thymosin and Stomach-Neoplasms

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Humans; Immunologic Factors; Precancerous Conditions; Stomach Neoplasms; Thymosin

Gastric cancer (GC) is histologically classified into intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), and the latter is poorly differentiated and highly metastatic. In this study, using quantitative real-time polymerase chain reaction, we described a complete protocol for in vivo CRISPR-Cas9-based knockout screening of essential genes for DGC metastasis. We functionally screened 30 candidate genes using our mouse DGC models lacking Smad4, p53, and E-cadherin. Pooled knockout mouse DGC cells were transplanted into a spleen of syngeneic immunocompetent mice to study clonal advantages in context of a complex process of liver metastasis. Tmsb4x (thymosin beta-4 X-linked), Hmox1, Ifitm3, Ldhb, and Itgb7 were identified as strong candidate genes that promote metastasis. In particular, Tmsb4x enhanced DGC metastasis and stomach organoid-generated tumor growth in in vivo transplantation models. Tmsb4x promoted tumor clonogenicity and anoikis resistance. In situ hybridization analysis showed that Tmsb4x is highly expressed in E-cadherin-negative mouse DGC models compared with mouse IGC and intestinal cancer models. E-cadherin deficiency also increased Tmsb4x expression in stomach organoids via Wnt signaling activation. Collectively, these results demonstrate that Tmsb4x promotes DGC metastasis. In addition, this experimental system will aid in the identification of novel target genes responsible for DGC metastasis.

Topics: Animals; Biomarkers, Tumor; CRISPR-Cas Systems; Disease Models, Animal; Gene Expression; Gene Knockout Techniques; Humans; Mice; Neoplasm Metastasis; Real-Time Polymerase Chain Reaction; Signal Transduction; Stomach Neoplasms; Thymosin

The thymosin beta 10 (TMSB10) was originally identified from the thymus, which plays a key role in the development of many cancers. However, the underlying molecular mechanisms of TMSB10 involved in GC have not been understood.. We sought to determine the expression of TMSB10 in human GC tissues and illustrate whether it is correlated with the clinical pathologic characteristics and prognosis in GC patients. Its roles and potential mechanisms in regulating tumor growth, invasion, and angiogenesis were evaluated by TMSB10 knockdown/overexpression of GC cells in vitro and ex vivo.. Marked overexpression of TMSB10 protein expression was observed in GC cells and tissues, which was associated with the advanced tumor stage and lymph nodes (LN) metastasis of GC patients. Furthermore, prognostic analysis showed that GC patients with high TMSB10 expression had a remarkably shorter survival and acted as an important factor for predicting poor overall survival in GC patients. Moreover, TMSB10 overexpression promoted, while TMSB10 knockdown the proliferation, EMT process, and angiogenesis of GC cells.. The study highlights that TMSB10 may hold promise as potential prognosis prediction biomarker for the diagnosis of GC and a potential therapeutic target, which will facilitate the development of a novel therapeutic strategy against GC.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Neoplasm Invasiveness; Neovascularization, Pathologic; Prognosis; Stomach Neoplasms; Thymosin

β-thymosin plays important roles in the development of the lymphatic system and the central nervous system in vertebrates. However, its role and function in invertebrates remain much less explored. Here, we firstly isolated a gene encoding β-thymosin in silkworm (Bombyx mori L.). Interestingly, this gene encodes two polypeptides, named as BmTHY1 and BmTHY2, via two different modes of RNA splicing. The recombinant proteins fused with an N-term GST tag were over-expressed in Escherichia coli (E. coli) and further purified to near homogenity to prepare mouse antibodies. The Western blot analysis showed that these proteins were expressed in various tissues and organs, as well as in different developmental stages. Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period. The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection. In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Bombyx; Cell Movement; Cell Proliferation; Cloning, Molecular; Computational Biology; Gene Expression Regulation, Developmental; Insect Proteins; Mice; Molecular Sequence Data; Organ Specificity; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Stomach Neoplasms; Thymosin; Tumor Cells, Cultured

Despite major advances in its diagnosis and treatment, gastric cancer (GC) remains a major life-threatening disease. Treatment of the disease is further aggravated by the lack of diagnostic biomarkers that can aid in the early detection of GC and promote its favorable prognosis. The present work aims to identify novel diagnostic biomarkers for GC.. The present work is a case-control study that focuses on proteomic analysis of serum from healthy volunteers and GC patients using ClinProt profiling technology based on mass spectrometry. A pattern of proteins/peptides with the ability to differentiate the studied populations was identified. Deregulated proteins/peptides differentially expressed in the serum of patients compared with healthy volunteers were identified by mass spectroscopy.. A pattern of proteins/peptides consisting of four protein/peptide peaks at m/z 1467, 1867, 2701, and 2094 was identified. These protein/peptide peaks were able to differentiate the studied populations with close to 100% sensitivity and specificity. Three of the deregulated proteins/peptides at m/z 1867, 2701, and 2094 were identified by mass spectroscopy (LTQ Orbitrap XL) as tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c₁ complex subunit 1, respectively.. The pattern of proteins/peptides identified in the present work shows great potential for GC diagnosis. Deregulated proteins of tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c₁ complex subunit 1 may be involved in the pathogenesis of GC and serve as potential serological diagnostic biomarkers.

