Chengyong Qin

Over-Expression of the Endocan Gene in Endothelial Cells from Hepatocellular Carcinoma is Associated with Angiogenesis and Tumour Invasion

Endocan plays a role in tumour angiogenesis and tumour growth. The aim of this study was to detect the expression of endocan in hepatocellular carcinoma (HCC) tumour-associated endothelial cells and to correlate endocan expression with clinicopathological parameters and tumour angiogenesis. Tumour tissues and surrounding non-cancerous hepatic parenchyma from 42 primary HCC patients were studied. Endothelial cells were isolated using magnetic microbeads conjugated with anti-CD31 and endocan expression was evaluated by real-time reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. Endocan was significantly over-expressed in endothelial cells isolated from HCC tumours compared with corresponding non-cancerous liver tissues. In addition, the endocan mRNA level was significantly correlated with the serum α-fetoprotein level, intra-tumoural microvessel density, vascular endothelial growth factor mRNA, and vascular and venous invasion. The over-expression of endocan in tumour endothelial cells was closely related to the process of angiogenesis and pathogenesis in HCC, and suggests that endocan might be a useful marker for HCC progression.

Expression of P-Glycoprotein and Multidrug Resistance-Associated Protein is Associated with Multidrug Resistance in Gastric Cancer

This study evaluated the sensitivities of gastric cancer cells to various chemotherapy drugs, and investigated the relationship between the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) and multidrug resistance. Drug sensitivities were determined using a methyltetrazolium assay: expression levels of P-gp and MRP were measured using immunohistochemistry. On purification culture, gastric cancer cells were found to be most sensitive to cisplatin, mitomycin and adriamycin, moderately sensitive to etoposide and 5-fluorouracil, and less sensitive to homocamptothecin and methotrexate, with sensitivities of 76.7%, 70.0%, 66.7%, 60.0%, 56.7%, 43.3% and 30.0%, respectively. Positive expression for P-gp and MRP in gastric cancer tissues was 41.7% and 29.2%, respectively;co-expression of P-gp and MRP in cancer tissue was 23%. The drug-resistant groups had higher positive expression of P-gp and MRP compared with the drug-sensitive groups. In conclusion, expression of P-gp and MRP seems to be associated with multidrug resistance in gastric cancer.

The miR-545/374a cluster encoded in the Ftx lncRNA is overexpressed in HBV-related hepatocellular carcinoma and promotes tumorigenesis and tumor progression.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). Previous studies have shown several long noncoding RNAs (lncRNAs) play various roles in HCC progression, but no research has focused on the expression pattern of microRNA clusters encoded in lncRNAs. The Ftx gene encodes a lncRNA which harbors 2 clusters of microRNAs in its introns, the miR-374b/421 cluster and the miR-545/374a cluster. To date, no research has focused on the role of the miR-545/374a and miR-374b/421 clusters in HBV-related HCC. In this study, 66 pairs of HBV-related HCC tissue and matched non-cancerous liver tissue specimens were analyzed for the expression of the Ftx microRNA clusters. Our results showed that the miR-545/374a cluster was upregulated in HBV-HCC tissue and significantly correlated with prognosis-related clinical features, including histological grade, metastasis and tumor capsule. Transfection studies with microRNA mimics and inhibitors revealed that miR-545/374a expression promoted in vitro cell proliferation, cell migration and invasion. The wild-type HBV-genome-containing plasmid or full-length HBx protein encoding plasmid was transfected into the Bel-7402 cell line and observed for their influence on miR-545/374a expression. We found that transfection of the HBV genome or HBx alone resulted in an increase in miR-545/374a expression. Next, by monitoring the expression of sera miR-545/374a before and after surgical tumor excision, we found serum miR-545/374a was tumor-derived and exhibited a sharp decrease 25 days after tumor excision. We also examined the gender-based difference in miR-545/374a expression among HCC patients and utilized microRNA target prediction software to find the targets of miR-545/374a. One of these targets, namely estrogen-related receptor gamma (ESRRG) was inversely correlated with miR-545 expression. In conclusion, the overexpression of miR-545/374a cluster located in the Ftx lncRNA is partially responsible for a poor prognosis, and monitoring sera levels of miR-545/374a may be a useful diagnostic marker for HCC.

