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Purpose: Prognostic markers discovery is a strategy for early diagnosis and individualization therapy for human cancer. In this study, we focus to integrate different methods to identify specific biomarker and elucidate its clinical significance.Experimental Design: A powerful tool named Digital Gene Expression Display online was applied to isolate differentially expressed genes correlated with gastric cancer. Matrix metalloproteinase 11 (MMP11) was selected and confirmed at both mRNA and protein level in 10 cell lines, 123 cases of tumor tissues, and 305 cases of gastric cancer serum specimen by semiquantitative PCR, immunohistochemistry staining, and ELISA techniques, respectively.Results: Our data showed that overexpression of MMP11 at mRNA and protein level was consistently detected in cell lines and primary tumors compared with matched normal tissues. Importantly, serum MMP11 levels were also significantly elevated in gastric cancer patients compared with those of the control subjects (P < 0.001), and the positive expression was well correlated with metastasis in gastric cancer patients (P = 0.009). Furthermore, we have shown that overexpression of MMP11 was associated with the malignant proliferation of AGS cells.Conclusions: Combination of gene expression profiling and specific clinical resource is a promising approach to validate gene expression patterns associated with malignant phenotype. As a secreted protein, MMP11 may play an important role in carcinogenesis and has potential implication as a biomarker for the diagnosis and prognosis of human cancers including gastric cancer.

Supplementary Data from Identification of Matrix Metalloproteinase 11 as a Predictive Tumor Marker in Serum Based on Gene Expression Profiling

Following standard treatments, the traditional model for enhancing anti-tumor immunity involves performing immune reconstitution (e.g., adoptive immune cell therapies or immunoenhancing drugs) to prevent recurrence. For patients with advanced non-small cell lung cancer, we report here on two objectives, the immunosenescence for advanced non-small cell lung cancer and hydrogen gas inhalation for immune reconstitution. From July 1st to September 25th, 2019, 20 non-small cell lung cancer patients were enrolled to evaluate the immunosenescence of peripheral blood lymphocyte subsets, including T cell, natural killer/natural killer T cell and gamma delta T cell. Two weeks of hydrogen inhalation was performed during the waiting period for treatment-related examination. All patients inhaled a mixture of hydrogen (66.7%) and oxygen (33.3%) with a gas flow rate of 3 L/min for 4 hours each day. None of the patients received any standard treatment during the hydrogen inhalation period. After pretreatment testing, major indexes of immunosenescence were observed. The abnormally higher indexes included exhausted cytotoxic T cells, senescent cytotoxic T cells, and killer Vδ1 cells. After 2 weeks of hydrogen therapy, the number of exhausted and senescent cytotoxic T cells decreased to within the normal range, and there was an increase in killer Vδ1 cells. The abnormally lower indexes included functional helper and cytotoxic T cells, Th1, total natural killer T cells, natural killer, and Vδ2 cells. After 2 weeks of hydrogen therapy, all six cell subsets increased to within the normal range. The current data indicate that the immunosenescence of advanced non-small cell lung cancer involves nearly all lymphocyte subsets, and 2 weeks of hydrogen treatment can significantly improve most of these indexes. The study was approved by the Ethics Committee of Fuda Cancer Hospital, Jinan University in China (approval No. Fuda20181207) on December 7th, 2018, and was registered on ClinicalTrials.gov (ID: NCT03818347) on January 24th, 2019.

The use of hydrogen for cancer control has made great progress in cytology and animal experiments. With the increasing number of hydrogen products on the market, larger numbers of advanced cancer patients have participated in clinical trials or received treatment at home after purchase. Our study reported a real-world survey from 82 patients with good cancer control using hydrogen products, including real world evidence from patients who received ineffective traditional treatment, patients who received traditional treatment that failed, or patients who refused traditional treatment. Two typical cases were reported herein. Subsequently, we included studies on the mechanism of hydrogen oncology. The mechanism of cancer control using hydrogen includes the inhibition of tumor cells and the activation of exhausted lymphocytes. Large-scale real world evidence has shown clinical value, and yet remains to be further developed and researched.

Chemotherapy, targeted therapy, and immunotherapy are used against advanced non-small cell lung cancer. A clinically efficacious method for relieving the adverse events associated of such therapies is lacking. Fifty-eight adult patients were enrolled in our trial to relieve pulmonary symptoms or the adverse events of drugs. Twenty patients who refused drug treatment were assigned equally and randomly to a hydrogen (H2)-only group and a control group. According to the results of tumor-gene mutations and drug-sensitivity tests, 10, 18, and 10 patients were enrolled into chemotherapy, targeted therapy, and immunotherapy groups in which these therapies were combined with H2-therapy, respectively. Patients underwent H2 inhalation for 4–5 hours per day for 5 months or stopped when cancer recurrence. Before study initiation, the demographics (except for tumor-mutation genes) and pulmonary symptoms (except for moderate cough) of the five groups showed no significant difference. During the first 5 months of treatment, the prevalence of symptoms of the control group increased gradually, whereas that of the four treatment groups decreased gradually. After 16 months of follow-up, progression-free survival of the control group was lower than that of the H2-only group, and significantly lower than that of H2 + chemotherapy, H2 + targeted therapy, and H2 + immunotherapy groups. In the combined-therapy groups, most drug-associated adverse events decreased gradually or even disappeared. H2 inhalation was first discovered in the clinic that can be used to control tumor progression and alleviate the adverse events of medications for patients with advanced non-small cell lung cancer. This study was approved by the Ethics Committee of Fuda Cancer Hospital of Jinan University on December 7, 2018 (approval No. Fuda20181207), and was registered at ClinicalTrials.gov (Identifier: NCT03818347) on January 28, 2019.

