Houjie Liang

Endostatin suppresses colorectal tumor-induced lymphangiogenesis by inhibiting expression of fibronectin extra domain A and integrin α9.

Endostatin is a natural occurring anti‐angiogenic peptide and has been shown to inhibit tumor lymphangiogenesis by suppressing the expression of tumor‐stimulating growth factors. We have previously shown that fibronectin alternative extra domain A (EDA) facilitates lymphangiogenesis of colorectal tumors. Since it is known that EDA interacts with integrin α9 in the lymphatic endothelial cells (LECs), we hypothesized that endostatin may target EDA‐integrin α9 pathway to inhibit colorectal tumor‐induced lymphangiogenesis. To test this hypothesis, we examined the effect of endostatin on EDA secreted by SW480 colorectal cancer cells and treated human LECs with different doses of endostatin in the presence of conditional medium from SW480 cells. We found that endostatin significantly reduced EDA secretion by SW480 cells and the expression of integrin α9 in LECs. Immunofluorescence studies showed that EDA and integrin α9 colocalized on the cell membrane of LECs and these colocalizations were dramatically reduced by endostatin. Co‐immunoprecipitation studies demonstrated that EDA interacted with integrin α9 in LECs, and showed that endostatin treatment inhibited the formation of EDA–integrin α9 complex in LECs. Furthermore, we found that the arrangement and polarity of LEC cytoskeletons were destroyed by endostatin substantially, leading to a reduced formation of tube‐like structures of LECs and a suppressed chemotaxis of LECs toward SW480 cells. Consistently, EDA and integrin α9 expressions as well as lymphangiogenesis were significantly suppressed by endostatin in colorectal cancer xenografts. In conclusion, our results suggest that endostatin reduces colorectal tumor‐induced lymphangiogenesis, at least in part, by inhibiting EDA‐integrin α9 pathway. J. Cell. Biochem. 112: 2106–2114, 2011.

ERCC5 promoter polymorphisms at -763 and +25 predict the response to oxaliplatin-based chemotherapy in patients with advanced colorectal cancer

2 This study aimed to investigate whether single nucleotide polymorphisms (SNPs) in the promoter of the excision repair cross complementation group 5 (ERCC5) gene influences response to oxaliplatin-based chemotherapy. Eighty-three patients with cytologically or histologically confirmed advanced colorectal cancer (CRC), at least one measurable lesion and underwent oxaliplatin-based chemotherapy were studied. To this end, six polymorphisms (-1415C>T, -763A>G, -413C>T, +25A>G, +202C>T, +372C>T) in the ERCC5 promoter were selected for investigation. Genomic DNA was obtained from peripheral blood cells, and polymerase chain reaction-ligation detection reaction was used to analyze these SNPs. The χ2 test or Fisher’s exact test was then used to investigate the association between polymorphisms and chemotherapy response. Our results showed that the response rate among patients with the -763GG genotype (72.7%) was significantly higher than that of other genotypes (22.2% for AA genotype, P=0.008 and 37.2% for AG genotype, P=0.046 respectively). In addition, the response rate among patients with the +25AA genotype (75%) was significantly higher than that of other genotypes (24.1% for GG genotype, P=0.004 and 35.7% for AG genotype, P=0.022 respectively). Patients with the -763A/+25G haplotype had a higher risk of non-response to oxaliplatin chemotherapy compared to those carrying the -763G/+25A haplotype (OR 2.672, 95% CI 1.353-5.278, P=0.004). However, no genetic variation was observed at site -413, and no significant association was found between the -1415C>T, +202C>T or +372C>T polymorphisms and chemotherapy response. Therefore, these data suggest that ERCC5 promoter polymorphisms at -763 and +25 may be important predictors of response to oxaliplatin chemotherapy.

Stable RNA interference of hexokinase II gene inhibits human colon cancer LoVo cell growth in vitro and in vivo.

