[Show abstract][Hide abstract] ABSTRACT: The Akt/PKB kinase family, including Akt1, 2 and 3, plays critical roles in regulating cell growth, proliferation, survival, metabolic and many other cellular activities. Recent evidence indicates that PKB/Akt is frequently constitutively active in many types of human cancer including gastric cancer. In the present study, we applied immunohistochemistry to tissue microarray to detect the expression of Akt1, followed by Akt1 small interference RNA (siRNA) to examine knock down of the Akt1 gene on the growth inhibition of human gastric cancer SGC7901 cells. Our results indicate that the expression of Akt1 was significantly increased in gastric cancer compared to normal gastric tissue and adjacent non-cancer tissue. The in vitro study shows that cell growth was significantly inhibited and G0/G1 arrest was observed in siRNA-Akt1-treated group. In vivo, the size of tumors was significantly smaller in SGC7901 subcutaneous mice model treated with siRNA-Akt1 than those treated with siRNA-nonsense and PBS. Our studies demonstrated siRNA-Akt1 can inhibit Akt1 expression, exerted growth inhibition effect on SGC7901 cells in vitro and in vivo. Suppression of Akt1 expression by siRNA could be a new strategy in gastric cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: Cyclooxygenase-2 (COX-2) and Protein kinase B (PKB/Akt) play a crucial role in the formation of many malignant tumors and have been shown to be the important therapeutic targets. In the present study, we examined immunohistochemical expression of phosphorylated Akt (p-Akt) and COX-2 in 45 gastric adenocarcinomas with different tumor grades. Then, adenovirus-mediated small hairpin RNA (shRNA) expression vectors rAd5-Akt1+COX-2 (rAd5-A+C) that target sequences of human COX-2 and Akt1 were used to examine the inhibitory effects on cell proliferation, invasion and apoptosis in SGC7901 gastric adenocarcinoma and U251 glioma cells. Cell growth was inhibited by over 70%, as indicated by a MTT assay, and was accompanied by G1/G0 phase arrest in the rAd5-A+C treated group, indicating poor cell growth activities. The number of cells invading through the matrigel in the rAd5-A+C treated group was significantly decreased (36.2+/-3.1) compared with that of the control group SGC7901 (105.0+/-4.0) and the nonsense sequence group rAd5-HK (102.5+/-6.4). In addition, the tumor volumes in the SGC7901 subcutaneous nude mouse model treated with rAd5-A+C was significantly smaller than those of the control group and nonsense sequence group rAd5-HK. When COX-2 and Akt1 were dramatically downregulated, Ki-67, CyclinD1, MMP-2, MMP-9 and Bcl-2 were also downregulated. Our results demonstrated that p-Akt and COX-2 were overexpressed in gastric adenocarcinomas and their expression levels were elevated with the ascending order of tumor malignancy; rAd5-A+C targeting COX-2 and Akt1 downregulated their expression significantly in a sequence-specific manner, exerting inhibitory effects on SGC7901 and U251 cell proliferation, invasion and apoptosis. In conclusion, our data suggest a novel mechanism for the regulation of malignant tumor cell growth and provide evidence for combined gene therapy for malignant tumors.
Technology in cancer research & treatment 12/2009; 8(6):467-78. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To construct a short hairpin RNA (shRNA) adenovirus vector targeting Akt1 (protein kinase B1, PKB1/Akt1) and cyclooxygenase-2 (COX-2) and study its effects on the growth of SGC-7901 human gastric adenocarcinoma cell.
Adenovirus pGSadeno-Akt1 + COX-2 (rAd5-A + C) vector was constructed and transfected into SGC-7901 cell. The proliferative activity of tumor cell was evaluated by MTT assay and flow cytometry in vitro. rAd5-HK and rAd5-A + C were injected into the established subcutaneous SGC-7901 gastric adenocarcinoma in nude mice. During the observation period of 21 days, tumor volume was measured every 3 days to further observe the anti-tumor effects of rAd5-A + C on SGC-7901 cell and cell situ apoptosis was detected by TUNEL assay.