Oligonucleotide primers for STAT3, EZH2, β-actin, reporter gene, EMSA, and siRNA of STAT3. Table S2. Correlation between STAT3 and EZH2 expression in GC. Table S3. STAT3 and EZH2 were associated with the clinical stage of the malignancy. Table S4. Multivariate analysis of factors associated with OS. Figure S1. The EZH2 promoter-Luc constructs designed for this study. (A) schematic map of the short and full-length EZH2 promoter-Luc constructs, the full length of construct contained three important STAT3 binding motifs (-1702/+52), the short construct (-1702/-447) didn’t contain this STAT3 binding region (-436/+52). (B) The promoter sequence of EZH2 was located from -436 to +52. The three STAT3-binding motifs are highlighted in blue. Figure S2. Deletion analysis of EZH2 promoter activity. (A) Schematic maps of the EZH2 promoter 5′-deletion constructs used as reporter plasmids. (B) Relative luciferase activity after transient transfection of the reporter plasmids into SGC7901 cells. The ratios between the luciferase activities induced by each reporter vector and pRL-TK were calculated. The data shown are means ± SE of triplicate experiments. Figure S3. Kaplan-Meier analysis showed the combination between p-STAT3 and EZH2 expression in overall survival of GC patients. Figure S4. Relative expression of STAT3 by knocking down with specific siRNA in SGC7901 by real-time PCR analysis (**p

The gene, collagen triple helix repeat containing 1 (CTHRC1), has been reported to increase in several kinds of human solid cancers and is associated with tumor invasion and metastasis. To date, the expression and function of CTHRC1 in gastric cancer (GC) have not been reported. The aim of this study was to investigate the expression levels and regulatory transcription mechanisms of CTHRC1 in GC. Immunohistochemical analysis revealed that CTHRC1 expression was markedly increased in carcinoma compared with normal gastric mucosa, chronic atrophic gastritis, and intestinal metaplasia (P

BACKGROUND Human beta-defensin-3 (HBD(3)) is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coli (E. coli) is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD(3) protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways. METHODS We first cloned the complementary DNA coding for the mature chain of HBD(3), inserted it into the vector PGEX-KG then transformed E. coli BL21 (DE3) with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-beta-D-galactopyranoside (IPTG) the transformed E. coli produced a recombinant glutathione S-transferase and HBD(3) (GST-HBD(3)) fusion protein. The fusion protein was treated with thrombin to produce pure HBD(3) protein then the antimicrobial activity of HBD(3) was evaluated in a liquid microdilution assay. RESULTS The fusion protein GST-HBD(3) was efficiently cleaved by thrombin and yielded HBD(3) that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 microg/ml. The E. coli strain expressing the recombinant protein did not grow slower than the empty vector strain. CONCLUSION Active HBD(3) in E. coli by expressing the recombinant protein GST-HBD(3) could be produced, and suicide did not occur in the E. coli strain expressing the recombinant protein.

OBJECTIVE Gastric cancer (GC) is a typical type of inflammation-related tumor. The p42.3 gene is shown to be highly expressed in GC, but its association with gastritis remains unknown. We aimed to explore the relationship between gastric inflammation and p42.3 gene in vitro and in vivo. METHODS Normal gastric epithelial cells (GES-1) were treated with Helicobacter pylori (H. pylori) and tumor necrosis factor (TNF)-α. Total cell mRNA and protein were extracted and collected, and polymerase chain reaction and Western blot were performed to determine the relative expression of p42.3 gene. In total, 291 biopsy samples from patients with chronic non-atrophic gastritis were collected and immunohistochemistry was used to measure the p42.3 protein expression. The association between p42.3 protein expression and the clinicopathological characteristics of these patients were analyzed. RESULTS Both H. pylori and TNF-α significantly enhanced the p42.3 protein expression in GES-1 cells in a time and dose-dependent manner. In addition, p42.3 gene expression was positively associated with the severity of gastric mucosal inflammation and H. pylori infection (P = 0.000). Its expression was significantly more common in severe gastric inflammation and in H. pylori-infected cases. CONCLUSION p42.3 gene expression is associated with gastric mucosal inflammation that can be upregulated by TNF-α and H. pylori infection.

Metastasis is a relatively early event and a major cause of death in gastric cancer (GC) patients. Gastrokine 1 (GKN1) is a stomach-specific protein that is normally expressed in gastric mucosa but not in primary tumors or cell lines. We and others have demonstrated that GKN1 inhibits cell growth; however, its role in metastasis is not clear. In this study, we explored the role of GKN1 in cell invasion. Immunohistochemistry (IHC) was used to measure the expression of GKN1 in precancerous lesions and in GCs. The cell invasion assay was employed to examine the effect of GKN1 on cell invasion. The molecular mechanism of GKN1 in inhibiting GC cell invasion in vitro was explored by western blotting. We noted a gradual decrease in GKN1 expression from normal mucosa to dysplastic gastric tissue to GC, and that low GKN1 expression was associated with metastasis (P=0.003). We showed that GKN1 inhibits cell invasion by downregulating MMP2 expression through the NF-κB pathway. These results provide molecular evidence that GKN1 inhibits metastasis in GC cells, and indicate that GKN1 is a potential novel therapeutic target for gastric cancer.

