one checked that BbKI stimulates the production of CINC-I in mice
(it ratifies to the IL-8 in human) . Studies demonstrate that IL-8 promotes the growth of melanomas (Haghnegahdar et al . , 2000) , it is a powerful angiogenic factor regulated for TNF-α and secreted for macrophages or monocytes when they were permeated the estroma (Torisu et al . , 2000). So, BbKI can increase the growth of the tumor through the activation of this citocina.
The material eluted with 35 minutes that he showed affinity the rBbKI-Sepharose demonstrated similarity with selectina of plaques (P-selectina) . P-selectina is a molecule of cellular dependent adhesion of Ca2 that mediates the interaction of neutrόfilos to plaques as well as to cells endoteliais (Buck, 1995) .
Mayer and collaborators (1998) demonstrated which samples of pots of patients with gastric cancer express high levels of P-selectinas when in the presence of it was permeated of leucocytes. Still, some studies demonstrate the high expression of P-selectinas in the plasma of patients with cancer (Blann et al . , 2001) . Meantime, Caine and, after they administered drugs anti- angiogenicas I eat collaborators (2005) marismatati and captopril, they demonstrated that these drugs did not modify the levels of P-selectina in the blood. Additionally it was demonstrated what in the absence of P-selectina, exist other mechanisms involved in the roller-bearing of leucocytes,
during the inflamMation (Ridger et al . , 2005) . Therefor, rBbKI can be recognizing P-selectinas, blockading the roller- bearing of neutrόfilos and, consequently interfering in the inflamMation. The same sequence of the amino-terminal region there presents similarity with the subunity a of the factor 3 induced by the hipoxia (It imagines 28) , what can contribute to the process angiogenico (revised by Browder et al., 2000; Raghunand et al . , 2003) . Therefor, while recognizing this factor BbKI can amplify the local action of pro-factors - angiogenicos .
We can still emphasize that the material eluted with 37 minutes showed similarity with the receiver containing power type imunoglobulin, family of molecules of cellular adhesion that have several functions (revised by Buck, 1995) . BbKI might be inhibitor the interaction of some member of this family with his ligant, if it turned in the dysfunction of processes anti-tumorals .
Effect of EcTI in models tumorals. The cellular adhesion of melan-a and Tm5 on IV collagen, fibronectin and vitronectin dose was inhibited by EcTI of form and dependent time. A possible action of EcTI would be of recognition to the receivers of the cellular membrane, and therefore to obstruct the adhesion. Integrins recognizes several structural proteins, but they act also like receivers
of proteases, like the metalo-proteases (MMPs) , the activity of MMP-2 was demonstrated to be a dependant of the interaction with avb3 (Hofmann et al., 2000). The interference in molecules involved in the cellular adhesion can be a person in charge in inhibition of the activation of MMP-2 for EcTI, as soon as in cells of melanoma, the expression of the MMP-2 can be regulated through the interaction of avb3 to the vitronectin (Albelda et al . , 1990) and for interaction homotipica when it mediated it it shears imunoglobulina MCAM (molecule of cellular adhesion of melanoma) MMP-2 (Xie et al . , 1997)
The action of EcTI in the decrease of the cellular growth was confirmed by tests of cellular proliferation and cellular metabolism, demonstrating to be more efficient than the chemotherapic drugs 5-FU in high concentrations. The swinging between cellular proliferation and cellular death controls the growth of the tumor, being that the increase of the proliferation can be a determinative one for malice (Reed, 1999) . EcTI interferes in this process causing a decrease in the cellular proliferation.
The results showed that EcTI induces increase of great percentage of fragmentation of the DNA of melan-a and Tm5. Several works demonstrate that the anchorage to MEC is crucial for progression of the cellular cycle between steps G1 ® S, phase of cells metabolically active in continuous
growth, and the phase of synthesis of DNA, respectively (Jogging, 1996; Lukashev and Web, 1998) .
