Olga Kim

GKN2 Contributes to the Homeostasis of Gastric Mucosa by Inhibiting GKN1 Activity

Gastrokine 1 (GKN1) plays an important role in maintaining gastric mucosa integrity. Here, we investigated whether gastrokine 2 (GKN2) contributes to the homeostasis of gastric epithelial cells by regulating GKN1 activity. We analyzed cell viability, proliferation, and death in AGS cells transfected with GKN1, GKN2, GKN1 plus GKN2 using MTT, BrdU incorporation, and apoptosis assays, respectively. In addition, the expression levels of the cell cycle‐ and apoptosis‐related proteins, miR‐185, DNMT1, and EZH2 were determined. We also compared the expression of GKN1, GKN2, and CagA in 50 non‐neoplastic gastric mucosae and measured GKN2 expression in 169 gastric cancers by immunohistochemistry. GKN2 inhibited anti‐proliferative and pro‐apoptotic activities, miR‐185 induction, and anti‐epigenetic modifications of GKN1. There was a positive correlation between GKN1 and GKN2 expression (P = 0.0074), and the expression of GKN1, but not GKN2, was significantly lower in Helicobacter pylori CagA‐positive gastric mucosa (P = 0.0013). Interestingly, ectopic GKN1 expression in AGS cells increased GKN2 mRNA and protein expression in a time‐dependent manner (P = 0.01). Loss of GKN2 expression was detected in 126 (74.6%) of 169 gastric cancers by immunohistochemical staining and was closely associated with GKN1 expression and differentiation of gastric cancer cells (P = 0.0002 and P = 0.0114, respectively). Overall, our data demonstrate that in the presence of GKN2, GKN1 loses its ability to decrease cell proliferation, induce apoptosis, and inhibit epigenetic alterations in gastric cancer cells. Thus, we conclude that GKN2 may contribute to the homeostasis of gastric epithelial cells by inhibiting GKN1 activity. J. Cell. Physiol. 229: 762–771, 2014.

Gastrokine 1 inhibits the carcinogenic potentials of Helicobacter pylori CagA

Helicobacter pylori CagA directly injected by the bacterium into epithelial cells via a type IV secretion system, leads to cellular changes such as morphology, apoptosis, proliferation and cell motility, and stimulates gastric carcinogenesis. We investigated the effects of cytotoxin-associated gene A (CagA) and gastrokine 1 (GKN1) on cell proliferation, apoptosis, reactive oxygen species (ROS) production, epithelial-mesenchymal transition (EMT) and cell migration in CagA- or GKN1-transfected gastric epithelial cells and mucosal tissues from humans and mice infected with H.pylori. On the molecular level, H.pylori CagA induced increased cell proliferation, ROS production, antiapoptotic activity, cell migration and invasion. Moreover, CagA induced activation of NF-κB and PI3K/Akt signaling pathways and EMT-related proteins. In addition, H.pylori CagA reduced GKN1 gene copy number and expression in gastric cells and mucosal tissues of humans and mice. However, GKN1 overexpression successfully suppressed the carcinogenic effects of CagA through binding to CagA. These results suggest that GKN1 might be a target to inhibit the effects from H.pylori CagA.

The Role of Gastrokine 1 in Gastric Cancer

Homeostatic imbalance between cell proliferation and death in gastric mucosal epithelia may lead to gastritis and gastric cancer. Despite abundant gastrokine 1 (GKN1) expression in the normal stomach, the loss of GKN1 expression is frequently detected in gastric mucosa infected with Helicobacter pylori, as well as in intestinal metaplasia and gastric cancer tissues, suggesting that GKN1 plays an important role in gastric mucosal defense, and the gene functions as a gastric tumor suppressor. In the stomach, GKN1 is involved in gastric mucosal inflammation by regulating cytokine production, the nuclear factor-κB signaling pathway, and cyclooxygenase-2 expression. GKN1 also inhibits the carcinogenic potential of H. pylori protein CagA by binding to it, and up-regulates antioxidant enzymes. In addition, GKN1 reduces cell viability, proliferation, and colony formation by inhibiting cell cycle progression and epigenetic modification by down-regulating the expression levels of DNMT1 and EZH2, and DNMT1 activity, and inducing apoptosis through the death receptor-dependent pathway. Furthermore, GKN1 also inhibits gastric cancer cell invasion and metastasis via coordinated regulation of epithelial mesenchymal transition-related protein expression, reactive oxygen species production, and PI3K/Akt signaling pathway activation. Although the modes of action of GKN1 have not been clearly described, recent limited evidence suggests that GKN1 acts as a gastric-specific tumor suppressor. This review aims to discuss, comment, and summarize the recent progress in the understanding of the role of GKN1 in gastric cancer development and progression.

Influence of the hTERT rs2736100 polymorphism on telomere length in gastric cancer.