Tx is a transforming gene cloned from a nasopharyngeal carcinoma (NPC) cell line CNE2. Sequence analysis revealed that Tx encoded an aberrant immunoglobulin kappa light chain, which is abnormally expressed in epithelial tumor cells and plays an important role in nasopharyngeal carcinogenesis. Bioinformatic analysis confirmed the presence of a single nucleotide polymorphism (SNP) site in the Tx gene as matched to the Cancer Genome Anatomy Project SNP clusters database, which predicted 8 candidate SNP sites. Distribution of the confirmed SNP site in the genomes of healthy individuals and NPC patients was analyzed by denaturing high performance liquid chromatography. Heteroduplex genotype (GC/CG) occurred in NPC patients with a frequency significantly higher (52.44%) than that detected in healthy individuals (33.75%). In contrast, homoduplex genotype (GG) was less frequent in NPC patients (31.70%) than in normal individuals (56.25%), suggesting that heteroduplex genotype of Tx gene might be a risk factor for NPC.

Microsatellite instability (MSI) is a hallmark of the DNA replication error phenotype, due to the inactivation of mismatch repair genes. MSI has been implicated in colon and many other gastrointestinal cancers. MSI usually can be analyzed by PCR amplification of microsatellite markers followed by electrophoresis and detected using autoradiography or fluorescence techniques. We report here a novel method for high-throughput detection of MSI using denaturing high performance liquid chromatography (DHPLC). Amplification of two mononucleotide markers (BAT25 and BAT26) by polymerase chain reaction (PCR) is followed by DHPLC analysis to display alteration in the length of repetitive sequences. These two markers were tested in 84 colorectal cancer samples confirmed to be 44 MSI-H and 40 MSI-L or MSS, previously defined by multiple microsatellite markers and/or by immunohistochemical analyses of MLH1 and MSH2 proteins. Among 44 MSI-H samples, sequence variations in BAT26 and BAT25 were detected in 44 (100%) and 43 (98%), respectively, while no sequence variation in the two markers was detected in 40 MSI-L or MSS samples. A total of 96 gastric cancers and their matched normal tissues were then analyzed for MSI-H using this method. Sequence variations in BAT26 and BAT25 were detected in nine (9.4%) samples. Seven of the nine cases were shown unstable at both BAT26 and BAT25; one each was unstable at BAT26 or BAT25. These results were confirmed by autoradiography analyses. Together, our results demonstrate high sensitivity and specificity of DHPLC in the analysis of sequence variations in BAT25 and BAT26 to determine MSI status. This simple, efficient, and high-throughput approach will facilitate analysis of MSI in large sample sets of any cancers.

Objective: To investigate the effect of adenovirus-mediated p53 (Adp53) transfer on thermosensitivity of human gastric carcinoma cell lines (BGC823). Methods: Two human gastric carcinoma cell lines with different p53 status, BGC823-wtp53 cell (abbreviate W) bearing the wilt-type p53 and BGC823-mutp53 cell (abbreviate M) bearing the mutant p53, were cultured with DMEM medium and were infected with Adp53 at a viral multiplicity of infection of 100 (1:100MOI) for 48h before heating. Cell cycle redistribution and apoptosis of two human gastric carcinoma cell lines in 24h at 37°C after heat treatment at 42°C for 2h or 43°C for 0.5h were analyzed by flow cytometry. Relative tumor volume growth curves were used in a nude mouse tumor model of the two cell lines following hyperthermia at 43 C for 0.5h after 48h intratumoral injection of 1×108 pfu of Adp53 to evaluate thermoenhancemet effectin vivo. Results:In vitro study showed that both W and M cells infected with Adp53 and treated with heating had strong arrest in G2 (after heating at 42°C for 2h, 34.0% of original population for W cells and 25.3% of original population for M cells) and produced obvious apoptotic response. The apoptosis rate showed 230% increased (for W cells) and 110% increase (for M cells) compared with heating only control.In vivo study showed that the growth of tumor of both W cells and M cells was significantly delayed by hyperthemia combining with Adp53 as compared to tumors receiving either treatment alone. Conclusion: This study demonstrated that Adp53 transfer increased cellular apoptosis and thermo- sensitivityin vitro and tumor thermosensitivityin vivo independent of cellular intrinsic p53 status. These results support the combined used of p53 gene therapy with hyperthermia in clinical trials.

Over several years, scientists investigating cancer have focused their efforts on elucidating the mechanisms underlying cancer metastasis, with the aim of finding a way to inhibit this process. These mechanisms, however, only explain the process of cancer metastasis, but do not explain why cancer would metastasize in the first place. Cancer metastasizes due to several factors, namely attack by the immune system, lack of oxygen and necessary nutrients, large amounts of lactic acid produced by glycolysis and increased cell death. Therefore, the majority of the presently available treatments for cancer also bear the potential to induce metastasis. Thus, it is crucial in medical practice to minimize the risk of cancer metastasis during a time when there are no effective means to inhibit this process.

