Effects of Cobra Cardiotoxins on Intracellular Calcium and the Contracture of Rat Cardiomyocytes Depend on Their Structural Types.

Cardiotoxins (CaTx) of the three-finger toxin family are one of the main components of cobra venoms. Depending on the structure of the N-terminal or the central polypeptide loop, they are classified into either group I and II or P- and S-types, respectively, and toxins of different groups or types interact with lipid membranes variably. While their main target in the organism is the cardiovascular system, there is no data on the effects of CaTxs from different groups or types on cardiomyocytes. To evaluate these effects, a fluorescence measurement of intracellular Ca2+ concentration and an assessment of the rat cardiomyocytes' shape were used. The obtained results showed that CaTxs of group I containing two adjacent proline residues in the N-terminal loop were less toxic to cardiomyocytes than group II toxins and that CaTxs of S-type were less active than P-type ones. The highest activity was observed for Naja oxiana cobra cardiotoxin 2, which is of P-type and belongs to group II. For the first time, the effects of CaTxs of different groups and types on the cardiomyocytes were studied, and the data obtained showed that the CaTx toxicity to cardiomyocytes depends on the structures both of the N-terminal and central polypeptide loops.

Functional and structural properties of cardiotoxin isomers produced by blocking negatively charged groups.

In this study, we investigated the functional roles of Asp40, Asp57, and C-terminal Asn60 in Naja atra cardiotoxin 3 (CTX3) structure and function by modifying these three carboxyl groups with semicarbazide. The conjugation of the carboxyl groups with semicarbazide produced two conformational isomers whose gross and fine structures were different from those of CTX3. The blocking of the carboxyl groups increased the structural flexibility of CTX3 in response to trifluoroethanol-induced effect. Despite presenting modest to no effect on decreasing the induction of permeability in zwitterionic phospholipid vesicles, the carboxyl group-modified CTX3 showed a marked reduction in its permeabilizing effect on anionic phospholipid vesicles in comparison to that of the native protein. Compared with native CTX3, carboxyl group-modified CTX3 exhibited lower activity in inducing membrane leakage in U937 cells. The CD spectra of lipid-bound toxins and the color transition of polydiacetylene/lipid assay showed that the membrane interaction mode of CTX3 was distinctly changed by the modification in the carboxyl groups. Given that the selective modification of Asp40 does not cause the conformational isomerization of CTX3, our data indicate that the carboxyl groups in Asp57 and Asn60 are essential in maintaining the structural topology of CTX3. Furthermore, modification of carboxyl groups changes the interdependence between the infrastructure and the global conformation of CTX3 in modulating membrane permeabilizing activity.

Negative inotropic mechanisms of ß-cardiotoxin in cardiomyocytes by depression of myofilament ATPase activity without activation of the classical ß-adrenergic pathway.

Beta-cardiotoxin (ß-CTX) from the king cobra venom (Ophiophagus hannah) was previously proposed as a novel ß-adrenergic blocker. However, the involvement of ß-adrenergic signaling by this compound has never been elucidated. The objectives of this study were to investigate the underlying mechanisms of ß-CTX as a ß-blocker and its association with the ß-adrenergic pathway. The effects of ß-CTX on isolated cardiac myocyte functions, calcium homeostasis, the phosphorylation level of targeted proteins, and the myofibrillar ATPase activity were studied. Healthy Sprague Dawley rats were used for cardiomyocytes isolation. Like propranolol, ß-CTX attenuated the cardiomyocyte inotropy and calcium transient alterations as induced by isoproterenol stimulation. In contrast, these effects were not observed in forskolin-treated cells. Interestingly, cardiomyocytes treated with ß-CTX showed no changes in phosphorylation level at any PKA-targeted sites in the myofilaments as demonstrated in Western blot analysis. The skinned fibers study revealed no change in myofilament kinetics by ß-CTX. However, this protein exhibited the direct inhibition of myofibrillar ATPase activity with calcium de-sensitization of the enzyme. In summary, the negative inotropic mechanism of ß-CTX was discovered. ß-CTX exhibits an atypical ß-blocker mechanism. These properties of ß-CTX may benefit in developing a novel agent aid to treat hypertrophic cardiomyopathy.

