lithium-chloride and Stomach-Neoplasms

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been reported in some cancer cells, including AGS human gastric adenocarcinoma cells. Reducing this resistance might shed light on the treatment of human gastric adenocarcinoma. In this study, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors can restore TRAIL responsiveness in gastric adenocarcinoma cells. The effect of two GSK-3 inhibitors, SB-415286, and LiCl, on apoptosis signaling of TRAIL in human gastric adenocarcinoma cell lines and primary gastric epithelial cells was analyzed. Both inhibitors can sensitize gastric adenocarcinoma cells, but not primary gastric epithelial cells, to TRAIL-induced apoptosis by increasing caspase-8 activity and its downstream signal transmission. Adding p53 siRNA can downregulate GSK-3 inhibitor-related sensitization to TRAIL-induced apoptosis and caspase-3 activity. GSK-3 inhibitors strongly activate the phosphorylation of JNK. Inhibition of JNK leads to earlier and more intense apoptosis, showing that the activation of JNK may provide anti-apoptotic equilibrium of pro-apoptotic cells. Our observations indicate that GSK-3 inhibitors can sentize AGS gastric adenocarcinoma cells to TRAIL-induced apoptosis. Therefore, in certain types of gastric adenocarcinoma, GSK-3 inhibitor might enhance the antitumor activity of TRAIL and mightbe a promising candidate for the treatment of certain types of gastric adenocarcinoma.

Topics: Adenocarcinoma; Aminophenols; Apoptosis; Cell Line, Tumor; Drug Screening Assays, Antitumor; Glycogen Synthase Kinase 3; Humans; Lithium Chloride; Maleimides; Protein Kinase Inhibitors; RNA, Small Interfering; Stomach Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53

Cancer stem cells (CSCs) are believed to contribute to the tumor growth in gastric carcinoma (GC), a common lethal malignancy. This study investigated the effect of aquaporin 3 (AQP3) on stem-like properties of human GC cells. Elevated AQP3 expression was associated with CD44 expression in human GC specimens. Expression of AQP3 and that of CD44 positively correlated with Lauren classification, lymph node metastasis, and lymphovascular invasion. Altering the AQP3 expression had pronounced effects on the tumorigenic potential and self-renewal capacity of the gastric cancer cell lines SGC7901, MGC803, and AGS, both in vitro and in vivo. Overexpression of AQP3 induced CD44 expression and activation of the β-catenin signaling pathway, whereas silencing AQP3 expression using short hairpin RNA had the opposite effect. Furthermore, pharmacological inhibition of GSK-3β using LiCl impaired the effect of AQP3 knockdown in CSCs, whereas the inhibition of the Wnt/β-catenin pathway by XAV939 blocked the effect of AQP3 overexpression. These results demonstrate that AQP3 promotes stem-like properties of human GC cells by activating the Wnt/GSK-3β/β-catenin signaling pathway.

Topics: Adult; Aged; Animals; Aquaporin 3; beta Catenin; Blotting, Western; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Heterocyclic Compounds, 3-Ring; Humans; Lithium Chloride; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Stomach Neoplasms; Transplantation, Heterologous; Wnt Signaling Pathway

Notch and Wnt signaling play critical roles in the regulation of development and diseases. Several studies have previously reported that Notch may be a therapeutic target in the treatment of various types of human cancer. In this study, we report that activation of Notch1 inhibits the proliferation of BGC-823 gastric cancer cells. However, the activation of the Wnt/β‑catenin signaling pathway promotes the growth of BGC-823 cells. Furthermore, the combinational activation of the two signaling pathways promotes the proliferation of BGC-823 cells. These data suggest that the activation of Wnt signaling overcomes the pro-apoptotic role of Notch in BGC-823 gastric cancer cells.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin E; Cyclin-Dependent Kinase 2; Doxycycline; Homeodomain Proteins; Humans; Lithium Chloride; Proto-Oncogene Proteins c-myc; Receptor, Notch1; Signal Transduction; Stomach Neoplasms; Transcription Factor HES-1; Wnt Proteins

