Crotamine-like from Southern Pacific rattlesnake (Crotalus oreganus helleri) Venom acts on human leukemia (K-562) cell lines and produces ultrastructural changes on mice adrenal gland.

Crotamine is a cationic, non-enzymatic, protein integrating a minor family of myotoxins, composed of 42 amino acid residues, described in Viperidae and Crotalidae snake's families that has been used in neuroscience research, drug progressing and molecular diversity reports. Crotamine-like protein (CLP) from C.o.helleri venom was isolated in fraction 5 from 7 peaks obtained by sulfopropyl waters protein pak cationic exchange column. In tricine-SDS-PAGE under non-reduced conditions this CLP showed a single band of ~8 kDa molecular weight. CLP induced toxicity of K-562 cells with a CC50 of 11.09 µM. In mice adrenal gland after 24 h of CLP injection, cortical cells exhibited swollen mitochondria with scarce tubular cristae, some elements of smooth and rough endoplasmic reticula, widened nuclear envelope, slightly osmiophilic lipid droplets, and autophagic vacuoles. In some areas cortical cells plasma membrane and endothelial walls disappeared, which indicated a necrosis process. In other areas, endothelial cell cytoplasm did not present the normal caveolae and pinocytotic vesicles. To our knowledge, this is the first report on mice adrenal gland damages, caused by the injection of CLP from rattlesnakes. Our results propose that adrenal cortex lesions may be significant in the envenoming etiopathogenesis by CLP.

Gene expression profiling of the venom gland from the Venezuelan mapanare (Bothrops colombiensis) using expressed sequence tags (ESTs).

BACKGROUND: Bothrops colombiensis is a highly dangerous pit viper and responsible for over 70% of snakebites in Venezuela. Although the composition in B. colombiensis venom has been identified using a proteome analysis, the venom gland transcriptome is currently lacking. RESULTS: We constructed a cDNA library from the venom gland of B. colombiensis, and a set of 729 high quality expressed sequence tags (ESTs) was identified. A total number of 344 ESTs (47.2% of total ESTs) was related to toxins. The most abundant toxin transcripts were metalloproteinases (37.5%), phospholipases A2s (PLA2, 29.7%), and serine proteinases (11.9%). Minor toxin transcripts were linked to waprins (5.5%), C-type lectins (4.1%), ATPases (2.9%), cysteine-rich secretory proteins (CRISP, 2.3%), snake venom vascular endothelium growth factors (svVEGF, 2.3%), L-amino acid oxidases (2%), and other putative toxins (1.7%). While 160 ESTs (22% of total ESTs) coded for translation proteins, regulatory proteins, ribosomal proteins, elongation factors, release factors, metabolic proteins, and immune response proteins. Other proteins detected in the transcriptome (87 ESTs, 11.9% of total ESTs) were undescribed proteins with unknown functions. The remaining 138 (18.9%) cDNAs had no match with known GenBank accessions. CONCLUSION: This study represents the analysis of transcript expressions and provides a physical resource of unique genes for further study of gene function and the development of novel molecules for medical applications.

The characterization of trans-pecos copperhead (Agkistrodon contortrix pictigaster) venom and isolation of two new dimeric disintegrins.

The vast amounts of toxins within the venom of snakes, while known to cause medical emergencies, display various biological functions. Trans-pecos copperhead (Agkistrodon contortrix pictigaster) crude venom separated by cation-exchange chromatography showed several fractions with fibrinolytic, hemorrhagic, gelatinase and platelet activities. Venom fractions 1, 2, 4, 5, and 12-17 contained fibrinolytic activity. Venom fractions 1, 2, 5 and 12-14 had hemorrhagic activity. Fractions 1, 2, 12, 13 and 17 contained gelatinase activity. Reverse-Phase C18 High Performance Liquid Chromatography was also used to purify and isolated disintegrins from this venom. Anti-platelet aggregation activity of the C18 fractions collected and performed on whole human blood showed that they inhibited platelet aggregation in presence of several agonists. Results from both SDS-PAGE and N-terminal sequencing determined that pictistatin 1 obtained from the Trans-Pecos copperhead venom was a dimeric disintegrin, and pictistatin 2 was a heterodimeric disintegrin. The molecules with anti-platelet activity could be considered in the development of more effective drugs, for numerous blood-related diseases such as stroke, heart attacks, thrombosis, and other medical conditions. In this study, we are presenting the first report of the purification, isolation, and partial characterization of two new dimeric disintegrins isolated from the venom of trans-pecos copperhead.