C9orf140 is a newly identified and characterized gene which is associated with cell proliferation and tumorigenicity. Expression of C9orf140 is upregulated in human gastric cancer and colorectal cancer (CRC); however, little is known about its role in CRC progression. We have investigated the clinical significance, biological effects and mechanisms of C9orf140 signaling. We found that the expression of C9orf140 is dramatically increased in a subset of CRC and correlates significantly with vascular invasion and lymph node metastasis. Our finding showed that knockdown of C9orf140 significantly reduced cell proliferation and invasion in vitro and dramatically increased overall survival and decreased lung metastasis in vivo. Conversely, overexpression of C9orf140 significantly increased lung metastasis and shortened overall survival when compared with control tumors. C9orf140-induced CRC cell invasion may depend on promoting the epithelial-mesenchymal transition progression. STAT5 may directly interact with the enhancer of zeste homolog 2 (EZH2) and β-catenin to enhance C9orf140 gene transactivation. Furthermore, C9orf140 may participate in cell invasion which is induced by STAT5, EZH2 or β-catenin activation. We describe the role of C9orf140 in CRC progression and find that C9orf140 overexpression may be regulated by STAT5, EZH2 and β-catenin interaction.

Objective: To evaluate the relationship between flow cytometric DNA ploidy, biological features and prognosis in patients with Stage II colorectal cancer. Methods: Nuclear DNA content, proliferation index and S-phase fraction were measured in a prospective series of 45 patients with curatively resected Stage II colorectal adenocarcinomas by means of flow cytometry using frozen tumor samples. Results: Of the 45 samples examined, 17 tumors (38%) were diploid and 28 (62%) aneuploid. The diploid tumors were significantly more common in the proximal colon than in the distal colon (67% vs. 23%; P 0.05). The proliferation index and S-phase fraction in the distal tumors were higher than those in the proximal tumors, but the difference was not significant (P>0.05). When the 5-year survival rate of patients with Stage II colorectal cancer was compared by the log rank test, a significant relationship between DNA ploidy status and disease free survival was observed in the group of all patients. Patients with DNA diploid tumors had a better disease free survival than those with DNA aneuploid tumors (P

Comparative genomic hybridization (CGH) analysis was used to identify chromosomal imbalances in 52 human primary hepatocellular carcinomas (HCCs). The most prominent changes were gains of part or all of chromosome arms 8q (83% of cases) and 1q (73%) and loss of 16q (63%). Other commonly overrepresented sites were 5p, 7q, and Xq. Recurrent sites of DNA sequence amplification included 8q23–24 (five cases) and 11q13–14 (four cases). Other frequently underrepresented sites were 4q, 8p, 16p, and 17p. Taken collectively, these findings and data from other CGH studies of HCCs define a subset of chromosome segments that are consistently over- or underrepresented and highlight sites of putative oncogenes and tumor suppressor genes, respectively, involved in hepatocellular oncogenesis. Loss of heterozygosity analysis with a panel of polymorphic microsatellite markers distributed along 16q defined a minimal region of chromosomal loss at 16q23.1–24.1, suggesting that this region harbors a tumor suppressor gene whose loss/inactivation may contribute to the pathogenesis of many HCCs. © 2000 Wiley-Liss, Inc.

A comparison has been made of performance of the neon-like chromium soft x-ray lasing at 28.5 nm driven by a double 900 ps pulse at 6 TW/cm2, with that driven by a double 200 ps pulse at similar irradiance. The double 200 ps pulse has been found to be much more efficient to drive x-ray lasing with higher intensity.

Four oligonucleotides (15 mer) 13–16 and their conjugates 17–20 in which psoralen was covalently linked to 5′ end were synthesized. Compounds 16 and 20 contain the sequence which is complementary to the sense strand covering the first four codons and the upstream sequence close to the ribosome binding site of c-Ha-ras mRNA. It was found that compounds 16 and 20 inhibited the growth of cells that had been transformed by the c-Ha- ras plasmid activated c-Ha- ras oncogene. Compound 20 that carried psoralen was more efficient in inhibiting the growth of transformed cells than compound 16 without psoralen, suggesting that the psoralen might have increased permeability of oligodeoxynucleotides. A model experiment of duplex formation was pertinent to the idea that the inhibitory effect was caused by RNase H activity.

This chapter introduces the current status of gene therapy and presents the antitumor mechanism of action, clinical trials and practice, manufacture process, quality control, gene therapy guideline, and intellectual property rights of the world’s first gene therapy drug recombinant human adenovirus-p53 injection trademarked as Gendicine approval in 2003.

