BTG2 Is Down-Regulated and Inhibits Cancer Stem Cell-Like Features of Side Population Cells in Hepatocellular Carcinoma.

BACKGROUND: Our previous study found that B cell translocation gene 2 (BTG2) was hyper-methylated and down-regulated in side population (SP) cells of hepatocellular carcinoma (HCC) cell line. However, its clinical significances and biological impacts on HCC SP cells remained unclear. AIMS: To investigate the prognostic value of BTG2 gene in HCC and its influences on cancer stem cells (CSCs)-like traits of HCC cell line SP cells. METHODS: BTG2 expression in human HCC and adjacent non-cancerous tissues was detected by immunohistochemical staining and quantitative real-time PCR, and also obtained from GEO and TCGA data. Its prognostic values were assessed. Its biological influences on HCC cell line SP cells were evaluated using cell viability, cell cycle, plate clone-forming assay, and chemoresistance in vitro and tumorigenicity in vivo. RESULTS: BTG2 expression was significantly suppressed in human HCC compared to adjacent non-cancerous tissues. BTG2 expression was correlated with TNM stage, tumor size and vascular invasion. Lower expression of BTG2 was associated with poorer overall survival and disease-free survival. In vitro, overexpression of BTG2 substantially suppressed cell proliferation and accumulation of HCC cell line SP cells in G0/G1 phase. Colony formation ability was markedly suppressed by BTG2 overexpression. Moreover, sensitivity of HCC cell line SP cells to 5-fluorouracil was substantially increased by overexpression of BTG2. Furthermore, tumorigenicity of HCC cell line SP cells transfected with BTG2 plasmids was significantly reduced in vivo. CONCLUSIONS: BTG2 gene could regulate the CSC-like traits of HCC cell line SP cells, and it represented as a molecular prognostic marker for HCC.

DNA methylation profiling identifies EYA4 gene as a prognostic molecular marker in hepatocellular carcinoma.

BACKGROUND: DNA hypermethylation plays important roles in carcinogenesis by silencing key genes. This study aims to identify pivotal genes in hepatocellular carcinoma (HCC) by DNA methylation microarray and to assess their prognostic values. MATERIALS AND METHODS: DNA methylation microarray was performed in 45 pairs of HCC and adjacent nontumorous tissues and six normal liver tissues to identify hypermethylated genes in HCC. Potential prognosis-related genes were selected among hypermethylated genes by analyzing influences of methylation levels on disease-free survival (DFS) and overall survival (OS) in 45 patients. Their prognostic values were validated in 154 patients with HCC (including the initial 45 patients) to determine the independent prognostic gene. RESULTS: Altogether, 54 CpG islands in 44 genes were hypermethylated in HCC compared with liver tissues. Among them, methylation levels of ERG and HOXA11 were inversely associated with DFS (both P < 0.050), and methylation levels of EYA4 were inversely related to DFS and OS (both P < 0.050). EYA4 expression was inversely related to tumor size (P < 0.050). Lower EYA4 expression and larger tumor size were independent predictors of both shorter DFS and OS, and higher Barcelona Clinic Liver Cancer (BCLC) staging was an independent predictor of shorter OS (all P < 0.050). CONCLUSIONS: EYA4 functions as a prognostic molecular marker in HCC. Its aberrant hypermethylation and subsequent down-regulation may promote tumor progression.

Analysis of the genome-wide DNA methylation profile of side population cells in hepatocellular carcinoma.

BACKGROUND: DNA methylation plays an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the DNA methylation profile of stem cells in hepatocellular carcinoma (HCC) remains unclear. AIMS: To investigate the genome-wide DNA methylation profile of side population (SP) cells of HCC, a special subpopulation of cells enriched with cancer stem cells, by DNA methylation microarray analysis and to analyze the functions and signal pathways of the aberrantly methylated genes in SP cells. METHODS: Side population cells were isolated from HCC cell lines Huh7 and PLC/PRF/5 using flow cytometry, and the tumorigenicity of these SP cells was assessed in NOD/SCID mice. The genome-wide DNA methylation status of SP cells and non-SP (NSP) cells was detected and compared by DNA methylation microarray analysis. Genes with differential methylation between SP and NSP cells were further analyzed for their functions and roles in related signaling pathways. RESULTS: Subcutaneous inoculation of 1 × 10(3) SP cells yielded tumors in 60 % NOD/SCID mice, whereas no tumor was developed after the inoculation of 1 × 10(6) NSP cells. Genome-wide DNA methylation microarray analysis showed that 72 and 181 genes were hypermethylated and hypomethylated, respectively, in both Huh7 and PLC/PRF/5 SP cells as compared with their corresponding NSP cells. Analyses of signaling pathways revealed that hypermethylated and hypomethylated genes were related to four and eight pathways, respectively. CONCLUSIONS: Hepatocellular carcinoma SP cells possessed a differential DNA methylation status compared with NSP cells, and the differentially methylated genes in SP cells were involved in 12 signaling pathways. Our results provide valuable clues for further investigations in elucidating the importance of epigenetic regulation in sustaining HCC SP cells and tumorigenesis.

