Preclinical studies of a novel snake venom-derived recombinant disintegrin with antitumor activity: A review.

Snake venoms consist of a complex mixture of many bioactive molecules. Among them are disintegrins, which are peptides without enzymatic activity, but with high binding affinity for integrins, transmembrane receptors that function to connect cells with components of the extracellular matrix. Integrin-mediated cell attachment is critical for cell migration and dissemination, as well as for signal transduction pathways involved in cell growth. During tumor development, integrins play key roles by supporting cancer cell proliferation, angiogenesis, and metastasis. The recognition that snake venom disintegrins can block integrin functions has spawned a number of studies to explore their cancer therapeutic potential. While dozens of different disintegrins have been isolated, none of them as yet has undergone clinical evaluation in cancer patients. Among the best-characterized and preclinically most advanced disintegrins is vicrostatin (VCN), a recombinant disintegrin that was rationally designed by fusing 62 N-terminal amino acids derived from the disintegrin contortrostatin with 6 C-terminal amino acids from echistatin, the disintegrins from another snake species. Bacterially produced VCN was shown to target multiple tumor-associated integrins, achieving potent anti-tumor and anti-angiogenic effects in in vitro and in vivo models in the absence of noticeable toxicity. This review will introduce the field of snake venom disintegrins as potential anticancer agents and illustrate the translational development and cancer-therapeutic potential of VCN as an example.

Methods for Evaluation of a Snake Venom-Derived Disintegrin in Animal Models of Human Cancer.

Integrin targeting has been shown to be an effective approach for anticancer therapy. We engineered a recombinant disintegrin, vicrostatin (VCN), that binds with high affinity and specificity to the Arg-Gly-Asp (RGD) class of integrins, including αvß3, αvß5, and α5ß1, involved in tumor invasion and metastasis. We used three different delivery modalities to examine anticancer activity of VCN in mouse models of human ovarian cancer, glioma, and prostate cancer. A female mouse model was used to examine the treatment of established ovarian cancer (OC) using VCN delivered intraperitoneally (IP) weekly either in saline or impregnated in a viscoelastic gel. SKOV3luc cells (a human OC cell line) were directly injected IP into immunodeficient mice. We also examined the antitumor activity of radioiodinated VCN delivered intravenously in a human glioma model in nude mice. We evaluated the effectiveness of 131I-VCN in combination with the DNA alkylating agent temozolomide in limiting glioma growth. Finally, treatment of a bone metastatic model of human prostate cancer (PC) in immunodeficient mice was examined using a liposomal formulation of VCN (LVCN) delivered intravenously. Human PC cells were suspended in a solution of Matrigel and injected into the left tibia of immunodeficient mice. Diameters of both the left and right (control) tibias were measured by caliper repeatedly after VCN treatment was initiated.

Preclinical evaluation of a 64Cu-labeled disintegrin for PET imaging of prostate cancer.

A novel recombinant disintegrin, vicrostatin (VCN), displays high binding affinity to a broad range of human integrins in substantial competitive biological advantage over other integrin-based antagonists. In this study, we synthesized a new 64Cu-labeled VCN probe and evaluated its imaging properties for prostate cancer in PC-3 tumor-bearing mice. Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar) was conjugated with PEG unit and followed by coupling with VCN. The precursor was then radiolabeled with positron emitter 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer to provide 64Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe (64Cu-Sar-RGD). 64Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of > 98%. The specific activity of 64Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64Cu-Sar-RGD, 64Cu-Sar-PEG-VCN produces higher tumor-to-muscle (T/M) imaging contrast ratios at 2 h (4.66 ± 0.34 vs. 2.88 ± 0.46) and 24 h (4.98 ± 0.80 vs. 3.22 ± 0.30) post-injection (pi) and similar tumor-to-liver ratios at 2 h (0.43 ± 0.09 vs. 0.37 ± 0.04) and 24 h (0.57 ± 0.13 vs. 0.52 ± 0.07) pi. The biodistribution results were consistent with the quantitative analysis of microPET imaging, demonstrating good T/M ratio (2.73 ± 0.36) of 64Cu-Sar-PEG-VCN at 48 h pi in PC-3 tumor xenografts. For both microPET and biodistribution studies at 48 h pi, the PC-3 tumor uptake of 64Cu-Sar-PEG-VCN is lower than that of 64Cu-Sar-RGD. 64Cu-Sar-PEG-VCN has the potential for in vivo imaging of prostate cancer with PET, which may provide a unique non-invasive method to quantitatively localize and characterize prostate cancer.