Wnt5a, a non-transforming Wnt family member, plays complicated roles in oncogenesis and cancer metastasis. However, Wnt5a signaling in gastric cancer progression remains poorly defined. In this study, we found that Wnt5a dose-dependently stimulated the migration of human gastric cancer cells (SGC-7901), with the maximal effect at 100 ng/mL, via enhancing phosphorylation of PI3K/Akt and GSK3β and activating RhoA. Pharmaceutical inhibition of PI3K with LY294002 or Akt siRNA significantly decreased Wnt5a-induced GSK3β phosphorylation and consequently cell migration. Additionally, GSK3β siRNA remarkably inhibited Wnt5a-induced RhoA activation, stress fiber formation and cell migration. Analogously, pre-treatment with LiCl, which induced phosphorylation of GSK3β at Ser9, increased Wnt5a-induced cell migration. Finally, ectopic expression of dominant negative RhoA (N19) suppressed Wnt5a-induced cell migration. Taken together, we demonstrated for the first time that Wnt5a promoted gastric cancer cell migration via the PI3K/Akt/GSK3β/RhoA signaling pathway. These findings could provide a rationale for designing new therapy targeting gastric cancer metastasis.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Chromones; Enzyme Activation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recombinant Proteins; rhoA GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Signal Transduction; Stomach Neoplasms; Wnt Proteins; Wnt-5a Protein

Thymosin beta-4 (Tβ4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tβ4 and it is associated with Tβ4-mediated migration in gastric cancer cells. Various expression level of Tβ4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tβ4. Cell migration was higher in SNU668 with a higher expression level of Tβ4 than that in SNU638 with a lower Tβ4. Although the level of phosphorylated(p)-GSK-3α (inactive), β-catenin, E-cadherin and E-cadherin:β-catenin complex was relatively higher, p-GSK-3β (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3α/β inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK-3α increased SNU638 cell migration in accordance with the reduction of E-cadherin:β-catenin complex formation through a decrease in β-catenin and E-cadherin. Expression level of GSK-3α/β, β-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tβ4-siRNA and by the treatment with acetylated-serine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tβ4, respectively. E-cadherin expression in SNU638 cells was decreased by β-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3α, β-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, β-catenin and E-cadherin could be negatively regulated by Tβ4-induced ERK phosphorylation. It suggests that Tβ4 could be a novel regulator to control Wnt signaling pathways.

Topics: Animals; beta Catenin; Butadienes; Cadherins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Glycogen Synthase Kinase 3; Humans; Lithium Chloride; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nitriles; Peptides; Phosphorylation; RNA Interference; RNA, Small Interfering; Stomach Neoplasms; Thymosin; Wnt Signaling Pathway

Cancer stem cells (CSCs) possess the ability of self-renewal and tumor initiation. Targeting key signaling pathways that are active in CSC self-renewal is one approach to cancer therapy. Abnormal activation of the Wnt/β-catenin pathway has been described in a wide variety of human cancers and in CSCs; however, the role of this pathway in gastric CSCs has not been reported. In our study, we investigated whether the Wnt/β-catenin pathway plays an important role in gastric CSCs. First, we isolated cancer stem-like cells (CSLCs) from the human gastric cancer cell line MKN-45 using tumorsphere cultures. We tested whether tumorsphere cells were CSLCs using the following three criteria: i) We identified that the expression of the CSC marker CD44 was significantly greater in tumorsphere cells compared to adherent cells; ii) compared with adherent cells, the floating tumorsphere cells had greater self-renewing capacity; iii) in vivo xenograft studies showed that tumorsphere cells generate larger tumors than adherent cells at the same number. In addition, we studied the mechanism(s) by which the canonical Wnt signaling pathway acts in CSLCs. Western blotting and real-time PCR showed that the expression levels of β-catenin and c-myc, cyclin d1 and axin 2 were downregulated/upregulated with the inhibition/activation of the Wnt pathway. The pathway blocked by DKK-1 caused a higher reduction in the self-renewing capacity of MKN-45 tumorsphere cells and the pathway activated by lithium chloride improved the self-renewal of CSLCs. In conclusion, our data suggested that the Wnt/β-catenin pathway is essential for the self-renewal of CSLCs in human gastric cancer.

Topics: Adjuvants, Immunologic; Animals; Axin Protein; beta Catenin; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Intercellular Signaling Peptides and Proteins; Lithium Chloride; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Proto-Oncogene Proteins c-myc; Spheroids, Cellular; Stomach Neoplasms; Transplantation, Heterologous; Wnt Signaling Pathway

Aberrant regulation of glycogen synthase kinase-3beta (GSK-3beta) 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-3beta phosphorylated at Tyr216 (pGSK-3beta) and its relationship with other tumor-associated proteins in human gastric cancers.. Immunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3beta inhibitor lithium chloride (LiCl) for immunoblot analysis.. We found that pGSK-3beta 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-3beta 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-3beta 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.. GSK-3beta activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3beta activation is a useful prognostic marker for the early-stage gastric cancer.