The purpose of this study is to investigate the effect of silencing hexokinase II (HK II) gene with RNA interference (RNAi) technique on colon cancer LoVo cell proliferation in vitro and in vivo. A short hairpin RNA (shRNA) eukaryotic expression vector against HK II gene was constructed, named as plasmid pGenesil-1-HK II, and transfected into LoVo cells. The expression of HK II gene was detected by RT-PCR and Western blot analysis respectively. Then, tumor colony formation was observed, and cell cycle was also assessed by flow cytometry and the contents of intracellular adenosine triphosphate (ATP) by high performance liquid chromatography (HPLC). Furthermore, LoVo cells were injected subcutaneously into nude mice. After a 4-week follow-up period, the sizes and weights of tumors were measured. Moreover, the expression of Ki67 protein was observed by immunohistochemical technique, and cell apoptosis by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Consequently, the expression of HK II gene was efficiently blocked by RNAi. Down-regulation of HK II gene expression significantly suppressed cloning efficiency and cell cycle of LoVo cell in vitro and tumor growth in vivo. Compared with untransfected LoVo cells, LoVo cells transfected with pGenesil-1-HK II plasmids showed significant decrease in the cellular ATP contents and Ki67 expression, and obvious increase in the apoptosis indexes. Our results suggest that HK II gene can act as a crucial therapeutic target for slowing colon cancer growth.

Colorectal tumor derived fibronectin alternatively spliced EDA domain exserts lymphangiogenic effect on human lymphatic endothelial cells

The objective of this study was to investigate whether tumor derived fibronectin alternatively spliced EDA domain has a lymphangiogenic potency on human lymphatic endothelial cells (LECs) in tumor generation to facilitate tumor lymphatic metastasis. LECs were cultured in three-dimentional culture system and treated with SW480 supernant which was highly rich in EDA, the result demonstrated that SW480 supernant could facilitate tube-like formations of LECs evidently when compared with controls. Integrinα9 was identified by immunofluorescence to be a specific receptor for EDA because we found co-locozation of EDA and integrinα9 on LECs as well as significant up-regulation of integrinα9 in SW480 supernant treated group. Westernblot and immunofluorescence revealed that EDA also had important roles accommodating the expressions of some key regulators of lymphangiogenesis such as Prox1 and F-actin so as to facilitate motility and sprouting of LECs. In addition, it had been confirmed that all of these effects could be inhibited markedly by EDA antibody (IST-9). Based on these findings, we assert that EDA derived from tumor cells has an important role in facilitating lymphangiogenesis of malignant tumor. Furthermore, EDA pathway may provide a potent target for tumor lymphatic metastasis therapy.

Phosphorylated Insulin-Like Growth Factor 1 Receptor is Implicated in Resistance to the Cytostatic Effect of Gefitinib in Colorectal Cancer Cells

IntroductionThe ability of certain cancer cells to maintain signaling via the phosphoinositide-3-kinase/Akt and/or Ras/mitogen-activated protein kinase (MAPK) pathways has been repeatedly involved in resistance to epidermal growth factor receptor (EGFR) inhibition.DiscussionWe investigated the potential mechanisms of the uncoupling of EGFR from its downstream signals in colorectal cancer (CRC) cells. Alternative growth factor receptors and regulation of downstream pathways in different gefitinib-responsive cell lines were determined. Basal insulin-like growth factor receptor-1β (IGFR-1β) phosphorylation was undetectable or present at very low levels in highly gefitinib-responsive cell lines and was present at strikingly high levels in less responsive cell lines. Further analysis of cell lines representing the most sensitive (Lovo), moderately sensitive (HT29), and most resistant (HCT116) strains was treated with an IGFR-1 inhibitor (AG1024), gefitinib, or both, revealing that elevated IGFR-1β phosphorylation can compensate for the loss of EGFR signaling function. Increased insulin-like growth factor II expression induced by gefitinib or heterodimerization of EGFR and IGFR-1β may trigger IGFR-1β signal transduction via activation of Akt and MAPK. In addition, high levels of EGFR and IGFR-1β phosphorylation were detected in CRC tumor tissue. We also showed that gefitinib- and/or AG1024-induced cytostatic effects could be mediated by glycogen synthase kinase-3β (GSK-3β) activation. Our data suggest that the crosstalk between EGFR and IGFR-1β signaling are likely to contribute to resistance of CRC cells to gefitinib and that measurement of GSK-3β activation may present a potential biomarker for evaluating the antitumor efficacy of receptor tyrosine kinase inhibition.