Accumulating evidence shows that microRNAs, functioning as either oncogenes or tumour suppressors by negatively regulating downstream target genes that are actively involved in tumour initiation and progression, may be promising biomarkers and therapy targets. Data mining through a microRNA chip database indicated that let-7c may be associated with tumour metastasis. Here, we confirmed that down-regulation of let-7c in primary cancer tissues was significantly associated with metastases, advanced TNM stages and poor survival of colorectal cancer patients. Moreover, ectopic expression of let-7c in a highly metastatic Lovo cell line remarkably suppressed cell migration and invasion in vitro by the down-regulation of K-RAS, MMP11 and PBX3, as well as tumour growth and metastases in vivo, whereas inhibition of let-7c in low-metastatic HT29 cells increased cell motility and invasion by the enhanced gene expression of K-RAS, MMP11 and PBX3. Interestingly, the luciferase reporters' activities with the 3'-UTRs of K-RAS, MMP11 and PBX3 were inhibited significantly by let-7c. Importantly, rescue experiments involving the over-expression of these genes without their 3'-UTRs completely reversed the effects of let-7c on tumour metastasis, both in vitro and in vivo. Finally, the levels of let-7c were inversely correlated with those of MMP11 and PBX3, but not with those of K-RAS. Taken together, these results demonstrate that let-7c, apart from its tumour growth suppression role, also functions as a tumour metastasis suppressor in colorectal cancer by directly destabilizing the mRNAs of MMP11 and PBX3 at least.

As a newly identified and characterized gene, p42.3 is associated with cell proliferation and tumorigenicity. The expression of p42.3 is upregulated in human gastric cancer (GC), but its underlying mechanisms of action are not well understood. MicroRNAs (miRNAs) are known to play vital regulatory roles in many cellular processes. Here we utilized bioinformatics and experimental approaches to investigate the regulatory relationship between miRNAs and the p42.3 gene. We showed that miR-29a could repress p42.3 expression at both the mRNA and protein levels via directly binding to its 3’UTR. Furthermore, an inverse relationship was observed between miR-29a and p42.3 expression in gastric cancer cell lines and GC tissue samples, especially in cases where p42.3 was downregulated. Taken together, we have elucidated previously unrecognized roles of miR-29a and indicated that miR-29a may function, at least partially, by targeting the p42.3 gene in human GC.

To investigate whether S100A14 and S100A4 expression correlates with metastatic potential and prognosis in colorectal cancer (CRC), we firstly used RT-PCR analysis to detect mRNA expression of S100A14 and S100A4 in 40 pairs of fresh tumor samples matched with adjacent normal tissues. We then evaluated the clinical significance of our findings with immunohistochemistry on 115 samples of formalin-fixed and paraffin-embedded tumors on tissue microarrays. Typically, we identified decreased S100A14 mRNA levels (52.5%, 21/40), and increased S100A4 mRNA levels (70.0%, 28/40) in primary CRC samples. In addition, down-regulated or absent S100A14 expression was detected in 56.5% of samples (65/115) and was correlated with poor differentiation (P=0.010). In contrast, overexpressed S100A4 was detected in 57.4% of samples (66/115) and was associated with lymph node metastasis (P=0.001). Simultaneous S100A14 low-expression and S100A4 high-expression was correlated with high CRC metastatic potential (P0.001). Taken together, the signature derived from the combined expression status of S100A14 and S100A4 could be a valuable prognostic indicator in CRC.

To investigate whether S 100A 14 and S 100A4 expression correlates with metastatic potential and prognosis in colorectal cancer (CRC), we firstly used RT-PCR analysis to detect mRNA expression of S 100A14 and S 100A4 in 40 pairs of fresh tumor samples matched with adjacent normal tissues. We then evaluated the clinical significance of our findings with immunohistochemistry on 115 samples of formalin-fixed and paraffin-embedded tumors on tissue micro-arrays. Typically, we identified decreased S100A14 mRNA levels (52.5%, 21/40), and increased S100A4 mRNA levels (70.0%, 28/40) in primary CRC samples. In addition, down-regulated or absent S100A14 expression was detected in 56.5% of samples (65/115) and was correlated with poor differentiation (P=0.010). In contrast, overexpressed S100A4 was detected in 57.4% of samples (66/115) and was associated with lymph node metastasis (P=0.001). Simultaneous S 100A 14 low-expression and S 100A4 high-expression was correlated with high CRC metastatic potential (P

To systematically understand the cellular and molecular mechanism of gastric cancer (GC) development and to discover early diagnosis and predictive biomarkers, which will be used for early diagnosis and novel treatment targets.70 mer 22 K-oligonucleotide microarrays and bioinformatic analysis were conducted to recognize gene expression profiles in GC and normal appearing tissue (NAT). The control group was collected from non-tumor patients including 20 specimen mixture as a common reference (CR) and 5 individuals as additional control. Our results showed that 837 different expression genes (DEGs) were identified in GC while 570 DEGs were in NATs by Bayesian analysis (P0.001, Fold change2.0) as compared respectively with CR. An interesting finding is that we identified 67 over-expressed genes in both GC and NAT tissues, and these gene expression alterations could not be detected by comparison of GC with NATs, which were normally used in routine experiment design. Most of these genes were involved in the control of cell proliferation, metabolism and differentiation.These differential expressed genes were confirmed at mRNA and protein levels in primary tumors using RT-PCR and immunohistochemistry (IHC). The results showed that three genes, EGR1, CYR61 and ADAMTS1 were over expressed in both GC and NATs at mRNA level. These results were consistent with oligo microarray data. Another interesting finding is that these three genes were also over-expressed in intestinal metaplasia (IM) and dysplasia (DYS), which indicated that these three genes might be potential biomakers for early detection of GC.Through the systematic analysis of gene expression profiles in GC tissues, NAT and CR normal tissues, we identified a group of genes over-expressed both in GC and precancerous lesions, which might be potential biomarkers for early GC diagnosis.

