A Study On Mechanism Of Cell Cycle Arrest In Hepatocellular Carcinoma Cells Induced By Melittin

Background:Hepatocellular carcinoma (HCC) is one of the most common cancer in China. Because of its high mortality,it is widely known as "the king of cancer". HCC usually develops in the presence of continuous inflammation and hepatocyte regeneration in the setting of chronic hepatitis and cirrhosis, although the molecular mechanisms linking chronic inflammation to malignant transformation remain to be further defined. Treatment of HCC is complex. Involvement of surgery,chemotherapy and radiotherapy, but up to now there has been no satisfying result. The majority of patients with HCC presented with an advanced stage beyond surgical treatment.In addition,chemotherapy and radiotherapy have limited efficacy in advanced hepatocellular carcinoma. So it is important to explore effective drugs and combination methods for therapy of this disease.The cell cycle consists of four distinct phases:G1,S,G2 and M phase. Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs), determine a cell's progress through the cell cycle. Two families of genes, the cip/kip family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of26 amino acid residues in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic. It has been reported that melittin has multiple effects, inculding antibacteria, antivirus and anti-inflammation, in various celltypes. We and others have shown that melittin can induce growth inhibition and apoptosis in various tumor cells.We also demonstrated that melittin can prevent liver cancer cell metastasis through inhibition of the Racl-dependent pathway and can Sensitize Human Hepatocellular Carcinoma Cells to TRAIL-induced Apoptosis by Activating CaMKⅡ-TAK1-JNK/p38 and Inhibiting IκBαKinase-NFκB. In our trial experiment,we observed that melittin can inhibit the growth of HCC at a relative lower concentration, meanwhile we couldn't observe obvious apoptosis or necrocytosis. However, the mechanisms of this effect of melittin have not been fully elucidated. So we design this study to uncover the underlying mechanism of this effect.We demonstrated that melittin can alter the cell cycle distribution in HCC cells,and the portio of S phase increased sharply after the melittin stimulation. We inferred that melittin may manipulate the expression of some important genes,which are indispensable in the control of cell cycle.Methods:(1)We first observe the anti-tumour activity of melittin on HCC cell line. MTT assay was used to determine the growth inhibition effect of melittin.(2)Apoptosis effect induced by melittin was carried out by flow cytometry. (3) Distribution of melittin-treated HCC cells in the different phases of the cell cycle was analyzed by FCM. (4) the expression of cell cycle regulatory protein such as CyclinA and CyclinDl were detected by Western blotting.Results:(1)On the concentration ranging from 0.5ug/ml to 5 ug/ml, melittin exhibited significant anti-hepatocarcinoma activity in a concerntration-dependent manner in vitro,and the growth inhibition rate is up to 50%.(2) Melittin on the concentrations of 0.5ug/ml to 5 ug/ml could induce slightly apoptosis of HCC cell lines,which cannot fully explain its anti-tumor effect at these concentrations.(3) The FACS analysis suggested melittin can cause S phase arrest in HCC cells, the number of cells in S phase increased after stimulated for 24h, consistently, the number of cells in the phase increased about 20% after the stimulation of melittin for 48 hours.(5)Stimulated by melittin for 30 minutes, the protein level of CyclinA and CyclinDl decreased sharply in HCC detected by western blotting analysis,while the expression of p27 was-upregulated up to five fold compared with blank.Conclusions:Melittin could cause S phase accumulation in HCC cells at relative lower concentration,which may partly contribute to its growth inhibitory effect.The cell cycle arrest was partly due to the change of protein level of CyclinA,CyclinDl and p27,but the further investigation is needed to uncover the underlying mechanism.