Shinne-Ren Lin

Separation and structure-function studies of Taiwan cobra cardiotoxins.

Six cardiotoxins (CTXs) and one cardiotoxin-like basic protein (CLBP) from Naja naja atra (Taiwan cobra) venom were separated by a SP-Sephadex C-25 column. CTXn and CTXI were well separated by eluting with ammonium acetate buffer, and the separation of CLBP from CTXIV and CTXV mixtures was achieved using sodium phosphate buffer. These findings suggest a differential interaction of CTXs with the chromatographic matrix using different buffer systems. Chemical modification studies on cationic residues of CTXI suggested that there was no single lysine or arginine residue exclusively responsible for its biological activity. Moreover, it was found that the cytotoxicity and hemolytic sites of CTXI could be dissociated by chemical modifications. It suggests the potentiality for preparing toxin derivatives in which a specific activity is retained.

p38 MAPK and NF-κB pathways are involved in naphtho[1,2-b] furan-4,5-dione induced anti-proliferation and apoptosis of human hepatoma cells

Naphtho[1,2-b] furan-4,5-dione (NFD) was investigated for its anti-proliferation effect on human hepatocellular carcinoma (HCC), Hep3B, HepG(2), and Huh-7 cells. The effect of NFD on inhibiting proliferation and apoptosis was correlated with up-regulation of pro-apoptotic protein and down-regulation of pro-survival proteins. Remarkably, we found that NFD inhibited the nuclear translocation of NF-kappaB, likely accounting for the down-regulation of pro-survival Bcl-2 family. Furthermore, suppression of p38 MAPK activity by a specific inhibitor significantly rescued the cell proliferation inhibited by NFD. These findings suggest that signaling imbalance between p38 MAPK and NF-kappaB by NFD results in the proliferative inhibition and apoptosis of HCC tumor cells.

Up-regulation of Bax and endonuclease G, and down-modulation of Bcl-XL involved in cardiotoxin III-induced apoptosis in K562 cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced K562 cell apoptosis was confirmed by DNA fragmentation (DNA ladder, sub-G1 formation) and phosphatidylserine (PS) externalization with an IC50 value of 1.7 µg/ml at 48 h. A mechanistic analysis demonstrated that CTX III-induced apoptotic cell death was accompanied by up-regulation of both Bax and endonuclease G (Endo G), and downregulation of Bcl-XL. CTX III had no effect on the levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. CTX III treatment caused loss of the mitochondrial membrane potential (Δψm), release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and -3. CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. However, CTX III did not generate reactive oxygen species (ROS) and antioxidants, including N-acetylcysteine and catalase, did not block CTX III-induced apoptosis in K562 cells. Modulation of Bax, Bcl-XL, and the Endo G proteins, release of mitochondrial cytochome c, and activation of caspase-3 and -9 all are involved in the CTX III-triggered apoptotic process in human leukemia K562 cells.

Naphtho[1,2-b]furan-4,5-dione induces apoptosis and S-phase arrest of MDA-MB-231 cells through JNK and ERK signaling activation.

Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits anti-carcinogenic effect. The results of present study showed that NFD inhibited the proliferation of breast cancer MDA-MB-231 cells through the induction of S-phase arrest and apoptosis. NFD-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclin A, cyclin B, and cyclin-dependent kinase (Cdk)2. NFD-induced apoptosis was characterized by increase of sub-G1 population, phosphatidylserine (PS) externalization, and activation of caspases. Moreover, up-regulation of Bad and down-regulation of Bcl-2, Bcl-X(L), and survivin led to the loss of mitochondrial membrane potential (DeltaPsim), the release of cytochrome c and sequential activation of caspase-9 and caspase-3. NFD activated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) in MDA-MB-231 cells. Inhibitors of JNK (SP600125) and ERK (PD98059), but not p38 MAPK (SB203580) suppressed NFD-induced S-phase arrest and apoptosis in MDA-MB-231 cells. Both SP600125 and PD98059 attenuated Bad up-regulation, and reversed down-regulation of Bcl-2, Bcl-X(L), survivin, cyclin A, cyclin B, and Cdk2 in NFD-treated cells. Taken together, our data show that JNK and ERK-signaling pathways play important roles in NFD-mediated S-phase arrest and apoptosis of MDA-MB-231 cells.

Cardiotoxin III induces apoptosis in K562 cells through a mitochondrial-mediated pathway.

