B chain is a functional subunit of beta-bungarotoxin for inducing apoptotic death of human neuroblastoma SK-N-SH cells.

beta-Bungarotoxin (beta-Bgt), a presynaptic phospholipase A(2) (PLA(2)) neurotoxin isolated from the venom of Bungarus multicinctus, consists of A chain and B chain. The goal of the present study is to explore the functional contribution of the two subunits to the toxicity of beta-Bgt. beta-Bgt was found to induce apoptotic death of SK-N-SH cells via elevating intracellular Ca(2+) and intracellular ROS production. Moreover, an activation of p38 MAPK was associated with the cytotoxicity of beta-Bgt. SB202190 (p38 MAPK inhibitor), N-acetylcysteine (antioxidant reagent), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) (Ca(2+) chelator) and the inhibitors of Ca(2+) release from intracellular depots (ruthenium red and 2-aminoethoxydiphenyl borate) effectively attenuated the cytotoxicity of beta-Bgt. In sharp contrast to the inability of A chain, B chain was able to induce cytotoxic effects on SK-N-SH cells as beta-Bgt did. Abolishment of PLA(2) activity did not significantly alter the cytotoxic activity of beta-Bgt. MK801 (an NMDA receptor antagonist), antibodies against NMDA receptor and 4-aminopyridine (a potassium channel blocker) markedly reduced the cytotoxic effects of beta-Bgt, B chain and catalytically inactivated beta-Bgt. Moreover, antibodies against NMDA receptor blocked the binding of rhodamine-labeled beta-Bgt to SK-N-SH cells. Taken together, our data indicate that B chain is a functional subunit responsible for the cytotoxicity of beta-Bgt, and suggest that the cytotoxicity of beta-Bgt is mediated by NMDA receptor and potassium conductance.     

DC-81-Indole conjugate agent induces mitochondria mediated apoptosis in human melanoma A375 cells

DC-81, an antitumor antibiotic produced by the Streptomyces species, belongs to pyrrolo[2,1-c] [1,4]benzodiazepine (PBD), which are potent inhibitors of nucleic acid synthesis. We previously reported an efficient synthesis of PBD hybrids linked with indole carboxylates. This is the first demonstration on the mechanism of the anticancer effect of PBD hybrid (IN6CPBD) agent on human melanoma A375 cells. IN6CPBD-treated cells exhibited higher cytotoxicity than DC-81 and displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, a degradation of caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage. Because degradative changes associated with apoptosis are often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in IN6CPBD-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with IN6CPBD resulted in the loss of mitochondrial membrane potential (DeltaPsimt), a decrease in intracellular pH (pHi), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and cytochrome c release. Collectively, our studies indicate that IN6CPBD induces apoptosis in A375 cells through a mitochondrial dysfunction pathway, leading to caspase-3 substrate PARP cleavage and subsequent apoptotic cell death.     

Synthesis and Anti-inflammatory Effect of Chalcones and Related Compounds

Purpose. Mast cell and neutrophil degradations are the important players in inflammatory disorders. Combined with potent inhibition of chemical mediators released from mast cells and neutrophil degranulations, it could be a promising anti-inflammatory agent. 2,5-Dihydroxychalcone has been reported as a potent chemical mediator and cyclooxygenase inhibitor. In an effort to continually develop potent anti-inflammatory agents, a novel series of chalcone, 2- and 3-hydroxychalcones, 2,5-dihydroxychalcones and flavanones were continually synthesized to evaluate their inhibitory effects on the activation of mast cells and neutrophils and the inhibitory effect on phlogist-induced hind-paw edema in mice. Methods. A series of chalcones and related compounds were prepared by Claisen-Schmidt condensation of appropriate acetophenones with appropriate aromatic aldehyde and the anti-inflammatory activities of these synthetic compounds were studied on inhibitory effects on the activation of mast cells and neutrophils. Results. Some chalcones showed strong inhibitory effects on the release of -glucuronidase and histamine from rat peritoneal mast cells stimulated with compound 48/80. Almost all chalcones and 4-hydroxyflavanone exhibited potent inhibitory effects on the release of -glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP). Some chalcones showed potent inhibitory effects on superoxide formation of rat neutrophils stimulated with fMLP/cytochalasin B (CB) or phorbol myristate acetate (PMA). 2,3-Dihydroxy-, 2,5-dihydroxy-4-chloro-, and 2,5-dihydroxychalcone showed remarkable inhibitory effects on hind-paw edema induced by polymyxin B in normal as well as in adrenalectomized mice. Conclusions. These results indicated that the anti-inflammatory effects of these compounds were mediated, at least partly, through the suppression of chemical mediators released from mast cells and neutrophils.     

Naja nigricollis CMS-9 enhances the mitochondria-mediated death pathway in adaphostin-treated human leukaemia U937 cells