Expression of Tiam1 and VEGF-C correlates with lymphangiogenesis in human colorectal carcinoma

Objective To investigate the relationship between Tiam1 and lymphangiogenesis in human colorectal carcinoma (CRC) tissues, as well as the expression of VEGF-C in a CRC cell line (HCT116) after knockdown of the Tiam1 gene with RNA interference (RNAi). Methods The expressions of Tiam1, Rac1, VEGF-C and Podoplanin in 50 samples of CRC were detected by immunohistochemical analysis. The lymph microvessel density (LMVD) in Podoplanin positive specimens was evaluated. The results were analyzed statistically to investigate the correlation of Tiam1, VEGF-C, lymph node metastasis and other clinicopathological parameters. An shRNA eukaryotic expression vector against Tiam1 gene was constructed and transfected into HCT116 cells. The expression of Tiam1 gene was assessed by RT-PCR and western blot analysis. Results In the specimens of CRC tissue, the positivity rate of Tiam1 and VEGF-C was 84% and 58%, respectively. The positivity rate of VEGF-C in the Tiam1 positive group (64.3%) was significantly higher than that in the Tiam1 negative group（25.0%）. The LMVD in the Tiam1 positive group（11.35±3.34） was significantly higher than that in the Tiam1 negative group（7.38±2.27）.In addition, the expression of the Tiam1 gene was efficiently blocked by RNAi. Down-regulation of Tiam1 gene expression significantly suppressed HCT116 cell growth in vitro. Compared with untransfected HCT116 cells, HCT116 cells transfected with pGenesil-1-Tiam1 plasmids showed a significant decrease in the expression of VEGF-C. Conclusions We suggest that the Tiam1 gene may act as a crucial therapeutic target for Lymphangiogenesis in CRC.

Downregulation of the Hexokinase II Gene Sensitizes Human Colon Cancer Cells to 5-Fluorouracil

Background: Extensive trials have indicated that cancer cells with high glycolytic activity exhibit decreased sensitivity to anticancer agents. Moreover, recent research has proven that specific inhibitors of hexokinase (HK) II, a key glycolytic enzyme, may enhance the activity of anticancer drugs. The aim of this study was to investigate the effect and mechanisms of HK II on chemosensitivity of a colon cancer cell line (LoVo) to 5-fluorouracil (5-FU). Methods: HK II gene expression was downregulated by RNA interference in the colon cancer cell line LoVo, which was detected by Western blot analysis. Then the IC50 value of 5-FU was determined in LoVo cells via MTT assay. In addition, cell apoptosis and mitochondrial membrane potential (MMP) were assessed by flow cytometry and caspase-3 activity by its substrate color reaction. Results: In LoVo cells, HK II downregulation resulted in a decreased IC50 value of 5-FU and increased apoptosis. Furthermore, HK II downregulation resulted in a decreased MPP and activation of caspase-3. Conclusion: Our findings suggest that targeting HK II may be beneficial for patients with colon cancer treated with 5-FU.