A Novel Venom-Derived Peptide for Brachytherapy of Glioblastoma: Preclinical Studies in Mice.

We developed a bacterial expression system to produce a recombinant disintegrin, vicrostatin (VCN), whose structure is based on a natural disintegrin isolated from southern copperhead snake venom. Our goal is to develop VCN for potential clinical translation as an anti-cancer agent. VCN is a peptide of 69 amino acids with a single tyrosine residue. We have employed VCN as integrin-targeted radionuclide therapy (brachytherapy) for treatment of glioblastoma (GBM, glioma). GBM is a deadly brain cancer that doesn't discriminate between sexes and knows no age limit. We established that the tyrosine residue in VCN can be radioiodinated with full retention of bioactivity. 131I-VCN was utilized for integrin-targeted radionuclide therapy using mouse models of glioma. The combination of radioiodinated VCN plus temozolomide (a DNA alkylating agent) significantly prolonged survival of glioma-bearing mice. We also obtained similar results using an immunocompetent mouse model and a murine glioma cell line. In summary, as demonstrated in studies reported here we have shown that VCN as targeted radionuclide therapy for GBM has significant translational potential for therapy of this deadly disease.

Multimeric disintegrin protein polymer fusions that target tumor vasculature.

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

Snake venom metalloproteinases.

Recent proteomic analyses of snake venoms show that metalloproteinases represent major components in most of the Crotalid and Viperid venoms. In this chapter we discuss the multiple activities of the SVMPs. In addition to hemorrhagic activity, members of the SVMP family also have fibrin(ogen)olytic activity, act as prothrombin activators, activate blood coagulation factor X, possess apoptotic activity, inhibit platelet aggregation, are pro-inflammatory and inactivate blood serine proteinase inhibitors. Clearly the SVMPs have multiple functions in addition to their well-known hemorrhagic activity. The realization that there are structural variations in the SVMPs and the early studies that led to their classification represents an important event in our understanding of the structural forms of the SVMPs. The SVMPs were subdivided into the P-I, P-II and P-III protein classes. The noticeable characteristic that distinguished the different classes was their size (molecular weight) differences and domain structure: Class I (P-I), the small SVMPs, have molecular masses of 20-30 kDa, contain only a pro domain and the proteinase domain; Class II (P-II), the medium size SVMPs, molecular masses of 30-60 kDa, contain the pro domain, proteinase domain and disintegrin domain; Class III (P-III), the large SVMPs, have molecular masses of 60-100 kDa, contain pro, proteinase, disintegrin-like and cysteine-rich domain structure. Another significant advance in the SVMP field was the characterization of the crystal structure of the first P-I class SVMP. The structures of other P-I SVMPs soon followed and the structures of P-III SVMPs have also been determined. The active site of the metalloproteinase domain has a consensus HEXXHXXGXXHD sequence and a Met-turn. The "Met-turn" structure contains a conserved Met residue that forms a hydrophobic basement for the three zinc-binding histidines in the consensus sequence.

Reversal of thienopyridine-induced platelet dysfunction following desmopressin administration.

