Kesen Xu

Integrin alphavbeta6 mediates the potential for colon cancer cells to colonize in and metastasize to the liver

Integrin alphavbeta6 (αvβ6) is correlated with colon cancer progression. To detect the effects of αvβ6 on liver metastasis, the specificity of αvβ6 against the monoclonal antibody (mAb) 2G2 was examined by immunoprecipitation. Integrin αvβ6‐immunoreactivity (IR) in liver metastasis tissues (63 cases) and colon carcinoma (358 cases) were examined. These results showed that αvβ6 was specifically recognized by the mAb 2G2, and that rates of αvβ6 positivity in liver metastatic tissues (71.4%, 45/63) were higher than that for primary colon cancer (34.0%, 122/358) (P < 0.01). Patients who were αvβ6‐positive had higher liver metastasis rates (17%, 21/122) than those who were αvβ6‐negative (only 3%, 7/236) (P < 0.01). To examine the underlying mechanisms associated with αvβ6 regulating colonic metastasis in the liver, experimental liver metastasis (intrasplenic injection of HT29 transfectants) and liver colonization assays (direct injection of WiDr transfectants into the liver) in nude mice were performed; these demonstrated that αvβ6 contributed to the promotion of the metastatic potential and the survival of cancer cells in the liver. Matrix metalloproteinase‐9 (MMP‐9) levels in the cultures of both HT29 and WiDr cells were detected by the Biotrak MMP‐9 activity assay system and gelatin zymography assay, and showed that suppression of αvβ6‐IR inhibited MMP‐9 activity and secretion. Transwell migration assay in vitro also showed that αvβ6 promoted migration on fibronectin for HT29/WiDr mock compared with HT29/WiDr antisense β6 transfects (P < 0.01). We concluded that αvβ6 may mediate the potential for colon cancer cells to colonize in and metastasize to the liver. The mechanisms that αvβ6 may be involved in include the promotion of MMP‐9 secretion, the enhancement of migration on fibronectin, and the survival of cancer cells in the liver. (Cancer Sci 2008; 99: 879–887)

Vascular endothelial growth factor (VEGF) enhances gastric carcinoma invasiveness via integrin alpha(v)beta6

Integrins play an important role in tumor metastasis induced by vascular endothelial growth factor (VEGF). However, in the case of gastric cancer, the precise role of VEGF in regulating integrin alphavbeta6 is unclear. In this study, we found that most of the alphavbeta6 integrin-positive gastric cancer tissues were also VEGF-positive. Furthermore, when gastric carcinoma cells were exposed to VEGF, expression of alphavbeta6 integrin was up-regulated and the extracellular signal-related kinase (ERK) pathway was activated. When integrin alphavbeta6 was blocked either with beta6 siRNA or anti-alphavbeta6 antibody, the migration of tumor cells normally induced by VEGF, as well as the activation of ERK, were markedly inhibited. Blocking the ERK signaling pathway significantly inhibited cell mobility. Taken together, the data suggest that VEGF is critical to the invasive process in human gastric cancer and that this occurs via up-regulation of integrin alphavbeta6 expression and activation of ERK.

PKC promotes the migration of colon cancer cells by regulating the internalization and recycling of integrin αvβ6

Recently published studies have suggested that integrin trafficking is necessary to support cell migration, but the role of internalization and recycling of integrin αvβ6 in colon cancer cells remained unclear. In our study, we demonstrated the existence of the integrin cycle and found that inhibition of ERK2 phosphorylation by PD98059 or deletion of the ERK2 direct binding site on the β6 cytoplasmic domain could interrupt the internalization of integrin αvβ6, but had no effect on its recycling. Furthermore, integrin αvβ6 trafficking played a key role in the migration of colon cancer cells towards fibronectin. Activation of PKC significantly accelerated the internalization and recycling of integrin αvβ6, which could facilitate rapid redistribution of integrin αvβ6 and increase cell motility. When colon cancer cells became crowded, the increase in αvβ6 levels at the cell surface was not accompanied by a change in total αvβ6 expression in cell lysates. This change may be due to a redistribution of αvβ6 in cell microstructures and a rapid cellular response towards the demands of migration.