High-mobility group box 1 protein activating nuclear factor-κB to upregulate vascular endothelial growth factor C is involved in lymphangiogenesis and lymphatic node metastasis in colon cancer

Objectives To investigate the roles of high-mobility group box 1 (HMGB1) protein in lymphangiogenesis and lymphatic node metastasis in colon cancer. Methods Archival tumour specimens from patients with colon cancer were analysed in this retrospective immunohistochemical study. HMGB1, vascular endothelial growth factor C (VEGF-C) and podoplanin protein levels were analysed immunohistochemically. In vitro studies using the colon cancer cell line HCT116 were also undertaken to investigate the relationship between HMGB1, VEGF-C and nuclear factor (NF)-κB. Results Specimens from 70 patients with colon cancer were reviewed. The presence of positive HMGB1 immunohistochemical staining significantly correlated with lymphatic microvessel density, lymph node metastasis and VEGF-C immunohistochemical staining in colon cancer specimens. The presence of positive VEGF-C immunohistochemical staining significantly correlated with lymph node metastasis. The in vitro studies demonstrated that HMGB1 upregulated VEGF-C mRNA and protein in a dose-dependent manner in HCT116 cells, and that this was mediated via NF-κB. Conclusions HMGB1 immunohistochemical staining was significantly associated with lymphangiogenesis and lymphatic node metastasis in colon cancer. There was evidence that HMGB1 upregulates VEGF-C by activating NF-κB in a colon cancer cell line.

Semaphorin3F Down-Regulates the Expression of Integrin αvβ3 and Sensitizes Multicellular Tumor Spheroids to Chemotherapy via the Neuropilin-2 Receptor in vitro

Background: Multicellular resistance (MCR), i.e. decreased sensitivity to anticancer drugs compared with common monolayer cell (MC) cultures, depends partly on tumor cell-cell adhesion. Previous studies have shown that anti-adhesive therapies, including integrin αv, β1 and αvβ3 targeting, induced apoptosis and reversed the sensitivity of MCR. Methods: A model of three-dimensional cell culture was used to establish HT29 multicellular spheroid cells (MCS) and explore the effect of semaphorin3F (Sema3F) on integrin-mediated cell-cell interactions in MCS of a human colorectal adenocarcinoma cell line (HT29) and sensitization of HT29 MCS to 5-fluorouracil and oxaliplatin via a decrease in integrin αvβ3. Results: Elevated expression of Sema3F led to the up-regulation neuropilin-2 (Nrp2) receptor expression and the down-regulation of integrin αvβ3 expression. Furthermore, short interfering RNA of Nrp2 could reverse MCR. Conclusion: Our study demonstrates that Sema3F can sensitize MCR by decreasing integrin αvβ3 expression via the Nrp2 receptor.

Multimodal therapy for adult Wilms' tumour: an experience from one centre.

IntroductionWilms’ tumour (WT) is very rare in adults but very common in children. Treatment guidelines for adult patients with WT are still insuffi cient. Some study groups recommend that therapeutic protocols for adults with WT (AWT) should follow the guidelines that have been established for children.ObjectiveTo describe the clinical and pathological characteristics of AWT as well as the treatment protocols and outcomes for AWT at our treatment centre.Material and methodsSeven patients (5 females and 2 males) were diagnosed with AWT in our hospital between 2002 and 2009. The tumours were staged and the patients were treated according to the paediatric regimen recommended by the National Wilms’ Tumor Study Group.ResultsThe median patient age at the time of diagnosis was 29 years (range, 16–37 years). Flank pain was the most common clinical presentation. One patient was in Stage I of disease development, two were in Stage II, two were in Stage III and two were in Stage IV. Anaplasia was present in 3 patients with Stage III or Stage IV disease. All of the patients but one underwent nephrectomy and 2 incomplete surgeries were performed. Seven patients received 2-drug or 3-drug chemotherapy (dactinomycin and vincristine and/or doxorubicin). Two patients with Stage III disease also received radiation therapy (a total dose of 3600 or 3960 cGy). Complete remission was achieved in 4 patients. Three patients (one with Stage III disease, 2 patients with Stage IV disease) died of their disease and those patients were all classifi ed with an unfavourable histological type called anaplasia. With a median follow-up of 53.5 months (range, 40–102 months), the 3-year and 5-year overall survival rates were 57.1% (95% confi dence interval, 20.4–93.8%).ConclusionsThe results of this report suggest that histological anaplasia might be an adverse prognostic factor for AWT. Proper application of the diagnostic and therapeutic regimens established for children may improve the prognosis of adult patients with WT.