Human beta-defensin-3 (HBD(3)) is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coli (E. coli) is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD(3) protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways.We first cloned the complementary DNA coding for the mature chain of HBD(3), inserted it into the vector PGEX-KG then transformed E. coli BL21 (DE3) with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-beta-D-galactopyranoside (IPTG) the transformed E. coli produced a recombinant glutathione S-transferase and HBD(3) (GST-HBD(3)) fusion protein. The fusion protein was treated with thrombin to produce pure HBD(3) protein then the antimicrobial activity of HBD(3) was evaluated in a liquid microdilution assay.The fusion protein GST-HBD(3) was efficiently cleaved by thrombin and yielded HBD(3) that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 microg/ml. The E. coli strain expressing the recombinant protein did not grow slower than the empty vector strain.Active HBD(3) in E. coli by expressing the recombinant protein GST-HBD(3) could be produced, and suicide did not occur in the E. coli strain expressing the recombinant protein.

Folate deficiency is associated with increased risk of many human diseases including several cancers. Folate receptor-alpha (FR-alpha) plays an important role in mediating influx of folates into cells and its expression can be induced by some environmental risk factors. Our previous study showed that decreased expression of FR-alpha occurs in human gastric cancer cell lines. In order to reveal the molecular mechanism of the difference of FR-alpha expression among these cell lines and analyze the relation between the mutations or/and polymorphisms of FR-alpha gene and gastric cancer, we screened the mutations of global FR-alpha gene in 12 gastric cancer cell lines and 28 gastric tumors and matched normal tissues by using PCR-DHPLC and DNA sequencing techniques, then further checked the exon2 of FR-alpha gene in 138 gastric tumors and matched normal tissues. Furthermore, we examined the relationship between two polymorphisms of FR-alpha A1314G and C1816delC and risk for gastric cancer in 296 cases and 354 age and sex-matched controls in northern China. We found that the allele frequency of FR-alpha1314A among cases was significantly higher than that among controls (0.186 vs. 0.143, P=0.014). Subjects with the A/G and A/A genotype had an increased risk for developing gastric cancer compared with the G/G genotype (OR=1.55; 95% CI=1.10-2.17). These results support the hypothesis that genetic polymorphism in the FR-alpha gene may contribute to susceptibility to carcinogenesis of the gastric cancer in the at-risk Chinese population.

AIM: To compare the gene expression between La (NO3) 3-exposed and control rats in vivo. METHODS: Rats were fed La (NO3) 3 once daily at a dose of 20 mg/kg for one month by gavage. Gene expression of hepatocytes was detected using mRNA differential display (DD) technique and cDNA microarray and compared between treated and control groups. RESULTS: Six differentially expressed sequence tags were cloned by DD, of which five were up regulated and one was down regulated in treated rats. Two sequences were determined. One band was novel. The other shared 100% sequence homology with AU080263 Sugano mouse brain mncb Mus musculus cDNA clone MNCb-5435 5’. With DNA microarray, 136 differentially expressed genes were identified including 131 over-expressed genes and 5 under-expressed genes. Most of these differentially expressed genes were cell signal and transmission genes, genes associated with metabolism, protein translation and synthesis. CONCLUSION: La (NO3) 3 could change the expression levels of some kinds of genes. Further analysis of the differentially expressed genes would be helpful for understanding the wide biological effect spectrum of rare earth elements.

To study the deletion of mitochondiral DNA in hepatocellular carcinoma and hepatocellular nodular hyperplasia and its significance in the development of cancer.Deleted mtDNA (CD-mtDNA) and wild type mtDNA (WT-mtDNA) were quantitatively analyzed by using real-time PCR in 27 hepatocellular carcinomas (HCC) and corresponding noncancerous liver tissues and 27 hepatocellular nodular hyperplasiae (HNH).A novel CD (4 981 bp) was detected in 85% (23/27) and 83%(22/27) of HCC and HNH tumor tissues, respectively, which were significantly higher than that in paired noncancerous liver tissues (57%, 15/27) (P0.05). The CD/WT-mtDNA ratio in HCC tumors was 0.00092 (median, interquartile range, 0.0001202-0.00105), which was significantly higher than that in paired noncancerous liver tissues (median, 0.000, quartile range, 0-0) (P=0.002, Mann-Whitney Test), and was 25 of times of that in HNH tissues (median, 0.0000374, quartile range, 0-0.0004225) (P=0.002, Mann-Whitney test).CD-mtDNA mutation plays an important role in the development and progression of HCC.

AIM: To better clarify the main target organs of dimethylarsinic acid toxicity and the role of metallothionein (MTs) in modifying dimethylarsinic acid (DMAA) toxicity. METHODS: MT-I/II null (MT- /-) mice and the corresponding wild-type mice (MT+/+), six in each group, were exposed to DMAA (0-750 mg/kg body weight) by a single oral injection. Twenty four hours later, the lungs, livers and kidneys were collected and undergone pathological analysis, induction of apoptotic cells as determined by TUNEL and MT concentration was detected by radio-immunoassay. RESULTS: Remarkable pathological lesions were observed at the doses ranging from 350 to 750 mg/kg body weight in the lungs, livers and kidneys and MT+/+ mice exhibited a relatively slight destruction when compared with that in dose matched MT- /- mice. The number of apoptotic cells was increased in a dose dependent manner in the lungs and livers in both types of mice. DMAA produced more necrotic cells rather than apoptotic cells at the highest dose of 750 mg/kg, however, no significant increase was observed in the kidney. Hepatic MT level in MT+/+ mice was significantly increased by DMAA in a dose-dependent manner and there was no detectable amount of hepatic MT in untreated MT-/- mice. CONCLUSION: DMAA treatment can lead to the induction of apoptosis and pathological damage in both types of mice. MT exhibits a protective effect against DMAA toxicity.