Norcantharidin induces HT-29 colon cancer cell apoptosis through the αvβ6–extracellular signal-related kinase signaling pathway

Norcantharidin has been used as an efficacious anticancer drug in China for many years, but its true mechanism remains poorly understood. Intriguingly, in an in vitro series study of anticancer drugs, we found that norcantharidin can effectively inhibit epithelial tumor cells from expressing integrin αvβ6. Our previous studies have confirmed that integrin αvβ6 is closely relevant to malignant epithelial cell tumor biology behavior, and it can promote cancer cells to invade and metastasize through a special αvβ6–extracellular signal‐related kinase (ERK) direct signaling pathway. In this study, we investigated the relationship between the norcantharidin anticancer mechanism and integrin αvβ6. After HT‐29 colon cancer cells were treated with norcantharidin, cell apoptosis increased remarkably. The expression of αvβ6 and the amount of p‐ERK decreased substantially; simultaneously, the linkage between αvβ6 and ERK was barely detectable. However, the expression of other integrins and the levels of mitogen‐activated protein kinase hardly changed. On these grounds, we presumed that norcantharidin induced HT‐29 colon cancer cell apoptosis through the αvβ6–ERK signaling pathway. This finding elicited a novel strategy for targeting the whole αvβ6–ERK signal pathway, rather than simply blocking the combining site of αvβ6–ERK in colon cancer treatment. (Cancer Sci 2009; 100: 2302–2308)

Establishment of a Rat Model of Portal Vein Ligation Combined with In Situ Splitting

Background Portal vein ligation (PVL) combined with in situ splitting (ISS) has been shown to induce remarkable liver regeneration in patients. The purpose of this study was to establish a model of PVL+ISS in rats for exploring the possible mechanisms of liver regeneration using these techniques. Materials and Methods Rats were randomly assigned to three experimental groups: selective PVL, selective PVL+ISS and sham operation. The hepatic regeneration rate (HRR), Ki-67, liver biochemical determinations and histopathology were assessed at 24, 48, and 72 h and 7 days after the operation. The microcirculation of the median lobes before and after ISS was examined by laser speckle contrast imaging. Meanwhile, cytokines such as TNF-α, IL-6, HGF and HSP70 in regenerating liver lobes at 24 h was investigated by RT-PCR and ELISA. Results The HRR of PVL+ISS was much higher than that of the PVL at 72 h and 7 days after surgery (p<0.01). The expression of Ki-67 in hepatocytes in the regenerating liver lobe was stronger in the PVL+ISS group than in the PVL group at 48 and 72 h (p<0.01). There was a significant reduction in microcirculation blood perfusion of the left median lobe before and after ISS. Liver biochemical determinations and histopathology demonstrated more severe hepatocyte injury in the PVL+ISS group. Both the mRNA levels of TNF-α and IL-6 and the protein levels of TNF-α, IL-6 and HGF in regenerating liver lobes were higher in the PVL+ISS than the PVL alone. Conclusions The higher HRR in the PVL+ISS compared with the PVL confirmed that we had successfully established a PVL+ISS model in rats. The possible mechanisms included the reduced microcirculation blood perfusion of the left median lobe and up-regulation of cytokines in the regenerating lobes after ISS.

Suppression of integrin αυβ6 by RNA interference in colon cancer cells inhibits extracellular matrix degradation through the MAPK pathway

Integrin αυβ6 plays a very important role in the progression of colon cancer cells and is now defined as a novel, independent prognostic indicator for aggressive colon cancer in humans. Herein, we use the RNA interfering technology to downregulate the expression of αυβ6 in colon cancer cells. Our data demonstrate that plasmid vector based shRNA can effectively down‐regulate αυβ6 expression in protein and mRNA levels. Supression of integrin αυβ6 inhibits the phosphorylation and nonphosphorylation level of ERK1/2, the secretion of uPA, pro‐MMP‐9 and pro‐MMP‐2 in tumor conditioned medium, and more important, inhibits MAPK‐dependent [3H] labeled collagen IV degradation via the plasminogen activation cascade. Our study demonstrates in vitro that supression of integrin αυβ6 inhibits extracellular matrix degradation through the MAPK pathway.

