Zhengfeng Yin

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

BackgroundCancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.MethodsHuman hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.ResultsThe PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.ConclusionsNonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

Isolation of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Using a Novel Cell Separation Strategy

Purpose: To establish a sensitive and specific isolation and enumeration system for circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC). Experimental Design: HCC cells were bound by biotinylated asialofetuin, a ligand of asialoglycoprotein receptor, and subsequently magnetically labeled by antibiotin antibody–coated magnetic beads, followed by magnetic separation. Isolated HCC cells were identified by immunofluorescence staining using Hep Par 1 antibody. The system was used to detect CTCs in 5 mL blood. Blood samples spiked with Hep3B cells (ranging from 10 to 810 cells) were used to determine recovery and sensitivity. Prevalence of CTCs was examined in samples from HCC patients, healthy volunteers, and patients with benign liver diseases or non-HCC cancers. CTC samples were also analyzed by FISH. Results: The average recovery was 61% or more at each spiking level. No healthy, benign liver disease or non-HCC cancer subjects had CTCs detected. CTCs were identified in 69 of 85 (81%) HCC patients, with an average of 19 ± 24 CTCs per 5 mL. Both the positivity rate and the number of CTCs were significantly correlated with tumor size, portal vein tumor thrombus, differentiation status, and the disease extent as classified by the TNM (tumor-node-metastasis) classification and the Milan criteria. HER-2 gene amplification and TP53 gene deletion were detected in CTCs. Conclusion: Our system provides a new tool allowing for highly sensitive and specific detection and genetic analysis of CTCs in HCC patients. It is likely clinically useful in diagnosis and monitoring of HCC and may have a role in clinical decision making. Clin Cancer Res; 17(11); 3783–93. ©2011 AACR.

Circulating tumor cells in hepatocellular carcinoma: detection techniques, clinical implications, and future perspectives.

Hepatocellular carcinoma (HCC) is a primary liver cancer with a huge challenge in terms of its complex etiology and its management. The fact that the most common site of early tumor recurrence in liver transplantation for HCC is the transplanted allograft strongly suggests that circulating tumor cells (CTCs) are really an active source of HCC metastasis or recurrence. In the past decade, with the tremendous progress in the technology of CTC detection, there is convincing evidence that CTCs have great potential as a marker for metastatic disease and poor prognosis in patients with a malignancy. Currently some interesting and encouraging results have been achieved in HCC CTC detection, although the knowledge about its clinical relevance in HCC is lagging behind other major tumor types. Here we will review existing and developing methodologies for CTC detection, discuss future perspectives, and describe the potential clinical impact of the identification and molecular characterization of CTC subset or circulating cancer stem cells in HCC patients. Particular attention is given to the results based on the HCC CTC study.

The biological and clinical importance of epithelial-mesenchymal transition in circulating tumor cells.

AbstractMovement of tumor cells from a primary tumor to a nonadjacent or distant site is a contiguous and complex process. Among the multiple natural cellular programs that promote initiation and progression of tumor metastasis, epithelial–mesenchymal transition (EMT) may play a key role in the ultimate generation of a metastatic foci. Acquisition of the EMT phenotype by tumor cells not only increases their migration and invasion potentials, thereby facilitating their ability to infiltrate blood vessels and to produce circulating tumor cells (CTCs), but also promotes survival of CTCs in the bloodstream and their ability to extravasate out of the circulatory system and invade proximal tissues. In organs distal to the primary tumor, the phenotypic switching mechanism of mesenchymal–epithelial transition (MET) enables CTCs to grow and colonize, enhancing the likelihood of establishing metastasis. In addition, CTCs that have undergone EMT attain increased resistance to chemotherapy and targeted therapy. CTCs with the EMT phenotype have become recognized as an active source of metastases, and targeting EMT/MET processes during the individual steps of tumor metastasis represents a promising new approach for alleviating cancer metastasis and recurrence. In this article, we focus on the biological and clinical importance of EMT and/or MET in CTCs during the individual steps of tumor metastasis, summarizing the recent findings of the regulatory roles played by EMT and/or MET in the generation, survival, and recolonization of CTCs and discussing the EMT-targeting strategies developed for tumor diagnosis as well as their potential for management of metastatic malignant diseases.