AIM To investigate the functional consequences of rs2736100 polymorphism in telomere length and examine its link to gastric cancer risk. METHODS Telomere length and human telomerase reverse transcriptase (hTERT) mRNA expression were measured in 35 gastric cancer tissues and 5 cell lines and correlated to rs2736100 polymorphism. The relationship between rs2736100 polymorphism and the risk of gastric cancer were examined in 243 gastric cancer patients and 246 healthy individuals. RESULTS The rs2736100 A allele carrier is closely associated with reduced hTERT mRNA expression and shortened telomere length in gastric cancer tissue and cell lines. When gastric cancers were stratified by histological subtype, telomere length and hTERT mRNA levels were significantly increased in those with the C/C genotype in intestinal-type gastric cancer, but not in diffuse-type gastric cancer. Interestingly, there was no significant difference in the genotype and allele frequencies of the rs2736100 polymorphism between the patients with gastric cancer and healthy controls. CONCLUSION The rs2736100 polymorphism of the hTERT gene is involved in the regulation of hTERT expression and telomere length, but not in the risk of gastric cancer.

NKX6.3 controls gastric differentiation and tumorigenesis

NKX6.3 transcription factor is known to be an important regulator in gastric mucosal epithelial differentiation. The present study aimed to investigate whether NKX6.3 acts as an essential tumor suppressor in gastric carcinogenesis. Absent or reduced protein expression and decreased DNA copy number and mRNA transcript of the NKX6.3 gene were frequently observed in gastric cancers. Overexpression of NKX6.3 in AGSNKX6.3 and MKN1NKX6.3 cells markedly arrested cell proliferation by inhibiting cell cycle progression and induced apoptosis through both death receptor- and mitochondrial-pathways. In addition, stable NKX6.3 transfectants increased the expression of gastric differentiation markers, including SOX2 and Muc5ac, and decreased the expression of intestinal differentiation markers, CDX2 and Muc2. In ChIP-cloning and sequencing analyses, NKX6.3 coordinated a repertoire of target genes, some of which are clearly associated with cell cycle, differentiation and death. In particular, NKX6.3 transcriptional factor was found to bind specifically to the upstream sequences of GKN1, a gastric-specific tumor suppressor, and dramatically increase expression of the latter. Furthermore, there was a positive correlation between NKX6.3 and GKN1 expression in non-cancerous gastric mucosae. Thus, these data suggest that NKX6.3 may control the fate of gastric mucosal cells and function as a gastric tumor suppressor.

NKX6.3 Is a Transcription Factor for Wnt/β-catenin and Rho-GTPase Signaling-Related Genes to Suppress Gastric Cancer Progression

Despite ongoing research and recent progress, the prognosis for patients with advanced gastric cancer remains poor. Wnt/β-catenin and Rho-GTPase signaling pathways are known to play essential roles in malignant transformation and progression of various tumors, including gastric cancer. Here, we identify that NKX6 transcription factor, locus 3 (NKX6.3) binds directly to specific promoter regions of Wnt/β-catenin and Rho-GTPase pathway-related genes, resulting in inhibition of cancer cell migration and invasion. Additionally, we find that the expression level of NKX6.3 is involved in regulation of gastric cancer progression and expression of Wnt/β-catenin and Rho-GTPase pathway-related genes in clinical samples. These results suggest that NKX6.3 prevents EMT and cell migration, implying that NKX6.3 inactivation might be one of the key mechanisms of gastric cancer cell invasion and metastasis.

Gastrokine 1 expression in the human gastric mucosa is closely associated with the degree of gastritis and DNA methylation.

Purpose Gastrokine 1 plays an important role in gastric mucosal defense. Additionally, the Gastrokine 1-miR-185-DNMT1 axis has been shown to suppress gastric carcinogenesis through regulation of epigenetic alteration. Here, we investigated the effects of Gastrokine 1 on DNA methylation and gastritis. Materials and Methods Expression of Gastrokine 1, DNMT1, EZH2, and c-Myc proteins, and the presence of Helicobacter pylori CagA protein were determined in 55 non-neoplastic gastric mucosal tissue samples by western blot analysis. The CpG island methylation phenotype was also examined using six markers (p16, hMLH1, CDH1, MINT1, MINT2 and MINT31) by methylation-specific polymerase chain reaction. Histological gastritis was assessed according to the updated Sydney classification system. Results Reduced Gastrokine 1 expression was found in 20 of the 55 (36.4%) gastric mucosal tissue samples and was closely associated with miR-185 expression. The Gastrokine 1 expression level was inversely correlated with that of DNMT1, EZH2, and c-Myc, and closely associated with the degree of gastritis. The H. pylori CagA protein was detected in 26 of the 55 (47.3%) gastric mucosal tissues and was positively associated with the expression of DNMT1, EZH2, and c-Myc. In addition, 30 (54.5%) and 23 (41.9%) of the gastric mucosal tissues could be classified as CpG island methylation phenotype-low and CpG island methylation phenotype-high, respectively. Reduced expression of Gastrokine 1 and miR-185, and increased expression of DNMT1, EZH2, and c-Myc were detected in the CpG island methylation phenotype-high gastric mucosa. Conclusions Gastrokine 1 has a crucial role in gastric inflammation and DNA methylation in gastric mucosa.

