Search

To investigate midkine (MK) protein expression in human hepatocellular carcinoma (HCC) and its relation to dissemination of cancer cells in blood. Circulating tumor cells were detected in preoperative blood samples using reverse transcription polymerase chain reaction (RT-PCR) for alpha-fetoprotein (AFP) mRNA. Midkine expression was immunohisto-chemically examined in surgically resected HCC tissues. Forty-one HCC patients with positive serum AFP were studied. Positive AFP mRNA expression was shown in 18 preoperative blood samples (43.9%) and midkine overexpression was detected in 29 excised tumors (70.7%). Among the 29 patients, 18 (62.1%) showed positive AFP mRNA in their preoperative blood samples. In contrast, the positive AFP mRNA expression was only observed in 3 out of 12 patients (25.0%) without aberrant midkine expression. Statistical analysis showed that abnormal midkine expression in the tumors was a variant significantly associated with the presence of tumor cells in the blood circulation (P

To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC).Peripheral blood samples were collected from healthy subjects, patients with HCC or various other cancers, and patients with hepatic lesions or hepatitis. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation. The remaining cells were cytocentrifuged on polylysine-coated slides. Isolated cells were treated by triple immunofluorescence staining with CD45 antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1 (CPS1), used as liver-specific markers, and costained with DAPI. The cell slide was imaged and stained tumor cells that met preset criteria were counted. Recovery, sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines. Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining, respectively.CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR(+) selection (Ps0.05). The expression rates of either ASGPR or CPS1 were different in various liver cancer cell lines, ranging between 18% and 99% for ASGPR and between 9% and 98% for CPS1. In both human HCC tissues and liver cancer cell lines, there were a few HCC cells that did not stain positive for ASGPR or CPS1. The mixture of monoclonal antibodies against ASGPR and CPS1 identified more HCC cells than either antibody alone. However, these antibodies did not detect any tumor cells in blood samples spiked with the human breast cancer cell line MCF-7 and the human renal cancer cell line A498. ASGPR(+) or/and CPS1(+) CTCs were detected in 29/32 (91%) patients with HCC, but not in patients with any other kind of cancer or any of the other test subjects. Furthermore, the improved method detected a higher CTC count in all patients examined than did the previous method (P = 0.001), and consistently achieved 12%-21% higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs.Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells.

To investigate the effects of icaritin, an active ingredient extracted from Epimedium Sagittatum (Sieb. et Zucc.), on CCl4-induced liver injury and its possible mechanisms. Hepatocytes isolated from Sprague-Dawley male rats were treated with 3 mmol/L CCl4 for 24 h to induce acute liver cell injury, then icaritin (0.1, 1, 10, 100 μmol/L, respectively) was administrated to the cells, and estrogen receptor antagonist ICI182,780 (1 μmol/L) was co-treated with 10 μmol/L icaritin. Biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), and superoxide dismutase (SOD)) and cell apoptosis were detected to evaluate the injury degree. Protein expressions of Bax, Bcl-2, liver fatty acid-binding protein (L-FABP), and peroxisome proliferator-activated receptor-α (PPAR-α) as well as reactive oxygen species (ROS) generation were determined by western blot. Icaritin alleviated CCl4-induced liver cell injury in a concentration-dependent manner and 10 μmol/L was the optimal concentration. Icaritin (10 μmol/L) significantly reduced activities of ALT, AST in cell culture medium and MDA level of the impaired liver cells, but increased the intercellular SOD activity. The apoptotic rate of the impaired liver cells was also decreased by icaritin (10 μmol/L) treatment. Icaritin might exert antioxidative and anti-apoptotic functions via estrogen-like effect, as the ratio of Bcl-2/Bax was significantly increased, while protein expressions of L-FABP and PPAR-α were markedly increased, and this function was blocked by the estrogen receptor antagonist ICI182,780 efficiently. Icaritin may be a promising drug candidate for acute liver injury benefiting from the antioxidative and anti-apoptotic functions via estrogen-like effect.