Suppression of cholangiocarcinoma cell growth by human umbilical cord mesenchymal stem cells: a possible role of Wnt and Akt signaling.

Emerging evidence indicates that human mesenchymal stem cells (hMSCs) can be recruited to tumor sites, and affect the growth of human malignancies. However, little is known about the underlying molecular mechanisms. Here, we observed the effects of hMSCs on the human cholangiocarcinoma cell line, HCCC-9810, using an animal transplantation model, and conditioned media from human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). Animal studies showed that hUC-MSCs can inhibit the growth of cholangiocarcinoma xenograft tumors. In cell culture, conditioned media from hUC-MSCs inhibited proliferation and induced apoptosis of tumor cells in a dose- and time-dependent manner. The proliferation inhibition rate increased from 6.21% to 49.86%, whereas the apoptosis rate increased from 9.3% to 48.1% when HCCC-9810 cells were cultured with 50% hUC-MSC conditioned media for 24 h. Immunoblot analysis showed that the expression of phosphor-PDK1 (Ser241), phosphor-Akt (Ser 437 and Thr308), phosphorylated glycogen synthase kinase 3β (phospho-GSK-3βSer9), β-catenin, cyclin-D1, and c-myc were down-regulated. We further demonstrated that CHIR99021, a GSK-3β inhibitor reversed the suppressive effects of hUC-MSCs on HCCC-9810 cells and increased the expression of β-catenin. The GSK-3β activator, sodium nitroprusside dehydrate (SNP), augmented the anti-tumor effects of hUC-MSCs and decreased the expression of β-catenin. IGF-1 acted as an Akt activator, and also reversed the suppressive effects of hUC-MSCs on HCCC-9810 cells. All these results suggest that hUC-MSCs could inhibit the malignant phenotype of HCCC-9810 human cholangiocarcinoma cell line. The cross-talk role of Wnt/β-catenin and PI3K/Akt signaling pathway, with GSK-3β as the key enzyme bridging these pathways, may contribute to the inhibition of cholangiocarcinoma cells by hUC-MSCs.

Synthetic chenodeoxycholic acid derivative, HS-1200, induces apoptosis of human hepatoma cells via a mitochondrial pathway.

We investigated whether HS-1200 has anti-proliferation effects on human hepatoma cells in vitro. Here, chromatin condensation, DNA ladder formation and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) were observed after treatment of HS-1200, indicating the occurrence of apoptotic cell death, which was associated with up-regulation of Bax, cleaved-caspase-3 and cleaved-caspase-9. Inhibition of caspase-9 rescued HS-1200-induced apoptosis. Furthermore, cells treated with HS-1200 showed a reduction in mitochondrial membrane potential (Deltapsi(m)) and caused cytochrome c release into the cytosol. The results indicated that synthetic chenodeoxycholic acid HS-1200 could induce cell apoptosis in BEL7402 human hepatoma cell line, via a Bax/cytochrome c/caspase-9 independent pathway. This study suggested that HS-1200 is potentially useful as an apoptosis inducer for the treatment of hepatocellular carcinoma.

Submucosal tunneling endoscopic resection for tumors of the esophagogastric junction

Abstract Background: For submucosal tumors (SMTs) originating from the muscularis propria (MP) layer of the esophagogastric junction (EGJ), submucosal tunneling endoscopic resection (STER) is now widely used, and it shows promise in overcoming the limitations of endoscopic submucosal dissection. Aims: This study aimed to evaluate the efficacy and safety of the STER technique for treating SMTs of the EGJ originating from the MP layer. Material and methods: From October 2011 to February 2014, 20 patients were enrolled for STER surgery. Results: The patients were categorized into three groups according to the tumor location. The esophagocardiac group had a lower complication rate (0/7) compared with the cardiac group (3/6) and the gastrocardiac group (3/7). The mean operation time in the esophagocardiac (83 ± 24 min) and cardiac (83 ± 55 min) groups was significantly shorter than that of the gastrocardiac group (145 ± 44 min) (P < 0.05). The en bloc resection rate was 100%, with no severe complications and no recurrence during the follow-up period. Conclusions: The STER technique appears to be a feasible and safe minimally invasive approach for SMTs originating from the MP layer of the EGJ, with satisfying en bloc resection, a short operation time, and low rates of severe complications.