After transfection of constructed adenovirus vector rAd5-A + C into SGC-7901 cell, cell proliferative activity in rAd5-A + C treatment group was significantly suppressed, and cell cycle indicated that control group SGC-7901 and no-load group rAd5-A + C cells in G0/G1, S and G2/M phases accounted for the total number of cells 49.8%, 35.2%, 15.0% and 50.8%, 36.5%, 12.7% respectively. While the treatment group rAd5-A + C in G0/ G1, S and G2/M phases accounted for the total number of cells 68.1%, 21.8% and 10.1% respectively. The tumor volume in treatment group was lower than that of control and no-load groups and the difference had statistical significance (F = 3.679, P = 0.043) and rAd5-A + C could induce the apoptosis of tumor cell.
Adenovirus-mediated Akt1 and COX-2 shRNA can inhibit the growth of SGC-7901 human gastric adenocarcinoma cell. It may provide a new strategy for gastric cancer gene therapy.
[Show abstract][Hide abstract] ABSTRACT: To construct a recombinant adenovirus vector that expresses small hairpin RNAs (shRNA) against COX-2, AKT1 and PIK3R1 gene and to evaluate its potential for suppressing the cell proliferation of human gastric adenocarcinoma SGC701 cell in vitro and in vivo, which will enable the development of a gene therapy protocol for the treatment of human gastric adenocarcinoma.
Three strips of shRNA targeting AKT1, COX-2 and PIK3R1, was subcloned into adenovirus expression vector. After verification, it was amplified and titered. The recombinant adenovirus expression vector was infected into human gastric adenocarcinoma SGC7901 cells in vitro and the infected cells were injected in nude mice. The mRNA and protein expression levels of AKT1, COX-2 and PIK3R1 were determined by real-time PCR and Western blot respectively. Cell proliferation in vitro was determined by methyl thiazolyltetrazolium (MTT) assay and flow cytometry, tumor growth in vivo was measured by volume of tumor in nude mice.
Restriction digestion and sequencing analysis showed that the rAd5-C-A-P adenovirus expression vector was constructed successfully. It significantly inhibited the expression of AKT1, COX-2 and PIK3R1, and cell growth was inhibited over 70% as indicated by MTT assay and accompanied with G0/G1 phase arrest. Cell growth on matrigel matrix showed that the rAd5-C-A-P transfected cells were detached from the matrix or grew in a scattered clustering pattern, indicating poor cell growth activities in 2-D matrigel. Tumor growth in nude mice in the C + A + P group was inhibited (P<0.01).
shRNA targeting COX-2, AKT1 and PIK3R1 down regulated significantly the expression of the three genes in a sequence-specific manner, exerted proliferation inhibition effect on SGC7901 cells in vitro and in vivo.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 08/2009; 26(4):383-7.
[Show abstract][Hide abstract] ABSTRACT: To construct a short hairpin RNA(shRNA) adenovirus vector targeting P85 and protein kinase B1 (PKB1/Akt1) and study its effects on the growth of SGC-7901 human gastric adenocarcinoma cells.
P85 and Akt1 shRNA expression frames were subcloned to pGSadeno adenovirus vector with homologous recombination technology to construct pGSadeno-P85 + Akt1 (rAd5-P + A) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells and then its titer and transfection efficiency were detected with fluorescent microscope. P85 and Akt1 mRNA protein expression was identified with real-time PCR and Western blot. The proliferative activity of tumor cells was evaluated with MTT assay and flow cytometry in vitro. rAd5-HK and rAd5-P + A mediated by adenovirus were injected into the established subcutaneous SGC-7901 gastric adenocarcinoma in nude mice. During the observation period of 21 days, tumor volume was measured every 3 days to further testify the anti-tumor effect of rAd5-P + A on the SGC-7901 gastric adenocarcinoma cells and cell in situ apoptosis was detected with TUNEL assay.