Gastrokine 1 inhibits gastrin-induced cell proliferation

BackgroundGastrokine 1 (GKN1) acts as a gastric tumor suppressor. Here, we investigated whether GKN1 contributes to the maintenance of gastric mucosal homeostasis by regulating gastrin-induced gastric epithelial cell growth.MethodsWe assessed the effects of gastrin and GKN1 on cell proliferation in stable AGSGKN1 and MKN1GKN1 gastric cancer cell lines and HFE-145 nonneoplastic epithelial cells. Cell viability and proliferation were analyzed by MTT and BrdU incorporation assays, respectively. Cell cycle and expression of growth factor receptors were examined by flow cytometry and Western blot analyses.ResultsGastrin treatment stimulated a significant time-dependent increase in cell viability and proliferation in AGSmock and MKN1mock, but not in HFE-145, AGSGKN1, and MKN1GKN1, cells, which stably expressed GKN1. Additionally, gastrin markedly increased the S-phase cell population, whereas GKN1 significantly inhibited the effect of gastrin by regulating the expression of G1/S cell-cycle regulators. Furthermore, gastrin induced activation of the NF-kB and β-catenin signaling pathways and increased the expression of CCKBR, EGFR, and c-Met in AGS and MKN1 cells. However, GKN1 completely suppressed these effects of gastrin via downregulation of gastrin/CCKBR/growth factor receptor expression. Moreover, GKN1 reduced gastrin and CCKBR mRNA expression in AGS and MKN1 cells, and there was an inverse correlation between GKN1 and gastrin, as well as between GKN1 and CCKBR mRNA expression in noncancerous gastric mucosae.ConclusionThese data suggest that GKN1 may contribute to the maintenance of gastric epithelial homeostasis and inhibit gastric carcinogenesis by downregulating the gastrin-CCKBR signaling pathway.

The single nucleotide polymorphism (SNP) of the estrogen receptor-β gene, rs1256049, is associated with knee osteoarthritis in Korean population

BACKGROUND Estrogens affect articular cartilage metabolism via estrogen receptors (ER) in chondrocytes and are believed to play an important role in the pathophysiology of osteoarthritis (OA). The aim of this study is to determine whether the single nucleotide polymorphism (SNP) of the estrogen receptor-β (ER-β) is associated with an increased susceptibility to knee OA. METHODS The possible influence of the SNP of the ER-β was investigated in 286 OA patients and 294 healthy subjects as controls. A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay and a PCR-single strand conformation polymorphism (SSCP) assay were used to identify the Rsa polymorphism genotype among healthy controls and OA patients, respectively. RESULTS For rs1256049 (Rsa), frequencies of genotypes GG, GA, and AA were 49.0% (144/294), 43.5% (128/294), and 7.5% (22/294) in healthy controls, and 35.3% (101/286), 45.5% (130/286), and 19.2% (55/286) in OA patients. Frequencies of alleles G and A among healthy controls were 70.7% (416/588) and 29.3% (172/588); whereas those among OA patients were 58.0% (332/572) and 42.0% (240/572). Statistically significant differences in allele and genotype frequencies of rs1256049 were observed between OA patients and controls (P<0.0001). In particular, the risk of OA was significantly increased in carriers with the rs1256049A allele and rs1256049 AA homozygotes. CONCLUSIONS These results suggest a close association of rs1256049 ER-β polymorphisms with susceptibility to OA in the Korean population. CLINICAL RELEVANCE The rs1256049 polymorphism of the estrogen receptor-β gene can potentially be used to identify genetically high-risk subgroup of osteoarthritis in advance and to understand pathogenesis of osteoarthritis.

GKN1 and miR-185 are associated with CpG island methylator phenotype in gastric cancers

Epigenetic modifications including aberrant DNA methylation play a significant role in cancer development. We investigated mRNA expression levels of GKN1, miR-185, DNMT1 and EZH2, as well as their association with the CpG island methylator phenotype (CIMP) in gastric cancers. Twenty-four incident gastric carcinomas were characterized for methylation status and mRNA expression levels of GKN1, miR-185, DNMT1 and EZH2 were examined. In gastric cancer, methylation frequencies in the Mint1, Mint2, E-CDH, hMLH1 and p16 genes were 41.7%, 41.7%, 29.2%, 25% and 4.2%, respectively. At least one gene was methylated in 19 (79.2%) cases. When the methylation status was classified as high (H) and low (L), 11 (45.8%) cases showed CIMP-H. Twentytwo (91.7%) of 24 gastric cancers showed decreased expression of GKN1 and miR-185, compared to samples from the corresponding non-cancerous gastric mucosa. Increased expression of DNMT1 and EZH2 was found in 21 (87.5%) gastric cancers. Statistically, there was a close association between the methylation status and expression levels of GKN1, miR-185, DNMT1 and EZH2 (P<0.05). These results suggest that GKN1 and miR-185 may be representative and predictive biomarkers for methylation status in gastric carcinogenesis.