Effect Of Chronic Alcohol Consumption On The Progression And Metastatic Potential Of Hepatocellular Carcinoma

1. Introduction Hepatocellular carcinoma(HCC) is the fifth most common cancer worldwide and the third in China. HCC accounts for up to 90% of primary liver cancers with a poor prognosis. The mortality rates are accordingly high in relation to incidence. There are more than 500,000 new HCC patients. The incidence of HCC is 30.3 of 100,000 in China with the third leading cause of death. But the cause of liver cancer and exact molecular mechanism still is not entirely clear.According to epidemiological surveys, chronic alcohol ingestion is an important environmental etiology among the main risk factors for HCC, and the researchers get more and more attention about it. It is sufficiently evidenced that alcohol is assosicated with all digestive tract tumors including the liver and breast tumors.Many epidemiological studies have investigated that alcohol also may influence the progression from an adenoma to a carcinoma and may favor the development of high-risk polyps or cancer among patients with adenomas. However, the risk of different alcohol consumption on HCC progression and angiogenesis is little known and mechanism is not clear. Angiogenesis, the formation of new blood vessels from endothelial precursors, is a prerequisite for growth and progression of solid malignancies. To grow beyond microscopic size, both primary tumors and their micrometastases must initiate angiogenesis. Recently, biologic markers such as vascular endothelial growth factor(VEGF) related to tumor neovascularization have been identified that may provide prognostically useful information in the cancer management, such as oral and oropharyngeal squamous cell carcinoma. VEGF has been identified to promote tumor angiogenesis by stimulating endothelial-cell proliferation and promoting vascularpermeability. In addition, the inflammatory tumor microenvironment has many roles in tumor progression and metastasis, including creation of a hypoxic environment, increased angiogenesis and invasion. Monocyte chemotactic protein-1(MCP-1) has been recognized as an angiogenic chemokine and identified correlatively with the invasion and metastasis of tumor. Reactive oxygen species(ROS) is present in all aerobic cells, the surplus or deficiency of antioxidant scavengers can break the balance and lead to oxidative stress. Role of reactive oxygen species(ROS) in the process of malignancy is identified. Their mechanisms include oxidative stress induced oxidative injury, inflammation and lipid peroxidation. The balance between ROS and antioxidant is important to control tumor growth. The surplus or deficiency of antioxidant scavengers can break the balance and lead to oxidative stress which has a vital role on tumor progression. Higher level of intracellular ROS is a common feature in alcohol-associated injury resulted from cytochrome P450 enzymes. Many studies have confirmed that there is close relation of ROS with NF-κB. It is an important nuclear transcription factor closely related to malignant biological behaviour. Its activation can promote expression of tumor metastasis-related genes such as cell adhesion molecules(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), and metalloproteinase-9(MMP-9), etc. The activation of NF-κB is also involved in the cell proliferation, differentiation, migration, adhesion ability and the regulation of cycle and differentiation; associated with tumorigenic, tumor growth and transfer and other relevant process. The objective of the present study was two-fold: 1) to investigate the alcohol consumption in HCC patients and analyze the correlation between alcohol intake and pathological features and tumor malignant; 2) to explore the impact of alcohol on the malignant progress of HCC in vivo and in vitro. Here, this study is expected to investigate the mechanism of alcohol in the malignant progression of liver tumor and provide a new way and approach for their treatment.2. Objectives(1) To investigate the alcohol consumption in HCC patients and analyze the correlation between alcohol intake and pathological features and tumor malignant. And mesuring VEGF and MCP-1 expression between different groups and the activiation of NF-κB pathway to explore the possible role of chronic alcohol consumption in the development of HCC.(2) To establish mice drinking mode in vivo, and observe the effect of alcohol feeding on Hep G2 transplanted tumor growth in nude mice and lung metastasis; In vitro to further observe the effect of alcohol on proliferation of Hep G2 cells, migration capacity and angiogenesis; and to explore its possible mechanism.3. Materials and methods Part Ⅰ: Chronic alcohol consumption enhances the progression and metastasis of human hepatocellular carcinoma The medical records of 52 HCC patients who had been consecutively admitted to first hospital of Anhui Medical University were retrospectively analyzed between January 2009 and December 2009. All patients were diagnosed by pathology without any anti-cancer treatment before operation. Statistic analysis is made on alcohol consumption in HCC patients and mesure the expression of CD34, VEGF, and MCP-1 and NF-κB used by immunohistochemistry. Part Ⅱ: Alcohol induces the malignant progress of liver tumor and its mechanism Nude mice aged 5–6 weeks were divided into two groups: control and alcohol group(n = 7). Mices were given 2 % ethanol in drinking water day and night. The control group was provided with regular drinking water only. Hep G2 cell was implanted subcutaneously in nude mice. The size of the tumors was monitored every 2 days. When the