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.

BPIQ, a novel synthetic quinoline derivative, inhibits growth and induces mitochondrial apoptosis of lung cancer cells in vitro and in zebrafish xenograft model.

BACKGROUND: 2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy] phenyl}-11H-indeno[1,2-c]quinolin-11-one (BPIQ) is a derivative from 6-arylindeno[1,2-c]quinoline. Our previous study showed the anti-cancer potential of BPIQ compared to its two analogues topotecan and irinotecan. In the study, the aim is to investigate the potency and the mechanism of BPIQ against lung cancer cells. METHODS: Both in vitro and zebrafish xenograft model were performed to examine the anti-lung cancer effect of BPIQ. Flow cytometer-based assays were performed for detecting apoptosis and cell cycle distribution. Western blot assay was used for detecting the changes of apoptotic and cell cycle-associated proteins. siRNA knockdown assay was performed for confirming the apoptotic role of Bim. RESULTS: Both in vitro and zebrafish xenograft model demonstrated the anti-lung cancer effect of BPIQ. BPIQ-induced proliferative inhibition of H1299 cells was achieved through the induction of G2/M-phase arrest and apoptosis. The results of Western blot showed that BPIQ-induced G2/M-phase arrest was associated with a marked decrease in the protein levels of cyclin B and cyclin-dependent kinase 1 (CDK1). The up-regulation of pro-apoptotic Bad, Bim and down-regulation of pro-survival XIAP and survivin was observed following BPIQ treatment. CONCLUSIONS: BPIQ-induced anti-lung cancer is involved in mitochondrial apoptosis. BPIQ could be a promising anti-lung cancer drug for further applications.

Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression.

Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression.

Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin.

Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity.

Inhibitory action of naphtho[1,2-b]furan-4,5-dione on hepatocyte growth factor-induced migration and invasion of MDA-MB-231 cells: mechanisms of action.

Naphtho[1,2-b]furan-4,5-dione (NFD), a bioactive component of Avicennia marina, has been shown to exhibit anticancer activity. The aim of the present study was to explore the effect of NFD on hepatocyte growth factor (HGF)-induced cell migration and invasion of MDA-MB-231 human breast cancer cells, as well as the underlying mechanism of action. Cell viability was determined using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay, western blot analysis was used to measure protein expression and cell migration and invasion were evaluated by the cell wound healing assay, Boyden chamber assay and gelatin zymography. When cells were treated with non-toxic concentrations of NFD (1-3 µmol/L, 24 h), NFD concentration-dependently inhibited HGF-promoted cell migration and invasion. Simultaneously, NFD efficiently suppressed c-Met phosphorylation and downstream activation of phosphatidylinositol 3-kinase (PI3K) and Akt. In addition, NFD inhibited the phosphorylation of IκB kinases and IκBα and nuclear translocation of nuclear factor (NF)-κB, as well as matrix metalloproteinase (MMP)-9 activity. Furthermore, the c-Met inhibitor PHA665752 (10 µmol/L) inhibited HGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of PI3K/Akt, suggesting that PI3K/Akt activation occur downstream of c-Met activation. In conclusion, the results of the present study suggest that NFD inhibits HGF-induced invasion and migration of MDA-MB-231 cells via HGF- and/or c-Met-mediated PI3K/Akt and NF-κB signalling pathways, leading to downregulation of MMP-9 expression and cell migration.

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.

Taiwan cobra phospholipase A2 suppresses ERK-mediated ADAM17 maturation, thus reducing secreted TNF-α production in human leukemia U937 cells.

The goal of this study was to explore the signaling pathway regulating the processing of proADAM17 into ADAM17 in Taiwan cobra phospholipase A2 (PLA2)-treated human leukemia U937 cells. PLA2 induced reactive oxygen species (ROS)-elicited p38 MAPK activation and ERK inactivation in U937 cells. Catalytically inactive bromophenacylated PLA2 (BPB-PLA2) and PLA2 mutants evoked Ca(2+)-mediated p38 MAPK activation, and the level of phosphorylated ERK remained unchanged. PLA2 treatment reduced mature ADAM17 expression and secreted TNF-α (sTNF-α) production. Co-treatment of SB202190 (p38 MAPK inhibitor) and catalytically inactive PLA2 increased ERK phosphorylation, ADAM17 maturation and sTNF-α production. Nevertheless, mRNA levels of ADAM17 and TNF-α were insignificantly altered after PLA2 and SB202190/BPB-PLA2 treatment. ADAM17 activity assay and knock-down of ADAM17 revealed that ADAM17 was involved in sTNF-α production. Restoration of ERK activation increased the processing of proADAM17 into ADAM17 in PLA2-treated cells, while inactivation of ERK reduced ADAM17 maturation in untreated and SB202190/BPB-PLA2-treated cells. Removal of cell surface heparan sulfate abrogated PLA2 and SB202190/BPB-PLA2 effect on ADAM17 maturation. Taken together, the present data reveal that PLA2 suppresses ERK-mediated ADAM17 maturation, thus reducing sTNF-α production in U937 cells. Moreover, the binding with heparan sulfate is crucial for the PLA2 effect.