The adenovirus vector rAd5-P + A was successfully constructed and it dramatically downregulated P85 and Akt1 mRNA expression in SGC-7901 gastric adenocarcinoma cells. Compared with a control group of SGC-7901 cells and cells transfected with general adenovirus rAd5-HK as control, P85 and Akt1 protein expression 48 h and 72 h after rAd5-P + A transfection was decreased by 57.5% and 63.7%, 67.8% and 75.6% with statistical significance (P = 0.005, P = 0.003). Cell proliferative activity in rAd5-P + A transfected cells was suppressed from the second day (P < 0.001) and the decreased P85 and Akt1 expression was accompanied by 5.9% -7.1% decrease of S phase fraction and 12.1% - 13.7% increase of G0/G1 phase. The tumor volume of rAd5-P + A treated group was smaller than that of the control and rAd5-HK group with statistical significance (F = 9.871, P = 0.025). Moreover, rAd5-P + A could induce cell in situ apoptosis.
Adenovirus-mediated targeting P85 and Akt1 shRNA can inhibit the growth of SGC-7901 human gastric adenocarcinoma cells and this may provide a new strategy of combination gene therapy in gastric adenocarcinoma.
Zhonghua nei ke za zhi [Chinese journal of internal medicine] 07/2009; 48(7):557-61.
[Show abstract][Hide abstract] ABSTRACT: To investigate abnormal protein expression of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in human gastric adenocarcinoma, and further reveal the clinical significance.
The MMP-9, VEGF and PCNA proteins expression was determined by immunohistochemistry staining in 45 gastric adenocarcinoma tissues, 45 adjacent specimens and 10 normal gastric mucosa tissues via tissue arrays accordingly. The relationship of these protein expression with differentiation degree, development and progression of gastric adenocarcinoma were also analyzed.
Positive rates of MMP-9, VEGF and PCNA in gastric adenocarcinoma, adjacent specimens and gastric normal mucosa were as follows: MMP-9, 82.2% (37/45), 64.4% (29/45), 30.0% (3/10) (P = 0.019); VEGF, 73.3% (33/45), 62.2% (28/45), 30.0% (3/10) (P = 0.029); PCNA, 84.4% (38/45), 71.1% (32/45), 10.0% (1/10), there were statistically significant difference (P = 0.001). The positive rates of MMP-9, VEGF and PCNA in well-differentiated adenocarcinoma, moderately differentiated adenocarcinoma and poorly differentiated adenocarcinoma were as follows: MMP-9, 70.0% (7/10), 80.0% (8/10), 88.0% (22/25), there were statistically significant difference (P = 0.015); VEGF, 50.0% (5/10), 60.0% (6/10), 88.0% (22/25), there were statistically significant difference (P = 0.000); PCNA, 60.0% (6/10), 90.0% (9/10), 92.0% (23/25), the difference is significant statistically (P = 0.004). The expression of MMP-9, VEGF and PCNA showed positive relationship with each other by rank correlation analysis (P < 0.05).
Tissue arrays technology is effective tool to analyze the expression of cancer related proteins in gastric adenocarcinoma. The expression of MMP-9, VEGF and PCNA proteins participates in the tumorigenesis and development process of gastric adenocarcinoma, and these can be used as indexes to evaluate prognosis in clinical.
Zhonghua nei ke za zhi [Chinese journal of internal medicine] 02/2009; 48(2):114-7.
[Show abstract][Hide abstract] ABSTRACT: To detect the expression of human beta-defensin 2 (HBD-2) and tumor necrosis factor-alpha (TNF-alpha) in the colonic mucosae of ulcerative colitis (UC) patients and diarrhea-predominant irritable bowel syndrome (IBS-D) patients and to evaluate the roles of HBD-2 and TNF-alpha in the pathogenesis and the relationship between them.