tumors grow, the volume was calculated every 3 days. At the end of experiment, animals were killed and the tumors were removed. Some of the tumor tissues were fixed for HE and immunohistochemical studies for expressions of CD34,VEGF and MCP-1. Meanwhile, the lung tissues also are removed to observe the metastasis. In vitro 0.2% alcohol stimulates cultured Hep G2 cells. Groups were divided as followed: control and ethanol group. ROS level in Hep G2 cells was detected by flow cytometry; cytoplasm and nucleus of NF-κB p65, IκBα and p-IκBα protein expression were detected by Western blot assay. ERK and JNK pathways were also analyzed by Western blot. VEGF and MCP-1 m RNA and protein expression in supernatant were respectively measured by RT-PCR and ELISA. MTT method, scratch test and Transwell method were used to detect cell proliferation and migration/invasion ability, and three-dimensional angiogenesis in vitro model was made to observe angiogenesis. In addition, to evaluate effect of PDTC, a specific inhibitor of NF-κB on the transplanted liver tumor growth, the nude mice was injected with PDTC(100 mg/kg) intraperitoneally and tumor volume was calculated every three days.4. Results Part Ⅰ There were 52 HCC cases enrolled in our study. 45 cases were male and 7 females, aged 22 to 74 years old, with a median age of 50 and the average age was 49.8 ± 12.1. 24 patients have ever intake alcohol compared with 28 never-drinking patients.The rates of TNM stage and vessel invasiveness were significantly higher in the patients with alcohol intake than without alcohol intake(76.9 % vs 35.9 %, P =0.01, 88.9 % vs 37.4 %, P =0.014, respectively). Alcohol drinkers who consumed ≥ 35 g and < 87.5 g of alcohol per day had a significantly higher risk of tumor progression than nondrinkers(OR =16.67 and OR =33.7, respectively).Compared with non-drinking HCC patients, overall survival rates of drinking HCC patients at 12 and 36 months were 75 % vs 57.15 % and 62.5 % vs 35.7 % respectively. Compared with liver cirrhosis groups and distant non-cancerous tissues, AMVD was remarkably increased in HCC tissues with no drinking(42.6 ± 4.82 vs 27.2 ± 1.48 and 42.6 ± 4.82 vs 16.2 ± 4.43, P <0.01). AMVDwas maximal in HCC tissues with alcohol consumption(61.0 ± 6.78).Compared with non-drinking HCC patients, the VEGF/VEGFR-2 and MCP-1/CCR2 expression were higher. NF-κB was strongly expressed in hepatocellular carcinoma tissue and mainly located in the cytoplasm and nucleus of cancer cells presented with pale yellow to brown granular substance. Compared with HCC patients with no alcohol intake, NF-κB was stained deeper in cytoplasm and nucleus of drinking HCC tissues. Part Ⅱ Establish a drinking mice model and successfully copy hepatocellular carcinoma transplanted tumor model in nude mice. The weight of transplanted tumor in alcohol is higher than water control group(0.95 ± 0.1 vs 0.38 ± 0.03, P <0.05); the metastasis rate in alcohol group was significantly higher than in water control group(P <0.01). The microvessel density in alcohol group was significantly higher than in the water control group(64.6 ± 8.92 vs 34.3 ± 3.83, P <0.05). In addition, the expression of VEGF and MCP-1 in alcohol group was higher than in water group and PDTC group; and their expression was downregulated in alcohol+PDTC group. There is no statistical difference of Hep G2 cell proliferation between groups at 24 and 48 h after alcohol stimulation. Alcohol significantly accelerated Hep G2 cell to central scratches at 24 h and enhanced migration ability from the lower chamber to the upper chamber. Using a 3-D tumor cells endothelial cell co-culture system in vitro, the average sprouting rate was 27.3 % in HUVEC culture system alone, while the rate was 42.9 % in HUVEC and Hep G2 co-culture system. Compared with control medium, 0.2 % alcohol stimulation increases the average sprouting rate to 67.9 %(P <0.05). VEGF and MCP-1 m RNA levels in alcohol group were markedly increased in time-dependent manner. VEGF and MCP-1 protein levels in supernatant were significantly higher than in the control group at 12, 24 and 48 h after ethanol stimulation. Hep G2 cells were cultured in vitro and stimulated by alcohol with significantly increasing levels of intracellular ROS levels. P65 protein in Hep G2 cellcytoplasm stimulated by alcohol was significantly lower than the control group, while P65 protein expression in the nucleus was significantly higher than the cytoplasm. IκBα expression after alcohol stimulation was significantly downregulated with higher level of phosphorylation protein. Phosphorylation of ERK and JNK in 0.2 % alcohol group was significantly higher than in the control group, with a peak at 24 h and total ERK protein levels did not change significantly. VEGF and MCP-1 expression in tumor xenografts with alcohol intake were significantly higher than in the control group and PDTC group. PDTC treatments could downreguate expression of VEGF and MCP-1 and significantly alleviate the growth rate of the tumor.5. Conclusions(1) Chronic alcohol consumption can enhance the malignant progression in HCC patients. In addition, alcohols may activiate NF-κB pathway and upregulate the expression of VEGF and MCP-1 and contribute to the development of HCC.(2) In vivo, mice tumor model simulating human chronic drinking was successfully established. Alcohol promotes the transplanted tumor growth in nude mice breaing Hep G2 cells and lung metastasis; In vitro, alcohol can enhance proliferation of Hep G2, migration capacity and angiogenesis; and promote the malignant progression of HCC possibly through ROS mediated activation of NF-κB pathway and dependent of VEGF and MCP-1.