| BackgroundHepatocellular carcinoma (HCC) is the most common cancer and the second most common cause of cancer death in our country, which may be an early warning of serious problems to people’s health. A number of epidemiological studies have shown that chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection are as the risk factor for hepatocellular carcinoma, and followed by alcoholism, aflatoxin contamination of food are closely related to HCC infections. Many molecules studies have been showed that the metastasis and recurrence dominant aetiological factor in hepatocellular carcinoma development, which was a multifactorial complex multi-step process that involves a complex series of intracellular changes. In addition, due to the hidden onset, low rates of early diagnosis, most patients diagnosed with stage 2 HCC tumors, which lost the effective treatment time. Nevertheless, despite substantial improvements in surgery and other drug treatments, overall 5-year survival rate of patients with HCC is very low. One of the most important reasons leading to poor survival rate could be related to high rate of recurrence and metastasis after curative resection and liver transplantation. The clinical treatment of hepatocellular carcinoma drugs targeting too single-minded, only for a certain factors in the mechanisms of hepatocellular carcinoma. In addition, patients prone to drug resistance and adverse reaction, as a whole cannot obtain satisfactory therapeutic effect. Therefore, developed new anti-cancer drug, which is an important theoretical significance and application value for a big country of HCC.Cathepsin S (CTSS/Cat S), a member of the lysosomal cysteine cathepsin family (Cat B, C, F, H, K, L, O, S, V, W and X) that is known to play important roles in cell proliferation, angiogenesis, tumor growth, and metastasis. CTSS first isolated from bovine lymph nodes and spleen by Kirschke in 1986. The CTSS are mainly distributed in the spleen, lymph nodes, heart, and antigen presenting cells, and CTSS, Cat H, Cat L, Cat B respectively 57%,41%,31% sequence homology. CTSS is one of the most important proteases, which mainly expression in the lymphoid organs. Previous studies have shown that CTSS is closely connected with human tumors, such as liver cancer, pancreatic cancer, breast cancer, pancreatic cancer etc. Previous studies have shown that CTSS are mainly located in lysosomes, which can be translocated to the cell surface and secreted into the extracellular milieu, which involved in the regulation of apoptosis, arterial extracellular matrix (ECM) fibronectin, collagen, elastin proteins, and matrix metalloproteinase-2 (MMP-2). In addition, silencing CTSS gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro. Therefore targeted inhibition of CTSS expression can significantly inhibit tumor angiogenesis. Claire Ward et suggest that inhibition of CTSS activity have a synergistic effect with anti-VEGF. It has been demonstrated that angiogenesis plays a key role in tumorigenesis, invasion and metastasis. Thus, inhibition of VEGF/VEGFR2 has been pursued as a therapeutic target in HCC. Chinese medicine is a rich resource in our country. Thus, the animal and plant extract bioactive component have been reported the role of anti-hepatoma angiogenesis, and widely used in clinical practice. Basis on preliminary studies, we chose Melittin monomer (bee venom effective active ingredient) as the object of study.Melittin an amphiphilic peptide (26 amino acid residues) isolated from honeybee Apis mellifera. Melittin also induces structural alterations of membranes including pore formation, fusion, and vesiculation. It has been reported that Melittin has multiple effects, including anti-bacteria,anti-virus,and anti-inflammation. It was demonstrated that Melittin can induce cell cycle arrest, growth inhibition and apoptosis in various tumor cells. However, the mechanisms of the anti-cancer effects of Melittin have not been fully elucidated.Our previous studies have demonstrated the effects of CTSS on the tumor and invasion of HCC cells. In addition, we showed that the CTSS-target siRNA inhibited expression of CTSS in human MHCC97-H cells, leading to suppression of cancer cell proliferation, invasion and angiogenesis, as well as inducing apoptosis. In recent years, it has been found that Melittin inhibits various HCC cell proliferations and induce apoptosis and inhibit tumor growth. This suggests that venom inhibits vascular endothelial cells to form new blood vessels. Therefore we further determined whether Melittin could inhibit CTSS-induced tumor growth and angiogenesis in vitro and in vivo.ObjectsTo investigate Melittin inhibits CTSS-induced tumor growth and angiogenesis through blocking VEGF-A/VEGFR2/MEK1/ERK1/2 pathway in liver cancer cells.Methods1. The expression levels of CTSS in different HCC cell lines was determined by RT-PCR and Western blot analysis, screening high expression CTSS of MHCC97-H hepatoma cell line.2. To establishment of the shRNA-CTSS cell line, MHCC97-H cells were stably transfected with either shRNA-CTSS or an empty vector.3. Determining MHCC97-H cell growth, invasion and angiogenesis with in vitro clonogenic assay, MTT assay, flow cytometry assay, Transwell chamber assay, scratches assay.4. Determining Melittin prevents stable transfected MHCC97-H cell growth, invasion and angiogenesis with in vitro clonogenic assay, MTT assay, Transwell chamber assay, scratches assay and ELSIA assay.5. HUVECs were transfection with CTSS-RNA(CTSS-HUVECs) or NC-RNA (Mock-HUVECs), determining Melittin suppresses CTSS-induced migration and invasion of HUVECs6. The in vivo models used were orthotopic transplanted nude-mouse tumor models of human hepatocellular carcinoma metastasis established with shRNA-CTSS/MHCC97-H cells or with Mock/MHCC97-H cells, determining Melittin inhibits CTSS-induced tumor growth and associated angiogenesis.Results1 RT-PCR and Western blot analysis were used to detect CTSS levels in 8 cell lines, of which higher levels were enriched in MHCC97-H cells compared with that in the other cell lines.2. shRNA-CTSS significantly inhibits the expression of CTSS in stable transfected MHCC97-H cell, and inhibit shRNA-CTSS/MHCC97-H cells proliferation, colonal formation, apoptosis, migration, and invasion, compared to controls (p< 0.05).3. Melittin prevents Mock/MHCC97-H cells invasion, metastasis and angiogenesis through inhibition the activity of CTSS. It can induce Mock/MHCC97-H cells colonal formation, migration, secretion of VEGF, and invasion, compared to controls (p< 0.05).4. Melittin inhibits the activation of CTSS-mediated signaling pathways in stable transfected MHCC97-H cells. The expression of the CTSS, VEGF-A, Ras, p-Raf, p-MEK1, and p-ERK1/2 proteins was significantly down-regulated as a result of Melittin treatment, compared to controls (p<0.05).5. Melittin inhibits CTSS-induced invasion and angiogenesis of HUVECs. CTSS-RNA significantly increased the expression of CTSS in HUVECs. It can induce HUVECs migration and angiogenesis with Melittin treatment, compared to controls (p<0.05).6. Melittin inhibits CTSS-induced tumor growth and associated angiogenesis in vivo. Melittin significant decrease in tumor weight and tumor volume and suppressed activation of CTSS, VEGFR2, VEGFA, p-Ras, p-Raf, p-MEK1, p-ERK1/2 proteins, compared to controls (p< 0.05). Furthermore Melittin inhibit percent positive CD34 cells in Mock/MHCC97-H nude mice, compared with controls (p< 0.05).ConclusionsSilence CTSS significantly affected the biological behavior of MHCC97-H cells; Melittin may inhibition of angiogenesis and tumor growth by regulation CTSS through blocking VEGF-A/VEGFR2/MEK1/ERK1/2 pathway, suggesting that Melittin may be a CTSS inhibitor, which is expected to become a potential treatment of liver cancer drugs. |
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