Cardiotoxin III induces c-jun N-terminal kinase-dependent apoptosis in HL-60 human leukaemia 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. The molecular effects of CTX III on HL-60 cells were dissected in the present study. We found that the antiproliferative action of CTX III on HL-60 cells was mediated through apoptosis, as characterized by an increase of sub G1 population, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. Upregulation of Bax, downregulation of Bcl-2, the release of mitochondrial cytochrome c to cytosol and the activations of capase-9 and -3 were noted, while CTX III had no appreciable effect on the levels of Bcl-X(L) and Bad proteins. Moreover, c-Jun N-terminal kinase (JNK) was activated shortly after CTX III treatment in HL-60 cells. Consistently, the SP600125 compound, an anthrapyrazolone inhibitor of JNK, suppressed apoptosis induced by CTX III. As expected, this JNK inhibitor also attenuated the modulation of Bax and Bcl-2, as well as the cytosolic appearance of cytochrome c and the activation of caspase-3 and caspase-9 that induced by CTX III. These findings suggest that CTX III can induce apoptosis in HL-60 cells via the mitochondrial caspase cascade and the activation of JNK is critical for the initiation of the apoptotic death of HL-60 cells.

Main-chain dynamics of cardiotoxin II from Taiwan cobra (Naja naja atra) as studied by carbon-13 NMR at natural abundance: delineation of the role of functionally important residues.

Cardiotoxin analogue II (CTX II) is an all beta-sheet, small molecular mass (6.8 kDa), basic protein possessing a wide array of biological properties. Nearly complete assignment of the protonated carbon resonances has been achieved by heteronuclear NMR experiments. The study shows that the correlation between the carbon-13 chemical shifts and CTX II structure is good in general, but interesting deviations are also noticed. To characterize the internal dynamics of CTX II, longitudinal, transverse relaxation rates and heteronuclear 13C{1H} NOEs were measured for alpha-carbons at natural abundance by two-dimensional NMR spectroscopy. Relaxation measurements were obtained in a 14.1 T spectrometer for 50 residues, which are evenly spread along the CTX II polypeptide chain. Except for five alpha-carbons, all data were analyzed from a simple two-parameter spectral density function using the model free approach of Lipari and Szabo. The microdynamical parameters (S2, taue, and Rex) were calculated with an overall rotational correlation time (taum) for the protein of 4.8 ns. For most residues, the alpha-carbons exhibit fast (taue < 30 ps) restricted libration motions (S2 = 0.79-0.89). The present study reveals that the functionally important residues located at the tips of the three loops are flexible, and the flexibility of residues in this region could be important in the binding of cardiotoxins to their putative "receptors" which are postulated to be located on the erythrocyte membrane. In addition, the results obtained in the present study support the earlier predictions on the relative role of the lysine residues in the erythrocyte lytic activity of cardiotoxins.