Identification of 14-3-3β in human gastric cancer cells and its potency as a diagnostic and prognostic biomarker

Gastric cancer is the second most common cause of cancer deaths worldwide and due to its poor prognosis, it is important that specific biomarkers are identified to enable its early detection. Through 2-D gel electrophoresis and MALDI-TOF-TOF-based proteomics approaches, we found that 14-3-3β, which was one of the proteins that were differentially expressed by 5-fluorouracil-treated gastric cancer SC-M1 cells, was upregulated in gastric cancer cells. 14-3-3β levels in tissues and serum were further validated in gastric cancer patients and controls. The results showed that 14-3-3β levels were elevated in tumor tissues (n=40) in comparison to normal tissues (n=40; p<0.01), and serum 14-3-3β levels in cancer patients (n=145) were also significantly higher than those in controls (n=63; p<0.0001). Elevated serum 14-3-3β levels highly correlated with the number of lymph node metastases, tumor size and a reduced survival rate. Moreover, overexpression of 14-3-3β enhanced the growth, invasiveness and migratory activities of tumor cells. Twenty-eight proteins involved in anti-apoptosis and tumor progression were also found to be differentially expressed in 14-3-3β-overexpressing gastric cancer cells. Overall, these results highlight the significance of 14-3-3β in gastric cancer cell progression and suggest that it has the potential to be used as a diagnostic and prognostic biomarker in gastric cancer.     

Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation

Recently (Cytometry 2003, 56A, 71-80), we reported that direct cell-to-cell contact is required for stimulating proliferation of bystander rat liver cells (WB-F344) cocultured with irradiated cells, and neither functional gap junction intercellular communication nor long-range extracellular factors appear to be involved in this proliferative bystander response (PBR). The molecular basis for this response is unknown. Confluent monolayers of WB-F344 cells were exposed to 5-Gray (Gy) of gamma-rays. Irradiated cells were mixed with unirradiated cells and co-cultured for 24 h. Cells were harvested and protein expression was examined using 2-DE. Protein expression was also determined in cultures of unirradiated and 5-Gy irradiated cells. Proteins were identified by MS. Nucleophosmin (NPM)-1, a multifunctional nucleolar protein, was more highly expressed in bystander cells than in either unirradiated or 5-Gy irradiated cells. Enolase-alpha, a glycolytic enzyme, was present in acidic and basic variants in unirradiated cells. In bystander and 5-Gy irradiated cells, the basic variant was weakly expressed, whereas the acidic variant was overwhelmingly present. These data indicate that the presence of irradiated cells can affect NPM-1 and enolase-alpha in adjacent bystander cells. These proteins appear to participate in molecular events related to the PBR and suggest that this response may involve cellular defense, proliferation, and metabolism.     

Involvement of ERK1/2 pathway in TGF-beta1-induced VEGF secretion in normal human cytotrophoblast cells

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the angiogenesis during the development of placenta, but the intracellular signaling mechanism by which TGF-beta1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to TGF-beta1 stimulated the secretion of the VEGF gene encoding vascular endothelial growth factor, which is a key factor in angiogenesis. Meanwhile, treatment of normal human cytotrophoblast cells with TGF-beta1-induced expression of HIF-1a, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. Our data indicated that TGF-beta1 induced extracellular signal- regulated kinase (ERK) 1/2 phosphorylation in normal human cytotrophoblast cells. Moreover, treating cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited TGF-beta1 stimulation of VEGF secretion and HIF-1a protein expression. These data indicated that in normal human cytotrophoblast cells, TGF-beta1 induced HIF-1a-mediated VEGF secretion, and TGF-beta1-stimulated-ERK1/2 activation may be involved in this process.     

