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To explore the role of hypoxia-inducible factor-1α (HIF-1α) in formation of multidrug resistance (MDR) induced by microenvironment and to find a new and effective molecular target on preventing and reversing chemoresistance in hepatocellular carcinoma (HCC). In HepG2 cells exposed to hypoxia, low glucose or transfected by plasmid pcDNA3/HBX, the expression of HIF-1α mRNA and protein was respectively detected using real-time fluorescent quantitative PCR and Westernblot technique and its expression localization was investigated by immunocytochemical technique. Plasmid pcDNA3/HIF-1α was transfected into HepG2 cells and then the expression of multidrug resistance related genes mdr1, multidrug resistance-associated protein 1 (MRP1) and lung resistance protein (LRP) in transfected cells was determined by the same methods. In HepG2 cells respectively exposed to hypoxia, low glucose or transfected by plasmid pcDNA3/HBX, HIF-1α was overexpressed at mRNA and protein levels to varying degrees and translocated into nucleus. The gene expression levels of mdr1, MRP1 and LRP in HepG2 cells transfected by plasmid pcDNA3/HIF-1α were respectively increased by 2.4±0.2, 2.2±0.3 and 2.3±0.4 folds as compared with those in non-transfected HepG2 cells (all P

Accumulating evidence demonstrates that the microenvironment of the host has an important effect on the chemoresistance of tumors. We also found that the formation of intrinsic multidrug resistance is related to environmental factors that are common with tumor growth of hepatocellular carcinoma. The aim of this study was to explore the molecular mechanisms by which multidrug resistance of hepatocellular carcinoma is induced by the microenvironment. In particular, the regulation of nuclear transcription factor (hypoxia-inducible factor-1α, HIF-1α) activation in the process of multidrug resistance formation was investigated.HepG2 cells were exposed to different microenvironmental conditions respectively, such as hypoxia, stimulation of glucose deprivation and transfection of plasmid PcDNA3/HBx. In the HepG2 cells, the expression of the related MDR proteins, HIF-1α protein expression and localization, activity of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) were detected. Specific inhibitor U0126 was used to block ERK/MAPK signal pathway, the alteration of HIF-1α and the related MDR proteins were investigated. Multivariate analysis of variance (MANOVA) repeated measures and one-way analysis of variance (ANOVA) followed by Tukey test or t-test were used to determine differences over time and effects of the treatments.The above three microenvironment factors increase the expression of the related MDR proteins (including P-gp, LRP, and MRP1) and induce MDR of HepG2 cells. HIF-1α was induced at the protein and mRNA levels and the nuclear translocation was also increased. The activity of ERK/MAPK was also increased in HepG2 cells. But when ERK/MAPK pathway was inhibited, the mRNA and protein expression of MDR1, MRP1, and LRP was to some extent decreased. Inhibition of ERK/MAPK significantly reduced activated HIF-1α protein and the nuclear translocation of HIF-1α, whereas HIF-1α mRNA levels were not affected.The microenvironmental factors could induce MDR of HepG2 cells by the activity of HIF-1α. The activity of HIF-1α is regulated by the ERK/MAPK pathway at the phosphorylation level. As an important nuclear transcription factor, HIF-1α controls the transcription of MDR-related genes and the synthesis of their corresponding proteins by ERK/MAPK signal pathway in HepG2 cells.