Liver metastases represent the major cause of death in patients with colorectal cancer (CRC). Recent studies have suggested that the chemotactic responses of tumor cells are necessary for metastatic spread to the liver, and CCL20 and CXCL8 have a strong association with CRC metastasis. The aim of our study was to identify the mechanisms by which CCL20 and CXCL8 synergize to promote metastatic progression and evaluated their potential as prognostic markers for CRC patients. The abilities of CCL20 and CXCL8 to promote CRC cell progression and epithelial–mesenchymal transition (EMT) phenotype were analyzed in vitro. Possible signaling pathways were investigated with specific pathway inhibitors and small interfering RNA (siRNA). 213 Patients with CRC who underwent surgery were enrolled for analysis of CCL20, CXCL8 and E-cadherin expressions in tumor tissues. Prognostic factors were then identified. CCL20 or CXCL8 alone was not sufficient to induce complete EMT in CRC cells, but both of them could coordinately induce EMT-like phenotype that was required to maintain CRC cell proliferation, migration and invasion. PI3K/AKT-ERK1/2 pathway crosstalk was demonstrated to be responsible for this process. Coexpression of CCL20 and CXCL8 was negatively correlated with E-cadherin expression in human CRC tissues. CRC patients with coexpression of CCL20 and CXCL8 were more likely to develop liver metastases and both coexpression was an independent high-risk factor for a most poor prognosis. CCL20 and CXCL8 synergize to promote CRC metastatic progression by coordinated induction of EMT via PI3K/AKT-ERK1/2 signaling axis. Detection of both coexpressions can be used to predict clinical outcomes in CRC patients.

Background OCT4 and BIRC5 are preferentially expressed in human cancer cells and mediate cancer cell survival and tumor maintenance. However, the molecular mechanism that regulates OCT4 and BIRC5 expression is not well characterized. Methods By manipulating OCT4 and BIRC5 expression in hepatocellular carcinoma (HCC) cell lines, the regulatory mechanism of OCT4 on BIRC5 and CCND1 were investigated. Results Increasing or decreasing OCT4 expression could enhance or suppress BIRC5 expression, respectively, by regulating the activity of BIRC5 promoter. Because there is no binding site for OCT4 within BIRC5 promoter, the effect of OCT4 on BIRC5 promoter is indirect. An octamer motif for OCT4 in the CCND1 promoter has directly and partly participated in the regulation of CCND1 promoter activity, suggesting that OCT4 also could upregulated the expression of CCND1. Co-suppression of OCT4 and BIRC5 induced cancer cell apoptosis and cell cycle arrest, thereby efficiently inhibiting the proliferative activity of cancer cells and suppressing the growth of HCC xenogrfts in nude mice. Conclusion OCT4 can upregulate BIRC5 and CCND1 expression by increasing their promoter activity. These factors collusively promotes HCC cell proliferation, and co-suppression of OCT4 and BIRC5 is potentially beneficial for HCC treatment.

Background OCT4 and Survivin are important factors for cancer cell proliferation, renewal and dedifferentiation, and correlate with resistance to radiotherapy and chemotherapy in most human cancers, but their regulatory mechanisms are not well known. Methodology/principal findings In this study, 50 patients with esophageal squamous cell carcinoma (ESCC) were retrospectively analyzed. OCT4 was expressed in 13 cases (26%), and survivin was positively expressed in 31 cases (62%), examined by immunochemistry. OCT4 was found to be an independent predictive factor for median survival time, and the patients from the subgroup with both high expression of OCT4 and Survivin had the worst prognosis investigated by log-rank test. To further explore the molecular regulatory mechanism between OCT4 and Survivin, we constructed the specific small hairpin RNA (shRNA)-expressing vectors targeting OCT4 or/and Survivin and manipulated the expression of OCT4 and Survivin. By Western blotting and RT-PCR, we found that OCT4 could up-regulate Survivin expression in the esophageal cancer cell lines Eca109 and TE1. Simultaneously knockdown of OCT4 and Survivin expression induced cell apoptosis and G2-phase decrease of cell cycle by flow cytometry, and finally exerted an enhanced anti-proliferation potency in Eca109 and TE1 cell lines by MTT assay. Conclusions This study shows that OCT4 and Survivin expression were correlated with poor survival in patients with ESCC. OCT4 and Survivin may be regarded as targets in ESCC biotherapy.