Topics: Adenoma; Aged; Biomarkers, Tumor; Carrier Proteins; Case-Control Studies; Female; Gene Expression; Humans; Male; Mass Spectrometry; Middle Aged; Protein Isoforms; Sensitivity and Specificity; Stomach Neoplasms; Thymosin; Tubulin

Thymosin β4 (Tβ4), a small acidic actin binding peptide, is overexpressed in a side population of cancer stem cells and CD133-positive colorectal cancer stem cells. In order to understand the relationship between Tβ4 and CD133, we studied the expression patterns of Tβ4 and CD133 in ovarian cancers. The expression patterns of Tβ4 and CD133 were studied in normal ovaries, primary ovarian cancers, metastatic ovarian cancers, primary stomach cancers, and normal stomachs by Western blot and immunohistochemistry. Expression patterns and co-localization of Tβ4 and CD133 were examined by immunofluorescence and confocal laser-scanning microscopy. Tβ4 is overexpressed in primary ovarian cancers, but not in primary stomach cancers, when compared with normal controls. However, Tβ4 levels in metastatic stomach cancers to the ovary are significantly upregulated compared with levels in normal stomachs and primary stomach cancers. These results suggest that Tβ4 levels are related to tumorigenesis in ovarian cancers and metastasis in stomach cancers. The expression of Tβ4 in normal ovaries and normal stomachs was weak, but was co-localized with CD133 expression. Tβ4 expression was also co-localized with CD133 expression in primary ovarian carcinomas, metastatic ovarian cancers from stomach cancers and primary stomach cancers. These data suggest that Tβ4 expression is strongly related to CD133 expression and is a characteristic of stem cells or cancer stem cells.

Topics: AC133 Antigen; Antigens, CD; Biomarkers, Tumor; Female; Glycoproteins; Humans; Immunohistochemistry; Neoplastic Stem Cells; Ovarian Neoplasms; Peptides; Stomach Neoplasms; Thymosin; Up-Regulation

Thymosin alpha 1 (Tα1) has been shown to have beneficial effects on numerous immune system parameters, but little is known about the effects of Tα1 on patients with gastric carcinoma. The objective of this study was to determine the effect of Tα1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro, and to evaluate its efficacy as an immunoregulatory factor in patients with gastric carcinoma. We compared the effect of Tα1 on the frequency of CD4+ and CD8+ T cells, especially the CD4+CD25+Foxp3+ Tregs in peripheral blood mononuclear cells (PBMCs) from gastric carcinoma patients (N = 35) and healthy donors (N = 22). We also analyzed the changes in the proliferation of PBMCs in response to treatment with Tα1, and examined the production of Th1, Th2, and Th17 cytokines by PBMCs and tumor-infiltrating lymphocytes. The treatment of PBMCs from gastric cancer patients, with Tα1 (50 µg/mL) alone increased the percentage of CD4+CD25+Foxp3+ (suppressive antitumor-specific Tregs) from 1.68 ± 0.697 to 2.19 ± 0.795% (P < 0.05). Our results indicate that Tα1 increases the percentage of Tregs and IL-1β, TNF-α, and IL-6 in vitro.

Topics: Adult; Aged; Antineoplastic Agents; Case-Control Studies; Cell Proliferation; Cytokines; Female; Flow Cytometry; Humans; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Stomach Neoplasms; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Th2 Cells; Thymalfasin; Thymosin; Young Adult

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

We aimed to investigate the sources of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and estimate the value of both ER subtypes in gastric adenocarcinoma and analyze the possible relationship of prothymosin alpha (ProTalpha) to ERs.. ERs at the mRNA and protein levels in matched advanced gastric adenocarcinomas and surrounding non-cancerous tissues were examined by using reverse transcription-polymerase chain reaction and immunohistochemical (IHC) methods. Cell proliferation related protein ProTalpha was also detected in IHC. The immunoreactive signal, corresponding to the proteins expression level, was quantitatively analyzed.. Both ERalpha and ERbeta mRNAs were detected in most of the cancer and matched normal tissues analyzed. At the protein level, the percentage of ERalpha and ERbeta positive cases changed. ERalpha immunoreactivity was only detected in poorly differentiated adenocarcinoma and ERalpha positive expression correlated with depth of invasion of the tumors. Compared with non-cancerous tissues, gastric tumors showed decreased ERbeta expression and lost ERbeta. Altered ERbeta in gastric adenocarcinoma correlated with decreased differentiation. And the tumors involved lymph node metastasis showed significantly lower expression level of ERbeta. ProTalpha in ERbeta-positive tumors showed higher expression than that in lost ERbeta tumors.. Altered expression of ERalpha and ERbeta in tumors compared with corresponding normal gastric tissues was more common in poorly differentiated adenocarcinomas and related to malignant properties, such as lymph node metastasis. Decreased ERbeta and increased ProTalpha expression in advanced gastric adenocarcinoma indicated that ERbeta may play an anti-proliferation role which is opposed to the role of ProTalpha in gastric epithelium.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Differentiation; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Prognosis; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Severity of Illness Index; Stomach Neoplasms; Thymosin