OSU-03012, a non-Cox inhibiting celecoxib derivative, induces apoptosis of human esophageal carcinoma cells through a p53/Bax/cytochrome c/caspase-9-dependent pathway.

OSU-03012 is a celecoxib derivative devoid of cyclooxygenase-2 inhibitory activity. It was previously reported to inhibit the growth of some tumor cells through the AKT-signaling pathway. In the current study, we assessed the ability of OSU-03012 to induce apoptosis in human esophageal carcinoma cells and the mechanism by which this occurs. A cell proliferation assay indicated that OSU-03012 inhibited the growth of human esophageal carcinoma cell lines with an IC50 below 2 &mgr;mol/l and had the most effective cytotoxicity against Eca-109 cells. Terminal deoxynucleotidyl transferase-mediated nick-end labeling assay and flow cytometry analysis showed that OSU-03012 could induce the apoptosis in Eca-109 cells. After treatment of Eca-109 cells with 2 &mgr;mol/l OSU-03012 for 24 h, the apoptosis index increased from 14.07 to 53.72%. OSU-03012 treatment resulted in a 30–40% decrease in the mitochondrial membrane potential and caused cytochrome c release into the cytosol. Further studies with caspase-9-specific and caspase-8-specific inhibitors (z-LEHDfmk and z-IETDfmk, respectively) pointed toward the involvement of the caspase-9 pathway, but not the caspase-8 pathway, in the execution of OSU-03012-induced apoptosis. Immunoblot analysis demonstrated that OSU-03012-induced cellular apoptosis was associated with upregulation of Bax, cleaved caspase-3, and cleaved caspase-9. Ser-15 of p53 was phosphorylated after 24 h of treatment of the cancer cells with OSU-03012. This increase in p53 was associated with the decrease in Bcl-2 and increase in Bax. An inhibitor of p53, pifithrin-&agr;, attenuated the anticancer effects of OSU-03012 and downregulated the expression of Bax and cleaved caspase-9. Altogether, our results show that OSU-03012 could induce apoptosis in human esophageal carcinoma cells through a p53/Bax/cytochrome c/caspase-9-dependent pathway.

SNAI1 promotes the development of HCC through the enhancement of proliferation and inhibition of apoptosis

SNAI1, a zinc‐finger transcription factor, plays an important role in the induction of epithelial–mesenchymal transition (EMT) in various cancers. However, the possible functions of SNAI1 in the proliferation and apoptosis of hepatocellular carcinoma have not been clearly identified. In this study, we investigated the effects and mechanisms of SNAI1 in the proliferation and apoptosis of hepatocellular carcinoma using clinical samples and cell lines. We found that SNAI1 is highly expressed in the tissues of liver cancer compared with adjacent nontumor tissues. SNAI1 is also highly expressed in the hepatoma cell lines HepG2, SMMC‐7721, and BEL‐7402 compared with the human normal liver cell line L02. We also observed that SNAI1 expression was correlated with distal metastasis, incomplete tumor capsule formation, and histological differentiation in hepatocellular carcinoma (HCC). Moreover, we demonstrated that knockdown of SNAI1 via lentiviral vectors of RNAi against SNAI inhibited cell proliferation by inducing G1 arrest, which was accompanied by the downregulation of cyclin D1 but not that of cyclin A. In addition, knockdown of SNAI1 promoted apoptosis by decreasing the expression of Bcl‐2. In conclusion, our findings revealed that SNAI1 is involved in the development of hepatocellular carcinoma via regulating the growth and apoptosis of tumor cells.

