Novel Anti-tumor Polypeptide From Meretrix Meretrix Linnaeus And The Underling Molecular Mechanisms

Cancer is a major public health problem in the world. Currently, one in eight deaths is due to cancer. Over the past decade, a number of chemotherapy agents have offered significant advantages in the treatment of cancer. However, the effects of these drugs are often considered ineffective or excessively toxic. Therefore, novel, highly effective and lowly toxic drugs are urgently required. The biological and chemical diversity of the marine environment is immeasurable and therefore is an extraordinary resource for the discovery of new anticancer drugs. The marine mollusk Meretrix meretrix Linnaeus is a traditional Chinese medicine used for cancer. The purpose of current study was to purify a novel anti-tumor polypeptide from Meretrix meretrix Linnaeus and elucidate the underling molecular mechanisms.A novel anticancer polypeptide was extracted and purified from the coelomic fluid by ammonium sulphate fractionation, ion exchange chromatography, gel filtration and reversed phase chromatography. It gave a single band with a molecular mass of 15 KDa by SDS-PAGE and this peptide was designated Mere15. The effect of Mere15 on viability of tumor cell lines as well as benign cell lines was assessed by MTT assay. Results showed that Mere15 exhibited anti-tumor activity to a wide variety of human cancer cells lines in a dose-dependent manner. However, Mere15 did not significantly decrease the viability of benign cells. Besides, Mere15 significantly suppressed the growth of human lung adenocarcinoma A549 xenograft in nude mice without any gross abnormalities.In order to elucidate the possible mechanism, morphological changes of cancer cells were observed by phase contrast microscope, scanning electron microscope and Hoechst 33342 staining. Mitochondrial membrane potential assay, Annexin V-FITC/PI assay and DNA ladder assay were performed to study the apoptotic effects. Western blot analysis was performed to determine the apoptosis pathways. Flow cytometry was performed to measure the cell cycle arrest and tubulin immunofluorescence was used to detect the tubulin polymerization. Results showed that Mere15 inhibited the growth of cancer cells associated with G2/M phase arrest followed by apoptosis, including membrane blebbing, loss of mitochondrial membrane potential, externalization of phosphatidylserine, chromosome condensation and DNA fragmentation. Western blot analysis showed that both the intrinsic pathway and the extrinsic pathway were involved in Mere15-induced apoptosis.