Transarterial Embolization Alone Or In Combination With Arsenic Trioxide: The Influence On Metastatic Potential Of Hepatocellular Carcinoma

Part 1Increased metastatic potential and epithelial-mesenchymal transition of hepatocellular carcinoma after hypoxia in McA-RH7777 cellsPurpose:Transarterial chemoembolization (TACE) has been established as the standard care for patients with nonsurgical hepatocellular carcinoma (HCC). But hypoxia could be induced after TACE, and that might by correlated with migration and invasion. In the study, we examined whether hypoxia enhances the metastatic potential of residual HCC and investigated the mechanisms related to epithelial-mesenchymal transition (EMT).Materials and methods:The hepatoma cell line McA-RH7777, which is marked by green fluorescent protein (GFP), was used in the study. Hypoxia was induced with cobalt chloride (CoCl2), and the effective concentration was determined using a Cell Counting Kit-8 assay (CCK-8). The invasion of cells cultured under hypoxia and normoxia was observed using the Transwell assay. Comparing the distance to quantify the migration ability of the cells by wound-healing assay. The molecular changes of tumor cells in hypoxia and normoxia were evaluated by Western blotting or Real-Time PCR.Results:Hypoxia was successful induced with 100μmol/L CoCl2 at 24h, as indicated by the increase in hypoxia-inducible factor-la (HIF-1α) expression, and no significant differences in cell proliferation and apoptotic rates between two groups at 24h. The invasion assays indicated that the number of invading hypoxic cells was significantly higher than that of normoxic cells (30.2±2.46 vs.20.4±1.89, P=0.013). Enhanced wound healing capacity of tumor cells was showed after hypoxia. Accompanying increase of HIF-la expression, the metastatic potential of hypoxic tumor cells was enhanced. The enhancement of metastatic potential was indicated by a significant reduction in the expression of E-cadherin and an up-regulation of N-cadherin and Vimentin, which results in EMT. The level of Twist mRNA in hypoxic cells was significant increased compared with the normoxic controls.Conclusion:The increased metastatic potential and EMT in McA-RH7777 cells were induced after hypoxia, which can upregulate of the Twist.Part 2Arsenic trioxide suppress epithelial-mesenchymal transition induced by hypoxia via inhibiting the transcription factor Twist in McA-RH7777 cellsPurpose:Arsenic trioxide (ATO) has been found effectively in several types of cancer cells, including acute promyelocytic leukemia, and recently in hepatocellular carcinoma (HCC). In this study, we investigated the influence of ATO on metastatic potential of McA-RH7777 cells and underlying molecular mechanisms in vitroMaterials and methods:The hepatoma cell line McA-RH7777, which is marked by green fluorescent protein (GFP), was used in the study. Cells growth was evaluated using a Cell Counting Kit-8 assay (CCK-8). Cells apoptosis was observed with transmission electron microscope (TEM) and analysed using flow cytometer (FCM). Hypoxia was induced with cobalt chloride (CoCl2,100 μmol/L), and cell migration and invasion were observed using wound-healing and Transwell assay. Then the molecular changes of E-cadherin, N-cadherin, and Vimentin of tumor cells were determined by western blot. The effects of ATO on Twist activity of the tumor cells were further analyzed.Results:ATO inhibited cells growth and induced apoptosis in a time-and dose-dependent manner. ATO significantly promoted apoptosis at 48h, whether or not the cells were hypoxic (23.95±1.44% vs.25.97±1.46%; P=1.000). No significant differences in apoptotic rates were showed between hypoxic and normoxic group treated with ATO (2 μmol/L) for 24h. The results of the wound-healing and Transwell assay showed that ATO markedly reduced cell migration and invasiveness which were enhanced by hypoxia. Western blot analysis revealed lower expression of the epithelial cell marker E-cadherin in the hypoxic cells (as indicated by the increase in HIF-la expression) than in the normoxic cells. In addition, the levels of the mesenchymal cell markers Vimentin and N-cadherin increased; however, ATO inhibited the phenotypic changes and expression of epithelial-mesenchymal transition (EMT) markers by suppressing Twist. The marked suppression effect of ATO on invasiveness and metastatic potential related to EMT was also shown by immunofluorescence.Conclusions:The results of this study demonstrated that ATO have the effect of antagonizing hepatocellular carcinoma cells migration and invasion, inhibiting EMT induced by hypoxia via down regulation of transcription factors Twist.Part 3Transarterial embolization alone or in combination with Arsenic trioxide:the influence on tumor metastatic potential in a rat HCC modelPurpose:In the animal study, we examined whether transarterial embolization (TAE) enhances the metastatic potential of hepatocellular carcinoma (HCC), and further investigated the role of ATO in regulating the invasive activity of HCC after TAE.Methods:All protocols met National Institutes of Health guidelines, and they were approved by the animal research committee of Fudan University. The hepatoma cell line McA-RH7777, which is marked by green fluorescent protein (GFP), was injected subcutaneously into the right hindlimb of each rat to induce solid tumors. Forty male buffalo rats implanted with McA-RH7777 tumor in the liver were randomly divided into four groups:TAE+ATO, TAE, ATO, and control. TAE procedures were performed by retrograde placement of a catheter into the gastroduodenal artery on the 14th day after implantation. The TAE+ATO and TAE groups received an intra-arterial injection of iodized oil (0.2 ml/kg) that had been mixed with double the volume of saline. Three days after the interventional procedures, groups A and C were treated with ATO (2 mg/kg) intraperitoneally once a day for five days, and then three times a week for another week. All of the control treatment received an equivalent amount of saline. Tumor volumes were measured before and after treatment using magnetic resonance imaging (MRI). On the 14th day after the interventional procedures, a second MRI examination was performed to evaluate the volume of the tumors. Five randomly selected rats from each group were euthanized, and the livers and lungs were harvested for histopathological studies. Lung metastases were observed using fluorescence imaging, and the molecular changes of residual tumor cells were evaluated by western blotting or immunohistochemistry. The remaining five rats in each group were kept for survival and side effects observation survival analysis.Results:None of the animals died during implantation or during the postoperative period. As shown on the pretreatment MR images, solitary liver tumors grew successfully in the left liver lobe of all 40 rats; the rate of tumorigenicity reached 100%. Tumor sizes on the T2WI image reached 8.16±0.09 mm in diameter (201.80±7.68 mm3) two weeks after implantation and showed low signals on T1WT and high signals on T2WI. TAE led to the enhanced expression of epithelial-mesenchymal transition (EMT) markers and promoted lung metastasis, despite inhibited tumor growth and a prolonged overall survival rate in the animals. The marked suppression effect of ATO on invasiveness and metastatic potential related to EMT was shown in tissue by western blotting and immunohistochemistry. Furthermore, ATO inhibited EMT by suppressing Twist.Conclusion:The metastatic potential of HCC was increased after TAE. In addition, ATO is an effective anticancer agent in combination with TAE in the treatment of HCC, by suppressing tumor progression and metastasis via arresting EMT by inhibiting the Twist activation.