Morulustatin, A Disintegrin that Inhibits ADP-Induced Platelet Aggregation, Isolated from the Mexican Tamaulipan Rock Rattlesnake (Crotalus lepidus morulus).

The Tamaulipan rock rattlesnake (Crotalus lepidus morulus) is a montane snake that occurs in the humid pine-oak forest and the upper cloud forest of the Sierra Madre Oriental in southwestern Tamaulipas, central Nuevo Leon, and southeastern Coahuila in Mexico. Venom from this rattlesnake was fractionated by High-Performance Liquid Chromatography for the purpose of discovering disintegrin molecules. Disintegrins are non-enzymatic, small molecular weight peptides that interfere with cell-cell and cell-matrix interactions by binding to various cell receptors. Eleven fractions were collected by anion exchange chromatography and pooled into six groups (I, II, III, IV, V, and VI). Proteins of the six groups were analyzed by SDS-PAGE and western blot using antibodies raised against a disintegrin. The antibodies recognized different protein bands in five (II, III, IV, V, and VI) of six groups in a molecular mass range of 7 to 105 kDa. Western blot analysis revealed fewer protein bands in the higher molecular mass range and two bands in the disintegrin weight range in group II compared with the other four groups. Proteins in group II were further separated into nine fractions using reverse phase C18 chromatography. Fraction 4 inhibited platelet aggregation and was named morulustatin, which exhibited a single band with a molecular mass of approximately 7 kDa. Mass spectrometry analysis of fraction 4 revealed the identification of disintegrin peptides LRPGAQCADGLCCDQCR (MH+ 2035.84) and AGEECDCGSPANCCDAATCK (MH+ 2328.82). Morulustatin inhibited ADP-induced platelet aggregation in human whole blood and was concentration-dependent with an IC50 of 89.5 nM ± 12.

Biological activities of a new crotamine-like peptide from Crotalus oreganus helleri on C2C12 and CHO cell lines, and ultrastructural changes on motor endplate and striated muscle.

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 µM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.

Anti-platelet effect of cumanastatin 1, a disintegrin isolated from venom of South American Crotalus rattlesnake.

Disintegrins have been previously described in the venom of several snake families inhibiting signal transduction, cell-cell interactions, and cell-matrix interactions and may have therapeutic potential in heart attacks, thrombotic diseases, and cancers. This investigation describes the first disintegrin isolated from South American Crotalus venom (Venezuelan rattlesnake Crotalus durissus cumanensis), which inhibits platelet adhesion to matrix proteins. C. d. cumanensis crude venom was first separated on a Sephadex G-100 column into 4 fractions (SI to SIV). Crude venom and SIII fraction significantly diminished platelet adhesion to fibrinogen (Fg) and to fibronectin (Fn). Anti-adhesive SIII fraction was further separated by DEAE-Sephacel followed by C-18 reverse phase high performance liquid chromatography (HPLC). The platelet anti-adhesive fraction obtained was designated as cumanastatin-1. This disintegrin has a mass of 7.442 kDa as determined by mass spectrometry (MALDI-TOF/TOF) and pI of 8.5. Cumanastatin-1 also inhibited ADP-induced platelet aggregation with an IC(50) of 158 nM. However, it did not significantly inhibit collagen and thrombin-induced platelet aggregation. Cumanastatin-1 considerably inhibited anti-alpha(IIb)beta(3) integrin binding to platelets in a dose-dependent manner; however, it did not present any effect on the alpha(5)beta(1) integrin or on P-selectin.

Colombistatin: a disintegrin isolated from the venom of the South American snake (Bothrops colombiensis) that effectively inhibits platelet aggregation and SK-Mel-28 cell adhesion.