1. The aim of the present study was to explore the effect of the Naja nigricollis phospholipase A(2) CMS-9 on adaphostin-induced death of human leukaemia U937 cells. 2. Leukaemia U937 cells (Bcr/Abl-negative cells) were treated with adaphostin (0-10 μmol/L) and CMS-9 (0-1 μmol/L). The effects of CMS-9, adaphostin and their combination on cell viability, the generation reactive oxygen species (ROS), [Ca(2+) ](i) , p38 mitogen-activated protein kinase (MAPK) activation, Akt and extracellular signal-regulated kinase (ERK) inactivation, mitochondrial membrane potential (ΔΨ(m) ) and Bcl-2 family proteins were analysed. 3. Both adaphostin and CMS-9 induced U937 cell apoptosis, characterized by dissipation of ΔΨ(m) and ROS generation. Combined treatment further increased ΔΨ(m) loss and reduced the viability of adaphostin-treated cells. Unlike in CMS-9-treated cells, in adaphostin-treated cells ROS-induced increases in [Ca(2+) ](i) were observed. CMS-9-induced ROS generation resulted in p38 MAPK activation, whereas adaphostin treatment elicited ROS/Ca(2+) -mediated inactivation of Akt and ERK. Moreover, Akt was found to be involved in ERK phosphorylation. Suppression of p38 MAPK activation blocked CMS-9-induced ΔΨ(m) loss and Bcl-xL downregulation. Overexpression of constitutively active Akt and mitogen-activated protein kinase kinase (MEK) 1 rescued adaphostin-induced ΔΨ(m) loss and Bcl-2 downregulation. Similarly, CMS-9 augmented adaphostin toxicity in human leukaemia K562 cells via increased mitochondrial alterations. 4. The results suggest that two distinct pathways mediate adaphostin- and CMS-9-induced mitochondrial damage (i.e. the ROS-Ca(2+) -Akt-ERK and ROS-p38 MAPK pathways, respectively). These distinct pathway explain the augmentation by CMS-9 of ΔΨ(m) loss and apoptosis in adaphostin-treated U937 cells.     

Helium-neon laser irradiation stimulates cell proliferation through photostimulatory effects in mitochondria

Previous reports have shown that cellular functions could be influenced by visual light (400-700 nm). Recent evidence indicates that cellular proliferation could be triggered by the interaction of a helium-neon laser (He-Ne laser, 632.8 nm) with the mitochondrial photoacceptor-cytochrome c oxidase. Our previous studies demonstrated that He-Ne irradiation induced an increase in cell proliferation, but not migration, in the melanoma cell line A2058 cell. The aim of this study was to investigate the underlying mechanisms involved in photostimulatory effects induced by an He-Ne laser. Using the A2058 cell as a model for cell proliferation, the photobiologic effects induced by an He-Ne laser were studied. He-Ne irradiation immediately induced an increase in mitochondrial membrane potential (delta psi(mt)), ATP, and cAMP via enhanced cytochrome c oxidase activity and promoted phosphorylation of Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) expressions. He-Ne irradiation-induced A2058 cell proliferation was significantly abrogated by the addition of delta psi(mt) and JNK inhibitors. Moreover, treatment with an He-Ne laser resulted in delayed effects on IL-8 and transforming growth factor-beta1 release from A2058 cells. These results suggest that He-Ne irradiation elicits photostimulatory effects in mitochondria processes, which involve JNK/AP-1 activation and enhanced growth factor release, and ultimately lead to A2058 cell proliferation.     

Genetic organization of Bungarus multicinctus protease inhibitor-like proteins

The structural organization of the genes encoding Bungarus multicinctus protease inhibitor-like proteins (PILPs), PILP-1, PILP-2 and PILP-3, are reported in this study. Unlike PILP-2 and PILP-3, recombinant PILP-1 exhibited inhibitory activity on trypsin. PILP genes and B chain genes shared identical organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 chain and B2 chain genes were absent in that of PILP genes. Noticeably, intronic insertion in the second intron of B chain genes appeared in the promoter region of PILP-1 gene, but not in that of PILP-2 and PILP-3 genes. Comparative analyses of PILP genes and B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that PILP genes and B chain genes originate from a common ancestor, and that accelerated evolution may diversify PILP and B chain genes as that proposed for snake venom phospholipase A(2), neurotoxin and cardiotoxin genes.     

A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1α-mediated signaling

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1α (SDF-1α) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1α-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1α-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1α-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency.     

Effect of cholesterol on membrane-damaging activity of Naja nigricollis toxin γ toward phospholipid vesicles

Membrane-damaging activity of Naja nigricollis toxin γ on phospholipid vesicles was attenuated by incorporation of cholesterol into phospholipid vesicles. Deprivation of cholesterol from erythrocyte membrane enhanced notably hemolytic activity of toxin γ, while the hemolytic activity of toxin γ on cholesterol-depleted erythrocytes decreased when cholesterol was restored to membrane. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles were modestly affected in the presence of cholesterol. Time-resolved fluorescence and Fourier transform infrared spectra showed that phospholipid-bound toxin γ and cholesterol/phospholipid-bound toxin γ did not adopt the same conformation. Moreover, geometrical arrangement of toxin γ in contact with phospholipid vesicles was different from that with cholesterol/phospholipid vesicles as evidenced by N-(fluorescein-5-thiocarbamoyl)-1, 2-dihexadecanoyl-phosphatidylethanolamine fluorescence enhancement and color transformation of phospholipid/polydiacetylene membrane assay. Taken together, our data show that ordered phospholipid phase arising from incorporation of cholesterol affects conformation and topographical arrangement of toxin γ on water–lipid interface, thus attenuating its membrane-damaging activity against phospholipid vesicles.     

Biological evaluation of an antibiotic DC-81-indole conjugate agent in human melanoma cell lines

Pyrrolo[2, 1-c][1, 4]benzodiazepines (PBDs) are potent inhibitors of nucleic acid synthesis because of their ability to recognize and bind to specific sequences of DNA and form a labile covalent adduct. DC-81, an antitumor antibiotic produced by Streptomyces species, is a PBD. We combined DC-81 and an indole carboxylate moiety to synthesize a hybrid designed to have much higher sequence selectivity in DNA interactivity. In this paper, the cytotoxic potency of the hybrid in human melanoma cell lines was studied. XTT assay demonstrated that the DC-81-indole conjugate possessed cytotoxicity against human melanoma cell lines.