ERK1/2 and Akt pathway activated during (3R,6R)-bassiatin(1)-induced apoptosis in MCF-7 cells

(3R,6R)-bassiatin(1) was isolated from the endogenous fungus, Fusarium oxysporum J8-1-2. Previous studies showed that (3R,6R)-bassiatin(1) has anti-oestrogen properties which make it cytotoxic to ER (oestrogen receptor)-positive breast cancer cells (MCF-7) by cell cycle arrest and induction of apoptosis. (3R,6R)-bassiatin(1) suppresses mRNA and protein expression of the ERα and oestrogen responsive genes of cyclin D1 and PR. We have investigated the interaction between (3R,6R)-bassiatin(1) and ERα and followed the roles of ERK (extracellular-signal-regulated kinase), Akt and GSK3β (glycogen synthase kinase 3β) during (3R,6R)-bassiatin(1)-induced apoptosis of MCF-7 cells. (3R,6R)-bassiatin(1) competed with E2 (17β-estradiol) for ERα active sites to inhibit ERα activation. However, while ERK1/2 and Akt were activated, GSK3β was inactivated during (3R,6R)-bassiatin(1)-induced apoptosis, suggesting that this compound is indeed an anti-oestrogen agent that can also activate the survival signalling pathway. Apoptosis caused by (3R,6R)-bassiatin(1) may be related to activation of ERK.     

Proteomic analysis of human gastric juice: a shotgun approach

Gastric juice is the most proximal fluid surrounding the stomach tissue. The analysis of gastric juice protein contents will thus be able to accurately reflect the pathophysiology of the stomach. This biological fluid is also a potential reservoir of secreted biomarkers in higher concentration as compared to the serum. Unlike the rest of the gastrointestinal fluids, there were very few studies reported on gastric juice proteome. To date, the proteins that routinely populate this biofluid are largely unknown. This is partly due to the technical difficulties in processing a sample that contains a collection of other gastrointestinal fluids, especially saliva. In this study, we attempt to profile the protein components of the gastric fluids from chronic gastritis patients using a direct shotgun proteomics approach. These data represent the first report of the proteome of human gastric juice with gastritis background.     

Interferon regulatory factor-1 mediates interferon-gamma-induced apoptosis in ovarian carcinoma cells

Interferon-gamma (IFN-gamma), as one of interferon family that regulates antiviral, antiproliferative, and immunomodulatory responses, has been implicated for the growth regulation of ovarian cancer cells. However, the molecular mechanisms are not yet fully defined. To analyze detailed mechanisms, the ovarian cancer cell lines (2774, PA-1, OVCAR-3, and SKOV-3) were treated with IFN-gamma. The growth of 2774 was most effectively suppressed than that of other cells in both time-course and dose-dependent experiments. The order of sensitivity in other cells was PA-1 > OVCAR-3 > SKOV-3 (not responded at all). The DNA fragmentation and DAPI staining assays suggested that the IFN-gamma-mediated cytotoxicity could be triggered by apoptosis. The treatment induced IFN regulatory factor-1 (IRF-1) in two IFN-gamma-sensitive cells (2774, PA-1), whereas IRF-1 was not induced in two IFN-gamma-resistant cells (OVCAR-3, SKOV-3). The levels of p53 and p21WAF1 were not strikingly changed in all four cells. Interestingly, the expression of interleukin-converting enzyme (ICE, or caspase-1) was increased by the treatment in a kinetically consistent manner to the induction of IRF-1. However, CD95 (Fas/APO-1) was not changed. Apoptosis was greatly induced, when IRF-1 was transiently expressed in PA-1 without the treatment of IFN-gamma. However, it was repressed when IRF-1 together with IRF-2, an antagonist of IRF-1, were coexpressed. In addition, the effect of IFN-gamma was reduced in the 2774 and PA-1 cells stably expressing either IRF-1 antisense or IRF-2 sense, as shown by the cytotoxicity and FACS analysis. Furthermore, the IFN-gamma-induced apoptosis was greatly reduced, when inhibitors of ICE were treated into PA-1 cells. Taken together, these results suggest that IRF-1 directly mediates the IFN-gamma-induced apoptosis via the activation of caspase-1 gene expression in IFN-gamma-sensitive ovarian cancer cells.     

Proteomic characterization of Her2/neu-overexpressing breast cancer cells

The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients.     

Chirality transferring [3,3] sigmatropic rearrangement of (1-Acyloxy-2-alkenyl)trianlkylsilane synthesis of optically active vinylsilane-containing alpha-amino acid

A vinylsilane-containing alpha-amino acid and alpha,alpha-disubstituted alpha-amino acid 2 having two contiguous asymmetric carbon centers at their alpha and beta positions were synthesized in an optically active form by ester-enolate Claisen rearrangement of the alpha-acyloxysilane 1 as the key step, where the chirality of an alpha-acyloxy-TBDMS group was completely transferred to the rearranged product.     