Snake venoms are complex mixtures of proteins, which affect the vital biologic systems of prey, as well as humans. Envenomation leads to immobilization by paralysis, cardiac, and circulatory failure. These same venom proteins that cause havoc in the physiologic system could be used as therapeutic agents. Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are non-enzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction. These proteins may have potential in the treatment of strokes, heart attacks, cancers, osteoporosis, and diabetes. The present study describes the isolation and characterization of a disintegrin (colombistatin) found in the venom of the Venezuelan snake mapanare (Bothrops colombiensis). Colombistatin was purified by a two-step high-performance liquid chromatography procedure, which included reverse phase C18 and size exclusion protein Pak 60. Colombistatin inhibited ADP-induced platelet aggregation, human urinary (T24) and skin melanoma (SK-Mel-28) cancer cell adhesion to fibronectin, and cell migration. Colombistatin contained 72 amino acids with a mass of 7.778 kDa as determined by mass spectrometry. Colombistatin could be used as a therapeutic tool in the treatment of melanoma cancers and also thrombotic diseases.

The action of Lonomin V (Lonomia achelous) on fibronectin functional properties.

Lonomia achelous caterpillar, Lepidoptera distributed along some South American countries, induces a hemorrhagic syndrome in people who come into contact with its bristles. A clinical characteristic in these patients is that fresh healed wounds are re-opened and bleed. In order to explain this symptomatology, we evaluated the effect of Lonomin V (a protein isolated from L. achelous hemolymph), on some functional properties of fibronectin, which in turn plays an important role in the hemostasis. The effect of Lonomin V on fibronectin was studied by SDS-PAGE in reduced condition, binding to gelatin and heparin, crosslinking to fibrin and platelet adhesion. Formation of degradation products of 120, 66, 50, 40 and 29 kDa, some of which retain affinity to heparin and gelatin were observed; however, the fibronectin degradation fragments presented a significant decrease of crosslinking capacity to fibrin and platelet adhesion, suggesting that the proteolysis of fibronectin by Lonomin V induces changes in its crosslinking sites and on platelet receptors. These findings might partially explain the wound dehiscence observed in the patients. Due to its effect on adhesive proteins with concomitant impairment of some functional properties, Lonomin V might be useful for cellular adhesion studies involved in hemostasis such as platelet adhesion.

Intraspecies differences in hemostatic venom activities of the South American rattlesnakes, Crotalus durissus cumanensis, as revealed by a range of protease inhibitors.

Crotalus durissus cumanensis is an endemic rattlesnake found in Venezuela and Colombia. In this study, a comparative analysis of hemorrhagic, coagulation and fibrino(geno)lytic activities in the presence or absence of protease inhibitors was performed with venoms of the same species Crotalus durissus cumanensis, from seven geographical regions of Venezuela (Lagunetica, Santa Teresa, Carrizales, Guarenas, Anzoátegui, Margarita and Maracay). Lagunetica, Carrizales and Anzoátegui venoms induced hemorrhagic activity. All venoms, except that of snakes from the Carrizales region presented thrombin-like activity, which was inhibited completely by phenylmethanesulfonyl fluoride and ethylene glycol-bis-N, N,N',N'-tetraacetic acid. This effect of the latter could be explained by the high chelant calcium effect, which is a cofactor for the fibrin polymerization process. Soybean trypsin inhibitor was effective on Santa Teresa venom. Antithrombin III/Hep complex and phenantroline partially inhibited this activity in all venoms except Margarita and Anzoátegui, respectively, which were not inhibited. Serine protease inhibitors were more effective against thrombin, kallikrein and plasmin-like amidolytic activities. Additionally, metalloprotease inhibitors significantly inhibited the t-PA-like amidolytic activity and completely the hemorrhagic and fibrino(geno)lytic activities. In conclusion, the thrombin-like activity observed in these venoms was partially reduced by serine protease inhibitors, indicating the possible presence of catalytic domains in those enzymes that do not interact with these inhibitors. Using protease inhibitors on venom hemostatic activities could contribute to our understanding of the active components of snake venom on the hemostatic system, and further reveal the intraspecies variation of venoms, which is important in the treatment of envenomation, and in addition represents an interesting model for the study of venom in hemostasis.

[Fibronectin. Structure and functions associated to hemostasis. Review]. / Fibronectina. Estructura y funciones asociadas a la hemostasia. Revisión.