Chronic infection with hepatitis B virus (HBV) is associated with the majority of cases of hepatocellular carcinoma (HCC) in China. Despite this, there is no effective method for the early detection of HBV-induced liver cancer. Aberrant fucosylation is known to occur during the development of HCC. We, therefore, developed a method of applying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the relationship between aberrant fucosylation, tumor genesis and progression of HBV-associated HCC, and to establish proteomic profiling of serum for early diagnosis of HCC. The MALDI-TOF MS was based on Lens culinaris agglutinin (LCA) lectin magnetic beads and their affinity for separation. The method was applied initially to a 'training' cohort of 111 serum samples obtained from subjects in China with no liver disease (n=26), chronic hepatitis B without cirrhosis (n=21), HBV-infected cirrhosis (n=32), or HBV-infected HCC (n=32). In contrast to previous findings, the results of our profiling analysis demonstrated defucosylation on some of the glycoproteins involved in HCC. HCC was then diagnostically classified in a 'blind test' cohort (n=96). In this group we demonstrated that, HCC could be distinguished from all serum samples, HBV-associated chronic liver disease, and HBV-associated cirrhosis with a sensitivity/specificity of 70%/70%, 78%/74%, and 81%/82%, respectively. When combined with serum alpha-fetoprotein detection (AFP>20 ng/mL), the sensitivity/specificity improved to 78%/88%, 85%/88%, and 89%/91%, respectively. In conclusion, serum glycoprotein fucosylation abnormalities have diverse forms in patients with HCC. MALDI-TOF MS profiling of aberrant serum fucosylated glycoproteins distinguished HCC from controls with high accuracy.

Icaritin is an active ingredient extracted from the plant Herba Epimedium Sagittatum (Sieb. et Zucc.) Maxim. The purpose of this study is to investigate the effects and mechanisms of icaritin-induced cell death in activated hepatic stellate cells (HSCs) and ameliorating the development of liver fibrosis in rats.: In vitro, icaritin-induced cell death rates in HSC-T6 (rat) and LX-2 (human) HSC lines as well as normal hepatocyte cell lines HL-7702 (L02) and WRL-68 were assayed by MTT method, and the apoptotic ratios were detected by both flow cytometry and the Annexin-V-FITC Apoptosis Detection Kit. A Whole Rat Genome Microarray Kit was used to identify expression of interest genes through fold-change screening. In vivo study, experimental liver fibrosis models were built by carbon tetrachloride (CCl(4)) or common bile duct ligation (CBDL) in Wistar rats. Icaritin (1mg/kg/day, three days a week) was administered by gastric gavage for four weeks (n=6 per group). At the end of the study, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) as well as the contents of hydroxyproline and collagen I in liver tissues were measured. Histopathological changes and the distribution of activated HSCs were observed in the liver tissues using hematoxyline-eosin (HE) staining and immunohistochemical staining for α-smooth muscle actin (α-SMA).Icaritin induced apoptosis in HSC-T6 and LX-2 in a concentration- and time-dependent manner with little toxicity to normal hepatocyte cell lines. The IC(50) of icaritin in HSC-T6 was 12.83 μM at 48 h. Apoptotic ratio of HSC-T6 treated with 24 μM icaritin was 20.19%, and the G2 phase of the cell cycle did not occur (P0.05). Gene analysis showed that icaritin up-regulated Bak-1, Bmf and Bax expression while significantly down-regulated Bcl-2 expression (vs. control group, P0.01). These results suggested that mitochondrial pathway played an important role in icaritin-induced apoptosis in activated HSCs. In vivo results showed that icaritin reduced the number of activated HSCs, and brought the elevated levels of AST, ALT, hydroxyproline and collagen I to normal or near normal values (vs. model group, P0.05).Icaritin can induce cell death in activated HSCs through mitochondria-mediated apoptosis, ameliorate the progression of hepatic fibrosis in rats, and could be a promising drug for treating liver fibrosis.

Background Human sulfatase 1 (hSulf-1) is a heparin-degrading endosulfatase that desulfates cell surface heparan sulfate proteoglycans (HSPGs) in extracellular matrix and negatively modulates heparin-binding growth factor and cytokine signaling in cell proliferation. But hSulf-1 function is more complicated, and its molecular mechanism has not been well known. Principal Findings To further investigate the functions of hSulf-1 gene in regulating the vascular endothelial growth factor receptor (VEGFR) signaling, a series of vectors expressing hSulf-1, hSulf-1 small hairpin RNA (shRNA) and VEGFR-2 shRNA were generated. hSulf-1 re-expression could downregualte the VEGFR-2 phosphorylation and inhibit cancer cell proliferation both in ovarian and hepatocellular cancer cell lines. Knockdown of hSulf-1 expression by hSulf-1 shRNA enhanced the recovery of high levels of phosphorylated VEGFR-2, and knockdown of VEGFR-2 expression by VEGFR-2 shRNA inhibited the proliferation activity of cancer cells in vitro to some extent. In human cancer xenografts in nude mice, tumor growth was inhibited markedly after injections of adenovirus expressing hSulf-1, with the tumor inhibition rates of 46.19% and 49.56% in ovarian and hepatocellular tumor models, respectively. hSulf-1 expression significantly reduced tumor microvessel density. Conclusions The results demonstrated that hSulf-1 re-expression both in ovarian and hepatocellular cancer cells induces antitumor efficacy by attenuating the phosphorylation of VEGFR-2 and suppressing angiogenesis. Therefore, hSulf-1-mediated antiproliferation and antiangiogenesis could be a reasonable approach for cancer therapy.

