AIM:To investigate the effects of 17-AAG on apoptosis and cell cycle of HCT-15 cells and to clar-ify the related mechanisms .METHODS: MTT method was employed to evaluate the inhibitory effects of 17-AAG with Aifferent time and different doses on the proliferation of HCT-15 cells.The cells were stained with Annexin V-FITC/propid-iumiodide and measured by flow cytometry .The expression of STAT3, cyclin D1, Cyt C, caspase 9 and caspase 3 at mR-NA and protein levels was determined by RT-PCR and Western blotting .RESULTS:Treatment with 17-AAG at concentra-tion of 1.25~20 mg/L for 24 h and 48 h significantly inhibited the activity of HCT-15 cells at both time-and concentra-tion-dependent manners .Treatment with 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h significantly in-duced apoptosis and cell cycle arrest of HCT-15 cells.The exposure of 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h to the HCT-15 cells significantly down-regulated the expression of STAT 3 and cyclin D1 at mRNA and pro-tein levels, but up-regulated Cyt C, caspase 9 and caspase 3 mRNA and protein in a concentration-dependent manner . CONCLUSION:17-AAG inhibits the cell activity , induces apoptosis and G 1 arrest by down-regulating the expression of cyclin D1, and promoting the mitochondria apoptosis through STAT 3 pathway.

Objective To construct and confirm the JC virus(JCV) T antigen expression plasmid using mouse keratin 19 (K19) promoter specific for the gastric epithelial cells.Methods The Ndel site was mutated by FCR with Bell insertion at both sides.The DNA fragment digested by Bcl Ⅰ was ligated with the plasmid containing K19 promoter via Bam Ⅰ site.The DNA sequence was confirmed by restriction enzyme digestion and direct DNA sequencing.Cytokeratin 19 protein was examined to screen gastric carcinoma cell for transfection of K19-JCV T antigen expression plasmid by immunohistochemistry.The Western blot was employed to detect the JCV T antigen expression in the gastric carcinoma transfectant.Results K19-JCV T antigen expressing plasmid was successfully constructed.The ACS strongly expressed cytokeratin 19 protein and was selected for the transfection of K19-JCV T antigen expressing plasmid.JCV T antigen was positively expressed in the AGS transfectant.Conclusion The synonymous mutation and compatible ligation are useful in the plasmid construction.The methy lation of restriction enzyme should be considered.It is meaning for the transgenic animal model of gastric carcinoma to successfully construct the JC virus T antigen expression plasmid in gastric mucosa.

Objective To clarify the clinicopathological significance and the reversing effects of BTG3 expression on the aggressive phenotype in gastric cancer. Methods BTG3 expression was detected in gastric cancer tissues by on tissue microarray and immunostaining. BTG3?expressing plasmid was transfected into MKN28 and MGC803 cells,the proliferation,cell cycle,differentiation and autophagy were analyzed by CCK?8,PI staining,alkaline phosphatase activity and GFP?LC?3B transfection,respectively. Results BTG3 overexpression inhibited cell proliferation,in?duced S/G2 arrest,differentiation and autophagy in both cells(P<0.05). BTG3 expression was decreased in gastric cancer in comparison with the adjacent mucosa(P<0.05),and positively correlated with venous invasion and dedifferentiation of the cancers(P<0.05). Conclusion BTG3 ex?pression contributes to gastric carcinogenesis and subsequent progression. BTG3 overexpression can reverse the aggressive phenotypes,which could be employed as a potential target for gene therapy of gastric cancer.

Objective To study the effect of ING5 gene on growth inhibition of gastric cancer. Methods ING5 expressing plasmid was transfect?ed into SGC?7901 cells. The cell viability was assessed by CCK?8,cell apoptosis and cycle was detected by flow cytometry analysis,the migration and invasion was evaluated by scratch and transwell,the expressions of mRNA and protein were determined by real?time quantitative PCR and West?ern blot respectively. Nude mice were implanted subcutaneously with SGC?7901 cells and tumor size and related protein were analyzed. Results After transfection of pEGFP?N1?ING5,the proliferation of gastric cancer SGC?7901 cells was significantly inhibited(P<0.05),the apoptosis was decreased(P<0.05),the percentage of G1 phase was increased and G2 phase was decreased(P<0.05),the ability of migration and invasion was reduced(P<0.05),the expression of NF?κB,PI3K,p?Akt1/2/3,Bcl?2,XIAP,β?catenin,c?myc mRNA,and protein levels were significantly in?creased(P<0.05),and the expression of Akt1/2/3、MMP9 mRNA and protein levels were significantly decreased(P<0.05). Conclusion The ING5gene inhibits the SGC?7901 cell proliferation,induces G1 arrest,inhibits the migration and invasion,and effectively regulates the related genes and proteins about cell proliferation,cell cycle and adhesion,but reduces apoptosis. ING5 can inhibit the evolution and development of gastric can?cer.