Adenosine diphosphate (ADP)-receptor antagonists are widely used for thrombus prevention, although reversing their platelet dysfunction is difficult. This study evaluated the ability of desmopressin to reverse clopidogrel-induced platelet dysfunction. Sprague-Dawley rats received either clopidogrel (30 mg/kg) or placebo, followed 4 h later by saline or desmopressin (0.15, 0.3, or 0.6 µg/kg). Bleeding times and platelet aggregation studies were subsequently performed. A bleeding time >25 min was considered "prolonged." The median bleeding time for clopidogrel-exposed rats was 21 min, vs. 6 min for controls (p < 0.01). Progressively higher doses of 1-deamino-8-D-arginine vasopressin (DDAVP) were associated with a reduced number of rats with prolonged bleeding time (p = 0.001). Higher doses of DDAVP were also associated with a reduction in the median (IQR) bleeding time; 29 (13.5-30) min in rats receiving clopidogrel without DDAVP vs. 19 (12-28) min in rats receiving clopidogrel and 0.6 µg/kg DDAVP. The step-wise dosing of DDAVP resulted in a 54 % reduction in meeting the endpoint of prolonged bleeding time (OR 0.46; p = 0.025; 95 % CI 0.23-0.91). Platelet aggregation was observed in all control rats, but only some of those clopidogrel-treated rats who received 0.6 µg/kg DDAVP. In this model of an ADP-receptor antagonist, DDAVP results in partial reversal of clopidogrel-induced platelet dysfunction.

Hirudin and s18886 maintain luminal patency after thrombolysis with alfimeprase.

BACKGROUND: An optimal strategy to improve reperfusion in patients with arterial occlusions is a recognized clinical need. We hypothesized that hirudin (thrombin inhibitor) and S18886 [S18, thromboxane A(2) receptor (TP) antagonist] would improve blood flow and reperfusion rates after thrombolysis with the direct-acting fibrinolytic enzyme alfimeprase. METHODS: In anesthetized beagles, carotid artery thrombosis was induced by electrolytic endothelial injury. After 30 minutes of occlusion, animals were administered vehicle, hirudin, and/or S18. Carotid artery blood flow was monitored for 90 minutes after the infusion of alfimeprase or recombinant tissue plasminogen activator (rt-PA). RESULTS: The onset to reperfusion was more rapid in animals treated with alfimeprase than in those treated with rt-PA. All the animals treated with hirudin + S18 + alfimeprase maintained vessel patency, and all vehicle-treated animals reoccluded. In animals treated with hirudin + S18 + alfimeprase, time to reocclusion and total reflow time after thrombolysis were longer compared with vehicle-treated animals. The quality and quantity of blood flow were most improved in animals treated with hirudin + S18 + alfimeprase. There were no significant differences in time to reocclusion, total reflow time, and quality and quantity of blood flow between vehicle + rt-PA-treated animals and hirudin + S18 + rt-PA-treated animals. CONCLUSIONS: Dual antithrombotic therapy with hirudin and S18 improves reperfusion after thrombolysis with alfimeprase but not rt-PA.

Contortrostatin, a homodimeric disintegrin isolated from snake venom inhibits herpes simplex virus entry and cell fusion.

BACKGROUND: Herpes simplex virus (HSV) causes significant health problems from periodical skin and corneal lesions to encephalitis. HSV entry provides a unique opportunity for therapeutic intervention. In this study, we evaluated contortrostatin (CN), an Arg-Gly-Asp motif containing disintegrin isolated from snake venom, as a novel therapeutic agent with ability to block HSV entry and related membrane fusion. METHODS: In vitro efficacy of CN against HSV was determined using an HSV type-1 (HSV-1) entry assay based on the measurement of ß-galactosidase reporter activity originating from the genome of a recombinant strain of HSV-1(KOS) gL86. HSV-1 glycoprotein-mediated cell-to-cell fusion was used to study the effect of CN on polykaryocyte formation. Primary as well as transformed cell lines were used for this study. RESULTS: Pre-treatment of Chinese hamster ovary (CHO-K1) cells expressing HSV-1 glycoprotein D receptors and primary cultures of human corneal fibroblasts (CF) with CN resulted in the inhibition of entry, cell-to-cell fusion, and polykaryocyte formation. Interestingly, a more pronounced anti-HSV-1 effect was observed in naturally susceptible CF than CHO-K1 cells. CONCLUSIONS: CN, a novel venom-derived peptide, exhibits the ability to block two key steps, entry and cell-to-cell fusion, in HSV infection. Showing strong promise for development as an anti-HSV agent, it also demonstrates better prophylactic efficacy in primary cells.

The disintegrin contortrostatin in combination with docetaxel is a potent inhibitor of prostate cancer in vitro and in vivo.