Immunohistochemistry was conducted on the biopsy samples of colonic mucosa from 30 UC patients, 20 IBS-D patients, and 10 normal persons to measure the expression of HBD-2 and TNF-alpha.
The expression levels of HBD-2 and TNF-alpha in the colonic mucosa of UC were both significantly higher than those in the colonic mucosa of IBS-D (z = -4.856, z = -3. 987, all P < 0.01) and in the normal colonic mucosa (z = -3.611, z = -3. 248, all P < 0.01). But no significant differences were found between the colonic mucosa of IBS-D and the normal colonic mucosa in the intensity of the expressions of HBD-2 and TNF-alpha (z = -0.373, z = -0.032, all P > 0.05). There were no significant differences in the intensity of the expressions of HBD-2 and TNF-alpha in colonic mucosa of among the mild, moderate, and severe UC (chi2 = 1.190, P > 0.05, chi2 = 1.672, P > 0. 05).
The expressions of HBD-2 and TNF-at are significantly increased in UC, but are not correlated with IBS-D and the severity of clinical symptoms of UC patients.
[Show abstract][Hide abstract] ABSTRACT: To observe the cyclooxygenase-2 (COX-2) expression and the cell proliferation and apoptosis of gastric adenocarcinoma cells after transiently transfecting gastric cancer cells using specific COX-2 small interfering RNA (siRN) and discuss the role of COX-2 in gastric tumorigenesis and the effect of RNA interference (RNAi) as anti-cancer gene therapy.
Three groups were included in the study, i. e. a COX-2 siRNA group, a non-sense siRNA group and a control group. Gastric adenocarcinoma cells SGC7901 were cultured at 37 degrees C in an atmosphere containing 5% CO2. 72 hours after transfecting SGC7901 cells with specific COX-2 siRNA or non-sense siRNA, RT-PCR was used to detect COX-2 mRNA, immunohistochemistry and Western blot were taken to detect the expression of COX-2 protein and flow cytometry was taken to detect the cell cycle and apoptosis. MTT method was used to detect the proliferation and activity of the cells every day for one week after transfection.
The expression of COX-2 mRNA and protein in the COX-2 siRNA group was obviously suppressed as compared with non-sense siRNA group and control group. No change was found between the non-sense siRNA group and the control group. The reduced expression of COX-2 could inhibit SGC7901 cells proliferation and induce cell apoptosis, but had no effect on cancer cell cycle.
The experimental results suggest that effectively inhibiting the expression of COX-2 can suppress the proliferation of gastric adenocarcinoma cell and promote the process of cancer cell apoptosis, so RNAi is a powerful tool of gene therapeutic method.
Zhonghua nei ke za zhi [Chinese journal of internal medicine] 03/2008; 47(2):129-32.
[Show abstract][Hide abstract] ABSTRACT: To investigate the association of serotonin transporter (SERT) gene polymorphism with irritable bowel syndrome (IBS).
The VNTRs and 5-HTTLPR polymorphism of SERT gene was assessed with polymerase chain reaction in 81 patients with IBS and 48 healthy subjects(HS).
Compared with HS, IBS patients have a greater frequency of STin2.12/10 genotype in VNTRs region and a smaller frequency of STin2.12/12 genotype, however no significant difference was found in polymorphism of this region among the patients with C-IBS, D-IBS and A-IBS. We also found that the 5-HTTLPR allele L/L genotype occurred with greater frequency in C-IBS patients than in other two subgroups, whereas the L/S genotype occurred with smaller frequency in C-IBS. The frequency of association between 12/12 genotype and L/L genotype (12/12-L/L) in C-IBS was significantly higher than those in A-IBS and HS.
The presence of STin2.12/10 genotype may be correlated with IBS. The presence of L/L genotype and 12/12-L/L genotype association in IBS patients carries an increased risk of C-IBS, whereas the presence of the L/S genotype carries an increased risk of D-IBS and A-IBS.
Zhonghua nei ke za zhi [Chinese journal of internal medicine] 07/2004; 43(6):439-41.