1. Cardiotoxin (CTX) III is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III on human leukaemia K562 cells.

Down-regulation of the JAK2/PI3K-mediated signaling activation is involved in Taiwan cobra cardiotoxin III-induced apoptosis of human breast MDA-MB-231 cancer cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. Exposure of MDA-MB-231 cells with 0.03, 0.09, and 0.15 microM of CTX III for 18 h, CTX III-induced cell apoptosis, as evidenced by accumulation of sub-G1 population, externalization of phosphatidylserine, loss of mitochondrial membrane potential (DeltaPsim) with subsequent release of cytochrome c, and activation of both capases-9 and caspase-3. This correlated with up-regulation in Bax and Bad, and down-regulation of various anti-apoptotic proteins, including Bcl-2, Bcl-X(L), and survivin in CTX III-treated cells. Mechanistic studies showed that CTX III suppressed the phosphorylation of JAK2, STAT3, Akt, and activation of PI3K. Moreover, the PI3K inhibitor wortmannin blocked activation of STAT3 and Akt without affecting the JAK2 activation, whereas JAK2 inhibitor AG490 suppressed the levels of phospho-STAT3, phospho-Akt, and PI3K, suggesting that PI3K activation occurs after JAK2 phosphorylation, and both PI3K and JAK2 kinases cooperate to mediate STAT3 and Akt phosphorylation. Both AG490 and wortmannin also led to up-regulation in Bax and Bad, and down-regulation of Bcl-2, Bcl-X(L), and survivin in MDA-MB-231 cells. Taken together, these results indicate that CTX III induces apoptosis in MDA-MB-231 cells via concomitant inactivation of the JAK2, STAT3, PI3K, and Akt signaling pathways.

Effects of Cardiotoxin III on NF-κB Function, Proliferation, and Apoptosis in Human Breast MCF-7 Cancer Cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced apoptosis in human breast MCF-7 cancer cells was confirmed by sub-G1 formation, phosphatidylserine (PS) externalization, and poly (ADP-ribose) polymerase (PARP) cleavage with an IC50 of 2 microg/ml at 48 h. Effects of CTX III on proliferation and apoptosis correlated with upregulation of Bax, and downregulation of Bcl-XL, Bcl-2, and XIAP, with no appreciable alteration on the protein levels of Bid, Bim, and survivin. CTX III treatment also caused release of mitochondrial cytochrome c to the cytosol, which led to subsequent activation of capase-9. Moreover, CTX III inhibited the nuclear factor-kappaB (NF-kappaB) activation through inhibition of IkappaB kinase (IkappaK) activity. Overall, our results indicate that CTX III downregulates NF-kappaB in MCF-7 cells, leading to the suppression of proliferation and induction of apoptosis. These findings suggest the molecular basis for CTX III-induced apoptotic death of MCF-7 cells.

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β -glucuronidase

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse β-glucuronidase (mβG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-β-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional β-glucuronidase (βG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (βG) on CT26 tumors. The fluorescent intensity in βG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral βG vector into carcinoma xenografts. Importantly, mβG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human βG also allowed in vivo imaging, demonstrating that human βG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

MECHANISMS OF CARDIOTOXIN III-INDUCED APOPTOSIS IN HUMAN COLORECTAL CANCER COLO205 CELLS

1 Cardiotoxin III (CTX III) is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III in human colorectal cancer Colo205 cells. 2 Cardiotoxin III‐induced Colo205 cell apoptosis was confirmed by DNA fragmentation (DNA ladder and sub‐G1 formation) with an IC50 of 4 mg/mL at 48 h. 3 Further mechanistic analysis demonstrate that CTX III induced the loss of mitochondrial membrane potential (Dym), cytochrome c release from mitochondria into the cytosol and activation of capase‐9, caspase 3, as well as markedly enhancing the expression of Bax, but not Bcl‐2, protein in the cells. Moreover, the CTX III‐induced apoptosis was significantly blocked by the broad‐spectrum caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone. 4 However, CTX III did not generate the formation of reactive oxygen species and anti‐oxidants, including N‐acetylcysteine, and catalase could not block CTX III‐induced apoptosis in the Colo205 cells. 5 Taken together, these results suggest that CTX III may induce apoptosis through a mitochondrial‐ and caspase‐dependent mechanism and alteration of Bax/Bcl‐2 ratio in human colorectal Colo205 cancer cells.

Structure-function studies on Taiwan cobra long neurotoxin homolog.

A novel long neurotoxin homolog was purified from Naja naja atra (Taiwan cobra) venom using the combination of ion exchange chromatography and reverse phase high performance liquid chromatography. The determined protein sequence was essentially the same as that deduced from the cDNA amplified by reverse transcriptase-polymerase chain reaction. The long neurotoxin homolog exhibited an activity that inhibited acetylcholine-induced muscle contractions, as with N. naja atra cobrotoxin. The degree of inhibition caused by the addition of long neurotoxin homolog was approximately 70% of that observed with the addition of cobrotoxin. Unlike the well-known short and long neurotoxins, this neurotoxin homolog contained two additional cysteine residues forming a disulfide linkage in the N-terminal region. Circular dichroism measurement and computer models of the neurotoxin reveal that its secondary structure was not abundant in beta-sheet as noted with short and long neurotoxins. This less ordered structure may be associated with the lower activity noted with the long neurotoxin homolog. Together with the finding that the known long neurotoxin homologs exclusively appear in the venoms of the Naja and Bungarus genera, the long neurotoxin homologs should represent an evolutionary branch from the long and short neurotoxins in the Elapidae family.