CCL20 and CXCL8 synergize to promote progression and poor survival outcome in patients with colorectal cancer by collaborative induction of the epithelial-mesenchymal transition.

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.

Detection of Circulating Tumor Cells in Hepatocellular Carcinoma Using Antibodies against Asialoglycoprotein Receptor, Carbamoyl Phosphate Synthetase 1 and Pan-Cytokeratin

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.

Icaritin induces cell death in activated hepatic stellate cells through mitochondrial activated apoptosis and ameliorates the development of liver fibrosis in rats

AIM OF THE STUDY 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. MATERIALS AND METHODS : 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). RESULTS 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 (P<0.05). Gene analysis showed that icaritin up-regulated Bak-1, Bmf and Bax expression while significantly down-regulated Bcl-2 expression (vs. control group, P<0.01). 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, P<0.05). CONCLUSIONS 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.

hSulf-1 Gene Exhibits Anticancer Efficacy through Negatively Regulating VEGFR-2 Signaling in Human Cancers

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.

CXCL8, overexpressed in colorectal cancer, enhances the resistance of colorectal cancer cells to anoikis

Anoikis is a form of apoptosis which occurs when anchorage-dependent cells either show loss of adhesion or inappropriate adhesion. Only a few cancer cells that detach from the primary site of the tumor acquire the ability to resist anoikis and form metastasis. The mechanism underlying the resistance of colorectal cancer (CRC) cells to anoikis remains unclear. Interleukin-8 (alternatively known as CXCL8) is associated with CRC angiogenesis and progression. Here, we found that a high abundance of CXCL8 or TOPK strongly correlated with poor overall and disease-free survival of 186 patients with CRC. A combination of high CXCL8 and high TOPK expressions had the worst prognosis. We showed that CXCL8 expression was negatively correlated with anoikis in CRC cells. CXCL8 treatment enhanced the resistance of CRC cells to apoptosis, which was accompanied by the increase of TOPK, and the activation of AKT and ERK. Moreover, we demonstrated that the inhibition of either ERK or AKT by specific chemical inhibitors attenuated the CXCL8-mediated resistance to anoikis. Treatment with AKT inhibitor abolished the effects of CXCL8 on TOPK expression, suggesting that TOPK was downstream of AKT in the process of anoikis. Taken together, we demonstrated that CXCL8 is strongly implicated in the resistance of CRC cells to anoikis, and that the AKT, TOPK and ERK pathway may be a potential therapeutic target for CRC.

Risk factors of intrahepatic cholangiocarcinoma in patients with hepatolithiasis: a case-control study

BACKGROUND Why 3.3% to 10% of all patients with hepatolithiasis develop intrahepatic cholangiocarcinoma (ICC) remains unknown. We carried out a hospital-based case-control study to identify risk factors for the development of ICC in patients with hepatolithiasis in China. METHODS Eighty-seven patients with pathologically diagnosed hepatolithiasis associated with ICC and 228 with hepatolithiasis alone matched by sex, age (+/-2 years), hospital admittance and place of residence were interviewed during the period of 2000-2008. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for each risk factor. RESULTS Among the patients with hepatolithiasis associated with ICC, the mean age was 57.7 years and 61.0% were female. Univariate analysis showed that the significant risk factors for ICC development in hepatolithiasis were smoking, family history of cancer, appendectomy during childhood (under age 20), and duration of symptoms >10 years. In multivariate stepwise logistic regression analysis, smoking (OR=1.931, 95% CI: 1.000-3.731), family history of cancer (OR=5.175, 95% CI: 1.216-22.022), and duration of symptoms >10 years (OR=2.348, 95% CI: 1.394-3.952) were independent factors. CONCLUSION Smoking, family history of cancer and duration of symptoms >10 years may be risk factors for ICC in patients with hepatolithiasis.