[Study on the specific immunity induced by dendritic cell vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell in mice].

OBJECTIVE: To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell of mice. METHODS: Mouse brain microvascular endothelial cell bEnd.3 was cultured and identified for preparation endothelial cell bEnd.3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R2) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd.3 antigen 4 times in 4 weeks (bEnd.3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD3+CD8+ surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot. RESULTS: The expression of VEGF-R2 and integrin αV gene in bEnd.3 cells were expressed highly. After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd.3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11 ± 0.13) cm³ versus (3.38 ± 0.34) cm³]. The CTL response of spleen lymphocytes in vitro showed that bEnd.3-DC cells could kill bEnd.3 cells, the special lysis rate was more than 60%. The percentage of CD3+CD8+ spleen lymphocytes in bEnd.3-DC group [(38.6 ± 0.7)%] was higher than those in other groups (P < 0.05). The titer of serum antibody of bEnd.3-DC group was 1:3200, while it was 1:800 in DC group and there were not any in PBS group. Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd.3 cell in bEnd.3-DC group. Western blot analysis revealed that there were specific bands at 220,000 (VEGF-R2). CONCLUSIONS: bEnd.3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humoral immunity are induced by bEnd.3-DC antigen which maybe have some antigens in bEnd.3 cells that reacts with endothelial cell proliferation-related antigens.

IL-32 expression indicates unfavorable prognosis in patients with colon cancer.

Recently, interleukin (IL)-32 has been demonstrated to represent a novel biomarker for evaluating the prognosis of patients with gastric and lung cancer; however, its clinical significance in colon cancer remains unknown. In the present study, the IL-32 expression in 60 patients with colon cancer was examined with an immunohistochemistry assay. IL-32 expression was determined in 37 (61.67%) patients with colon cancer. Additionally, IL-32 was associated with tumor size and Dukes' stage. By using the Kaplan-Meier method, patients with positive IL-32 expression had shorter overall survival time, compared with those with negative IL-32 expression. Multivariate analysis indicated that IL-32 could be an independent prognostic factor in patients with colon cancer; therefore, IL-32 may be a novel prognostic biomarker and therapeutic target for colon cancer.

Radical gastrectomy with hepatoarterial catheter implantation for late-stage gastric cancer.

AIM: To determine the optimal type of surgery for late-stage gastric cancer with hepatic metastases. METHODS: We retrospectively analyzed 49 gastrectomies for late-stage gastric cancer conducted in the First Hospital Affiliated to Henan University of Science and Technology between September 2003 and September 2010. All gastrectomy operations were divided into two groups: radical resection (gastrectomy and simultaneous resection of hepatic metastases, n = 31), and palliative resection (gastrectomy without hepatic resection, n = 18). All 49 patients had chemotherapy catheter implantation in the hepatic artery via the gastroduodenal artery. Postoperative complications and cumulative survival rates of the two groups were compared and analyzed. RESULTS: There was no significant difference in the number of perioperative complications between the radical and palliative resection groups (6 and 3 cases, respectively, P > 0.05). The incidence of long-term complications including ileus (3 in the radical resection and 2 in the palliative resection groups) and anastomosis (2 cases in each group) was not significantly different (P > 0.05). The cumulative survival rate was significantly lower in the palliative resection group (P < 0.05). CONCLUSION: Radical gastrectomy with resection of hepatic metastases and hepatoarterial catheter implantation is the recommended surgery for late-stage gastric cancer patients with hepatic metastases.

IL-35 over-expression is associated with genesis of gastric cancer.

Overexpression of interleukin (IL)-35 has been found in a variety of malignancies, but the expression status in gastric cancer has yet to be elucidated clearly. In the present study, positive expression of EBI3 and p35 was 63.3% and 70.0% of cases, respectively. EBI3 expression was strongly related with larger tumor size and invasion depth (P<0.05). Similarly, expression of p35 was also correlated with larger tumor size (P<0.05). These results indicate that IL-35 might be involved in growth of gastric cancer. Interestingly, EBI3 and p35 expressions were positive correlated with Ki-67 expression. Moreover, EBI3 immunoreactivity was associated with Bcl-2 staining. Our data suggest IL-35 is correlated with genesis of gastric cancer by regulating growth and apoptosis.