A variety of events prune direct the cellular death between them the cellular adhesion (Vaux and Korsmeyer, 1999) . Integrins has important paper in the process of anoikis, in other words, cellular death provoked through the loss of anchorage (Parise et al . , 2000)., while using agents citotoxics, Hoyt and collaborators (1996) demonstrated that the adhesion of cells to the MEC and the activation of integrins abolish the cellular death induced by the citotoxidade . Additionally the desadesao for long period turns in the activation of caspases 8 and 9 and consequently active the caspase efetora 3, inducing to apoptosis (Bonfoco et al., 2000). Therefor, EcTI can induce the apoptosis for process of anoikis, obstructing the adhesion of cells and consequently provoking the cellular death, as soon as it causes decrease of the number of cells of steps G1 ® S and I increase in the phase of fragmentation.
Besides, the induction of the cellular death caused by EcTI can be also resulting from his effect on the plasmina and metalo-proteases, as soon as, recent studies demonstrated which resistant cells to the cellular death regulated for p53, factor of transcription that regulates the cellular cycle, repair of the DNA and PKC, express high levels of MMP- 9, MMP-10 and metaloprotease-12 (MMP-12) (Meyer et al . ,
2005) . The importance of metalo-proteases in melanoma is confirmed by the results of Toschi and collaborators (2000) superexpresaram the wild gene of p53 in cells of melanoma, they showed a decrease of the invasion. The authors make a list of this effect at the levels of MMP-2 secretado for cells .
In the experiments in living creature EcTI caused decrease of the volume tumoral being more efficient than the BbCI, over there, of the overlife to be superior to the animals that received PBS (not showed data) . Probably this decrease took place in reply to the inhibition of the proliferation of Tm5.
In cellular strain of melanoma, they have been correlated the production of MMP-2 and MMP-9 by the invasive potential (Vaisanen et al . , 1996). Besides, inhibitor of metaloprotease type 2 (TIMP-2) and inhibitor synthetic they reduced the growth and metastasis of melanoma (Montgomery et al., 1994). Consequently, the reduction of the volume tumoral caused by EcTI can be resulting from his activity anti- proteolϊtica.
Gelatinase 2 can affect the growth of the tumor through the clivagem of ICAM-I for MMP-9, increasing the resistance of the tumor cell to agents citotόxicos (Fiore et al., 2002). So, while inhibitor the activation of pro-one - MMP-9, EcTI
can obstruct the hydrolysis of ICAM-I, reducing the resistance of the tumor cells.
Another explanation is based on the action of EcTI on the cellular death. Many tumors resist apoptosis because of increasing the tax of efetores anti-apoptόticos regarding the pro-ones - apoptόticos (Evan and Vousden, 2001) . Plasmina can activate NF-kB in monόcitos (Syrovets et al., 2001), NF-kB is a pro-efetor - apoptόtico, i.e. it induces the expression of inhibitor proteins of the apoptosis, which are tied and inhibited certainties caspases, when was known as proteins that promote the cellular survival (Ghosh and Karin, 2002) . Through the inativaςao of plasmina, EcTI of an indirect form can inhibit the expression of NF-kB and of caspases, consequently promoting the apoptosis. The cellular proliferation is maintained in a rich environment in inflamMatory cells, growth factors, estroma activate, agents who promote damages to the DNA, potencializando and/or promoting the risks neoplasicos
(revised by Coussens and Werb, 2002) . Plasmina and gelatinases can activate and/or degrade mediators of the inflammation who stimulate tumor development (revised by Syrovets and Simmet, 2004; Bjδrkund and Koivunen, 2005). Therefore, thinking that EcTI inhibits these proteases, a paper is possible on these mediators.
The results obtained with the affinity chromatography EcTI-Sepharose and analysis in database were demonstrated by it what the material eluted with 31 minutes, similarity has 1 with inositol 1, 4, 5-trifosfato kinase (IP3) , it is released in the citosol and it signals the liberation of Ca2 essentially for function of several physiologic functions (Jogging, 1996) . So being, EcTI can be involved in the liberation of Ca2.