Objective to explore the role of Beclin 1 in gastric carcinogenesis and subsequent progression. Methods MkN28 cells were trans-fected with Beclin 1-expressing plasmid,and then the proliferation and cell cycle was measured by CCk-8 and PI staining. Beclin 1 expression was examined by immunohistochemistry and in situ hybridization on tissue microarrays containing gastric cancers,adjacent non-neoplastic mucosa,and metastatic lymph node. the correlation with the tumorgenesis,clinicopathological and prognostic parameters was analyzed. Results Beclin 1 overex-pression resulted in G2 arrest of MkN28 cells and reduced the proliferation. Beclin 1 mRNA was highly expressed in gastric cancer than matched mu-cosa by ISH(P < 0.05). Beclin 1 was highly expressed in male than female patients with gastric cancer(P < 0.05). the elder patients with gastric cancer had higher Beclin 1 expression than younger ones(P < 0.05). the diffuse-type carcinomas showed less Beclin 1 expression than intestinal and mixed type ones(P < 0.05). kaplan-Meier analysis indicated that Beclin 1 expression was positively correlated to favorable prognosis of the can-cer patients(P < 0.05). Conclusion Beclin 1 expression is closely linked to pathogenesis,metastasis and differentiation of gastric cancer. Beclin 1 might be employed to indicate the favorable prognosis of gastric cancer patients and regarded as a target of gene therapy.

Objective To investigate the effects of inhibitor of growth 5 (ING5) gene on the proliferation, apoptosis, migration and cell cycle of human breast cancer Bcap-37 cells.Methods The eukaryotic ING5-expressing plasmid and GFP-empty plasmid were steadily transfected in Bcap-37 cells, the expression of green fluorescent protein was measured with fluorescence microscopy, and the high expression of ING5 was measured by real time-PCR. Bcap-37-ING5 cells served as the experimental group, Bcap-37-GFP cells as the mock group and Bcap-37 as the control group. The effects of ING5 on the proliferation were detected by MTT, the cell cycle and apoptosis were detected by Flow cytometry, and the cell migration was detected by cell wound scratch assay and Transwell experiment.Results Bcap-37 cell lines steadily expressing ING5 protein with GFP-tag were acquired by stable transfection. ING5 over-expression inhibited the proliferation and led to G2 arrest of Bcap-37 cells, increased cells apoptosis and decreased the cell migration ability (P<0.05).Conclusion ING5 over-expression may have reverse effect for malignant phenotype of breast cancer cells, and may be employed to indicate the biomarker of prognosis of breast cancer patients and regarded as a target of gene therapy.

Objective Rho signaling component α-catulin,is a cytoskeletal linker protein and plays an important role in apoptotic and senescence resistance,cytoskeletal reorganization,mobility,invasion and epithelial to mesenchymal transition(EMt)of cancer cells. Methods Here,we ex-amined α-catulin expression in squamous epithelium,dysplasia and cancer of head and neck on tissue microarrays by immunostaining. Its expres-sion was compared with clinicopathological parameters and survival rate of cancers. Results It was found that α-catulin expression level was signifi-cantly higher in primary cancers than that in normal squamous epithelium and dysplasia(P < 0.05),but not significantly correlated with aggressive behaviors or adverse prognosis of HNSCC patients(P > 0.05). Cox′s proportional hazard model indicated that distant metastasis and tNM staging were independent prognostic factors for overall survival of the patients with head and neck squamous carcinoma(HNSCC,P < 0.05). Conclusion these findings suggested that up-regulated expression of α-catulin protein may play an important role in the pathogenesis of HNSCC,which might be employed as a potential marker for tumorgenesis of HNSCCs.

