Induction of CTX-d from cobra venoms on NB4 apoptosis and its mechanism / 中草药

ABSTRACT

Objective To investigate the effects of CTX-d from venoms of cobra (Naja naja atra) on inducing NB4 apoptosis and its mechanism. Methods MTT was used to detect the antitumor effect of CTX-d in vitro; Electron microscope and flow cytometry were used to observe the apoptotic inducing effect of CTX-d in NB4 cells; Mitochondrial transmembrane potential change (??m) was analyzed by flow cytometry; The levels of caspase-9, caspase-3, and cytochrome C in the cytosol fraction were analyzed by Western blotting. Results The IC50 values of CTX-d affected on NB4 cell for 6 and 12 h were 1.8 and 1.35 ?g/mL, respectively. CTX-d could induce morphological changes, such as condensed chromatin and swelling mitochondria in NB4 cells. Analyzed by flow cytometry, CTX-d induced apoptosis in NB4 cells evidenced by increasing sub G1 cell population in a dose- and time-dependent manner. The mitochondrial membrane potential of NB4 cells had already decreased when incubated with CTX-d (1.0 ?g/mL) for 0.5 h, and cytochrome C in the cytosol was detected simultaneously, which indicated the release of cytochrome C from mitochondria to cytosol. The caspase-9 was activated initially at 1 h after 1.0 ?g/mL CTX-d treatment, whereas the cleavage of caspase-3 was detected at 0.5 h. This suggested that some other mechanism may be involved in caspase-3 activation. Conclusion The results suggest that the loss of mitochondrial membrane potential and the release of cytochrome C from the mitochondria into the cytosol are the early events of CTX-d on NB4 apoptosis. Once release into the cytosol, cytochrome C precedes the activation of caspase-9 and -3 to leading to the apoptosis and there are maybe some other mechanism involved in caspase-3 activation.