And the material eluted in 35 minutes native showed similarity with receivers of cells "killer" type - lectin (Smyth et al., 2002) and with proteins involved in progression neoplasic 1 (Sun et al . , 1994) (It imagines 31). So, EcTI can be inducing the decrease of the growth tumoral through the recognition of proteins that contribute to the growth of the tumor.
Effect of rBbCI on the metabolism of strain tumorals human .
The activity showed for rBbCI in the analyzed strain demonstrates efficient inhibition regarding the chemotherapic drugs 5-FU (You imagine 32-39) . In the strain of gastric cancer, the acid pH and the presence of pepsin in the stomach they can activate the precursors of cisteinoproteases for active form (Pagano et al . , 1989) . Several works make a list of the presence of the catepsina L, besides the potential of
invasion, as well as, the regulation for cistatinas (Saleh et al., 2003; Kielan et al . , 2004).
In cancer of colon, Herszenyi and collaborators (1999) noticed in tissues of patients with cancer, that catepsina L was one of the relevant factors of short survival and which high levels of this enzyme were correlated by high risk of death. Zajc and collaborators (2002) , compared the expression of catepsina L in -strain of breast cancer with different potential invasive, MCF-7 went to less invasive strain when active and bigger concentration inhibitor is presenting less concentration of catepsina L. Therefor we can emphasize the paper of rBbCI on the inhibitory activity of the catepsina L in these types of tumors .
Additionally catepsina G and elastase induced the formation of multicellular spheroids in the strain of breast cancer (MCF-7) , being able to be contributing to the dissemination of cellular lodgers (Yui et al., 2005), once what experiments in survive they demonstrated that the intravenous injection of these lodgers turns in the formation of tumors of form more efficient than the scattered cells (Albini et al . , 1987). These results demonstrate another possible mechanism of rBbCI .
On the other side, since anti al-anti-trypsin was able to inhibit the liberation of the factor of growth - a in MCF- 7, while trypsin inhibitor of soy (BBI) did not affect this
activity (Yavelow et al., 1997), rBbCI be able to cause inhibition of growth factors, as soon as it inhibits elastase and it does not interfere in the activity of trypsin and chimotrypsin. The effect in the decrease of the metabolism of the inhibitor rBbCI in the strain THP-I and K562 can have taken place as a consequence of the blockade of proteases secreted for neutrophils, like HNE and catepsina G.
Effect of rBbKI on the metabolism of strain tumorals human
In the invasive strain Hs746T, rBbKI in concentrations more lifted up after incubation of 24 hours it was more efficient in the inhibition of the cellular metabolism, when compared with the strain not - metastatica MKN-28. Nagahara and collaborators (2005) , compared the expression of the gene of calicrein, KLK6, and noticed that his expression is proportional to the bad diagnosis of patients with gastric cancer and, while silencing the gene in the strain MKN-28, it took place to reduction of the cellular proliferation. In contrast, the calicreins can regulate negatively the growth of the tumor, I eat it hK3 it was able to inhibit the growth of the strain of breast cancer MCF-7 through the regulation of hormones (Lai et al . , 1996), meantime the same calicrein promoted angiogenesis (Derynck et al . , 2001). In accordance with the present invention, the results in strain
of breast cancer show the ability of rBbKI of inhibitor the metabolism, through other mechanisms and/or on different proteases .
The high expression of trypsin and matrilysine (metaloprotease-7 [MMP-7] ) activated for trypsin it was identified in cancer of colon. These factors are connected with the short time of life of patients with this type of tumor (Yamamoto et al . , 2003). So, rBbKI can be used by it like tool of study in the strain of cancer of colon due to inhibition of trypsin.