Previous study showed that allitridi markedly suppresses cellular proliferation, blocks cell cycle at G(1) phase, and induces cell apoptosis of human gastric cancer BGC823 cell. During the investigation of its molecular mechanisms and target genes, the authors found that protein expression of cyclin D1 and p27(Kip1) play an important role in an allitridi-induced G(1) arrest. This study was designed to explore the effect of allitridi on the expression of cyclin D1 and p27(Kip1) in BGC823 cells.After total RNA and total protein in allitridi-treated (25 microg/ml) and allitridi-untreated BGC823 cells were abstained, Western blot analysis was performed to determine the protein levels of cyclin D1 and p27(Kip1), and RT-PCR was performed to determine the mRNA levels of cyclin D1 and p27(Kip1).After treated with 25 microg/ml allitridi for 24 hours, the protein levels of cyclin D1 of BGC823 cells were downregulated and the protein levels of p27(Kip1) were upregulated, and this changes were more obvious after 48 hours,while their mRNA levels remained unchanged.Allitridi may influence the protein expression of cyclin D1 and p27(Kip1) in BGC823 cells, while do not influence their mRNA transcription.

Transporter associated with antigen processing(TAP) participates in immune surveillance, so it is probably relevant to carcinogenesis. Investigation of expression of TAP and its clinical significance in childhood acute leukemia will be helpful to clarify pathogenesis and to develop immunotherapy strategy.RT-PCR analysis was used to detect the expression of TAP1 and TAP2 in leukemia cells from bone marrow in 34 inpatients with primary acute lymphoblastic leukemia (ALL), 15 inpatients with relapsed ALL, 20 inpatients with acute medullary leukemia (AML), and 20 surgical inpatients without systematic disorders as control. And then,the absorbance (A) values of expanded bands were measured by digital imaging analyzer and relative A values were worked out based on A value of GAPDH (positive inside control).The relative A values of TAP1(0.448+/-0.167 and 0.169+/-0.021,respectively) and TAP2(0.196+/-0.180 and 0.112+/-0.020, respectively) in primary ALL group and relapsed ALL group were lower than those in control group (P0.01). The A values of LMP2, LMP7, and PA28alpha (991.4+/-532.7, 686.3+/-663.8, and 2022.3+/-1622.3, respectively) in relapsed ALL group were lower than those in control group (P less than 0.01,0.01 and 0.05, respectively). The A value of LMP2 in relapsed group was lower than that in control (P0.01). The A values of LMP7 for the cases with no remission and relapse were both lower than that for the cases with constant complete remission in primary ALL group (P0.01). The relative A value of TAP2 in AML group was lower than that in control group (P0.05). The relative A value of TAP1 in relapsed ALL group was lower than that in primary ALL group (P0.05). In primary ALL group, the relative A value of TAP1 (0.215+/-0.159) for cases with relapse (6/34 cases) was lower than that (0.462+/-0.189) for those with constant complete remission (24/34 cases) (P0.05).There exists decreased expression of TAP in both childhood ALL and AML, which probably contributes to the escape of leukemia cells from immune surveillance. Decreased expression of TAP1 subunit is probably related to the relapse of ALL.

To clarify the frequency of mtDNA 4 977 bp deletion in tumor cell lines, solid tumors, patient's serum of gastric tumor and to find a easy and exact method to diagnose gastric tumor.Primer-shift PCR method was used in 13 gastric tumor cell lines, 52 cases of gastric fresh tumor tissues matched the adjacent normal tissues, 40 cases of patient's serum of gastric tumor and 40 cases of normal serums for analysis of mtDNA deletion.Frequency of 4 977 bp mtDNA deletion was detected in 12 of 13 (92.3%) tumor cell lines, 38 of 52 (73.1%) cases of solid tumor tissues, 27 of 52 (52%) adjacent normal tissues, 17 of 40 (42.5%) patient's serum and 8 of 40 (20%) normal serums. Further more, in 2 of 10 pairs microdissected specimens, we found this deletion occurred not only in primary tumor but also in intestinal metaplasia comparing with no deletion was found in normal tissues. The frequency of this deletion was statistically significantly higher in the gastric tumor tissues and serums than in the adjacent normal tissues and serums. A good correlation between the deletion and young age of patients was representative in our data.mtDNA 4 977 bp deletion maybe play an important role in the carcinogenesis of human gastric mucous and maybe has happened in the tumor cell malignant transformation. To detect this deletion in patient's serum is a easy and exact method and maybe become a potential tumor marker.