Blocking of negative charged carboxyl groups converts Naja atra neurotoxin to cardiotoxin-like protein.

Naja atra cobrotoxin and cardiotoxin 3 (CTX3) exhibit neurotoxicity and cytotoxicity, respectively. In the present study, we aimed to investigate whether the carboxyl groups of cobrotoxin play a role in structural constraints, thereby preventing cobrotoxin from exhibiting cytotoxic activity. Six of the seven carboxyl groups in cobrotoxin were conjugated with semicarbazide. Measurement of circular dichroism spectra and Trp fluorescence quenching showed that the gross conformation of semicarbazide-modified cobrotoxin (SEM-cobrotoxin) and cobrotoxin differed. In sharp contrast to cobrotoxin, SEM-cobrotoxin demonstrated membrane-damaging activity and cytotoxicity, which are feature more characteristic of CTX3. Furthermore, both SEM-cobrotoxin and CTX3 induced cell death through AMPK activation. Analyses of the interaction between polydiacetylene/lipid vesicles and fluorescence-labeled lipids revealed that SEM-cobrotoxin and cobrotoxin adopted different membrane-bound states. The structural characteristics of SEM-cobrotoxin were similar to those of CTX3, including trifluoroethanol (TFE)-induced structural transformation and membrane binding-induced conformational change. Conversely, cobrotoxin was insensitive to the TFE-induced effect. Collectively, the data of this study indicate that blocking negatively charged residues confers cobrotoxin with membrane-damaging activity and cytotoxicity. The findings also suggest that the structural constraints imposed by carboxyl groups control the functional properties of snake venom α-neurotoxins during the divergent evolution of snake venom neurotoxins and cardiotoxins.

Status of Asp29 and Asp40 in the Interaction of Naja atra Cardiotoxins with Lipid Bilayers.

It is widely accepted that snake venom cardiotoxins (CTXs) target the plasma membranes of cells. In the present study, we investigated the role of Asp residues in the interaction of Naja atra cardiotoxin 1 (CTX1) and cardiotoxin 3 (CTX3) with phospholipid bilayers using chemical modification. CTX1 contains three Asp residues at positions 29, 40, and 57; CTX3 contains two Asp residues at positions 40 and 57. Compared to Asp29 and Asp40, Asp57 was sparingly modified with semi-carbazide, as revealed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass and mass/mass analyses. Thus, semi-carbazide-modified CTX1 (SEM-CTX1) mainly contained modified Asp29 and Asp40, while SEM-CTX3 contained modified Asp40. Compared to that of native toxins, trifluoroethanol easily induced structural transition of SEM-CTX1 and SEM-CTX3, suggesting that the structural flexibility of CTXs was constrained by Asp40. Modification of Asp29 and Asp40 markedly promoted the ability of CTX1 to induce permeability of cell membranes and lipid vesicles; CTX3 and SEM-CTX3 showed similar membrane-damaging activity. Modification of Asp residues did not affect the membrane-binding capability of CTXs. Circular dichroism spectra of SEM-CTX3 and CTX3 were similar, while the gross conformation of SEM-CTX1 was distinct from that of CTX1. The interaction of CTX1 with membrane was distinctly changed by Asp modification. Collectively, our data suggest that Asp29 of CTX1 suppresses the optimization of membrane-bound conformation to a fully active state and that the function of Asp40 in the structural constraints of CTX1 and CTX3 is not important for the manifestation of membrane-perturbing activity.

Cardiotoxins from Cobra Naja oxiana Change the Force of Contraction and the Character of Rhythmoinotropic Phenomena in the Rat Myocardium.