Topics: Biomarkers, Tumor; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor Proteins; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Prognosis; Stomach Neoplasms; Tumor Cells, Cultured

The regulation of β-catenin activation by glycogen synthase kinase-3β (GSK-3β) in cancer has been shown to be cell type-specific. This study was performed to investigate the relationship between activated GSK-3β (phosphorylated at Tyr216) and β-catenin in gastric cancer. Immunohistochemical tissue array analysis of 278 human gastric carcinoma specimens showed positive immunoreactivity for activated GSK-3β in 44% of the samples, whereas membranous β-catenin and nuclear β-catenin were observed in 19% and 20% of the samples, respectively. However, GSK-3β activation was not correlated with the expression of either membranous β-catenin or nuclear β-catenin. Moreover, SNU gastric cancer cell lines over-expressing kinase dead GSK-3β and the same cells treated with a GSK-3β inhibitor showed that GSK-3β inhibition did not alter either the protein expression or transcriptional activity of β-catenin. In addition, GSK-3β activation was positively correlated with the expressions of anti-adenomatous polyposis coli (p = 0.002), p16 (p < 0.001), p21 (p < 0.001), p27 (p = 0.001), and p53 (p = 0.013). On the other hand, the nuclear expression of β-catenin was positively correlated with those of Bcl-2 (p = 0.025) and cyclin D1 (p = 0.043), but these expressions were not correlated with GSK-3β activation. Thus, the GSK-3β pathway seems to function in gastric cancer cells without involving the β-catenin pathway.

Topics: beta Catenin; Blotting, Western; Cell Line, Tumor; Chi-Square Distribution; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunohistochemistry; Lithium Chloride; Stomach Neoplasms; Tissue Array Analysis

Cysteine proteases like cathepsins are widely distributed proteolytic enzymes and form tight equimolar complexes with cystatins at their active sites. Among cystatins, CST1, encoding cystatin SN, is a member of the type 2 salivary cystatin family found in a variety of fluids and secretions, including plasma, tears, and saliva. CST1 was identified as an upregulated gene in gastric cancer tissues compared to noncancerous regions using our Affymetrix GeneChip microarray.. The upregulation of CST1 in gastric cancer was analyzed using RT-PCR (n=15), immnohistochemistry, and clinicopathological (n=77) analysis. CST1-siRNA was used for the suppression of CST1 gene expression and cathepsin proteolytic activity was assayed.. CST1 was upregulated in cancerous lesions of gastric cancer tissues compared to noncancerous regions and clinicopathological analysis showed a significant correlation between high expression of CST1 and pTNM stage (p=0.044). In CST1-siRNA transfected cells, cell proliferation was reduced and the proteolytic activity of cathepsins was increased.. CST1 might be highly involved in gastric tumorigenesis and regulate the proteolytic activity of cysteine proteases.

Topics: Cathepsins; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lithium Chloride; Male; Reverse Transcriptase Polymerase Chain Reaction; Salivary Cystatins; Stomach Neoplasms; Up-Regulation

Cyclooxygenase-2 (COX-2) expression is a marker of poor prognosis in gastric cancer patients, and its inhibition suppresses gastric tumorigenesis in experimental animal models. The mechanism that leads to COX-2 overexpression in this tumor type is unknown. We have now shown that inhibition of phosphatidylinositol 3-kinase by LY294002 suppresses both basal and phorbol myristate acetate-induced COX-2 expression in TMK-1 and MKN-28 gastric cancer cells. Furthermore, inhibition of glycogen synthase kinase-3beta (GSK-3beta) by SB415286 induced expression of COX-2 mRNA and protein as well as the enzyme activity in the gastric cancer cells. The effect of SB415286 was confirmed by the use of two additional GSK-3beta inhibitors, lithium chloride and SB216763. SB415286 had a modest 1.6-fold stimulatory effect on a 2-kb COX-2 promoter reporter construct, but more importantly, it was shown to block the decay of COX-2 mRNA. In contrast to modulation of phosphatidylinositol 3-kinase/Akt/GSK-3beta pathway, inhibitors of mitogen-activated protein kinases (MEK 1/2, p38, JNK) or the mammalian target of rapamycin did not alter COX-2 expression in gastric cancer cells. Our data show that inhibition of GSK-3beta stimulates COX-2 expression in gastric cancer cells, which seems to be primarily facilitated via an increase in mRNA stability and to a lesser extent through enhanced transcription.

Topics: Aminophenols; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Chromones; Cyclooxygenase 2; Dinoprostone; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Lithium Chloride; Maleimides; Models, Biological; Models, Statistical; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Signal Transduction; Stomach Neoplasms; Tetradecanoylphorbol Acetate; Time Factors; Transfection