Naphtho[1,2-b]furan-4,5-dione inhibits MDA-MB-231 cell migration and invasion by suppressing Src-mediated signaling pathways.

Naphtho[1,2-b]furan-4,5-dione (NFD), a bioactive component of Avicennia marina, has been demonstrated to display anti-cancer activity. Breast cancer is a highly malignant carcinoma and most deaths of breast cancer are caused by metastasis. In this study, we showed that NFD blocked migration and invasion of MDA-MB-231 breast cancer cells without affecting apoptosis or growth arrest. NFD caused significant block of Src kinase activity in MDA-MB-231 cells. Moreover, NFD treatment was correlated with reduced phosphorylation of FAK at Tyr 576/577, 861 and 925 sites, p130(Cas) at Tyr 410, and paxillin at Tyr 118. NFD also suppressed the activation of phosphatidylinositol 3-kinase/Akt. Consistent with inhibition of these signaling pathways and invasion, NFD reduced the expression of matrix metalloproteinase-9. Furthermore, Src antagonist PP2 caused a significant decrease in the phosphorylation of FAK, p130(Cas), paxillin, and PI3K/Akt. Our findings provide evidences that NFD inhibits Src-mediated signaling pathways involved in controlling breast cancer migration and invasion, suggesting that it has a therapeutic potential in breast cancer treatment.

Furano-1,2-Naphthoquinone Inhibits Src and PI3K/Akt Signaling Pathways in Ca9-22 Human Oral Squamous Carcinoma Cells.

Furano-1,2-naphthoquinone (FNQ), a biologically active component ofAvicennia marina, has been demonstrated to display anticancer activity. FNQ exerted cytotoxicity with the G2/M cell cycle arrest and apoptosis in Ca9-22 cells. FNQ-induced G2/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (CDK) 1 and 2 with concomitant induction of p27. FNQ-induced apoptosis was accompanied by Bax and Bad upregulation, and the downregulation of Bcl-2, Bcl-XL, Mcl-1, and X-linked inhibitor of apoptosis (XIAP), resulting in cytochrome C release and sequential activation of caspase-9 and caspase-3. Mechanistic studies showed that FNQ suppressed Src phosphorylation, PI3K, and Akt activation in Ca9-22 cells. Moreover, the Src inhibitor PP2 reduced the phosphorylation of Src and activation of PI3K/Akt, which was comparable with FNQ treatment. The combined treatment of FNQ with PP2 enhanced the cell cycle arrest and apoptosis and also led to the downregulation of Bcl-XL, Mcl-1, XIAP, cyclin A, cyclin B, CDK1, and CDK2 and upregulation of p27, Bax, and Bad. These findings suggest that FNQ-mediated cytotoxicity of Ca9-22 cells is related with the G2/M cell cycle arrest and apoptosis via inactivation of Src and PI3K/Akt-mediated signaling pathways.

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.

Cross talk between p38MAPK and ERK is mediated through MAPK-mediated protein phosphatase 2A catalytic subunit α and MAPK phosphatase-1 expression in human leukemia U937 cells.

This study explores the signaling transduction cascade of ERK and p38 MAPK on regulating MAPK phosphatase-1 (MKP-1) and protein phosphatase 2A catalytic subunit α (PP2Acα) expression in caffeine-treated human leukemia U937 cells. Caffeine induced an increase in the intracellular Ca(2+) concentration and ROS generation leading to p38 MAPK activation and ERK inactivation, respectively. Caffeine treatment elicited MKP-1 down-regulation and PP2Acα up-regulation. The transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) abolished the caffeine effect on MKP-1 and PP2Acα expression. Caffeine repressed ERK-mediated c-Fos phosphorylation but evoked p38 MAPK-mediated CREB phosphorylation. Knockdown of c-Fos and CREB by siRNA showed that c-Fos and CREB were responsible for MKP-1 and PP2Acα expression, respectively. Promoter and chromatin immunoprecipitating assay supported the role of c-Fos and CREB in regulating MKP-1 and PP2Acα expression. Moreover, transfection of dominant negative MKP-1 cDNA led to p38 MAPK activation and PP2Acα down-regulation in U937 cells, while PP2A inhibitor attenuated caffeine-induced ERK inactivation and MKP-1 down-regulation. Taken together, our data indicate that a reciprocal relationship between ERK-mediated MKP-1 expression and p38 MAPK-mediated PP2Acα expression crucially regulates ERK and p38 MAPK phosphorylation in U937 cells.