Fibronectin is an adhesive glycoprotein present in a soluble form in plasma and in an insoluble form in the extracellular matrix of many tissues. The human plasma level of this protein is about 300 +/- 100 microg/mL. It is synthesized and secreted by a wide variety of cells, consequently is one of the components of greater distribution in the body that participates in the biochemical reactions of diverse physiological and pathological processes. Due to the presence of multifunctional domains in its structure, fibronectin interacts with diverse components of the coagulation and fibrinolysis. It may bind to collagen, fibrinogen, fibrin, heparin, factor XIII and platelets, among others, regulating processes of importance in hemostasis such as: platelet adhesion and aggregation, tissue remodelling during wound healing, and activation of fibrinolysis by the plasminogen activators.

The binding effectiveness of anti-r-disintegrin polyclonal antibodies against disintegrins and PII and PIII metalloproteases: An immunological survey of type A, B and A+B venoms from Mohave rattlesnakes.

Snake venoms are known to have different venom compositions and toxicity, but differences can also be found within populations of the same species contributing to the complexity of treatment of envenomated victims. One of the first well-documented intraspecies venom variations comes from the Mohave rattlesnake (Crotalus scutulatus scutulatus). Initially, three types of venoms were described; type A venom is the most toxic as a result of ~45% Mojave toxin in the venom composition, type B lacks the Mojave toxin but contains over 50% of snake venom metalloproteases (SVMPs). Also, type A+B venom contains a combination of Mojave toxin and SVMP. The use of an anti-disintegrin antibody in a simple Enzyme-Linked Immunosorbent Assay (ELISA) can be used to identify the difference between the venoms of the type A, B, and A+B Mohave rattlesnakes. This study implements the use of an anti-recombinant disintegrin polyclonal antibody (ARDPA) for the detection of disintegrins and ADAMs (a disintegrin and metalloproteases) in individual crude snake venoms of Mohave rattlesnakes (Crotalus scutulatus scutulatus) of varying geographical locations. After correlation with Western blots, coagulation activity and LD50 data, it was determined that the antibody allows for a quick and cost-efficient identification of venom types.

Degradation of extracellular matrix proteins (fibronectin, vitronectin and laminin) by serine-proteinases isolated from Lonomia achelous caterpillar hemolymph.

Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.

Functional characterizations of venom phenotypes in the eastern diamondback rattlesnake (Crotalus adamanteus) and evidence for expression-driven divergence in toxic activities among populations.

Phenotypes frequently vary across and within species. The connection between specific phenotypic effects and function, however, is less understood despite being essential to our understanding of the adaptive process. Snake venoms are ideal for identifying functionally important phenotypic variation because venom variation is common, and venoms can be functionally characterized through simple assays and toxicity measurements. Previous work with the eastern diamondback rattlesnake (Crotalus adamanteus) used multivariate statistical approaches to identify six unique venom phenotypes. We functionally characterized hemolytic, gelatinase, fibrinogenolytic, and coagulant activity for all six phenotypes, as well as one additional venom, to determine if the statistically significant differences in toxin expression levels previously documented corresponded to differences in venom activity. In general, statistical differences in toxin expression predicted the identified functional differences, or lack thereof, in toxic activity, demonstrating that the statistical approach used to characterize C. adamanteus venoms was a fair representation of biologically meaningful differences. Minor differences in activity not accounted for by the statistical model may be the result of amino-acid differences and/or post-translational modifications, but overall we were able to link variation in protein expression levels to variation in function as predicted by multivariate statistical approaches.

Expression, purification, and analysis of three recombinant ECD disintegrins (r-colombistatins) from P-III class snake venom metalloproteinases affecting platelet aggregation and SK-MEL-28 cell adhesion.