So, the action of the rBbKI on different strain tumorals, it demonstrates a better efficiency for inhibition of the invasive strain. The effect probably is resulting from the inhibitory activity on proteases, according to the works that demonstrate the superexpression of proteases in the invasive strain. Besides, inhibitor showed effect dose and dependent time, this action differentiates it of the chemotherapic drugs 5FU.
Effect of EcTI on the metabolism of strain tumorals human
The EcTI showed important inhibitory action in the cellular metabolism (You imagine 32-39) . Several studies correlate the invasive potential with the presence of different proteases in gastric tumors.
Park and collaborators (1997) demonstrated what Hs746T definite caprioles, to capriole and MMP-2 and his invasive potential was reduced by the presence of Anti-whoops. High levels of MMP-2 and MMP-9 are associated by the low overlife in patients by gastric cancer (Sier et al., 1996; Bjόrrklund and Koivunen, 2005) .
Ahmed and collaborators (2003) demonstrated that the reduction in the activity of the Erk/MAP kinase in HCT 116 (cancer of colon - more invasive strain) with antibody to capriole the activation of pro-one - MMP-9 was abolished, consequently a decrease of the adhesion took place, and to to break the complex uPAR/bl there took place blockade of the activation of pro--MMP-9 and pro--MMP-2. So, EcTI can inhibit the road of the Erk/MAP kinase, inhibitor the activation of pro-one - MMP-9, it can still interact with the chain of bl integrina interfering in the activation of the pro-ones - gelatinases and or through the inativaςao of plasmina.
In cancer of colon, the increase of the concentration of the components of the system plasminogenio / plasmina and of the gelatinases they are associated by worse prediction and invasive potential (Zeng et al., 1996; Herszenyi et al . , 2000; Yang et al . , 2000; Berger, 2002).
The action of EcTI on the metabolism of the strain of breast cancer was similar to that obtained one with cancer of colon. The strain metastatica SKBR-3 definite MMP-9, while
the strain MCF-7 not - metastatica not expresses gelatinases. Different groups demonstrated the importance of the expression of gelatinases whoops and inhibitor of ativador of plasminogenio type 1 (PAI-I) in breast cancer, confirming the action on invasion and metastasis (Baramova et al., 1997; Janicke et al . , 2001; Bjδrklund and Koivunen, 2005). In such a way, that the efficiency of EcTI on the inhibition of the invasive strain of cancer of colon and of breast, can have taken place through his action on plasmina and gelatinases . In cells leucemicas, EcTI was more efficient than 5-FU especially with 24 hours of incubation. Investigators suggest that the gelatinases are directly involved in the leukemia, making a list of his paper in the angiogenesis, one of the diagnoses of the leukemia (revised by Klein et al . , 2004) . In 2000, Gazzanelli and collaborators demonstrated which expression and/or liberation of metalo-proteases can be regulated by the induction of the apoptosis, so EcTI, while inducing the cellular death, it can inhibit the activation of metalo-proteases . Though the activity of 5-FU in tumors is quite recognized, the resistance is frequently pointed out to this chemotherapic drugs. Strategies as co-treatment with other chemical agents or changes have been developed to increase the activity of 5-FU (Bajetta, 1997; Klampfer et al . , 2005).
Several studies are in development for identification of new biomarcadores, therapeutic targets and rational development of drugs to be used together. The effect of the combined therapy of EcTI and 5-FU or carboplatinum reduces the necessary dose for inhibition of the metabolism and the action together was more efficient (You imagine 40-43) .
Though knowledges of the analysis farmacocinetica of the study of combined therapy are necessary, the therapy with inhibitors will be able to contribute to reduce the toxic effects caused for 5 -FU or carboplatinum.
The mechanism for which the chemotherapic drugs 5-FU acts is different from inhibitors in accordance with the present invention.
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herein incorporated by reference in their entirety. Several modifications and variations of the present invention will be evident to those skilled in the art unless being apart from the scope and the spirit of the present invention. While this invention has been described in terms of the specific preferred embodiment, this application should be understood as non limited by such specific embodiment but completely within specifications of appended claims.