Young San Ko

A hypoxia-dependent upregulation of hypoxia-inducible factor-1 by nuclear factor-κB promotes gastric tumour growth and angiogenesis

Background:The underlying mechanisms involved in the activation of hypoxia-inducible factor-1 (HIF-1) in gastric cancer remain unclear. As nuclear factor-κB (NF-κB) as well as HIF-1 have been implicated in angiogenesis of various cancers, we investigated their relationship in gastric cancer.Methods:Nuclear expressions of HIF-1α and NF-κB/RelA were assessed in 251 human gastric carcinoma specimens by immunohistochemical tissue array analysis. Stable human gastric cancer cells, infected with a retroviral vector containing super-suppressive mutant form of IκBα (IκBαM), were used for animal studies as well as cell culture experiments. Xenografted tumours were measured and IκBαM effects on angiogenesis and HIF-1α activation were assessed by immunohistochemistry, western blotting, luciferase reporter assay, and semiquantitative reverse transcription–polymerase chain reaction. In addition, NF-κB effects on the HIF-1α degradation and synthesis were examined.Results:Hypoxia-inducible factor-1α activation positively correlated with RelA activation in clinical gastric cancer samples (P<0.001). The IκBαM overexpression suppressed tumour growth, microvessel density, and HIF-1α activation in xenografted tumours. Cell culture experiments showed that hypoxia-induced HIF-1α expression was reduced by NF-κB inhibition under hypoxic conditions at the translational level.Conclusion:The hypoxia-dependent activation of the NF-κB/HIF-1α/VEGF pathway contributes, at least in part, to gastric cancer promotion via enhancement of angiogenesis.

The forkhead transcription factor FOXO1 mediates cisplatin resistance in gastric cancer cells by activating phosphoinositide 3-kinase/Akt pathway.

BackgroundCisplatin (CDDP) is one of the most important chemotherapeutic agents in the treatment of advanced gastric cancer, but its efficacy is limited by CDDP resistance. Because the transcription factor FOXO1 is related to chemoresistance in various cancer cells, we investigated the function of FOXO1 in CDDP resistance in human gastric cancer cells.MethodsHuman gastric cancer cell lines MKN45 and SNU-601 were used. FOXO1 activation was modulated by transfection of FOXO1 AAA mutant gene or FOXO1 shRNA. The effects of FOXO1 on cell growth and CDDP cytotoxicity were assessed by crystal violet assay. Protein expressions of FOXO1, p110α, pAkt, and Akt were analyzed by Western blotting, and FOXO1 mRNA expression was evaluated by semiquantitative reverse transcription-polymerase chain reaction. FOXO1 activity was determined by luciferase reporter assay, and cell apoptosis was assessed by DAPI staining and Western blotting for PARP cleavage.ResultsCisplatin treatment induced FOXO1 expression and activation in both gastric cancer cell lines. FOXO1 overexpression increased the CDDP resistance without changes in cell growth, whereas FOXO1 silencing enhanced CDDP cytotoxicity along with apoptotic characteristics. Both constitutive and CDDP-induced FOXO1 activations were accompanied by an increase in p110α and pAkt expression. Furthermore, Akt inhibition by LY294002 treatment restored the CDDP cytotoxicity that was suppressed by FOXO1 overexpression.ConclusionFOXO1 inhibits CDDP-induced apoptosis in gastric cancer cells via activating PI3K/Akt pathway. Thus, FOXO1 may be an useful pharmacological indicator to predict CDDP efficacy in gastric cancer treatment.

Constitutive phosphorylation of the FOXO1 transcription factor in gastric cancer cells correlates with microvessel area and the expressions of angiogenesis-related molecules.