To analyze the effect of different hepatic vascular exclusions for massive hemorrhage in hepatectomy.The clinical data of 2238 cases with hepatectomy treated from January 1995 to August 2009 was analyzed retrospectively in the cause of massive hemorrhage (blood loss ≥ 1000 ml), blood loss during liver resection and massive hemorrhage incidence with different methods of hepatic vascular exclusion.Among 2238 cases received hepatectomy, 215 cases (9.6%) had massive hemorrhage because of portal vein tumor thrombus extraction (26.0%), extensive adhesions around the tumor (24.7%), section of liver hemorrhage (23.7%), hepatic vascular injury (15.8%), and tumor rupture (9.8%). Among 2182 cases received hepatectomy without portal vein tumor thrombus extraction, 159 cases (7.3%) had massive hemorrhage, 1257 cases (57.6%) which blood loss were less than 400 ml. Hepatectomy with different hepatic vascular exclusion methods had different blood loss and massive hemorrhage incidence.Pringle combined with clamping infrahepatic vena cava method and the liver double-hanging maneuver through the retrohepatic avascular tunnel on the right of the inferior vena cava method can reduce blood loss and massive hemorrhage incidence in hepatectomy more effectively, especially for huge liver tumor resection.

To evaluate the impact of preoperative three-dimensional visualization and virtual liver surgery planning on hepatic resection.All relevant structures (livers, portal vein, hepatic veins, and tumors) were extracted from multislice CT scans of 142 cases treated from May 2007 to May 2009. By the liver surgery planning system software Liv 1.0, reconstruction and image analysis of the relevant structures was performed and virtual resections of liver were carried out. Data were correlated to intraoperative findings.(1) Three-dimensional visualization revealed the spatial relationship of tumors to the intrahepatic vascular system, thus giving impressions how the neoplasms were situated. Virtual tumor resections corresponded to the intraoperative findings. (2) With the planning, an intended resection could be performed virtually and optimal identification of resection margins could be achieved. The ischemia and congestion territory within the remaining liver parenchyma could be calculated. Simulation resections could avoid liver parenchyma over resection and maintain a sufficient amount of liver tissue to sustain hepatic function. Virtual simulations of tumor resection were used successfully to plan of surgical procedures in the hepatic tumors. Hepatectomy was performed in 29 cases after virtual tumor resections but seemed impossible with conventional CT scan. Resection plans of 92 cases were optimized after virtual resections. (3) The mean liver volume of patients with primary hepatocellular carcinoma measured by the software and the real resected was (477 +/- 223) ml and (451 +/- 209) ml respectively. Comparison by means of linear regression analysis between volume measurement on the software and the real resected showed a nearly ideal correlation coefficient (R = 0.922, P0.01). The mean error was 6.1%.The three-dimensional tumor visualization and virtual simulation of tumor resections of the software Liv 1.0 provide an important reference for a valuable planning of complex hepatic resections. It is not only benefit to improve the predictability and security of hepatectomy but also helpful to improve the success rate of complex hepatic resections.

To elucidate intracellular signal pathway in formation of multidrug resistance (MDR) of hepatocellular carcinoma (HCC) induced by its microenvironment, and to explore the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process.Activity of ERK/MAPK was examined by Western blot technique through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein in HepG2 cells exposed to hypoxia, low glucose or transfected by plasmid pcDNA3/HBX. After being treated by the specific ERK/MAPK pathway inhibitor U0126, Western blot technique was used to analyze the alterations of the expression of P-gp, MRP1, LRP and HIF-1alpha at protein level. RT-PCR was used to analyze the alterations of the expression of HIF-1alpha mRNA. Cellular location of HIF-1alpha protein was determined by immunocytochemistry after being treated by U0126.The activations of ERK/MAPK determined by the ratio of phosphorylated ERK/MAPK to the total ERK/MAPK were increased in varying degrees in HepG2 cells respectively exposed to different microenvironment. After being treated by U0126 for 12 h, the expressions of mdr1, MRP1, LRP genes and protein in those cells were decreased to some extent. However, the gene expression of HIF-1alpha was not influenced and only its protein was decreased. HIF-1alpha protein was reversely translocated into cytoplasm from nucleus after being treated by U0126.ERK/MAPK pathway is involved in the course of the formation of MDR of HCC induced by microenvironment.

