Vitamin D analog EB1089 induces apoptosis in a subpopulation of SGC-7901 gastric cancer cells through a mitochondrial-dependent apoptotic pathway.

Gastric cancer is the second leading cause of cancer death worldwide. Cancer stem-like side population (SP) cells may be important factors that hinder efficacy of chemopreventative and chemotherapeutic approaches in gastric cancer. EB1089 is an antitumor agent that has been used in many cancers; however, no reports to date have determined the effects of EB1089 in gastric cancer. In our study, SP and main population (MP) cells were isolated from 4 gastric cancer cell lines in different stages of differentiation by flow cytometry (FCM) and confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. EB1089 decreased the proliferation, increased apoptosis, and induced mitochondrial damage in the SP cells isolated from 1 cell type (SGC-7901), but not MP cells, through increased Bax and decreased Bcl-2 and Bcl-xL protein expression. This protein expression pattern induced the activation of caspase-3 and caspase-9. The effects of EB1089 on SGC-7901 SP cells were blocked by treating cells with vitamin D receceptor (VDR) siRNA or butin (an inhibitor of the mitochondrial apoptosis pathway). Our results suggest that EB1089 targets SGC-7901 SP cells through a mitochondrial apoptosis pathway. However, further studies are needed to elucidate the signal transduction between VDR and the mitochondrial apoptosis pathway.

Hypermethylation and aberrant expression of secreted frizzled-related protein genes in pancreatic cancer.

AIM: To determine the methylation status and aberrant expression of some secreted frizzled-related protein (SFRP) genes in pancreatic cancer and explore their role in pancreatic carcinogenesis. METHODS: Methylation status and expression of SFRP genes were detected by methylation-specific PCR (MSPCR) and reverse-transcription PCR (RT-PCR) respectively. RESULTS: The frequencies of methylation for SFRP genes 1, 2, 4, 5 were 70%, 48.3%, 60% and 76.7% in pancreatic cancer samples, and 21.7%, 20%, 10% and 36.7% in matched cancer adjacent normal tissue samples, respectively (c2 = 28.23, P < 0.0001 for SFRP gene 1; c2 = 10.71, P = 0.001 for SFRP gene 2; c2 = 32.97, P < 0.0001 for SFRP gene 4; c2 = 19.55, P < 0.0001 for SFRP gene 5). Expression loss of SFRP genes 1, 2, 4 and 5 was found in 65%, 40%, 55% and 71.7% of 60 pancreatic cancer samples, and 25%, 15%, 18.3% and 31.7% of matched cancer adjacent normal tissue samples, respectively (c2 = 19.39, P < 0.0001 for SFRP gene 1; c2 = 9.40, P = 0.002 for SFRP gene 2; c2 = 17.37, P < 0.0001 for SFRP gene 4; c2 = 19.22, P < 0.0001 for SFRP gene 5). SFRP gene 1 was methylated but not expressed in PC-3 and PANC-1, SFRP gene 2 was methylated but not expressed in PANC-1 and CFPAC-1, SFRP gene 4 was methylated but not expressed in PC-3, and SFRP gene 5 was methylated but not expressed in CFPAC-1. CONCLUSION: Hypermethylation and aberrant expression of SFRP genes are common in pancreatic cancer, which may be involved in pancreatic carcino-genesis.

Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer.