Ginsenoside Rg1 inhibits tumor necrosis factor-alpha (TNF-alpha)-induced human arterial smooth muscle cells (HASMCs) proliferation

In China, the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The aim of this study was to determine the effects of ginsenoside Rg1 on the proliferation and molecular mechanism in cultured human arterial vascular smooth muscle cell (HASMC) induced by tumor necrosis factor-alpha (TNF-alpha). It was shown that ginsenoside Rg1 significantly inhibited TNF-alpha-induced HASMC proliferation in a dose-dependent manner. Treatment with ginsenoside Rg1, which blocked the cell cycle in the G1-phase, induced a downregulation of cyclin D1 and an upregulation in the expression of p53, p21(WAF/CIP1), and p27(KIP1). MEK inhibitors PD98059, U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not p38-inhibitor SB203580 or JNK-inhibitor SP600125 significantly aggravated ginsenoside Rg1-inhibited HASMC proliferation. Ginsenoside Rg1 markedly inactivated the extracellular signal-regulated kinases (ERK1/2) and protein kinase B (PKB), indicating that the inhibition of ginsenoside Rg1 on HASMC proliferation was associated with ERK and PI3K/PKB pathways. The inactivation of ERK and PI3K/PKB pathways and modulation of cell-cycle proteins by ginsenoside Rg1 may be of importance in inhibition of HASMCs proliferation.     

Overexpression of regucalcin suppresses cell response for tumor necrosis factor-alpha or transforming growth factor-beta1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.     

Upregulation of plasma C9 protein in gastric cancer patients

Gastric cancer is one of the leading causes of cancer‐related deaths worldwide. Current biomarkers used in the clinic do not have sufficient sensitivity for gastric cancer detection. To discover new and better biomarkers, protein profiling on plasma samples from 25 normal, 15 early‐stage and 21 late‐stage cancer was performed using an iTRAQ‐LC‐MS/MS approach. The level of C9 protein was found to be significantly higher in gastric cancer compared with normal subjects. Immunoblotting data revealed a congruent trend with iTRAQ results. The discriminatory power of C9 between normal and cancer states was not due to inter‐patient variations and was independent from gastritis and Helicobacter pylori status of the patients. C9 overexpression could also be detected in a panel of gastric cancer cell lines and their conditioned media compared with normal cells, implying that higher C9 levels in plasma of cancer patients could be attributed to the presence of gastric tumor. A subsequent blind test study on a total of 119 plasma samples showed that the sensitivity of C9 could be as high as 90% at a specificity of 74%. Hence, C9 is a potentially useful biomarker for gastric cancer detection.     

High frequency occurrence of 1-OPRD variant of PRNP gene in gastric cancer cell lines and Chinese population with gastric cancer

The prion protein gene PRNP encodes PrPc and PrPsc, causing a number of neurological disorders. Approximately 10-15% of human prion disease is inherited and more than 20 pathogenic mutations have been found. Most of the genetic alterations are point mutations, with the exception of genetic insertions of one to nine extra octapeptide repeats occurring in the important octapeptide-coding region. Our previous work showed that PrPc was overexpressed in gastric cancer. We wondered whether mutations of PrPc existed in human gastric cancer. DNA sequencing and gel electrophoresis were used to determine the possible mutation of PrPc in patients and cell lines of gastric cancer. We found that 1-OPRD (one octapeptide-repeat deletion) homozygosity or heterozygosity exists in several gastric cancer cell lines, e.g. MKN28 and KatoIII are homozygous for 1-OPRD, and SGC7901 and BGC-823 are heterozygous for 1-OPRD. The mutation frequency in tissues of gastric cancer cases is significantly higher than that in the common population (p<0.05). All positive cases in gastric cancer were found to be heterozygous for 1-OPRD. Further study of the variant may be helpful in understanding the mechanisms of occurrence and development of clinical gastric carcinoma as well as the biology of the mysterious gene PRNP.     

Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.