Brazilein suppresses migration and invasion of MDA-MB-231 breast cancer cells.

Brazilein, a bioactive compound isolated from Caesalpinia sappan L., has long been used in oriental folk medicines. Cancer metastasis is a primary cause of cancer death. However, the anti-metastatic effects of brazilein remain elusive. In this study, we found that brazilein inhibited human breast cancer MDA-MB-231 cell migration and invasion using wound-healing assay and Boyden chamber assay. The results of Western blot, gelatin zymography and reversed transcription-PCR analysis showed that brazilein suppressed matrix metalloproteinase-2 (MMP-2) expression in a concentration-dependent manner. Brazilein also decreased the nuclear protein level of nuclear factor kappaB (NF-κB). Brazilein potently suppressed the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), phosphatidylinositide-3-kinase (PI3K) and Akt, but did not affect phosphorylation of extracellular signal regulating kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK). Additionally, treatment of SB203580 (p38 MAPK inhibitor) or wortmannin (PI3K inhibitor) resulted in a reduced activity and expression of MMP-2 as well as inhibition on cell migration and invasion in MDA-MB-231 cells. Taken together, these results suggest that brazilein inhibition of MDA-MB-231 cells may be mediated through inactivation of both PI3K/Akt and p38 MAPK signaling pathways, leading to inhibitory effect on NF-κB activation. Consequently, brazilein suppresses MMP-2 expression, and thus confers anti-migration and anti-invasion of MDA-MB-231 cells.

Cardiotoxin III inhibits proliferation and migration of oral cancer cells through MAPK and MMP signaling.

Cardiotoxin III (CTXIII), isolated from the snake venom of Formosan cobra Naja naja atra, has previously been found to induce apoptosis in many types of cancer. Early metastasis is typical for the progression of oral cancer. To modulate the cell migration behavior of oral cancer is one of the oral cancer therapies. In this study, the possible modulating effect of CTXIII on oral cancer migration is addressed. In the example of oral squamous carcinoma Ca9-22 cells, the cell viability was decreased by CTXIII treatment in a dose-responsive manner. In wound-healing assay, the cell migration of Ca9-22 cells was attenuated by CTXIII in a dose- and time-responsive manner. After CTXIII treatment, the MMP-2 and MMP-9 protein expressions were downregulated, and the phosphorylation of JNK and p38-MAPK was increased independent of ERK phosphorylation. In conclusion, CTXIII has antiproliferative and -migrating effects on oral cancer cells involving the p38-MAPK and MMP-2/-9 pathways.

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.

Inhibition of EGF/EGFR activation with naphtho[1,2-b]furan-4,5-dione blocks migration and invasion of MDA-MB-231 cells.

Naphtho[1,2-b]furan-4,5-dione (NFD), a bioactive component of Avicennia marina, has been demonstrated to display anti-cancer activity. Activation of epidermal growth factor receptor (EGFR)-induced signaling pathway has been correlated with cancer metastasis in various tumors, including breast carcinoma. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of NFD on cell migration and invasion. NFD suppressed EGF-mediated protein levels of c-Jun and c-Fos, and reduced MMP-9 expression and activity, concomitantly with a marked inhibition on cell migration and invasion without obvious cellular cytotoxicity. NFD abrogated EGF-induced phosphorylation of EGF receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K)/Akt. The specific PI3K inhibitor, wortmannin, blocked significantly EGF-induced cell migration and invasion. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of PI3K/Akt, suggesting that PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that NFD inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of NFD as a potent anti-metastatic agent in MDA-MB-231 cells.

Cardiotoxin III suppresses MDA-MB-231 cell metastasis through the inhibition of EGF/EGFR-mediated signaling pathway.

Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis in various tumors. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of CTX III on cell migration. CTX III inhibited the EGF-induced activation of matrix metalloproteinase-9 (MMP-9), and further suppressed cell invasion and migration without obvious cellular cytotoxicity. CTX III suppressed EGF-induced nuclear factor-kappaB (NF-κB) nuclear translocation and also abrogated the EGF-induced phosphorylation of EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt, and extracellular regulated kinase (ERK)1/2. In addition, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an up-stream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by EGF. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-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 occur downstream of EGFR activation. These findings suggest that CTX III inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt, ERK1/2, and NF-κB signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of CTX III as a potent anti-metastatic agent in MDA-MB-231 cells.

Quercetin modulates activities of Taiwan cobra phospholipase A2 via its effects on membrane structure and membrane-bound mode of phospholipase A2.