Ursodeoxycholic acid induces apoptosis in hepatocellular carcinoma xenografts in mice

AIM To evaluate the efficacy of ursodeoxycholic acid (UDCA) as a chemotherapeutic agent for the treatment of hepatocellular carcinoma (HCC). METHODS BALB/c nude mice were randomized into four groups 24 h before subcutaneous injection of hepatocarcinoma BEL7402 cells suspended in phosphate buffered saline (PBS) into the right flank. The control group (n = 10) was fed a standard diet while treatment groups (n = 10 each) were fed a standard daily diet supplemented with different concentrations of UDCA (30, 50 and 70 mg/kg per day) for 21 d. Tumor growth was measured once each week, and tumor volume (V) was calculated with the following equation: V = (L × W(2)) × 0.52, where L is the length and W is the width of the xenograft. After 21 d, mice were killed under ether anesthesia, and tumors were excised and weighed. Apoptosis was evaluated through detection of DNA fragmentation with gel electrophoresis and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Western blot analysis was performed to determine the expression of apoptosis-related proteins BAX, BCL2, APAF1, cleaved caspase-9, and cleaved caspase-3. RESULTS UDCA suppressed tumor growth relative to controls. The mean tumor volumes were the following: control, 1090 ± 89 mm(3); 30 mg/kg per day, 612 ± 46 mm(3); 50 mg/kg per day, 563 ± 38 mm(3); and 70 mg/kg per day, 221 ± 26 mm(3). Decreased tumor volumes reached statistical significance relative to control xenografts (30 mg/kg per day, P < 0.05; 50 mg/kg per day, P < 0.05; 70 mg/kg per day, P < 0.01). Increasing concentrations of UDCA led to increased DNA fragmentation observed on gel electrophoresis and in the TUNEL assay (control, 1.6% ± 0.3%; 30 mg/kg per day, 2.9% ± 0.5%; 50 mg/kg per day, 3.15% ± 0.7%, and 70 mg/kg per day, 4.86% ± 0.9%). Western blot analysis revealed increased expression of BAX, APAF1, cleaved-caspase-9 and cleaved-caspase-3 proteins, which induce apoptosis, but decreased expression of BCL2 protein, which is an inhibitor of apoptosis, following administration of UDCA. CONCLUSION UDCA suppresses growth of BEL7402 hepatocellular carcinoma cells in vivo, in part through apoptosis induction, and is thus a candidate for therapeutic treatment of HCC.

MAPK/p38 regulation of cytoskeleton rearrangement accelerates induction of macrophage activation by TLR4, but not TLR3

Toll-like receptor 3 (TLR3) and TLR4 utilize adaptor proteins to activate mitogen-activated protein kinase (MAPK), resulting in the acute but transient inflammatory response aimed at the clearance of pathogens. In the present study, it was demonstrated that macrophage activation by lipopolysaccharide (LPS) or poly(I:C), leading to changes in cell morphology, differed significantly between the mouse macrophage cell line RAW264.7 and mouse primary peritoneal macrophages. Moreover, the expression of α- and β-tubulin was markedly decreased following LPS stimulation. By contrast, α- and β-tubulin expression were only mildly increased following poly(I:C) treatment. However, the expression of β-actin and GAPDH was not significantly affected. Furthermore, it was verified that vincristine pretreatment abrogated the cytoskeleton rearrangement and decreased the synthesis and secretion of proinflammatory cytokines and migration of macrophages caused by LPS. Finally, it was observed that the MAPK/p38 signaling pathway regulating cytoskeleton rearrangement may participate in LPS-induced macrophage cytokine production and migration. Overall, the findings of the present study indicated that MAPK/p38 regulation of the cytoskeleton, particularly tubulin proteins, plays an important role in LPS-induced inflammatory responses via alleviating the synthesis and secretion of proinflammatory cytokines and inhibiting the migration of macrophages.