The Antitumor Activity And Mechanism Of HN-1 From Amolops Hainanensis On Breast Cancer

In recent years,cancer has been the first killer of human with the increasing morbidity and mortality.In women worldwide,breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death.Presently,breast carcinoma is mainly controlled by precise surgery,chemo-and radio therapy,which provide relatively favourable prognosis for long-term survival,but it is still the most deadly cancer in women because of advanced tumor growth or distant metastasis.Chemotherapy is one of the mainstay medical disciplines specifically devoted to cure cancer.Conventional chemotherapeutic agents are cytotoxic by interfering with cell division.Thereby they usually associated with significant and cumulative systematic toxicities,and more likelihood that cancer cells become chemo-resistant.Therefore,new breakthroughs are expected in the drug therapy of tumors.Anticancer peptides(ACPs)are the bioactive peptide with antitumor activity and found in many organisms,including mammals,amphibians,insects,plants and microorganisms.ACPs have been suggested as promising agents for antitumor therapy due to their numerous advantages over other chemical agents such as their low molecular masses,relatively simple structures,greater specific cytotoxicity to tumor cells over healthy cells,fewer adverse reactions,ease of absorption,a variety of routes of administration and low risk for inducing multi-drug resistance.Besides,ACPs can improve the curative effect of tumor by combinating with other traditional cancer treatments or anti-cancer molecules.Herein,lately the immunostimulatory agents including cytokines and antibodies,especially the latter commonly called "checkpoint inhibitors'',have been garnering significant attention for their impressive anti-tumor therapeutics.Moreover,great interest has been drawn to develop combinatorial immunotherapeutic interventions by combining chemo-or radiotherapeutic regimens with immunotherapy.In current work,we studied the mechanism of anticancer and immunomodulatory of anticancer peptide HN-1,providing some theoretical basis for research and development of peptide-based anticancer drugs.The main work of this thesis is as follow:(1)HN-1 was determined to possess anticancer activity and neglectable cytotoxicity in vitro and in vivo.By in vitro screening the antitumor effect of HN-1,we discovered that HN-1 displayed anticancer activity against various cancer cell lines.MCF-7 showed the most sensitivity to HN-1 treatment.Moreover,HN-1 also exerted remarkable cytotoxicity against MDR human breast cancer cell line MCF-7/ADR.However,HN-1 showed almost ignorable cytotoxicity on normal cells.Furthermore,HN-1 notably inhibited tumor growth in a xenograft breast tumor model with negligible toxicity.The antitumor efficacy of HN-1 in vivo was next evaluated on nude mice xenografted with human MCF-7 cells.Administration of HN-1 every other day led to a significant decrease in tumor volume,and H&E staining showed no pathological toxicity and adverse effects to the main organs.(2)HN-1 induced caspase-independent apoptosis in vitro and in vivo.HN-1 induced apoptosis of MCF-7 cells.Then ELISA and Western blot proved that HN-1 induced MCF-7 apoptosis without caspase activation.Moreover,HN-1 activates a p53/Bax/Bcl-2 signaling cascade that triggers AIF mediated apoptosis.The results of Western blot,siRNA,flow cytometry and immunofluorescence showed that HN-1 increased p53 expression,accompanied by induction of Bax and reduction of Bcl-2 which induced the destruction of ??m.Then HN-1 induced nuclear translocation of AIF that trigger chromatin condensation and DNA fragmentation.And ROS-MAPK and NF-?B signaling pathways also participated in caspase-independent apoptosis caused by HN-1.(3)HN-1 induced anti-cancer immunity in a 4T1 murine breast cancer model without systemic toxicity.4T1 breast cancer models were established a in BALB/c mice,and HN-1 effectively suppressed the tumor growth and spleen index.The results of flow cytometry,ELISA and Immunohistochemistry showed that HN-1 significantly increased CD4-T cells percentage in spleen,regulated the levels of cancer immunity-associated cytokines/chemokines,regulated the infiltrating of CD4+T cells and macrophage in solid tumors and reduced the blood capillaries in HN-1-treating tumor sections.(4)HN-1 showed safe biodistribution in a 4T1 murine breast cancer model.The results of small animal in vivo imaging system showed that FITC-HN-1 fluorescence was observed exemplarily in tumor,spleen,intestine,kidney and bladder.The results revealed HN-1's good breast tumor target and low drug accumulation in normal tissues.Altogether,our findings illuminated the antineoplastic molecular mechanism of HN-1 and induced anti-cancer immunity in a 4T1 murine breast cancer model.These findings provided a new template for development of antineoplastic agents against breast cancer and more drug targets for research and development of peptide-based anti cancer drugs.