BACKGROUND: There are few available treatments for hormone refractory prostate cancer. Through the inhibition of integrins, contortrostatin (CN) effects tumor cell growth directly as well as through the inhibition of angiogenesis. The effect of CN in combination with docetaxel on prostate cancer cell lines in vitro and in vivo is evaluated in the present study. METHODS: FACS analysis of integrin expression, assessment of CN and docetaxel exposure on viability of plated cancer cells, and scratch test migration analysis were performed on PC-3 prostate cancer cells. CN and docetaxel inhibition of both PC-3 and CWR-22 prostate cancer cell lines were evaluated in a mouse xenograft bone model. Angiogenic activity in tumors were assessed using IHC with antibodies to CD31. RESULTS: Cell culture experiments indicate that the combination of docetaxel and CN inhibits growth in an additive fashion. FACS analysis of PC-3 cells shows expression of alpha5beta1 and alphavbeta5 integrins, but little expression of the alphavbeta3. CN showed complete inhibition of PC-3 migration in cultures grown on matrigel plates. In mice xenograft bone models, CN with docetaxel showed increased inhibition of both PC-3 and CWR-22 derived tumors. Analysis of treated xenograft tumors showed significantly decreased expression of CD31 indicating suppression of angiogenesis.

Vicrostatin - an anti-invasive multi-integrin targeting chimeric disintegrin with tumor anti-angiogenic and pro-apoptotic activities.

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., alphavbeta3, alphavbeta5, and alpha5beta1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.

Fibrolase: trials and tribulations.

Fibrolase is the fibrinolytic enzyme isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria including reverse phase HPLC, molecular sieve chromatography and SDS-PAGE. The purified enzyme is a zinc metalloproteinase containing one mole of zinc. It is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. Fibrolase shares a significant degree of homology with enzymes of the reprolysin sub-family of metalloproteinases including an active site homology of close to 100%; it is rapidly inhibited by chelating agents such as EDTA, and by alpha2-macroglobulin (α2Μ). The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(ß)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin, cleaving the α-chain more rapidly than the ß-chain. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phaase II stroke trial, and Nuvelo dropped further development in 2008.

The use of pepsin in receptor internalization assays.

For internalization experiments that use fluorescent antibody (Ab) staining to distinguish between inside versus outside cellular localization of various receptor targeting ligands, it is critical that there be efficient removal of all residual surface-bound fluorescent Ab. To achieve this, a fluorescent Ab removal technique is commonly employed in receptor internalization assays that utilizes low pH glycine-based buffers to wash off the residual non-internalized fluorescent Ab retained on cell surfaces. In this study, we highlight the shortcomings of this technique and propose an alternative in situ proteolytic approach that we found to be non-deleterious to the cells and significantly more effective in removing the residual fluorescence resulting from non-internalized surface-bound Ab.

Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7 A resolution.

Disintegrins are a family of small (4-14 kDa) proteins that bind to another class of proteins, integrins. Therefore, as integrin inhibitors, they can be exploited as anticancer and antiplatelet agents. Acostatin, an alphabeta heterodimeric disintegrin, has been isolated from the venom of Southern copperhead (Agkistrodon contortrix contortrix). The three-dimensional structure of acostatin has been determined by macromolecular crystallography using the molecular-replacement method. The asymmetric unit of the acostatin crystals consists of two heterodimers. The structure has been refined to an R(work) and R(free) of 18.6% and 21.5%, respectively, using all data in the 20-1.7 A resolution range. The structure of all subunits is similar and is well ordered into N-terminal and C-terminal clusters with four intramolecular disulfide bonds. The overall fold consists of short beta-sheets, each of which is formed by a pair of antiparallel beta-strands connected by beta-turns and flexible loops of different lengths. Conformational flexibility is found in the RGD loops and in the C-terminal segment. The interaction of two N-terminal clusters via two intermolecular disulfide bridges anchors the alphabeta chains of the acostatin dimers. The C-terminal clusters of the heterodimer project in opposite directions and form a larger angle between them in comparison with other dimeric disintegrins. Extensive interactions are observed between two heterodimers, revealing an alphabetabetaalpha acostatin tetramer. Further experiments are required to identify whether the alphabetabetaalpha acostatin complex plays a functional role in vivo.

