The Anti-HCC Effect Of A Novel CD13 Small Molecule Inhibitor And Associated Mechanisms

ObjectHepatocellular carcinoma(HCC)is the fifth most common malignancy worldwide and ranks second among all the cancer-related mortalities,thus HCC is a serious threat to human life and health.At present,the incidence of liver cancer in the world is on the rise,the World Health Organization recently published "Global Cancer Report" shows that new cases of liver cancer and the number of deaths rank first in China around the world,accounting for about 50%.At present,the clinical treatment for liver cancer is mainly through surgical resection,chemical therapy,radiation therapy,biological therapy.However,owing to the high recurrence rate,metastasis rate,poor prognosis and other factors,the survival rate of patients with liver cancer is very low.Therefore,it is urgent to further improve the mechanism of liver cancer,to clarify its pathogenesis,to provide new ideas and treatment strategies for the clinical treatment of liver cancer.CD 13,also known as aminopeptidase N(APN),is a zinc-dependent membrane-bound type ? metalloproteinase that could bind to glycoproteins which are excised from the N-terminus of the oligopeptide.CD 13 is expressed in a variety of tissues,such as kidney,intestinal epithelial cells,nerve cells(synaptic membrane and pericytes),myeloid cells(monocytes,macrophages and dendritic cells),fibroblasts as well as endothelial cells.At the same time,CD 13 is highly expressed in acute myeloid leukemia cells and multiple solid tumor tissues(such as squamous cell carcinoma of the head and neck,lung cancer,liver cancer,breast cancer,ovarian cancer,colon cancer,etc.).Thus,the molecule is determined as a tumor marker or a tumor cell surface antigen.CD 13 exerts several biological functions,and it is involved in a variety of physiological and pathological processes,and can be also used as a marker of liver cancer stem cells.Therefore,using CD 13 inhibitors with poor selectivity and poor tissue sensitivity may affect the normal function of tissue,and even cause a variety of complications.For this reason,we should develop a specific targeting inhibitors for the clinical treatment,which is the biggest challenge of CD 13 inhibitor's research and development.Based on the above phenomenon,through in vitro and in vivo experiments,we studied the role of the new CD 13 small molecule inhibitor 4.1-3k in the treatment of HCC and elucidated its internal mechanism by taking the advantage of molecular biology,which provided a useful reference to clinical liver cancer treatment and new targeted therapy drug research.Methods1.Using FACs to detect the expression of CD13 in several HCC cell lines.2.MTT assay was used to detect proliferation of hepatocellular carcinoma cell treated with CD13 inhibitor,4.1-3k.Cell cycle and apoptosis were detected by PI staining,Annexin V/PI staining.RT-PCR was used to detect the expression of apoptosis gene Bcl-2,Bax in HCC cells treated with CD 13 inhibitor.3.Transwell was used to detect the migration of hepatocellular carcinoma cell treated with CD 13 inhibitor.RT-PCR was used to detect the expression of metalloproteinase MMP2 and MMP9 in HCC cells after treatment with 4.1-3k.4.Hepatocellular carcinoma cells were treated with CD 13 inhibitor at different dose,and then supernatant were extracted.Angiogenesis was detected by HUVEC.Real-time quantitative PCR was used to detect the expression of genes related with angiogenesis(VEGF and MCP-1)in HCC cells.5.Hepatocellular carcinoma cells were treated with CD 13 inhibitor,and then total proteins were extracted.Western blot was detected the activation of MAPK?NF-?B and STAT3 signalings.The expression of inflammatory factor IL-6 and IL-10 was assayed by Real-time quantitative PCR.6.In DEN/CCL4-induced C57BL/6 mice,HBV C57BL/6 mice,tumor-bearing C57BL/6 mice,CD 13 expression in different tissue was detect by FACs.Then survival after the treatment of CD 13 inhibitor in tumor-bearing nude mice was calculated.7.Lung metastatic progress was detected in tumor-bearing BALB/c mice after the treatment of CD 13 inhibitor.Results1.The expression of CD 13 differ in different carcinoma cells,and CD 13 expression in liver cancer cells with S antigen or HBV genome(eg,PLC/PRF/5 and HepG2.2.15 cells)was higher than that in HBV-free cell lines.2.HCC cells,especially HepG2.2.15,displayed significantly decreased proliferation ability after treated with CD 13 inhibitor,especially 4.1-3k.Cell apoptosis was elevated by CD 13 inhibitor disposed.CD 13 inhibitor could promote cell apoptosis,especially 4.1-3k,while cell cycle was not affected by 4.1-3k.3.Under the treatment of CD13 inhibitor,liver cancer cell migration was significantly decreased,and 4.1-3k performed a better effect than Bestatin.At the same time,the expression of metalloproteinase MMP2 and MMP9 decreased after treated with CD 13 inhibitor.4.After the treatment with CD 13 inhibitor,the formation of angiogenesis was significantly inhibited.4.1-3k exerted a better activity than Bestatin in CD 13 highly expressed hepatocellular carcinoma cell lines.At the same time,angiogenesis-related genes VEGF and MCP-1 were down-regulated in liver cancer cells treated with CD 13 inhibitor.5.CD13 inhibitor treatment could restrain MAPK,NF-?B and STAT3 signaling and the expression of related-genes was decreased in liver cancer cells.At the same time,inflammatory factor IL-6 and IL-10 was down-regulated in liver cancer cells treated with CD 13 inhibitor.6.In DEN/CCL4 induced model and HBV model,CD 13 was highly expressed in immunosupressive cells,such as Treg and MDSC.After treatment of CD 13 inhibitor,the growth of tumor in tumor-bearing nude mice was inhibited and the survival time was prolonged.7.The number of pulmonary nodules was significantly reduced by CD 13 inhibitor treatment in the BALB/c lung metastatic model mice,and 4.1-3k was more meaningful than Bestatin.ConclusionIn this study,we studied the anti-hepatocarcinogenesis of CD 13 inhibitor 4.1-3k and elucidated its intrinsic mechanisms.We found that 4.1-3k could promote tumor cell apoptosis and inhibited tumor cell proliferation,migration and angiogenesis,thus affected the formation and progression of tumor.In addition,CD 13 inhibitor has an inhibitory effect on the tumor development in tumor-bearing mice and tumor metastasis,so that increased survival of tumor-bearing mice.Therefore,CD 13 can be used as a therapeutic target for the treatment of liver cancer,and 4.1-3k might be a candidate for developing new drugs.These findings provided basic data for developing more effective treatment of liver cancer new drugs.