Prothymosin-alpha and ERp57 were previously identified as markers for gastric metaplasia in a mouse model of Helicobacter-induced gastric metaplasia and neoplasia. In this paper we assess whether the expression of these putative biomarkers in humans is correlated with gastric metaplasia and adenocarcinoma and clinical outcomes.. Eight tissue microarrays, containing 749 paraffin-embedded tissue cores from 164 gastric cancer patients, were stained for prothymosin-alpha and ERp57 by horseradish peroxidase immunohistochemical techniques. The proportion of stained cells per core was quantitated using the Ariol SL-50 automated image analysis system.. Prothymosin-alpha stained a significantly higher percentage of nuclei in cancer and metastases compared with normal gastric mucosa. ERp57 staining was significantly decreased in cancer and metastases compared with both normal gastric mucosa and metaplasias. ERp57 expression also correlated with greater depth of tumor invasion and advanced stage of disease. Kaplan-Meier survival analysis determined that tumors with the highest quartile of ERp57 expression were statistically associated with longer postoperative survival. A Cox proportional hazard analysis showed that maintenance of ERp57 expression was associated with longer postoperative survival.. These results suggest that although prothymosin-alpha is overexpressed in gastric adenocarcinoma, it is not associated with alterations in survival. In contrast, loss of ERp57 expression correlated with more aggressive disease and could provide useful prognostic information for gastric cancer patients.

Topics: Adenocarcinoma; Biomarkers, Tumor; Down-Regulation; Endoplasmic Reticulum; Gastric Mucosa; Humans; Immunohistochemistry; Metaplasia; Molecular Chaperones; Prognosis; Protein Array Analysis; Protein Disulfide-Isomerases; Protein Precursors; Retrospective Studies; Stomach; Stomach Neoplasms; Survival Rate; Thymosin

The emergence of oxyntic atrophy and metaplastic cell lineages in response to chronic Helicobacter pylori infection predisposes to gastric neoplasia. We have described a trefoil factor family 2 (TFF2; spasmolytic polypeptide) expressing metaplasia (SPEM) associated with gastric neoplasia in both rodent and human fundus. To examine the relationship of SPEM to the neoplastic process in the H. felis -infected C57BL/6 mouse, we have now studied the association of SPEM-related transcripts with preneoplasia.. SPEM-related transcripts were identified by microarray analysis of amplified cRNA from SPEM, and surface mucous cells were isolated by laser capture microdissection from the same gastric sections from male C57BL/6 mice infected with H. felis for 6 months. Expression of SPEM-related transcripts was assessed by in situ hybridization and quantitative RT-PCR, as well as immunohistochemistry for prothymosin alpha.. Eleven SPEM-related transcripts were identified as detectable only in SPEM. The expression of the SPEM-related transcripts was validated by in situ hybridization and quantitative PCR. One transcript, the noncoding RNA Xist, was only identified in SPEM cells from the infected male mice. Ten of the 11 transcripts as well as TFF2 were also expressed in regions of gastritis cystica profunda. Immunocytochemistry for one of the identified proteins, prothymosin alpha, demonstrated prominent nuclear staining in SPEM and gastritis cystica profunda.. The expression of SPEM-related transcripts in regions of gastritis cystica profunda suggests that SPEM represents a precursor lineage for the development of dysplasia in this animal model of gastric carcinogenesis.

Topics: Animals; Helicobacter felis; Helicobacter Infections; Immunohistochemistry; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Male; Metaplasia; Mice; Mice, Inbred C57BL; Mucins; Muscle Proteins; Neuropeptides; Oligonucleotide Array Sequence Analysis; Peptides; Polymerase Chain Reaction; Precancerous Conditions; Protein Precursors; RNA, Messenger; Stomach Neoplasms; Thymosin; Trefoil Factor-2; Trefoil Factor-3

Thymosin alpha1 is a cleavage product of prothymosin alpha. Expression of prothymosin alpha increases during cell proliferation. Thymosin alpha1, measurable in plasma, may be an indicator of cell proliferation especially if a cancer is present. In this report we investigated the relation between the clinical behavior of gastric cancer and the plasma thymosin alpha1 level. Plasma thymosin alpha1 was measured in 52 gastric cancer patients using a newly developed radioimmunoassay. Twenty-one tumors and lymph nodes were examined for thymosin alpha1 using immunohistochemistry. The plasma thymosin alpha1 level was higher in gastric cancer patients than in normal volunteers; and it was higher in patients with lymph node involvement than in those with negative nodes. Immunohistochemical study of thymosin alpha1 showed positivity in 52% of gastric cancers and 100% of lymph node metastasis. We concluded that a high level of plasma thymosin alpha1 suggests aggressive behavior of a gastric cancer, such as lymph node involvement.

Topics: Biomarkers, Tumor; Cell Division; Coloring Agents; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Predictive Value of Tests; Protein Precursors; Radioimmunoassay; Statistics as Topic; Stomach Neoplasms; Thymalfasin; Thymosin