Crotalid venoms are rich sources of components that affect the hemostatic system. Snake venom metalloproteinases are zinc-dependent enzymes responsible for hemorrhage that also interfere with hemostasis. The disintegrin domain is a part of snake venom metalloproteinases, which involves the binding of integrin receptors. Integrins play an essential role in cancer survival and invasion, and they have been major targets for drug development and design. Both native and recombinant disintegrins have been widely investigated for their anti-cancer activities in biological systems as well as in vitro and in vivo systems. Here, three new cDNAs encoding ECD disintegrin-like domains of metalloproteinase precursor sequences obtained from a Venezuelan mapanare (Bothrops colombiensis) venom gland cDNA library have been cloned. Three different N- and C-terminal truncated ECD disintegrin-like domains of metalloproteinases named colombistatins 2, 3, and 4 were amplified by PCR, cloned into a pGEX-4T-1 vector, expressed in Escherichia coli BL21, and tested for inhibition of platelet aggregation and inhibition of adhesion of human skin melanoma (SK-Mel-28) cancer cell lines on collagen I. Purified recombinant colombistatins 2, 3, and 4 were able to inhibit ristocetin- and collagen-induced platelet aggregation. r-Colombistatins 2 showed the most potent inhibiting SK-Mel-28 cancer cells adhesion to collagen. These results suggest that colombistatins may have utility in the development of therapeutic tools in the treatment of melanoma cancers and also thrombotic diseases.

Biological and biochemical characterization of venom from the broad-banded copperhead (Agkistrodon contortrix laticinctus): isolation of two new dimeric disintegrins.

Disintegrins represent a family of effective cell-cell and cell-matrix inhibitors by binding to integrin receptors. Integrins are heterodimeric, transmembrane receptors that are the bridges for these cell interactions. Disintegrins have been shown to have many therapeutic implications for the treatment of strokes, heart attacks, and cancer. Two novel heterodimeric disintegrins were isolated from the venom of the broad-banded copperhead (Agkistrodon contortrix laticinctus). Crude venom separated by cation-exchange chromatography resulted in several fractions possessing hemorrhagic, fibrinolytic, gelatinase, and platelet activities. Venom fractions 2-3 and 17-19 showed fibrinolytic activity. Fractions 2-6, 8-11, and 16-21 had hemorrhagic activity. Gelatinase activity was found in fractions 3, 11, and 19. The isolation of laticinstatins 1 and 2 was accomplished by fractionating crude venom using reverse phase chromatography. Data from both SDS-PAGE and N-terminal sequencing determined that laticinstatins 1 and 2 were heterodimeric disintegrins, and both were assayed for their ability to inhibit platelet aggregation in human whole blood. Future functional evaluation of snake venom disintegrins shows considerable promise for elucidating the biochemical mechanisms of integrin-ligand interactions that will allow the development of adequate medications for hemostatic pathologies such as thrombosis, stroke, and cerebral and cardiac accidents. In this study, we are presenting the first report of the purification, and partial characterization of two new dimeric disintegrins isolated from the venom of broad-banded copperhead snakes.

Prevalence and factors associated with intimate partner violence among women in Recife/Pernambuco, Brazil.

Intimate partner violence is an important cause of morbidity and mortality among women. Although there are no official statistics, data reveal a high prevalence worldwide. This study aimed to estimate the prevalence and factors associated with intimate partner violence among women in a community in Recife, Pernambuco. A cross-sectional cohort study was conducted with 245 women in the 15 to 49-year age bracket. A questionnaire with sociodemographic variables was used, together with the WHO Violence Against Women (VAW) study tools and the Self-Reporting Questionnaire (SRQ-20). The participants all signed an informed consent form. The prevalence of intimate partner violence was classified by type of violence: emotional - 52.7%; physical - 46.1 %; and sexual - 13.6%. Bivariate analysis revealed an association between experiencing violence with not having a partner (p = 0.001) and drug use (p ≤ 0.001). In multivariate analysis, the variables were strongly associated with the outcome: sexual intercourse for fear (OR 5.58); depressive-anxious mood (OR 2.69); drug use (OR 2.57). A high prevalence of intimate partner violence in the community, especially emotional violence, emerges as an important finding, indicating the need for care in prevention and the overall health of this population.

Functional analysis of four single (RGDWL, RGDWM, RGDWP, RGDMN) and two double (RGDNM, RGDMP) mutants: The importance of methionine (M) in the functional potency of recombinant mojastin (r-Moj).