Anti-angiogenesis and RGD-containing snake venom disintegrins.

Angiogenesis is the fundamental process by which new blood vessels are formed. Extensive research has shown that this event can be co-opted by tumors to ensure their growth, survival and metastasis. The study of tumor angiogenesis therefore represents a promising area of research for development of anti-cancer therapeutics. Integrins, a family of cell surface molecules, are a major target of interest as they are known to play a vital role in pathological angiogenesis. Remarkably, small disulfide-rich peptides known as disintegrins, isolated from the venoms of various snake species have been found to bind integrins with extremely high affinity and block their function. Disintegrins are capable of inhibiting several aspects of tumor cell behavior both in vitro and in vivo, including adhesion, migration, invasion, metastasis and angiogenesis. In this review, we will briefly discuss tumor angiogenesis and molecules implicated in the angiogenic process, with a special focus on the role of integrins. We will also discuss therapeutic approaches towards the treatment of tumor angiogenesis, including non-integrin-targeted agents currently in clinical trials. We will summarize the major findings from studies using disintegrins to target integrin-associated angiogenesis in cancer models. Finally, we will present results obtained in our laboratory using the novel dimeric disintegrin, contortrostatin (CN), in studies of endothelial cells and models of breast, ovarian and prostate cancer. In summary, disintegrins represent an exciting new class of molecules that can potentially be used in a clinical setting to inhibit angiogenesis and augment conventional chemotherapeutic agents in the treatment of cancer.

The role of contortrostatin, a snake venom disintegrin, in the inhibition of tumor progression and prolongation of survival in a rodent glioma model.

OBJECT: Malignant gliomas are not curable because of diffuse brain invasion. The tumor cells invade the surrounding brain tissue without a clear tumor-brain demarcation line, making complete resection impossible. Therapy aimed at inhibition of invasion is crucial not only for prevention of tumor spread, but also for selectively blocking migrating cells that may be more resistant to chemotherapy and radiation. Recently, investigations have shown that the snake venom disintegrin contortrostatin specifically binds to certain integrins on the surface of glioma cells and thereby inhibits their interaction with the extracellular matrix (ECM), resulting in a blockage of cell motility and invasiveness. To translate these in vitro findings into clinical settings, the authors examined the effect of contortrostatin on glioma progression in a rodent model. METHODS: Athymic mice were intracranially or subcutaneously injected with U87 glioma cells, and the effect of intratumorally administered contortrostatin on tumor progression and animal survival was then studied. In addition, the authors evaluated the pharmacological safety of contortrostatin use in the brains of tumor-free animals. CONCLUSIONS: The results demonstrate that contortrostatin is able to inhibit tumor growth and angiogenesis and to prolong survival in a rodent glioma model. Moreover, contortrostatin appears to be well tolerated by the animal and lacks obvious neurotoxic side effects. Thus, contortrostatin may have potential as a novel therapeutic agent for the treatment of malignant gliomas.

Potent mimicry of fibronectin-induced intracellular signaling in glioma cells by the homodimeric snake venom disintegrin contortrostatin.

OBJECTIVE: The snake venom disintegrin contortrostatin (CN) is able to inhibit tumor progression and angiogenesis in vivo and therefore is of considerable interest as a potential antitumor drug. CN specifically binds to certain integrins on the tumor cell and angiogenic endothelial cell surface and inhibits their interaction with the extracellular matrix, resulting in blockage of cell motility and invasiveness. To understand the molecular consequences of CN binding to integrins, we set out to investigate and compare the effects of CN and fibronectin (FN) on integrin-induced signaling and the resulting alteration in cellular cytoskeletal morphology. METHODS: Two different malignant glioma cell lines were exposed to soluble or immobilized CN, FN, or both, and the consequences for intracellular signaling and cellular adhesion to matrix were investigated. RESULTS: CN binding to integrins can mimic the intracellular signaling cascade evoked by FN, because the phosphorylation of the key signaling proteins focal adhesion kinase, paxillin, and p130 Crk-associated substrate and the association of Src with focal adhesion kinase are similar. However, CN is at least one order of magnitude more potent than FN. When soluble CN is added to cells that are already attached to an FN-coated matrix, it effectively disrupts the binding of integrin to FN, leading to a decrease in integrin signaling, which, in turn, results in the disruption of the cytoskeleton and cellular detachment. CONCLUSION: Our results provide a mechanistic explanation of how soluble CN might block cellular migration and invasion, namely, by disrupting and preventing the binding of integrins to the extracellular matrix. We envision that this property of CN could be used in the treatment of gliomas, namely, by intratumoral infusion of CN to prevent glioma and endothelial cell interactions with the extracellular matrix, leading to inhibition of cell invasion.