The goal of the present study is to elucidate the mechanism of quercetin on modulating Naja naja atra phospholipase A2 (PLA2) activities. Sphingomyelin inhibited PLA2 enzymatic activity and membrane-damaging activity against egg yolk phosphatidylcholine (EYPC), while cholesterol and quercetin abrogated the sphingomeyelin inhibitory effect. Quercetin incorporation led to a reduction in PLA2 enzymatic activity and membrane-damaging activity toward EYPC/sphingomyelin/cholesterol vesicles. Both cholesterol and quercetin increased detergent resistance and reduced membrane fluidity of EYPC/sphingomyelin vesicles. Quercetin reduced detergent insolubility but increased ordered lipid packing of EYPC/sphingomyelin/cholesterol vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that quercetin altered the membrane-bound mode of PLA2 differently upon absorption onto the membrane bilayers of different lipid compositions. However, 8-anilinonaphthalene sulphonate-binding assay revealed that quercetin marginally affected the interaction between active site of PLA2 with phospholipid vesicles. Collectively, our data indicate that membrane-inserted quercetin modulates PLA2 interfacial activity and membrane-damaging activity via its effects on membrane structure and membrane-bound mode of PLA2.

Naja naja atra and Naja nigricollis cardiotoxins induce fusion of Escherichia coli and Staphylococcus aureus membrane-mimicking liposomes.

Our previous studies showed that the bactericidal effect of Naja naja atra cardiotoxin 3 (CTX3) and Naja nigricollis toxin γ was associated with their membrane-damaging activity. To elucidate the mechanism responsible for CTX3- and toxin γ-induced membrane permeability, we investigated the interacted mode of CTX3 and toxin γ with model membrane of Escherichia coli (phosphatidylethanolamine (PE)/phosphatidylglycerol (PG), mol/mol, 75/25) and Staphylococcus aureus (PG/cardiolipin, mol/mol, 60/40) in this study. Membrane-damaging activity of toxin γ on PE/PG and PG/cardiolipin vesicles were similar, while CTX3-induced leakage of PG/cardiolipin vesicles was notably higher than that of PE/PG vesicles. Noticeably, fusogenic activity of CTX3 and toxin γ on the phospholipid vesicles correlated positively with their membrane-damaging activity. Unlike toxin γ, CTX3 induced increasingly leakage and fusion of phospholipid vesicles with increased cardiolipin content. Changes in membrane fluidity and lipid packing occurred with the binding of CTX3 and toxin γ with vesicles, reflecting the penetration of toxin molecules into membrane bilayers. Consistent with the finding that PE/PG and PG/cardiolipin vesicles induced differently conformational changes of CTX3 and toxin γ, CTX3 and toxin γ adopted different membrane bound-mode upon absorption onto either PE/PG or PG/cardiolipin vesicles. Taken together, our data indicate that membrane-bound mode and membrane-perturbing effect of CTX3 and toxin γ in concert with targeted membrane compositions determine their fusogenicity and membrane-damaging activity, and suggest a causal relationship between bactericidal activity and fusogenicity of CTX3 and toxin γ.

Guanidination of notexin promotes its phospholipase A(2) activity-independent fusogenicity on vesicles with lipid-supplied negative curvature.

To address the requirement of phospholipase A(2) (PLA(2)) activity in membrane fusion events and membrane perturbation activity of notexin and guanidinated notexin (Gu-notexin), the present study was conducted. Notexin and Gu-notexin did not show PLA(2) activity after the removal of Ca(2+) with EDTA. Metal-free notexin and Gu-notexin were found to induce membrane leakage and fusion of phospholipid vesicles. Fusogenic activity of native and modified notexin correlated positively with their membrane-damaging activity underlying the deprivation of PLA(2) activity. Compared with Ca(2+)-bound Gu-notexin, fusogenicity of metal-free Gu-notexin was notably increased by incorporation of cholesterol, cholesterol sulfate, phosphatidylethanolamine, α-tocopherol and phosphatidic acid that supplied negative curvature into phospholipid bilayer. The ability of Gu-notexin to induce membrane fusion of vesicles with lipid-supplied negative curvature was higher than that of notexin regardless of the absence or presence of Ca(2+). Consistently, metal-free Gu-notexin markedly induced membrane fusion of red blood cells (RBCs) compared with metal-free notexin, and fusion activity of metal-free Gu-notexin on cholesterol-depleted RBCs notably reduced. Compared with notexin, Gu-notexin highly induced uptake of calcein-loaded phosphatidylcholine (PC)/cholesterol and PC/cholesterol sulfate vesicles by K562 cells in the presence of EDTA. Taken together, our data suggest that notexin and Gu-notexin could induce vesicle leakage and fusion via a PLA(2) activity-independent mechanism, and guanidination promotes PLA(2) activity-independent fusogenicity of notexin on vesicles with lipid-supplied negative curvature.