AIM: To identify the methylation of secreted frizzled-related protein 1 (SFRP1) in gastric cancer and to investigate the aberrant expression of SFRP1 and its correlation with the clinical pathological features of patients. METHODS: We determined SFRP1 methylation and SFRP1 mRNA expression in 3 gastric cancer cell lines SGC-7901, BGC-823, HGC-27, from 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens by methylation-specific (MSP) PCR and RT-PCR respectively. Fisher's exact test was used to analyze the statistical association between clinical pathological data and aberrant expression of SFRP1. RESULTS: In 3 cancer cell lines, BGC-823 and HGC-27 had methylated SFRP1 and lost SFRP1 mRNA expression. After treatment of BGC-823 and HGC-27 with the demethylating agent, 5-aza-2'-deoxycytidine, SFRP1 was re-expressed. In 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens, hypermethylation of SFRP1 was detected in 23 (44%) and 8 (15%) specimens respectively (chi(2) = 10.34, P < 0.01). Loss of SFRP1 expression was detected in 17(33%) and 6 (12%) specimens respectively (chi(2) = 6.75, P < 0.01). There was a significant correlation between SFRP1 hypermethylation and SFRP1 expression loss. SFRP1 expression was also correlated significantly with tumor stage and lymph node status, but not with patient sex, age and histological type. CONCLUSION: SFRP1 inactivation is a common and early event caused mainly by hypermethylation in gastric cancer. SFRP1 expression loss may be correlated with tumor metastasis in primary gastric cancer.

[The role of transforming growth factor-beta1/Smad3 signaling in bronchiolitis obliterans following lung transplantation].

OBJECTIVE: To investigate the mechanism underlying bronchiolitis obliterans (OB) following lung transplantation and the significance of transforming growth factor (TGF)-beta1/Smad3 signal pathway in this pathological process. METHODS: The tracheas of BALB/c mice were transplanted into the subcutaneous tissues of a Smad3ex8/ex8 gene knock-out Swiss black mouse and a Smad3 wild-type Swiss black mouse. Forty-two days later the tracheas were taken out. Immunocytochemistry was used to detect the alpha-smooth muscle actin (alphaSMA), a marker of fibroblast-myofibroblast differentiation. The tracheas of Smad3 knock-out and wild type mice were taken out, broken to pieces, and cultured to obtain the fibroblasts. The tracheal fibroblasts in primary culture were treated with TGF-beta1. The activation of Smad3 molecules was investigated with immunocytochemistry, Western blotting and DNA electrophoresis mobility gel shift assay (EMSA). Immunocytochemistry staining was also employed to detect the cytoskeletal polymerization and alphaSMA immunofluorescence after incubation with TGF-beta1; Western blotting and RT-PCR was conducted to detect the difference of alphaSMA at transcriptional and protein level. RESULTS: The number of alphaSMA positive myofibroblasts was great in the experimental OB models produced be transplantation of heterogeneous trachea from Smad3 wild type mice and was very small in the OB model produced be transplantation of heterogeneous trachea from Smad3 knock-out mice (t = 2.125, P = 0.040). Western blotting showed that in vitro experiment showed that phosphorylation of Smad3 protein was increased in the fibroblasts treated with TGF-beta1 and was almost absent in those not treated with TGF-beta1. EMSA showed that DNA binding was increased in the fibroblasts treated with TGF-beta1 and was almost absent in those not treated with TGF-beta1. Immunofluorescence staining showed that the cytoplasm of the fibroblasts not treated with TGF-beta1 was Smad3 positive, however, the nuclei of the fibroblasts treated with TGF-beta1 was Smad3 positive. RT-PCR showed that the alphaSMA mRNA expression level in the Smad3 wild-type fibroblasts was increased after treated with TGF-beta1, and was significantly higher than in the Smad3 knock-out fibroblasts treated with TGF-beta1 (t = 2.080, P = 0.027). Western blotting showed that the alphaSMA protein expression level in the Smad3 wild-type fibroblasts was increased after treatment with TGF-beta1, and was significantly higher than that of the Smad3 knock-out fibroblasts (t = 1.982, P = 0.032). CONCLUSION: TGFbeta1 promotes the production of alphaSMA protein and transformation of fibroblasts into myofibroblasts through the Smad3 dependent signal pathway, thus resulting in the development of bronchiolitis obliterans.

[The mechanism of transforming growth factor beta1 in myofibroblast differentiation].