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and gastric cancer susceptibility

AIM To identify the association between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and gastric cancer (GC) susceptibility. METHODS Systematic searches were performed on the electronic databases PubMed, ISI, Web of knowledge, CNKI and Wanfang, as well as manual searching of the references of the identified articles. A total of 26 papers were included in this meta-analysis. Overall and subgroup analyses were performed. Odds ratio (OR) and 95%CI were used to evaluate the associations between MTHFR polymorphisms and GC risk. The I (2) statistics were used to evaluate between-study heterogeneity. Sensitivity analysis was also performed. RESULTS Increased risk was found for the MTHFR C677T polymorphism under four genetic models (TT + CT vs CC: OR = 1.23, P = 0.002; T vs C: OR = 1.15, P = 0.001; TT vs CC: OR = 1.37, P = 0.0005; TT vs CT + CC: OR = 1.17, P = 0.0008). Subgroup analysis by ethnicity suggested that C677T polymorphism conferred a risk of GC in eastern but not in western populations. Stratification by tumor site showed an association between the C677T polymorphism and gastric cardia cancer and non-cardia GC in the worldwide population and in eastern populations. Regardless of comparisons with controls or diffuse-type GC, a positive association was found for the C677T polymorphism and an increased risk of intestinal-type GC in the whole population and in western populations. With regard to the A1298C polymorphism, we found that genotype CC was significantly decreased and conferred protection against GC in eastern populations (CC vs AA: OR = 0.44, P = 0.03; CC vs AC + AA: OR = 0.46, P = 0.04). CONCLUSION MTHFR C677T polymorphism is a risk factor for GC, and the A1298C polymorphism may be a protective factor against GC in eastern populations.

Integrin αvβ6 and matrix metalloproteinase 9 correlate with survival in gastric cancer.

AIM To investigate the expression of integrin αvβ6 and matrix metalloproteinase 9 (MMP-9), their association with prognostic factors and to assess their predictive role in gastric cancer patients. METHODS Immunohistochemistry was used to determine the expressions of integrin αvβ6 and MMP-9 in 126 specimens from patients with primary gastric carcinoma. Associations between immunohistochemical staining and various clinic pathologic variables of tissue specimens were evaluated by the χ(2) test and Fishers exact test. Expression correlation of αvβ6 and MMP-9 was assessed using bivariate correlation analysis. The patients were followed-up every 3 mo in the first two years and at least every 6 mo afterwards, with a median follow-up of 56 mo (ranging from 2 mo to 94 mo). Four different combinations of αvβ6 and MMP-9 levels (that is, both markers positive, both markers negative, αvβ6 positive with MMP-9 negative, and αvβ6 negative with MMP-9 positive) were evaluated for their relative effect on survival. The difference in survival curves was evaluated with a log-rank test. Survival analysis was conducted using the Kaplan-Meier survival and Cox proportional hazards model analysis. RESULTS The expressions of integrin αvβ6 and MMP-9 were investigated in 126 cases, among which 34.92% were positive for αvβ6 expression, and 42.06% for MMP-9 expression. The expression of αvβ6 was associated with Lauren type, differentiation, N stage, and TNM stage (the P values were 0.006, 0.038, 0.016, and 0.002, respectively). While MMP-9 expression was associated with differentiation, T stage, N stage, and TNM stage (the P values were 0.039, 0.014, 0.033, and 0.008, respectively). The positive correlation between αvβ6 and MMP-9 in gastric cancer was confirmed by a correlation analysis. The Kaplan-Meier survival analysis showed that patients with expression of αvβ6 or MMP-9 alone died earlier than those with negative expression and that patients who were both αvβ6 and MMP-9 positive had a shorter overall survival than those with the opposite pattern (both αvβ6 and MMP-9 negative) (P = 0.000). A Cox model indicated that positive expression of αvβ6 and MMP-9, diffuse Lauren type, as well as a senior grade of N stage, M stage, and TNM stage were predictors of a poor prognosis in univariate analysis. Only αvβ6 and MMP-9 retained their significance when adjustments were made for other known prognostic factors in multivariate analysis (RR = 2.632, P = 0.003 and RR = 1.813, P = 0.007). CONCLUSION The expression of αvβ6 and MMP-9 are closely correlated, and the combinational pattern of αvβ6 and MMP-9 can serve as a more effective prognostic index for gastric cancer patients.