The study of the influence of cobra Naja oxiana cardiotoxins on the contractility of the rat papillary muscles and its rhythmoinotropic characteristics has shown that the presence of toxins induces a slight contractility decrease in the stimulation frequency range up to 0.1 Hz. In the stimulation frequency range from 0.1 to 0.5 Hz, a positive inotropic effect is found. However, the positive inotropic effect is replaced by a negative one with further increase in the frequency up to 3 Hz. In the presence of cardiotoxins, the positive force-frequency relationship in the region of 1-3 Hz, characteristic of healthy rat myocardium, disappears and the relationship becomes completely negative. L-type calcium channel blocker nifedipine does not affect the changes induced by toxins, while a high concentration (10 mM) of calcium prevents the effects of cardiotoxins on the muscle. The results obtained show that the impairment of the force-frequency relationship occurs long before the development of irreversible damage in the myocardium and may be the first sign of the pathological action of cardiotoxins.

Monitoring the Disulfide Bonds of Folding Isomers of Synthetic CTX A3 Polypeptide Using MS-Based Technology.

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide demethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a ß-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.

Muscle regeneration is disrupted by cancer cachexia without loss of muscle stem cell potential.

Cancer cachexia is a severe, debilitating condition characterized by progressive body wasting associated with remarkable loss of skeletal muscle weight. It has been reported that cancer cachexia disturbs the regenerative ability of skeletal muscle, but the cellular mechanisms are still unknown. Here, we investigated the skeletal muscle regenerative process in mouse colon-26 (C26) tumor cell-bearing mice as a C26 cancer cachexia model. Although the proliferation and differentiation abilities of muscle stem cells derived from the C26 tumor cell-bearing mice were sustained in vitro, the proliferation and differentiation were severely impaired in the cachexic mice. The numbers of both macrophages and mesenchymal progenitors, which are critical players in muscle regeneration, were reduced in the cancer cachexic mice, indicating that the skeletal muscle regeneration process was disrupted by cancer cachexia. Furthermore, the number of infiltrated neutrophils was also reduced in cancer cachexia mice 24 hours after muscle injury, and the expression of critical chemokines for muscle regeneration was reduced in cancer cachexia model mice compared to control mice. Collectively, although the ability to regeneration of MuSCs was retained, cancer cachexia disturbed skeletal muscle regenerative ability by inhibiting the orchestrated muscle regeneration processes.

Progranulin deficiency leads to prolonged persistence of macrophages, accompanied with myofiber hypertrophy in regenerating muscle.

Skeletal muscle has an ability to regenerate in response to injury due to the presence of satellite cells. Injury in skeletal muscle causes infiltration of pro-inflammatory macrophages (M1 macrophages) to remove necrotic myofibers, followed by their differentiation into anti-inflammatory macrophages (M2 macrophages) to terminate the inflammation. Since both M1 and M2 macrophages play important roles, coordinated regulation of their kinetics is important to complete muscle regeneration successfully. Progranulin (PGRN) is a pluripotent growth factor, having a protective role against the inflamed tissue. In the central nervous system, PGRN regulates inflammation by inhibiting the activation of microglia. Here we used muscle injury model of PGRN-knockout (PGRN-KO) mice to elucidate whether it has a role in the kinetics of macrophages during muscle regeneration. We found the prolonged persistence of macrophages at the late phase of regeneration in PGRN-KO mice, and these macrophages were suggested to be M2 macrophages since this was accompanied with an increased CD206 expression. We also observed muscle hypertrophy in PGRN-KO mice at the late stage of muscle regeneration. Since M2 macrophages are known to have a role in maturation of myofibers, this muscle hypertrophy may be due to the presence of increased number of M2 macrophages. Our results suggest that PGRN plays a role in the regulation of kinetics of macrophages for the systemic progress of muscle regeneration.

Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana.

Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small ß-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of 13C,15N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

Taiwan cobra cardiotoxin III suppresses EGF/EGFR-mediated epithelial-to-mesenchymal transition and invasion of human breast cancer MDA-MB-231 cells.