Peutz-Jeghers syndrome (PJS) is an autosomal dominantly inherited disease. Fragile histidine triad (FHIT) gene is an important tumor suppressor gene at the fragile sites region of 3p14. The authors' previous study suggested that PJS patients might have a susceptible gene at the region of 3p14.2. This study was designed to reveal the relationship between the variant of FHIT gene in PJS and its canceration.Mutations of FHIT gene in 15 PJS patients and 20 unaffected members in 6 PJS families were determined using denaturing high-performance liquid chromatography (DHPLC), polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and DNA sequencing techniques.A non-sense mutation and a frame-shift mutation were identified at codon 54(GAA to TAA) (exon 6) which led to the change of the amino acid from glutamic acid (Glu) to stop codon, and a guanine insertion at codon 62 in exon 6 resulting in a premature stop codon TGA at codon 111 in one PJS patient. A homozygous deletion and a synonymous mutation were detected in exon 8. The homozygous deletion of exon 8 in FHIT gene was found in two polyps tissues and two cancerous tissues. And in 3 sporadic cases, the patients and their mothers have the same bands of SSCP and the same elution profiles of DHPLC when exon 8 was amplified. The DNA sequencing result showed that a synonymous mutation (polymorphism) occurred at codon 98 [CAT (H)--CAC (H)], this mutation resulted in no change of amino acid. In addition, one base substitute from A to G mutation at 5'end, +42 nucleotide in intron 6 of FHIT gene was detected in seven patients and two unaffected members.PJS patients have low frequency point mutation of FHIT gene and their cancerous tissues had homozygous deletions in FHIT gene. This study indicated that the mutations and deletions of FHIT gene in PJS may play a role in the development of PJS and their cancerations.

Recently, a sensitive and accurate technique called denaturing high-performance liquid chromatography (DHPLC) has been used in gene mutation screening, but several factors, especially DNA polymerase were discovered to affect the resolution of DHPLC technique. In order to find one or more suitable polymerases for DHPLC analysis, we designed this study to evaluate the influence of non-proofreading and proofreading DNA polymerase on mutation screening with DHPLC.Three fragments which were 268 bp, 317 bp, and 590 bp in size were chosen for DHPLC analysis. To evaluate the negative influence of polymerase on DHPLC resolution, the same fragments were amplified by using 11 kinds of DNA polymerases [6 brands of Taq, 1 kind of Taq gold and, 4 sorts of proofreading DNA polymerase (Pfu and Vent)] and those PCR products were analyzed by DHPLC. The chromatographical profile were identified and compared.There were two kinds of negative influences on DHPLC analysis chromatography profiles. There were no or tiny influences induced by proofreading or Taq gold polymerases.Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase. Especially, Pfu DNA ought to be preferentially selected to amplify GC-rich small fragments or other influence-prone fragments when mutation is detected by DHPLC analysis.

Extensive epidemiologic studies indicated protective effects of consumption of garlic on reducing human gastric cancer (HGC) incidence. Diallyl trisulfide (DATS), a critical organic allyl sulfur component of garlic, was reported to have chemopreventive effects in inhibiting tumor process. We used DATS to treat HGC cell line BGC823 cells, and showed that DATS induces G1/S arrest and apoptosis in BGC823 cells demonstrated by a flow cytometric analysis. To further isolate DATS inducible differentially expressed genes in BGC823 cells, we combined a highly specific subtractive hybridization of cDNA representational difference analysis (cDNA RDA) with a sensitive bidirectional radioactive detection of mRNA differential display (mRNA DD) to develop a subtractive hybridization differential display (SHDD) method. This modified method adopted a first round of bidirectional subtractive hybridization between two sample cDNAs and a second round of bidirectional subtractive hybridization between the two resultant first-round difference products. Bidirectional subtractive hybridizations magnified the differences between the two sample cDNAs and favored isolating mRNA species with very small expression differences. We employed the SHDD method to detect DATS inducible differentially expressed genes in BGC823 cells. A total of 14 cDNA fragments (11 upregulated and 3 downregulated by DATS treatment) were isolated and confirmed by reverse Northern blot analysis. Our data show that SHDD is a powerful technique for identifying differentially expressed mRNA species between two sample cDNAs and provide useful cellular and molecular information for understanding the effects of garlic against human gastric cancer.

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). BamH I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72 h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

We have introduced point mutations in v-rasH to study their effects on biochemical and biological properties of the ras-encoded protein p21. Several of these mutant proteins do not bind GTP and thus lack GTPase activity, while others were shown to have their GTP binding reduced. We have introduced these ras mutants into NIH 3T3 fibroblastoid cells to study major parameters of clinical importance which are associated with neoplastic transformation, particularly MHC expression in cells, metastasis and tumorigenesis in both nude mice and immune competent mice. Our data show that certain mutations in v-ras differentially affect the expression of the transformed phenotype. Mutant ras molecules deficient in GTP binding fail to generate rapidly progressing tumors in immune competent mice, and not all morphologically transformed cells were capable of experimental metastasis. Cells transformed by certain v-ras mutants form tumors in immunocompetent mice and show reduced expression of MHC class-1 antigens. Other cells are morphologically transformed and tumorigenic in athymic nude mice, but fail to form tumors in normal mice and show levels of MHC class-1 antigen expression similar to non-transformed 3T3 cells. The inverse relationship between MHC class-1-antigen expression and the degree of transformation in fibroblastoid cells suggests that the ras gene product could be involved in regulating MHC expression.

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.