OBJECTIVE: To investigate the mechanism underlying myofibroblast differentiation induced by transforming growth factor (TGF) beta1 in obliterative bronchiolitis following lung transplantation. METHODS: Heterotopic tracheal transplantation was performed in Smad3 wild-type and knock-out mice to simulate the lung transplantation in human. Murine tracheal fibroblasts cultivated in primary culture were used for in vitro study. Immunohistochemistry, immunocytochemistry, Western Blotting, RT-PCR and DNA electrophoresis mobility gel shift assay were conducted to detect the expression of alpha-smooth muscle actin (alphaSMA), the marker of fibroblast-myofibroblast differentiation, and the activation of Smad3, p38 and ERK1/2. RESULTS: In affected airways of experimental obliterative bronchiolitis, abundant expression of alphaSMA were found. In vitro study for tracheal fibroblasts, the activation of Smad3 by TGF-beta1 presents as three major forms, phosphorylation, nuclear translocation and DNA binding. In Smad3 wild-type fibroblasts, TGF-beta1 induces the increase of the myofibroblasts transformation, characterized by the elevation of alphaSMA, both at transcription and protein level. While in Smad3 knock-out fibroblasts, the transformation of myofibroblasts induced by TGF-beta1 is significantly decreased (t = 2.080, P = 0.027; t = 1.982, P = 0.032), but not completely abolished. Further study in Smad3-deficient fibroblasts demonstrates that p38 and ERK1/2 could be activated by TGF-beta1 and result in fibroblast differentiation. CONCLUSIONS: TGF-beta1 could promote the transformation of fibroblasts into myofibroblasts in Smad3 dependent and independent signal pathways, especially the Smad3 dependent path, and result in the development of obliterative bronchiolitis.

Roles of programmed cell death protein 5 in inflammation and cancer (Review).

PDCD5 (programmed cell death 5) is an apoptosis related gene cloned in 1999 from a human leukemic cell line. PDCD5 protein containing 125 amino acid (aa) residues sharing significant homology to the corresponding proteins of species. Decreased expression of PDCD5 has been found in many human tumors, including breast, gastric cancer, astrocytic glioma, chronic myelogenous leukemia and hepatocellular carcinoma. In recent years, increased number of studies have shown the functions and mechanisms of PDCD5 protein in cancer cells, such as paraptosis, cell cycle and immunoregulation. In the present review, we provide a comprehensive review on the role of PDCD5 in cancer tissues and cells. This review summarizes the recent studies of the roles of PDCD5 in inflammation and cancer. We mainly focus on discoveries related to molecular mechanisms of PDCD5 protein. We also discuss some discrepancies between the current studies. Overall, the current available data will open new perspectives for a better understanding of PDCD5 in cancer.

PDCD2 and NCoR1 as putative tumor suppressors in gastric gastrointestinal stromal tumors.

PURPOSE: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract. Previously, PDCD2 (programmed cell death protein 2) has been identified as a putative tumor suppressor in gastric cancer. As yet, however, no reports on PDCD2 expression and its physical interactor NCoR1 (nuclear receptor co-repressor), and their effects in GIST have been reported. METHODS: The expression of PDCD2 and NCoR1 was assessed in 43 primary gastric GIST and normal gastric tissue samples using Western blotting and quantitative real-time PCR. Next, associations between PDCD2 and NCoR1 expression and various clinicopathological features, including survival, were determined. To assess the effects of PDCD2 and NCoR1 expression in vitro, two GIST-derived cell lines (GIST-T1 and GIST882) were (co-)transfected with the expression vectors pEGFP-N1-PDCD2 and pcDNA3.1-NCoR1, after which the cells were subjected to CCK-8, PI staining and Annexin V-FITC/PI double staining assays, respectively. Finally, the mechanisms of action of PDCD2 and NCoR1 in GIST-derived cells were determined using immunoprecipitation and Western blotting assays. RESULTS: We found that the PDCD2 and NCoR1 protein levels were lower in gastric GIST tissues than in normal gastric tissues. The PDCD2 and NCoR1 expression levels were found to be significantly associated with the survival of the patients. Through exogenous expression analyses, we found that PDCD2 and NCoR1 can decrease proliferation, and increase apoptosis and G1 cell cycle arrest, in GIST-derived cells. Furthermore, we found that PDCD2 and NCoR1 can activate Smad2 and Smad3. CONCLUSIONS: Our data indicate that both PDCD2 and NCoR1 may act as tumor suppressors in GIST cells through the Smad signaling pathway.