Breast cancer is a highly malignant carcinoma and most deaths of breast cancer are caused by metastasis. The epithelial-to-mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis. CTX III, a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity; however, the effect of CTX III on the EMT of cancer cells remains elusive. CTX III treatment resulted in morphological changes from elongated and spindle shape to rounded and epithelial-like shape, induced upregulation of E-cadherin and concurrent downregulation of N-cadherin and Vimentin protein levels, corresponding to observed decreases in cell migration and invasion. CTX III treatment also decreased the expression of Snail and Twist in EGF-induced MDA-MB-231 cells. Concurrently, CTX III efficiently inhibited the EGFR phosphorylation and downstream activation of phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2. The EGFR specific inhibitor AG1478 significantly suppressed ERK1/2 and Akt phosphorylation, cell migration and invasion, as well as the expressional changes associated with EMT markers in EGF-induced MDA-MB-231 cells. CTX III inhibitory effect on EGF-evoked invasion of MDA-MB-231 cells is mediated through suppressing EGF/EGFR activation and EMT process.

Cardiotoxin III Inhibits Hepatocyte Growth Factor-Induced Epithelial-Mesenchymal Transition and Suppresses Invasion of MDA-MB-231 Cells.

The epithelial-mesenchymal transition (EMT) is the first step required for breast cancer to initiate metastasis. In this study, hepatocyte growth factor (HGF) was used as a metastatic inducer of MDA-MB-231 cells. Cardiotoxin III (CTX III) inhibited HGF-induced morphological changes and upregulation of E-cadherin with the concomitant decrease in N-cadherin and Vimentin protein levels, resulting in inhibition of cell migration and invasion. CTX III-induced downregulation of transcription factors, Snail, Twist, and Slug, in MDA-MB-231 cells. CTX III suppressed c-Met phosphorylation and downstream activation of phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2. The c-Met specific inhibitor PHA665752 attenuated ERK1/2 and Akt phosphorylation, cell migration and invasion, as well as the expressional changes of EMT markers induced by HGF. Taken together, our data suggest that CTX III suppresses HGF/c-Met-induced cell migration and invasion by reversing EMT, which involves the inactivation of the HGF/c-Met-mediated ERK1/2 and PI3K/Akt pathways in MDA-MB-231 cells.

Cytolytic Activity and Molecular Feature of Cardiotoxin and Cardiotoxin-like Basic Protein: The Electrostatic Potential Field Is an Important Factor for Cell Lytic Activity.

BACKGROUND/AIM: Cardiotoxin (CT) is a well-known cell lytic protein and has been purified from cobra venom. Cardiotoxin-like basic protein (CLBP) has two amino acid insertions and does not exhibit cell lytic activity. The molecular features of these CT family proteins were examined in the present study using molecular modeling and molecular simulation techniques. MATERIALS AND METHODS: Molecular models of CT and CLBP were constructed based on the X-ray data of Naja mossambica mossambica CT VII4 (Protein Data Bank ID: 1CDT). The structural features of these models were examined using molecular orbital and electrostatic potential parameters. RESULTS: The stereo-hydrophobicities and molecular torsions of CT and CLBP, which are indexes of structural features, were similar. Electrostatic potential fields (ESP) differed between CT and CLBP and this was considered one of the critical factors in molecular titer. CONCLUSION: The distribution of ESP fields may affect the cytolytic activity of the CT family.

Antiproliferative activity of cobra venom cytotoxins.

Cytotoxins (or cardiotoxins, CTs) are small rigid membrane-active proteins of the three-finger toxin (TFT) family. They comprise about 60 amino acid residues, stabilized by four disulphide bridges. CTs, the most abundant proteins in cobra venom are able to kill cancer cells in a dose and time-dependent manner. The present review summarizes the current data on the molecular pathways of cancer cell death, induced by CTs. A relationship between structural characteristics of CTs and mechanism of their antiproliferative activity is reviewed as well. The CT molecules are rigid and their structure does not change significantly, when they interact with their molecular targets. This rigidity facilitates identification of molecular entities, responsible for antiproliferative activity of the toxins. We demonstrate that consideration of a net electrostatic charge and recently introduced HTL (Hydrophobicity of the Tips of the Loops) score allows distinguishing between the two mechanisms of cell death. The first is related to membrane destabilization by the toxins. The second involves their capture inside the cells, followed by interrogation into signal cascades mediated by the proteins, essential for cell life. Via addressing to antibacterial activity of CTs, which is supposed to arise from the plasma membrane damage, we demonstrate that, if membrane deterioration is involved in malignant cell death, the toxic activity of CTs correlates with their HTL scores and net electrostatic charge. We assume the relationship found may be used for rational design of antiproliferative compounds.