BackgroundAlthough FOXO transcription factors may have an anti-angiogenic role, little is known about their role in tumor angiogenesis. The present study was performed to investigate the correlation between the constitutive expression of phosphorylated FOXO1 (pFOXO1) and angiogenesis in gastric cancer.MethodsImmunohistochemistry was performed on tissue array slides containing 272 gastric carcinoma specimens, and the correlations between the cytoplasmic pFOXO1 expression in gastric cancer cells and CD34-immunopositive microvessel area (MVA) or the expressions of angiogenesis-related molecules were analyzed. In vitro analyses with Western blotting and semiquantitative reverse transcription-polymerase chain reaction were performed using the stable SNU-638 gastric cancer cell line transfected with lentivirus-delivered FOXO1 short hairpin RNA.ResultsThe cytoplasmic expression of pFOXO1 in tumor cells was observed in 85% of gastric carcinoma cases, and was found to be positively associated with higher MVA (P = 0.048). Moreover, pFOXO1 expression was positively correlated with the expressions of several angiogenesis-related proteins, including hypoxia inducible factor-1α (HIF-1α, P = 0.003), vessel endothelial growth factor (P = 0.004), phosphorylated protein kinase B (P < 0.001), and nuclear factor-κB (P = 0.040). In contrast, the expression of pFOXO1 was not correlated with that of phosphorylated signal transducer and activator of transcription 3 or β-catenin. In addition, cell culture experiments showed that FOXO1 suppression increased the mRNA and protein expressions of HIF-1α.ConclusionOur results suggest that pFOXO1 expression in cancer cells plays a role in gastric cancer angiogenesis via mechanisms involving various angiogenesis-related molecules. Animal experiments are needed to confirm the anti-angiogenic role of FOXO1 in human gastric cancer.

Nuclear factor-κB dependency of doxorubicin sensitivity in gastric cancer cells is determined by manganese superoxide dismutase expression

The role of nuclear factor‐κB (NF‐κB) activation in cancer cell apoptosis appears to be tailored specifically for each cell type and the type of NF‐κB inducer. The present study aimed to determine whether or not NF‐κB activation is associated with chemosensitivity to doxorubicin (DOX) using the DOX‐sensitive SNU‐601 and DOX‐resistant SNU‐216 gastric cancer cell lines. The effect of NF‐κB activation on DOX (1 µg/mL) sensitivity was analyzed after the suppression of NF‐κB activation using transfection of the super‐suppressive mutant form of IκBα (mIκBα) or pretreatment with pyrrolidine dithiocarbamate. In addition, the association between NF‐κB and manganese superoxide dismutase (MnSOD) in relation to DOX sensitivity was analyzed after the modulation of MnSOD expression. The NF‐κB activity was much higher in DOX‐resistant SNU‐216 cells than in DOX‐sensitive SNU‐601 cells before and after DOX treatment. Overexpression of mIκBα or pyrrolidine dithiocarbamate pretreatment decreased the DOX resistance in SNU‐601 cells with low MnSOD expression, but not in SNU‐216 cells with high MnSOD expression. In comparison, the overexpression of MnSOD, which also suppressed NF‐κB activation in both cell lines, increased DOX resistance in SNU‐601 cells. Blocking of MnSOD expression using RNA interference techniques increased DOX sensitivity in SNU‐216 cells, which was further augmented by the additional inhibition of NF‐κB activity. Our results showed that whether NF‐κB contributes to DOX sensitivity in gastric cancer cells is determined by the level of MnSOD expression. Thus, targeting both MnSOD and NF‐κB may be helpful for increasing the efficacy of DOX treatment of DOX‐resistant SNU gastric cancer cells. (Cancer Sci 2008; 99: 1117–1124)

Loss of FOXO1 promotes gastric tumour growth and metastasis through upregulation of human epidermal growth factor receptor 2/neu expression.

Background:The biological significance of FOXO1, a member of the forkhead box O transcription factor family, in gastric cancer (GC) remains unclear. The present study provides direct evidence of the role of FOXO1 in tumour growth and metastasis of GC in relation to human epidermal growth factor receptor 2 (HER2).Methods:The expressions of FOXO1 and HER2 were modulated in GC cell lines (SNU-638, MKN45, SNU-216 and NCI-N87) by stable transfections. The effects of transfection on GC phenotypes were evaluated in vitro and in animal models. In addition, the relationship between FOXO1 and HER2 was analysed using GC clinical specimens, cell lines and xenografts.Results:FOXO1 silencing in GC cells increased colony formation and mesenchymal transition in vitro, as well as tumour growth and metastasis in nude mice, whereas HER2 silencing induced the opposite results.. Furthermore, an inverse relationship between FOXO1 and HER2 was found in clinical specimens of GC, GC cells and GC xenograft tumours. Although a negative crosstalk between these two molecules was shown, double knockdown of both FOXO1 and HER2 in GC cells revealed that HER2 silencing reversed the FOXO1 shRNA-induced migration and invasion even without the FOXO1 restoration.Conclusions:Our results indicate that loss of FOXO1 promotes GC growth and metastasis by upregulating HER2 expression and that the HER2 expression is more critical to the induction of GC cell metastasis. The present study provides evidence that the FOXO1/HER2 pathway may regulate GC progression in a subgroup of GC patients.