Objective To explore the effects and molecular mechanisms of suberoylanilide hydroxamic acid (SAHA) on ovarian carcinoma. Methods (1)Two groups of ovarian carcinoma cell lines (SKOV3 and SKOV3/DDP, HO8910 and HO8910-PM) were exposed to SAHA (1, 3, 5 and 7μmol/L SAHA,group 1-group 4). CCK-8 method was employed to eval-uate the inhibitory effects of SAHA.(2)Ovarian cancer cell lines treated with SAHA (2 or 5μmol/L SAHA) were used as 1 and 2 groups. Flow cytometry was performed following staining with Annexin V-FITC and PI for cell cycle and apoptosis.(3) Reverse transcription polymerase chain reaction (RT-PCR) and Western blot assay were used to assess the mRNA and pro-tein expression levels of phenotypic correlation factor. Results (1)After 48 h of SAHA treatment,the OD value of SKOV3, SKOV3/DDP,and HO8910 showed a trend of gradually reduce (P<0.05).(2)The apoptotic rates were significantly higher in SAHA 1 and SAHA 2 groups than those of control group (P<0.05). Compared with control group, after 48 h of SAHA treat-ment,S phase and G2/M phase of SKOV3 and SKOV3/DDP cells increased;G0/G1 phase of HO8910 and HO8910-PM cells increased in SAHA 1 and 2 groups (P<0.05).(3)The expression levels of CyclinB1 and Cdc2 (p34) mRNA were significant-ly lower in SAHA 1 and 2 groups than those of control group,while the expression levels of Caspase-3,p21 and p53 mRNA expression were significantly higher in SAHA 1 and 2 groups than those of control group. Furthermore,the expression of Ac-Histone H3,Ac-Histone H4,p53 protein were markedly improved,and CyclinB1,Cdc2(p34) protein decreased in SAHA 1-4 groups. Conclusion SAHA may suppress cell growth, induce apoptosis and cause cycle arrest in ovarian carcinoma cells by promoting histone acetylation or modulating their phenotype-related proteins of Caspase-3, p53, CyclinB1 and Cdc2(p34).

OBJECTIVETo investigate the role of matrix metalloproteinase-7 (MMP-7) expression in caricinogenesis and progression of gastric cancer.

METHODSWe studied MMP-7 expression and microvessel density (MVD) in adjacent mucosa and primary foci of 113 cases of gastric cancer by streptavidin-biotin-immunoperoxidase method using anti-MMP-7 and anti-CD34 antibodies. MMP-7 expression and mean MVD were compared with clinicopathological features of gastric cancer, with the relationship between MMP-7 expression and MVD concerned in gastric cancer.

RESULTSMMP-7 showed positive expression in adjacent mucosa of gastric cancer (29.20%, 33/113), less than that in gastric cancer (69.03%, 78/113). MMP-7 expression in primary foci of gastric cancer was positively correlated with tumor size, invasive depth, metastasis and TNM staging (P<0.05), but not with differentiation or growth pattern of gastric cancer (P>0.05). Positive correlation of mean MVD with tumor size, invasive depth, metastasis and TNM staging was found (P<0.05), despite no relationship between mean MVD and differentiation of gastric cancer (P>0.05). Mean MVD was dependent on MMP-7 expression in gastric cancer (P<0.05).

CONCLUSIONUpregulated expression of MMP-7 played an important role in carcinogenesis and progression by participating in growth, invasion, metastasis and angiogenesis of gastric cancer. MMP-7 expression could be regarded as an effective and objective marker to reflect the biological behaviors of gastric cancer.

Biomarkers, Tumor ; metabolism ; Humans ; Lymphatic Metastasis ; Matrix Metalloproteinase 7 ; metabolism ; Neoplasm Invasiveness ; Neoplasm Staging ; Neovascularization, Pathologic ; pathology ; Stomach Neoplasms ; blood supply ; metabolism ; pathology

BACKGROUNDIt has been known that Kai1 is one of transmembrane 4 superfamily regulating cell proliferation, adhesion and mobility and its down-regulated expression is closely correlated with progression and prognosis of tumor. Fas ligand (FasL) is one of tumor necrosis factor/nerve growth factor family activating caspase-3, a key proteinase in cell apoptosis and leading immune escape in tumor cells. The aim of this study is to investigate the expression of Kai1 and FasL in non-small cell lung cancer (NSCLC) and to explore their roles and relationship in carcinogenesis and progression of NSCLC.

METHODSKai1 and FasL expressions were examined in 79 NSCLC tissues and their adjacent normal lung tissues by SP immunohistochemistry. Their expressions were compared with clinicopathological features of NSCLC. The relationship between Kai1 and FasL expressions was also analyzed in NSCLC.

RESULTSKai1 expression in NSCLC tissue was remarkably lower than that in their adjacent tissue (55.7% vs 82.6%, P < 0.05). However, it was the converse for FasL (73.4% vs 52.2%, P < 0.05). Kai1 expression was not correlated with age and gender of NSCLC patients, tumor location or histological classification (P > 0.05), but negatively with lymph node metastasis and clinicopathological staging and positively with differentiation degree (P < 0.05). FasL expression was not correlated with age and gender of the patients, tumor location or histological classification (P > 0.05), but positively with lymph node metastasis and negatively with differentiation degree (P < 0.05). Kai1 expression was closely correlated with FasL expression in NSCLC (P < 0.05).

CONCLUSIONSDown-regulation of Kai1 expression and up-regulation of FasL may play important roles in carcinogenesis and progression of NSCLC. Kai1 and FasL could be considered as molecular markers to reflect pathobiological behavior of NSCLC. Additionally, close correlation of FasL expression with Kai1 expression in NSCLC provides a novel insight into the regulatory effects of FasL expression on Kai1 expression.