Myofiber-specific inhibition of TGFß signaling protects skeletal muscle from injury and dystrophic disease in mice.

Muscular dystrophy (MD) is a disease characterized by skeletal muscle necrosis and the progressive accumulation of fibrotic tissue. While transforming growth factor (TGF)-ß has emerged as central effector of MD and fibrotic disease, the cell types in diseased muscle that underlie TGFß-dependent pathology have not been segregated. Here, we generated transgenic mice with myofiber-specific inhibition of TGFß signaling owing to expression of a TGFß type II receptor dominant-negative (dnTGFßRII) truncation mutant. Expression of dnTGFßRII in myofibers mitigated the dystrophic phenotype observed in δ-sarcoglycan-null (Sgcd(-/-)) mice through a mechanism involving reduced myofiber membrane fragility. The dnTGFßRII transgene also reduced muscle injury and improved muscle regeneration after cardiotoxin injury, as well as increased satellite cell numbers and activity. An unbiased global expression analysis revealed a number of potential mechanisms for dnTGFßRII-mediated protection, one of which was induction of the antioxidant protein metallothionein (Mt). Indeed, TGFß directly inhibited Mt gene expression in vitro, the dnTGFßRII transgene conferred protection against reactive oxygen species accumulation in dystrophic muscle and treatment with Mt mimetics protected skeletal muscle upon injury in vivo and improved the membrane stability of dystrophic myofibers. Hence, our results show that the myofibers are central mediators of the deleterious effects associated with TGFß signaling in MD.

Cardiotoxin III suppresses hepatocyte growth factor-stimulated migration and invasion of MDA-MB-231 cells.

The hepatocyte growth factor (HGF)/c-Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA-MB-231 cells. When cells were treated with non-toxic doses of CTX III, CTX III inhibited the HGF-promoted cell migration and invasion. CTX III significantly suppressed the HGF-induced c-Met phosphorylation and downstream activation of phosphatidylinositol 3-kinase (PI3k)/Akt and extracellular signal-regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF-κB) as well as a reduction of matrix metalloproteinase-9 (MMP-9) activity. Furthermore, the c-Met inhibitor PHA665752 inhibited HGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c-Met activation. Taken together, these findings suggest that CTX III inhibits the HGF-induced invasion and migration of MDA-MB-231 cells via HGF/c-Met-dependent PI3K/Akt, ERK1/2 and NF-κB signalling pathways, leading to the downregulation of MMP-9 expression.

Design of a truncated cardiotoxin-I analogue with potent insulinotropic activity.

Insulin secretion by pancreatic ß-cells in response to glucose or other secretagogues is tightly coupled to membrane potential. Various studies have highlighted the prospect of enhancing insulin secretion in a glucose-dependent manner by blocking voltage-gated potassium channels (K(v)) and calcium-activated potassium channels (K(Ca)). Such strategy is expected to present a lower risk for hypoglycemic events compared to KATP channel blockers. Our group recently reported the discovery of a new insulinotropic agent, cardiotoxin-I (CTX-I), from the Naja kaouthia snake venom. In the present study, we report the design and synthesis of [Lys(52)]CTX-I(41-60) via structure-guided modification, a truncated, equipotent analogue of CTX-I, and demonstrate, using various pharmacological inhibitors, that this derivative probably exerts its action through Kv channels. This new analogue could represent a useful pharmacological tool to study ß-cell physiology or even open a new therapeutic avenue for the treatment of type 2 diabetes.

Cobra cardiotoxins: membrane interactions and pharmacological potential.