Action of fenretinide (4-HPR) on ovarian cancer and endothelial cells.

BACKGROUND: Fenretinide (4-HPR) is a synthetic retinoid that has been reported to inhibit the growth of cancer cell lines in vitro. MATERIALS AND METHODS: We examined the effect of 4-HPR on ovarian cancer (OVCAR-5) cell proliferation, viability and invasion using standard techniques. We also examined the action of 4-HPR on the actin cytoskeleton using immunocytochemistry, and on phosphorylation of focal adhesion kinase (FAK) using immunoprecipitation and phosphotyrosine immunoblotting. We then examined the activity of 4-HPR on endothelial cells using the tube formation assay on Matrigel. RESULTS: 4-HPR inhibited OVCAR-5 cell proliferation and viability at concentrations higher than 1 microM, with 70-90% growth inhibition at 10 microM. 4-HPR (1 microM) significantly inhibited OVCAR-5 invasion after 3 days preincubation. In view of the importance of the cytoskeleton in cell motility, we examined the action of 4-HPR on the actin cytoskeleton and on FAK phosphorylation. In OVCAR-5 cells treated with 1 mM fenretinide for 3 days, actin cytoskeleton stress fibers were disrupted and FAK tyrosine phosphorylation was elevated dose-dependently. Endothelial cells treated with 1 microM 4-HPR failed to form tubes, but formed small cellular aggregates. CONCLUSION: Fenretinide has anti-tumor activity by acting on the actin cytoskeleton and by regulating FAK tyrosine phosphorylation. 4-HPR also inhibits endothelial cell tube formation, a major step in angiogenesis.

Contortrostatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity.

Disintegrins are soluble peptides found in snake venom. They bind to Arg-Gly-Asp (RGD)-responsive integrins with high affinity (nM range) and block integrin function. Contortrostatin (CN), the disintegrin from southern copperhead venom, is a homodimer with a molecular weight of 13,500. Each chain contains 65 amino acids with an Arg-Gly-Asp motif. CN has anti-invasive and anti-adhesive activity on tumor cells and endothelial cells in vitro, and binds to integrins alphavbeta3, alphavbeta5, and/or alpha5beta1. In vivo studies using the human metastatic breast cancer cell line MDA-MB-435, in an orthotopic xenograft model in nude mice, revealed that CN has potent anti-tumor and anti-angiogenic activity. Recent studies have employed an intravenous liposomal delivery procedure. Liposomal delivery of CN has also been shown to provide effective in vivo anti-tumor and anti-angiogenic activity in a human ovarian cancer animal model.

Development of a novel recombinant disintegrin, contortrostatin, as an effective anti-tumor and anti-angiogenic agent.

Contortrostatin (CN) (Mr 13,500 Da) is a novel homodimeric disintegrin isolated from the venom of Agkistrodon contortrix contortrix (Southern Copperhead) snake and displays two RGD motifs (one on each chain), which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. In previous studies, we have shown that native CN administered in a liposomal formulation exhibits potent anti-angiogenic and tumor growth inhibitory activities. Current isolation of the protein from crude venom is difficult and prohibitively expensive for translation into the clinic. In this report, we describe a method amenable to large-scale production of a soluble monomeric form of recombinant CN with biologic activity; the protein is expressed directly in the cytoplasm of an engineered bacterial system with an expression yield of approximately 20 mg/l of culture. We present here the in vitro assays as well as the anti-tumor and anti-angiogenic evaluation of liposomal recombinant CN in an orthotopic, xenograft model of human breast cancer.