PPARγ activation reduces ischemia/reperfusion‑induced metastasis in a murine model of hepatocellular carcinoma

Ischemia/reperfusion (I/R) injury during liver resection or transplantation for the treatment of hepatocellular carcinoma (HCC) may increase the risk of metastasis. Peroxisome proliferator-activated receptor-γ (PPARγ) activation has been observed to exert a protective effect against hepatic I/R injury. However, whether PPARγ activation exerts a protective effect against I/R-associated liver metastasis remains unknown. Therefore, the present study aimed to investigate the effects of the PPAR agonist rosiglitazone and the specific PPARγ antagonist GW9662 on tumor metastasis following hepatic I/R. An experimental mouse model of hepatic I/R-induced HCC metastasis was designed in order to determine the effects of I/R on tumor metastasis in the liver. Four groups were established: Sham, control (I/R), rosiglitazone (Ro) and rosiglitazone with GW9662 (Ro + GW) groups. In the latter two groups, the treatments were administered intravenously 1 h prior to the induction of ischemia. Tumor load was measured 12 days after the procedure. Furthermore, tissue analyses were conducted to determine the expression levels of alanine aminotransferase, myeloperoxidase (MPO), matrix metalloproteinase (MMP)-9, vascular cell adhesion molecule (VCAM)-1, nuclear factor (NF)-κB and PPARγ. Rosiglitazone pretreatment appeared to significantly mitigate hepatic I/R injury, as indicated by serological and histological analysis. The levels of VCAM-1, MPO and MMP-9 expression in the Ro group were significantly reduced at 8 h following ischemia compared with those in the control and Ro + GW groups. In addition, rosiglitazone inhibited the I/R-induced activation of NF-κB, and GW9662 attenuated the inhibitory effect. To the best of our knowledge, the present study is the first to report on the expression and the functional roles of PPARγ in I/R-associated metastasis. Short-term treatment of mice with rosiglitazone, a potent PPARγ agonist, confers protective effects against hepatic I/R-associated metastasis. Thus, PPARγ may be a potential therapeutic target for the protection of the liver against I/R-associated metastasis.

Gene transfer of antisense B7.1 attenuates acute rejection against liver allografts in rats.

ABSTRACT Objective: Blockade of CD80-CD28 costimulatory pathway induces unresponsiveness of T cells to alloantigens and protects allografts against immune rejection. The aim of this study was to investigate whether downregulating the expression of B7.1 (CD80) in the donor livers by antisense B7.1 gene transfer could attenuate the acute immune rejection against liver allografts in rats. Methods: The liver grafts from 60 Dark Agouti rats were intraportally perfused with antisense B7.1 expression vector, before they were transplanted into Lewis rats. Empty vector pcDNA3 served as control to be perfused into livers of another group of 60 Dark Agouti rats. The orthotopic liver transplantation was performed. The rats were randomly sacrificed at scheduled time points to collect liver allografts, or monitored to record the survival rate. The livers were histologically examined to calculate Banff rejection activity index, or subjected to Western blot analysis or immunohistochemistry for examining the expression of B7.1, or counting CD4+ and CD8+ cells. Results: Antisense gene transfer resulted in markedly downregulation of B7.1 in the donor livers, attenuated acute immune rejection against liver allografts, prolonged the survival time of rats, and decreased the number of infiltrating CD4+ and CD8+ cells in livers. Conclusions: Blocking expression of B7.1 in liver by antisense gene therapy may represent a potential strategy to attenuate acute rejection against liver allografts.