Natural polycationic membrane-active peptides typically lack disulfide bonds and exhibit fusion, cell-penetrating, antimicrobial activities. They are mostly unordered in solution, but adopt a helical structure, when bound to phospholipid membranes. Structurally different are cardiotoxins (or cytotoxins, CTs) from cobra venom. They are fully ß- structured molecules, characterized by the three-finger fold (TFF). Affinity of CTs to lipid bilayer was shown to depend on amino acid sequence in the tips of the three loops. In the present review, CT-membrane interactions are analyzed through the prism of data on binding of the toxins to phospholipid liposomes and detergent micelles, as well as their structural and computational studies in membrane mimicking environments. We assess different hydrophobicity scales to compare membrane partitioning of various CTs and their membrane effects. A comparison of hydrophobic/hydrophilic properties of CTs and linear polycationic peptides provides a key to their biological activity and creates a fundamental basis for rational design of new membrane-interacting compounds, including new promising drugs. For instance, from the viewpoint of the data obtained on model lipid membranes, cytotoxic activity of CTs against cancer cells is discussed.

Inhibition of Src activation with cardiotoxin III blocks migration and invasion of MDA-MB-231 cells.

Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been demonstrated to display anticancer activity. Breast cancer is a highly malignant carcinoma and most deaths of breast cancer are caused by metastasis. In this study, we show that CTX III blocks migration and invasion of MDA-MB-231 breast cancer cells without affecting apoptosis or cell cycle arrest. CTX III caused significant block of Src kinase activity in MDA-MB-231 cells. Moreover, CTX III treatment was correlated with reduced phosphorylation of FAK at Tyr576, 861 and 925 sites, p130(Cas) at Tyr410, and paxillin at Tyr118. CTX III also suppressed the activation of extracellular signal-regulated kinase1/2 and phosphatidylinositol 3-kinase/Akt. Consistent with inhibition of these signaling pathways and invasion, CTX III inhibited the expression of matrix metalloproteinase-9. In addition, Src specific inhibitor PP2 caused a significant decrease in the phosphorylation of FAK, p130(Cas), paxillin, PI3K/Akt, and ERK1/2. Taken together, CTX III significantly inhibited phosphorylation of Src and downstream molecules as well as cell migration and invasion. Our findings provide evidences that CTX III inhibits Src-mediated signaling pathways involved in controlling MDA-MB-231 cell migration and invasion, suggesting that it has therapeutic potential in breast cancer treatment.

Fusogenicity of Naja naja atra cardiotoxin-like basic protein on sphingomyelin vesicles containing oxidized phosphatidylcholine and cholesterol.

This study investigated the effect of oxidized phosphatidylcholine (oxPC) and cholesterol (Chol) on Naja naja atra cardiotoxin-like basic protein (CLBP)-induced fusion and leakage in sphingomyelin (SM) vesicles. Compared with those on PC/SM/Chol vesicles, CLBP showed a lower activity to induce membrane permeability but a higher fusogenicity on oxPC/SM/Chol vesicles. A reduction in inner-leaflet fusion elucidated that CLBP fusogenicity was not in parallel to its membrane-leakage activity on oxPC/SM/Chol vesicles. The lipid domain formed by Chol and SM supported CLBP fusogenicity on oxPC/SM/Chol vesicles, while oxPC altered the interacted mode of CLBP with oxPC/SM/Chol vesicles as evidenced by Fourier transform infrared spectra analyses and colorimetric phospholipid/polydiacetylene membrane assay. Although CLBP showed similar binding affinity with PC/SM/Chol and oxPC/SM/Chol vesicles, the binding capability of CLBP with PC/SM/Chol and oxPC/SM/Chol vesicles was affected differently by NaCl. This emphasized that CLBP adopted different membrane interaction modes upon binding with PC/SM/Chol and oxPC/SM/Chol vesicles. CLBP induced fusion in vesicles containing oxPC bearing the aldehyde group, and aldehyde scavenger methoxyamine abrogated the CLBP ability to induce oxPC/SM/Chol fusion. Taken together, our data indicate that Chol and oxPC bearing aldehyde group alter the CLBP membrane-binding mode, leading to fusogenicity promotion while reducing the membrane-damaging activity of CLBP.