Role of cancer stem cell marker CD44 in gastric cancer: a meta-analysis.

Cluster of differentiation 44 (CD44), a principal cell surface receptor for hyaluronic acid, has been implicated in tumorigenesis and metastasis. However, the relationship between CD44 expression and the patients with gastric cancer remains controversial. A meta-analysis was performed to quantitatively review the correlation of CD44 expression with the clinicopathological data of the patients with gastric cancer. We conducted a final analysis of the patients from 18 studies. Combined odds ratios (OR) suggested that CD44 expression was related with stage, tumor size, and LN metastasis of gastric cancer, and CD44v6 was related with LN metastasis, lymphatic invasion, and venous invasion. Our results suggested that CD44 and CD44v6 expression could be used to predict the metastasis of gastric cancer.

The expression of ARHI in pT2a and pT2b stage gastric cancer and its clinical significance.

ARHI is a novel tumor suppressor gene located on chromosome 1p31. Downregulation of ARHI expression has been detected in many types of cancer. However, the effects of ARHI in gastric cancer remain unclear. The aim of this study was to identify the relationship between ARHI expression and gastric cancer clinicopathological features. In this study, 81 pT2 stage gastric cancer specimens were subclassified by pT2a and pT2b stage. ARHI mRNA and protein levels were evaluated by real-time PCR and western blot analysis, respectively. Methylation plays an important role in suppressor gene silencing. We utilized methylation-specific PCR to identify the status of CpG islands in the ARHI gene. We used immunohistochemistry to determine the expression of the protein and analyzed clinicopathological features. The levels of ARHI mRNA in gastric cancer were lower compared to normal tissues (P<0.01). Similarly, the levels of ARHI protein in the cancer specimens were lower (P<0.05). DNA hypermethylation was identified in 79.1% of gastric cancer specimens without ARHI expression. Immunohistochemistry results were significantly correlated with the pT2 category (P<0.05). The cumulative survival rate of patients with ARHI expression was significantly higher compared to those without ARHI expression (P<0.05). ARHI as a suppressor is not only an important factor in the pathogenesis of gastric cancer, but also a potential factor for tumor aggravation. ARHI expression in gastric cancer can be employed to indicate favorable prognosis for the disease.

Inhibition of proliferation, viability, migration and invasion of gastric cancer cells by Aurora-A deletion.

Accumulating evidence has demonstrated Aurora-A to be frequently overexpressed in many cancers, including gastric cancer. In order to study the effects of Aurora-A on gastric cancer cells, we detected the changes of cell phenotype after treatment with Aurora-A specific small interference RNA (siRNA). In addition, VX-680 was used simultaneously. RT-PCR and western-blot were used to determine the level of Aurora-A mRNA and protein in cells, including GES-1, SGC-7901, SGC-7901 lines treated with VX-680, SGC-7901 treated with DMSO, SGC- 7901 interfered using siRNA and SGC-7901 interfered using scrambled RNAi. MTT, PI staining and transwell assays were used respectively to analyze proliferation, viability, and migration and invasion of the cells. The results showed that deletion of Aurora-A may inhibit proliferation and induce G1 arrest. The transwell assay indicated that Aurora-A may promote metastasis of gastric cancer. Collectively, our findings support Aurora-A as an oncogene in gastric cancer. Deletion of Aurora-A may have potential as a therapeutic method for gastric cancer.