Purification And Characterization Of A Potent Anti-tumor Protein From Meretrix Meretrix Linnaeus

Most of current anti-tumor chemotherapies acted against normal mammalian cells, consequently causing severe side effects. Marine organisms represented an essentially unexploited reservoir for products of potentially biological and pharmacological interest. So far, literature on natural products derived from marine organisms was characterized by low cytotoxity and new mechanisms. Meretrix Meretrix Linnaeus was a traditional Chinese drug with anti-tumor activities. In the present studies, an anti-tumor protein was purified from the cavity fluid of Meretrix Meretrix, and the possible anticancer pharmacological action modes were also investigated.The purification procedure consisted basically of ammonium sulphate fractionation, two cycles of ion exchange chromatography and hydrophobic interaction chromatography. During the process, MTT colorimetric method was employed to trace the active component. The highly purified protein, designated MML, is a single band of 40 kD revealed by SDS-PAGE. It displayed remarkable in vitro toxicity to the tumor cells BEL-7402, but no toxicity to normal fibroblasts 3T3.The experiment revealed that the IC50 on BEL-7402 was about 52μg/mL.The cell death induced by MML was characteristic of cell morphological change and became apparent at nanomolar concentrations. Multiple approaches, including scanning electron microscopy, Hoechst33342/ PI dual staining, LDH leakage, cell cycle arrest assay, tubulin immunofluorescence and DNA ladder, were employed to investigate the possible pharmacological anti-tumor action modes of MML. The results showed that MML treatment could change the permeability of cell membrane, and the growth inhibition effect was perhaps correlated with the arrest of these cells in G2/M and tubulin depolymerization.