Research On The Anti-Tumor Mechanism Of Musca Domestica Pupae To Hepg2Cells

Lectins, a class of carbohydrate-binding non-immunogen proteins, possess the ability to agglutinate cells or precipitate polysaccharides. Lectins are found in all kinds of species and in a diversous of tissues of higher organisms. In recent years, some lectins are famous for their antitumor activity, immunomodulatory activity, antibacterial and other activities.A new lectin (42kDa) from Musca domestica pupae (MPL-3) was purified from Musca domestica pupa by affinity chromatography column (Sepharose4B) and anion exchange chromatography (DEAE). MPL-3has been known to inhibit proliferation in tumor cells. However, the molecular mechanism involved in MPL-induced cell apoptosis is still unclear.In this study, MPL-3inhibited HepG2cells proliferation in a dose-dependent manner with48h of46.10μg/mL (IC50). The results of HE staining and Hoechst33342staining showed that MPL-3induced a typical apoptosis in HepG2cells, such as nuclear chromatin condensation and cell rounding. Flow cytometry analysis also confirmed that apoptosis was involed in HepG2cells and showed the cell was arrested in S phase. MPL-3evoked reactive oxygen species (ROS) accumulation, and MPL-induced cell apoptosis was prevented by NAC (an inhibitor of ROS), suggesting involvement of ROS generation in MPL-induced HepG2cells apoptosis. MPL-3also induced an activation of phospho-JNK, and MPL-induced cell apoptosis also was prevented by SP600125(an inhibitor of JNK). Western blotting results revealed that MPL-3also inhibited the NF-κB/p65translocation from cytoplasm into nucleus, which was confirmed by immunofluorescence staining. The phosphorylation of JNK could be blocked by NAC, but not PDTC (an inhibitor of NF-κB); SP600125and NAC reversed the change of NF-κB; PDTC (an inhibitor of NF-κB) and SP600125had no significant effect on the ROS formation. Simultaneously, the expressions of FLIP and Bcl-2(target genes of NF-κB) were down-regulated, and the caspase-8and caspase-3were then activated.Taken together, the molecular regulation of MPL-3on HepG2cells apoptosis may be performed of intracellular ROS formation, activation of JNK phosphorylation, inhibition of the translocation of NF-κB from cytoplasm into nucleus, suppression of the FLIP and Bcl-2expression and activation of caspase cascade.