Constitutive activation of glycogen synthase kinase-3β correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer

BackgroundAberrant regulation of glycogen synthase kinase-3β (GSK-3β) has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3β phosphorylated at Tyr216 (pGSK-3β) and its relationship with other tumor-associated proteins in human gastric cancers.MethodsImmunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3β inhibitor lithium chloride (LiCl) for immunoblot analysis.ResultsWe found that pGSK-3β was expressed in 129 (46%) of 281 cases examined, and was higher in the early-stages of pathologic tumor-node-metastasis (P < 0.001). The expression of pGSK-3β inversely correlated with lymphatic invasion (P < 0.001) and lymph node metastasis (P < 0.001) and correlated with a longer patient survival (P < 0.001). In addition, pGSK-3β expression positively correlated with that of p16, p21, p27, p53, APC, PTEN, MGMT, SMAD4, or KAI1 (P < 0.05), but not with that of cyclin D1. This was confirmed by immunoblot analysis using SNU-668 gastric cancer cells treated with LiCl.ConclusionsGSK-3β activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3β activation is a useful prognostic marker for the early-stage gastric cancer.

A synergistic interaction between transcription factors nuclear factor-κB and signal transducers and activators of transcription 3 promotes gastric cancer cell migration and invasion

BackgroundThe transcription factor nuclear factor-κB (NF-κB) has been implicated in gastric cancer metastasis, but the underlying molecular mechanisms remain unclear. We investigated the role of the interaction between NF-κB and signal transducers and activators of transcription 3 (STAT3) in controlling metastatic potential of gastric cancer cells.MethodsImmunohistochemistry for NF-κB p65 (RelA), phospho-Tyr705-STAT3 (pSTAT3), or matrix metalloproteinase 9 (MMP9) was performed on tissue array slides containing 255 gastric carcinoma specimens. NF-κB inhibition in SNU-638 and MKN1 gastric cancer cell lines were performed by transduction with a retroviral vector containing NF-κB repressor mutant of IκBα, and STAT3 was silenced by RNA interference. We also did luciferase reporter assay, double immunofluorescence staining and immunoblotting. Cell migration and invasion were determined by wound-healing assay and invasion assay, respectively.ResultsNF-κB and STAT3 were constitutively activated and were positively correlated (P = 0.038) in gastric cancer tissue specimens. In cell culture experiments, NF-κB inhibition reduced STAT3 expression and activation, whereas STAT3 silencing did not affect NF-κB activation. Moreover, both NF-κB inhibition and STAT3 silencing decreased gastric cancer cell migration and invasion in a synergistic manner. In addition, both NF-κB activation and STAT3 activation were positively correlated with MMP9 in gastric cancer tissues (P = 0.001 and P = 0.022, respectively), decreased E-cadherin expression and increased Snail and MMP9 expressions in cultured cells.ConclusionNF-κB and STAT3 are positively associated and synergistically contribute to the metastatic potential of gastric cancer cells. Thus, dual use of NF-κB and STAT3 inhibitors may enhance the efficacy of the anti-metastatic treatment of gastric cancer.

Forkhead Transcription Factor FOXO1 Inhibits Angiogenesis in Gastric Cancer in Relation to SIRT1.