We have demonstrated in previous studies that a single amino acid change can alter the activity of the recombinant disintegrin r-Moj. In this study, four r-Moj recombinants containing single mutations (r-Moj-WL, r-Moj-WM, r-Moj-WP, r-Moj-MN) and two containing double mutations (r-Moj-MP and r-Moj-NM) at the binding loop were produced, purified, and tested. All r-Moj-W_, r-Moj-M_, and r-Moj-NM mutant peptides inhibited platelet aggregation at higher potency than r-Moj-D_ mutants. Five of the seven r-Moj peptides inhibited angiogenesis at different levels. Two of the mutant peptides with a methionine at the second position carboxyl of the RGD (r-Moj-WM and r-Moj-NM) were the strongest angiogenesis inhibitors, with r-Moj-WM being the most potent. Recombinant r-Moj-MP and r-Moj-WN failed to inhibit angiogenesis. Only the r-Moj-MP mutant peptide induced apoptosis of SK-Mel-28 cells significantly (p = 0.001). This was confirmed by chromatin condensation. Proliferation of SK-Mel-28 cells was inhibited at high levels (>70%) by all r-Moj mutant peptides. Recombinant r-Moj-MN and r-Moj-WN failed to inhibit cell migration significantly (p > 0.5). Recombinant r-Moj-NM was the strongest cell migration inhibitor (98% ± 0.69), followed by r-Moj-MP (80% ± 2.87), and r-Moj-WM (61.8% ± 5.45). The lowest inhibitor was r-Moj-WL (50% ± 12.16). Our functional data suggest that the most potent r-Moj disintegrins contain a methionine in the first or the second position carboxyl to the RGD.

Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): A second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells.

Disintegrins are small peptides produced in viper venom that act as integrin antagonists. When bound to integrins, disintegrins induce altered cellular behaviors, such as apoptotic induction. Disintegrins with RGDDL or RGDDM motifs induce apoptosis of normal and cancer cells. We hypothesized that a second aspartate (D) carboxyl to the RGD is sufficient to induce apoptosis. Five recombinant mojastin D mutants were produced by site-directed mutagenesis (r-Moj-DA, r-Moj-DG, r-Moj-DL, r-Moj-DN, and r-Moj-DV). Stable αv integrin knockdown and shRNA scrambled control SK-Mel-28 cell lines were produced to test a second hypothesis: r-Moj-D_ peptides bind to αv integrin. Only r-Moj-DL, r-Moj-DM, and r-Moj-DN induced apoptosis of SK-Mel-28 cells (at 29.4%, 25.6%, and 36.2%, respectively). Apoptotic induction was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown (to 2%, 17%, and 2%, respectively), but not in SK-Mel-28 cells with a stable scrambled shRNA. All six r-Moj-D_ peptides inhibited cell proliferation; ranging from 49.56% (r-Moj-DN) to 75.6% (r-Moj-DA). Cell proliferation inhibition by r-Moj-D_ peptides was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown. All six r-Moj-D_ peptides inhibited SK-Mel-28 cell migration at high levels (69%-100%). As a consequence, rac-1 mRNA expression levels were significantly reduced as early as 1 h after treatment, suggesting that rac-1 is involved in the cell migration activity of SK-Mel-28.

Inhibition of pancreatic tumoral cells by snake venom disintegrins.

Pancreatic cancer often has a poor prognosis, even when diagnosed early. Pancreatic cancer typically spreads rapidly and is rarely detected in its early stages, which is a major reason it is a leading cause of cancer death. Signs and symptoms may not appear until pancreatic cancer is quite advanced, and complete surgical removal is not possible. Furthermore, pancreatic cancer responds poorly to most chemotherapeutic agents. The importance of integrins in several cell types that affect tumor progression has made them an appealing target for cancer therapy. Some of the proteins found in the snake venom present a great potential as anti-tumor agents. In this study, we summarize the activity of two integrins antagonist, recombinant disintegrins mojastin 1 and viridistatin 2, on human pancreatic carcinoma cell line (BXPC-3). Both recombinant disintegrins inhibited some essential aspects of the metastasis process such as proliferation, adhesion, migration, and survival through apoptosis, making these proteins prominent candidates for the development of drugs for the treatment of pancreatic cancer.