AIM: To investigate expression and significance of inhibitor of apoptosis protein survivin in hepatocellular carcinoma (HCC). METHODS: The expression of survivin and vascular endothelial growth factor (VEGF) was investigated in 38 cases of HCC tissues and 38 liver cirrhosis tissues by immunohistochemistry and Western blot. The relationship between the expression of survivin and clinicopathological factors of HCC was analyzed. RESULTS: Survivin protein was detected in 23 (60.5%) of 38 HCCs and 3 (7.9%) of 38 liver cirrhosis tissues. In 23 cases of HCC which expressed survivin, the expression of VEGF was positive in 18 cases and slight positive or negative in 5 cases. While in 15 cases of HCC which did not express survivin, 12 cases did not express or slightly expressed, and 3 cases expressed VEGF. In liver cirrhosis tissues, the expression of VEGF was as follows: 24 cases were negative, 10 cases were weak positive and 4 cases were strong positive. The expression of survivin was coincident with the expression of VEGF in HCC (P < 0.01). The expression of survivin in HCC had no relationship with the patients’ age, gender, tumor size and differentiation level of HCC, while it was related to the metastasis of HCC. The protein quantitative analysis by Western blot also showed that overexpression of survivin in HCC was closely correlated to the expression of VEGF (P < 0.01). Furthermore, stronger expression of survivin and VEGF was also found in patients with metastasis rather than in those with no metastasis (P < 0.01). CONCLUSION: Survivin plays a pivotal role in the metastasis of HCC, and it has some correlation with tumorigenesis. The expression of survivin in the primary lesion is very useful as an indicator for metastasis and prognosis of HCC. It could become a new target of gene therapy of HCC.

To explore the mechanism of multidrug resistance of hepatocellular carcinoma induced by hypoxia and the potential role of hypoxia-inducible factor-1 alpha (HIF-1 alpha) and multidrug resistance related genes.Human hepatocarcinoma cell lines HepG2 cells were exposed to hypoxia and were transfected by plasmid HIF-1 alpha/PCDNA3, respectively. The expressions of multidrug resistance gene (mdr1), multidrug resistance protein (MRP1), and lung resistance protein (LRP) gene at the mRNA and the protein levels in the above two groups were respectively analyzed by real-time fluorescent quantitative PCR and Western-blot technique.In the hypoxia group, the expressions of mdr1, MRP1 and LRP were stepped up correlating to the degree of hypoxia, especially the prominent increase in the expression of MRP1. Furthermore, they were synchronous with the changes of the expression of HIF-1 alpha. Also the increased expression of mdr1, MRP1, and LRP gene was observed in transfected HepG2 cells by plasmid HIF-1 alpha/PCDNA3.Resistance of hepatocellular carcinoma to chemotherapeutics could be induced by hypoxia. HIF-1 alpha may be critical to the upregulation of the expression of the related multidrug resistance genes induced by hypoxia. HIF-1 alpha and these related multidrug resistance genes could be potential molecular targets for reversing multidrug resistance of hepatocellular carcinoma.

AIM: To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes: multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process. METHODS: A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure. RESULTS: Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P < 0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold). Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the transfected cells decreased to some extent. Compared with controls, a significant decrease in MDR-1 mRNA (53.3%), MRP-1 mRNA (59.7%) as well as LRP mRNA (56.4%) was observed in the U0126 treated transfected cells after 12 h. Western blot also demonstrated that the protein expression of these MDR associated genes slightly reduced after treated with U0126 for 12 h (MDR-1 40.1%, MRP-1 29.4%, LRP35.7%). This change was accompanied with the rise of cell apoptosis ratio confirmed by Annexin V-PI detection. The apoptosis index of U0126-treated cells increased by 1.28 fold, compared with that of transfected cells. Obviously, the MDR phenotype of these cells was obviously related with increased activities of the ERK/MAPK pathway. CONCLUSION: HBx protein might be one of the causes for the occurrence of MDR in HCC, and ERK/MAPK pathway might be involved in this change.