Purpose We previously reported that forkhead transcription factors of the O class 1 (FOXO1) expression in gastric cancer (GC) was associated with angiogenesis-related molecules. However, there is little experimental evidence for the direct role of FOXO1 in GC. In the present study, we investigated the effect of FOXO1 on the tumorigenesis and angiogenesis in GC and its relationship with SIRT1. Materials and Methods Stable GC cell lines (SNU-638 and SNU-601) infected with a lentivirus containing FOXO1 shRNA were established for animal studies as well as cell culture experiments. We used xenograft tumors in nude mice to evaluate the effect of FOXO1 silencing on tumor growth and angiogenesis. In addition, we examined the association between FOXO1 and SIRT1 by immunohistochemical tissue array analysis of 471 human GC specimens and Western blot analysis of xenografted tumor tissues. Results In cell culture, FOXO1 silencing enhanced hypoxia inducible factor-1α (HIF-1α) expression and GC cell growth under hypoxic conditions, but not under normoxic conditions. The xenograft study showed that FOXO1 downregulation enhanced tumor growth, microvessel areas, HIF-1α activation and vascular endothelial growth factor (VEGF) expression. In addition, inactivated FOXO1 expression was associated with SIRT1 expression in human GC tissues and xenograft tumor tissues. Conclusion Our results indicate that FOXO1 inhibits GC growth and angiogenesis under hypoxic conditions via inactivation of the HIF-1α–VEGF pathway, possibly in association with SIRT1. Thus, development of treatment modalities aiming at this pathway might be useful for treating GC.

Forkhead transcription factor FOXO1 inhibits nuclear factor‐κB in gastric cancer

FOXO1, a forkhead box O (FOXO) transcription factor, and nuclear factor‐κB (NF‐κB) are prognostically significant transcription factors in gastric cancer. As their relationship has been inconsistent depending on the cell type, we aimed to investigate whether FOXO1 is associated with NF‐κB p65 (RelA) in gastric cancer. Immunohistochemistry was performed on tissue array slides containing 298 gastric carcinoma specimens. We found that the cytoplasmic expression of pFOXO1, the inactive form of FOXO1, was positively correlated with nuclear RelA expression (p = 0.024). In addition, the expressions of pFOXO1 and RelA were positively related with cyclin D1 expression (p = 0.014 and p = 0.001, respectively) and Ki‐67 labeling index (p = 0.025 and p = 0.017, respectively). However, they did not show association with the expressions of cyclin E, p53 and pRb. Cell culture experiments showed that FOXO1 overexpression by transfection of FOXO1 AAA mutant gene suppressed NF‐κB activation in SNU‐484 gastric cancer cells. These results suggest that FOXO1 and NF‐κB are negatively associated and that FOXO1 is a negative upstream regulator of NF‐κB in gastric cancer.

Impairment of nuclear factor‐κB activation increased glutamate excitotoxicity in a motoneuron–neuroblastoma hybrid cell line expressing mutant (G93A) Cu/Zn‐superoxide dismutase

Mutations in the superoxide dismutase 1 (SOD1) gene are linked to glutamate excitotoxicity in familial amyotrophic lateral sclerosis (fALS), but the underlying mechanism remains unclear. We investigated whether nuclear factor‐κB (NF‐κB) activation is involved in glutamate excitotoxicity by using motor neuron–neuroblastoma hybrid cells that expressed a mutant (G93A) SOD1 (mtSOD1) or wild‐type SOD1 (wtSOD1). MtSOD1 cells were more vulnerable to glutamate excitotoxicity than wtSOD1 cells and showed higher NF‐κB activity, higher nuclear cRel expression, and lower nuclear RelA expression under basal conditions. Glutamate treatment increased NF‐κB activation along with nuclear expressions of RelA and cRel in wtSOD1 cells but induced only weak nuclear RelA expression in mtSOD1 cells. Suppression of NF‐κB activation using transfection of the superrepressive mutant form of IκBα (mIκBα) inhibited nuclear RelA expression in both types of SOD1 cells, which increased glutamate excitotoxicity in wtSOD1 cells but not in mtSOD1 cells. Furthermore, immunohistochemistry confirmed stronger RelA immunoreactivity in the nuclei of motor neurons of spinal cord in wild‐type SOD1 transgenic mice than in those in SOD1 G93A transgenic mice. In addition, we found that glutamate treatment decreased XIAP expression and increased caspase‐3 activity in mtSOD1 cells and mIκBα‐overexpressing wtSOD1 cells. Our results suggest that glutamate excitotoxicity in motor neurons of SOD1‐linked fALS is attributable, at least in part, to the impairment of IκBα‐dependent RelA activation and subsequent apoptosis mediated by XIAP inhibition and caspase‐3 activation.