The Venom Of The Spider Macrothele Raveni Inhibits Cell Growth Through Triggering Cell Apoptosis

[Objective] To analyze the cytotoxic activity of the venom of the spider Macrothele raveni and to elucidate its molecular mechanisms。[Methods] Seven cell lines, K562,normal human lymphocytes,HBL-100,MDA-MB-231,HepG2,A549 and HEK-293, were used in this study.①The cell growth inhibition of the venom on the cells was assayed by CCK-8 regeant;②Cell morphological changes under the venom treatment would be observed under fluorescentmicroscope by using DAPI staining;③DNA ladder detection was used to show the DNA fragmentation of venom treated cells;④Annexin V-FITC/PI dual staining was also applied to determined the cell apopotosis ratio;⑤The activities of caspase2,3,6,8,9 were messured by caspase activity assay kit;⑥The expression level of PARP was determined by western blotting;⑦The protein expression difference between venom and mock treated cells were analyzed by SILAC strategy.[Results] This venom potently inhibited the growth of K562,normal human lymphocytes,HBL-100,MDA-MB-231,HepG2,A549,HEK-293 in a dose and time dependent manner; With the trentment of the venom, the cells would undergo typical morphological changes of apoptosis, such as cell condensation,DNA fragmentation,apoptotic bodies, et.al, indicating that the venom was able to triger cell apoptosis. By using Annexin V-FITC and PI dual staining, it was revealed that more than 90% cells were apoptotic with the treatment of 10μg/mL venom. Machenically, at least two caspases, caspase 3 and 8 have been observed to be activated as cells treated with the venom. In addition, PARP, one of the caspase 3 substrates, was cleavaged. This means that the caspase cascade would involve in the cell apoptosis induced by the venom. By using protemocis study,36 apotosis related proteins were also identified.[Conclusion] The venom of spider Macrothele raveni potently and selectively suppresses the growth of K562,normal human lymphocytes,HBL-100,MDA-MB-231,HepG2,A549,HEK-293 cells by inducing apoptosis. Caspase signaling pathway might be one of the machenisms.