| Cytotoxins are the major active proteins found in cobra venoms and constitute up to about 40% of total protein in crude venom. They are composed of 60-63 amino acid residues with the mol. wt 6-7kDa. They exhibit a wide array of biological activities, such as (1) depolarize and contract excitable tissues: skeletal, cardiac muscle and nerve; (2) lyse tumor cells; (3) inhibit the activity of enzymes, such as the Na+,K+-ATPase, Ca2+-ATPase, protein kinase C(PKC). Various snake species in various districts contain different types of cytotoxin isoforms. It was found that 2-5 cytotoxin isoforms could be simultaneously isolated from the venom of a single snake species. A lot of studies have verified that CTX isoforms exhibit antitumor effects in vitro and in vivo, wherase they all put emphasis on the study of one certain CTX and did not compare antitumor effects and toxicities of several CTX isoforms from a single snake species. In this study, we isolated and purified four CTX isoforms from the venom of Naja atra and compared their antitumor effects(zw vitro and in vivo) and toxicities in order to sift out one high-efficient and low-toxic CTX. Isolation and Purification of CTXIn this study, the crude venom of cobra venom(Naja atra) was isolatedon SP-Sephadex C-25 cation-exchange column and fractionated into 14 protein peaks. The tenth ~ thirteenth fractions could induce the rat isolated LangendorfFs preparations to decrease their contracture amplitudes, ultimately leading to complete cardiac arrest. They acted on the rat isolated phrenic nerve-diaphragm preparations and indicated no Nerve-Muscle block but could inhibit direct contracture of diaphragm. Fraction X~XIII were further purified by Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography once each, exhibited no detectable phospholipase A2 (PLA2) activity with the pH-stat technique. They migrated as a single band on SDS-polyacrylmide gel electrophoresis (SDS-PAGE) by Coomassie brilliant blue stains which showed that they were homogeneous with one band respectively. The mol. wt of them were calculated to be 7.284, 7.332, 7.235 and 7.381 kDa orderly by Image Master YDS. The results showed that the fraction X~XIII were CTXs and then we named them CTX-X-XIII respectively. Assay oftoxicityAssays of toxicities of CTX were used , one based on approximate LD50 (i.v.) values in mice by Meier's method, the other on the minimal lethal dose inducing 100% death of animals(MLD). The i.v. LD50 values in mice of CTX-X~XIII showed to be 1.16 ?0.12, 1.80 ?0.21, 0.75 + 0.21, 1.85?.15ug/g body weight orderly. Assay of MLD wasperformed by administering CTX-X~XIII into rats (i.v.) at constant velocity. The MLD values showed to be 183 ?18, 310 ?26, 224 ?16, 343?8u,g/kg orderly and had significant difference in analysis of variance (PO.001). Assay of cytotoxicity in vitroThe inhibitions of CTX-X~XHI on cultured Bel7404 cells and HL-60 human leukemia cells were observed and the IC5o values were assayed by SRB(Sulforhodamine B) method. The results appeared that CTX-X~ XIII potently inhibited cell proliferation of Bel7404 and HL-60 cells. Furthermore, it showed to be good dose-effect relationship, their IC5o(24h) values for inhibition of Bel74o4 cell proliferation, in increasing order, were CTX-XII(1.12u.g/ml) < X(2.22ug/ml) < XIII(2.46ug/ml) < XI(2.97ug/ml). The IC50(24h) values for inhibition of HL-60 cell proliferation , in increasing order, were CTX-XII( 1.6 lug/ml) < X(1.97ug/ml) < XIII(2.40ug/ml) < XI(2.6lug/ml). As a result, the cytotoxicity of CTX-X梄III to cultured tumor cells, in decreasing order, was CTX- XII >X>XIII>XI. Antitumor activity in vivoThe antitumor effects in vivo by administering CTX-XII and CTX-XIII intraperitoneally on uterus cervix cancer U14-bearing mice and on Ehrilich ascites carcenoma EAC-bearing mice were observed. The results showed that the antitumor effect of CTX-XIII was greaterthan that of CTX-XILFour purified CTX isoforms from the Naja atra venom containing no PLA2 activity... |
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