To investigate the killing effect of herpes simplex virus thymidine kinase gene expression actuated by the promoter of the vascular endothelial growth factor gene on human hepatocellular tumor cells under hypoxic condition.Recombinant adenoviral vectors, AdVEGF-tk and AdVEGF-GFP, were constructed with HSV-tk or GFP under the control of VEGF promoter through AdEasy system. Then GFP expression in hepatoma cell line HepG2 and normal liver cell line L02 transfected with AdVEGF-GFP were observed under fluorescence microscope, and the sensitivity to GCV of the AdVEGF-tk-transfected cells under normoxia or hypoxia condition were monitored by MTT method.GFP expression actuated by VEGF promoter was detected in sporadic L02 cells, but in almost all HepG2 cells after transfected with AdVEGF-GFP. With GCV at 10 micro g/ml and MOI at 100, L02 cells were insensitive to GCV under oxic condition, but more than 70% L02 cells were killed under hypoxic condition. Moreover, HepG2 cells infected with AdVEGF-tk showed the increased GCV sensitivity under hypoxia (over 80% killed) as compared with normoxia (over 60% killed) conditions.Hypoxia enhances the GCV sensitivity of human hepatocellular tumor cells infected with recombinant AdVEGF-tk under the control of VEGF promoter.

To investigate the correlation between midkine (MK) protein expression with local infiltration and metastasis in human hepatocellular carcinoma (HCC).Immunohistochemical and Western Blot analysis for MK were performed on samples of tumor tissue and the paratumor tissue from HCC and benign liver tumors.The overexpression of MK protein determined by immunohistochemical analysis was similar to that by Western Blot analysis. No specific positivity was detected in either benign liver tumor tissue or normal liver tissue, but most of HCC tissue showed a positive reaction to MK immunostain. No correlation between MK expression and other clinicopathological features in MK negative or positive HCC cases was found. Yet, the overexpression rate of MK protein in HCC with intra-hepatic metastasis was significantly higher than that in HCC without intra-hepatic metastasis.In human hepatocellular carcinoma, MK overexpressed at protein level may very well be closely related to local infiltration and metastasis.

Background Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs. Methods The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients. Results ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects. Conclusions Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection.

mda-7/IL-24 shows tumor-suppressor activity in a broad spectrum of human cancer cells. However, the molecular mechanism by which mda-7/IL-24 induces apoptosis is not well understood and most likely involves different pathways depending on the tumor. We examined the apoptotic effect of the adenovirus-mediated mda-7/IL-24 (Ad.mda-7) on human HepG2 hepatoma cells. We found that blocking the endoplasmic reticulum (ER) stress inhibited apoptosis induced by Ad.mda-7 and down-regulated the expression of caspase-12, Bax and caspase-3. The treatment of subcutaneous tumor xenografts of HepG2 cells with Ad.mda-7 inhibited tumor growth and angiogenesis. As in the in vitro studies, we found that blocking ER stress prevented Ad.mda-7 from inducing apoptosis in liver cancer cells in vivo. Our studies suggest that Ad.mda-7 induces apoptosis of HepG2 cells mainly through activation of the ER stress pathway.

AIM: To examine the effect of troglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) ligand, on the proliferation and apoptosis of human liver cancer cells. METHODS: Liver cancer cell line HepG2 was cultured and treated with troglitazone. Cell proliferation was detected by 3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay; apoptosis was detected by flow cytometry and terminal deoxynucleotidyl transferase-mediated nick end labeling of DNA fragmentation sites (TUNEL) assay; and apoptosis-related protein was detected by immunocytochemistry and Western blotting. RESULTS: Troglitazone inhibited growth and induced apoptosis of HepG2 cells in a dose-dependent manner, and induced activation of caspase-3 expression. Troglitazone not only drove apoptosis-inhibiting factor survivin to translocate incompletely from the nucleus to the cytoplasm, but also inhibited expression of survivin, while it did not affect expression of apoptosis-promoting factor Bax. CONCLUSION: PPARγ ligands inhibit growth and induce apoptosis of liver cancer cells, and may have applications for the prevention and treatment of liver cancer.