Expression of annexin A3 in gastric cancer and its correlation with proliferation and apoptosis.

Annexin A3 has been identified as a novel biomarker in different types of cancers. However, little is known about its clinical significances and and biological roles in gastric cancer. In this study, we assessed annexin A3 expression in 80 patients with gastric cancer and explore its correlation with prognosis Moreover, correlations with Ki-67, Bcl-2 and Bax were also investigated. Expression of annexin A3 was increased in gastric cancer compared with that in normal gastric tissues. Annexin A3 expression was significantly associated with tumor volume and TNM stage (p<0.05). and inversely correlation with prognosis of patients. More interestingly, expression of annexin A3 was positive correlated with Ki-67 and Bcl-2 expression. Our study showed annexin A3 might be a potential prognostic marker for gastric cancer and involved in tumorigenesis by regulating apoptosis and proliferation.

Sorafenib enhances the chemotherapeutic efficacy of S-1 against hepatocellular carcinoma through downregulation of transcription factor E2F-1.

PURPOSE: Sorafenib and S-1 (one mixed formulation containing 5-FU prodrug and dihydropyrimidine dehydrogenase inhibitor) were two effective agents against hepatocellular carcinoma (HCC), but whether they had synergistic effects remained unclear. The present study aimed at evaluating their synergistic effects against HCC and its mechanisms. METHODS: Inhibitory effects of sorafenib, 5-FU and their combination on HCC cells PLC/PRF/5 and SK-HEP-1 were evaluated. Expressions of transcription factor E2F-1 and its downstream thymidylate synthetase (TS) in the treated cells were determined using real-time PCR and Western blot. In vivo anti-tumoral efficacy of S-1 plus sorafenib on HCC was evaluated in NOD/SCID mice. E2F-1 and TS expressions in tumors were determined by immunohistochemical staining. RESULTS: Sorafenib inhibited growth of HCC cells in dose-dependent manner, with IC50 of 5.4 ± 0.3 µmol/L for PLC/PRF/5 and 5.3 ± 0.5 µmol/L for SK-HEP-1. Sorafenib (1 µmol/L) enhanced inhibitory efficacy of 5-FU on HCC cells in vitro, dropping IC50 of 5-FU from 167.7 ± 12.1 to 105.4 ± 8.4 µmol/L for PLC/PRF/5 and 115 ± 10.2 to 82 ± 7.4 µmol/L for SK-HEP-1 (both p < 0.01). Sorafenib downregulated E2F-1 and TS expressions on HCC cells, and its combination with 5-FU yielded a synergistic downregulation of TS expression on HCC cells. In NOD/SCID mice with subcutaneously inoculated HCC, sorafenib combined with S-1 yielded greater inhibition on tumor growth and remarkable TS suppression when compared with sorafenib or S-1 alone (all p < 0.05). CONCLUSIONS: Sorafenib enhanced therapeutic efficacy of 5-FU/S-1 against HCC through downregulation of E2F-1 and TS expressions. Sorafenib combined with S-1 might represent as valuable therapeutic regimen against HCC.

[Study on the specific immunity of dendritic cell vaccine loading human umbilical vein endothelial cell antigen against U14 cervical cancer].

AIM: To explore the specific immunity of dendritic cell (DC) vaccine loading human umbilical vein endothelial cell (HUVEC) antigen.against U14 cervical cancer of mice. METHODS: Primary HUVECs were cultured, identified and made into antigen. The BALB/c mice were immunized with DCs loading HUVEC antigen 4 times a week. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, CTL response of spleen lymphocytes in vitro, the percentage of CD3(+)CD8(+) surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and Western blot. RESULTS: HUVECs with high purity were successfully cultured and by identified by immunocytochemistry and RT-PCR. After the vaccine was injected into mice, the tumors of mice in PBS group grew faster than the those in other groups. The tumors of mice in HUVEC-DC groups grew slowly and disappeared after 2 weeks and The tumors of mice in DC group disappeared after 3 weeks. The CTL response of spleen lymphocytes in vitro showed that HUVEC-DC-T cells could kill HUVEC cells. The percentage of CD3(+)CD8(+) surface markers of spleen lymphocytes in HUVEC-DC group was higher than that in other groups. The titer of serum antibody was 1:800. immunocytochemistry analysis indicated HUVEC-DC group had specific antigen-antibody reaction to HUVECs through and Western blot analysis revealed there were specific bands at 130 KDa and 220 KDa. CONCLUSION: HUVEC-DC vaccine and DC vaccine can inhibit the tumor growth of U14 cervical cancer of mice. The special cellular and humoral immunity are induced by HUVEC-DC vaccine. Furthermore, some antigens in HUVECs maybe have special immune reaction with integrin alphav and VEGF-R2.