Role Of Chemokines And Their Receptors In Hepatocellular Carcinoma Invading Portal Vein

Hepatocellular carcinoma (HCC) is the fifth most common neoplasm in the world, and the third most common cause of cancer-related death. Although clinical advances have been made in the fields of medical imaging, surgery, regional cancer therapy and biotherapy, the overall outcome of patients with HCC remains poor. The major obstacle to effective treatment is the high rate of metastasis and recurrence.HCC is characterized by its propensity for vascular invasion. HCC trends to invade portal vein, while it rarely invades hepatic vein. The presence of portal vein invasion is the most consistently reported major risk factor for recurrence after resection of HCC. It is widely accepted that intrahepatic metastasis through the portal venous system is an important mechanism for intrahepatic recurrence. Invasion of the portal vein is an important biological feature of HCC and a key prognostic factor that influences therapeutic effects and survival of patients. Therefore, research on the mechanism of portal vein invasion by HCC is pivotal to improve the therapeutic effects.Many factors have been demonstrated to be involved in portal vein invasion by HCC, which include oncogenes, proteases, adhesion molecules, motility stimulators and angiogenesis. However, all the factors could not explain the tendency of HCC invading portal vein.The interaction between tumor and host has consistently existed. The specific organ microenvironment determines the extent of cancer cell proliferation, angiogenesis, invasion, and survival. Therefore, both tumor and host should be considered in clarifying the mechanism of portal vein invasion by HCC.The microenvironments between portal area and central vein area are different. Regional distribution of chemokines was found in portal area. In the non-inflamed human liver, several chemokines, including regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and IL-8, can be detected in portal area and the expression of these chemokines increases markedly with inflammation.Chemokines are a family of chemoattractant cytokines that cause the directed migration of leukocytes. Chemokine signalling results in the transcription of target genes that are involved in cell invasion, motility, interactions with the extracellular matrix (ECM) and survival. Recent reports suggest their roles in cancer invasion and metastasis. To date, very few data are available about the effects of chemokines on human HCC migration and invasion.Part I : Expression of chemokine receptors and their ligands inhepatoma cells and tissuesThe aim of this study is to detect expression of chemokine receptors and their ligands in hepatoma cells and tissues. Gene expression profile of chemokine receptors was determined by RT-PCR, and then the expression and distribution of the typical chemokine receptors in HCC and adjacent liver tissues was investigated by RT-PCR, Western blot and immunohistochemical staining. The expression and distribution of their ligands were detected by immunohistochemical staining. Our results showed that hepatoma cells expressed several chemokine receptors. The expression profile of chemokine receptors in different hepatoma cells was varied. Among 19 chemokine receptors, CCR1 was detected in all examined hepatoma cell lines, and the expression level in metastatic cell lines was higher than that in poor metastatic cell lines. CCR1 mRNA and protein could be detected in HCC and adjacent liver tissues. The expression level of CCR1 was up-regulated in HCC tissues compared with that in adjacent liver tissues. Immunohistochemical staining revealed that CCR1 protein was mainly located on the membrane of HCC cells. The expression level of CCR1 mRNA in HCC was significantly correlated with absence of tumor capsule (P<0.05) and portal vein invasion (P<0.01) , while was not correlated with gender, age, serum AFP, tumor size or TNM staging. The moderate to strong staining of CCR1 protein was noted in 80.0% HCC specimens with portal vein invasion, which was significantly higher than that in HCC without portal vein invasion (PO.05). RANTES, MlP-la, the ligands of CCR1, were detected in inflammatory cells in the portal area in adjacent liver tissues. In conclusion, HCC cells consistently express CCR1 chemokine receptor. The expression level of CCR1 is correlated with HCC invasiveness, the ligands of CCR1 were expressed majorly in portal area in adjacent liver tissues. The findings indicate that CCR1 may play an important role in HCCinvading portal vein and serve as a potential marker to evaluate the invasiveness and prognoses of HCC.Part II: Effects of chemokines on hepatoma cells migrationand invasionWe have demonstrated that CCR1 consistently expressed in HCC cells both in vitro and in vivo, and the ligands of CCR1 were distributed in portal area in adjacent liver tissues. To investigate the effects of chemokines on HCC migration and invasion, the ability of migration, adhesion and degeneration of hepatoma cells was evaluated after chemokines exposure. Both RANTES and MlP-la showed strong chemotactic effects on MHCC97H cells in chemotaxis assay, and could prominently promote the polymerization of F-actin and formation of pseudopodia in the studies of fluorescent staining by confocal microscopy and flow cytometry. Pretreatment with antibody of CCR1 or RANTES, MlP-la could antagonize the above effects. Chemoinvasion assay showed that RANTES, MlP-la could increase the invasiveness of hepatoma cells. RANTES, MBP-la pretreatment enhanced the binding of liver cancer cell lines to human umbilical vein endothelial cells and fibronectin. In gelatin zymography assays, we found RANTES caused an increase in the activity of MMP-2 and MMP-9. The present study revealed that chemokines in portal area could regulate HCC migration direction and invasiveness. Blocking the interaction of chemokines and their receptors could inhibit the invasive and metastatic potential of HCC.Part IE: Molecular mechanisms of chemokines regulating HCCinvasivenessPrecious studies have demonstrated that chemokines in portal area could regulate HCC migration direction and enhance invasiveness of HCC. The purpose of the present study was to investigate the molecular mechanisms of chemokines regulating HCC invasive potential. The different gene expression profile between MHCC97H cells with chemokine stimulation and MHCC97H cells without chemokine stimulation was identified by the GEArray Q series human tumor metastasis gene array. Realtime PCR was used to further verify some genes expression. Among the 99 metastasis related genes, 33 genes were up-regulated in MHCC97H cells after...