COX-2 expression in gastric cancer and its relationship with angiogenesis using tissue microarray

AIM: To explore the expression and clinicopathological significance of cyclooxygenase-2 (COX-2) and microvessel density (MVD) in gastric carcinogenesis, and to investigate their roles in the invasion and the relationship between biological behaviors and prognosis of gastric cancer. METHODS: Using Envision immunohistochemistry, COX-2 and CD34 expressions in gastric cancer tissue array were examined. MVD was counted and the relationship between the biological behaviors and prognosis was analyzed. RESULTS: The expression of COX-2 in gastric cancer tissue was significantly higher than that in normal mucosa (χ2 = 12.191, P < 0.05). The over-expression of COX-2 in gastric cancer was obviously related to metastasis and depth of invasion (χ2 = 6.315, P < 0.05), but not related to the histological type and Borrmann type (χ2 = 5.391 and χ2 = 2.228, respectively). Moreover, MVD in gastric cancer tissues was significantly higher than that in the normal mucosa (65.49 ± 20.64 vs 36.21 ± 18.47, t/F = 7.53, P < 0. 05). MVD was related to the histologic type and metastasis (t/F = 3.68 and t/F = 4.214, respectively, P < 0. 05), but not related to the depth of invasion and Borrmann type (t/F = 0.583 and t/F = 0.459, respectively). MVD in COX-2-positive tissues was markedly higher compared to COX-2-negative tissues, indicating a positive correlation between COX-2 expression and MVD (t = 13.12, P < 0. 05). CONCLUSION: Tissue microarray (TMA) is a powerful tool for rapid identifi cation of the molecular alterations in gastric cancer. COX-2 expression, via inducingangiogenesis, may play an important role in gastric carcinogenesis. It could be served as a determinant factor for clinical prognosis and curative effect.

COX-2 expression and tumor angiogenesis in colorectal cancer

AIM: Cyclooxygenase-2 (COX-2) is one of the rate-limiting enzymes in metabolism of arachidonic acid, and COX-2 inhibitors demonstrate preventive effects on cancer,especially on colorectal cancer. The underlying mechanism remains unclear. The aim of this study was to illustrate the relationship between angiogenesis and COX-2 in carcinogenesis of colorectal cancer.METHODS: One hundred and seventy patients with colorectal cancer were enrolled in our study from January 1993 to September 2001 in School of Oncology, PekingUniversity. COX-2 and VEGF expression were detected with the immunohistochemistry (IHC) technique. IHC assays were carried out with the aid of tissue microarray (TMA)procedure. Specimens from 35 of these patients were examined with reverse transcriptase PCR (RT-PCR).RESULTS: COX-2 and VEGF expressions were stronger in colorectal cancer than those in the corresponding normal tissues, at both protein and mRNA levels. One hundred patients were eligible for analysis after IHC assay of COX-2 and VEGF. The positive rate of VEGF was much higher in COX-2 positive group (47/85) than in COX-2 negative group (x^2 = 4.181, P= 0.041). The result was further verified by the result of RT-PCR (x^2 = 8.517, P = 0.003). Correlation coefficient was 0.409 after Spearman correlation analysis (P=0.015).CONCLUSION: COX-2 may be involved in the course of tumor angiogenesis of colorectal cancer and acts through VEGF.

AEG1 induces COX-2 expression via activation of NF-κB and AP-1 in hepatoma HepG2 cells

Objective： The aim of this study was to investigate whether astrecyte elevated gene 1 （AEG1） regulates COX-2 expression in human hepatoma HepG2 cells and related pathways involved in this process. Methods： Human hepatoma HepG2 cells were transfected with pcDNA3.1（-）-AEG1 plasmid or psilencer2.0-AEGl-shRNA1 plasmid to up/down-regulate AEG1 expression, pcDNA3.1（-） and psilencer 2.0 empty vector plasmids were transfected respectively as control. Real-time RT-PCR was carried out to measure the expression levels of AEG1 and COX-2 mRNA. The expression levels of AEG1 and COX-2 protein were detected by Western blot. NF-KB signaling was blocked by PDTC, and AP-1 signaling was blocked by curcumin. Results： AEG1 mRNA and protein levels were increased after pcDNA3.1（-）-AEG1 transfection, and decreased after psilencer2.0-AEGl-shRNAs transfection. COX-2 mRNA and protein levels were increased in AEGl-overexpressing cells and decreased in AEGl-knockdown cells. Phosphorylations of p65 and c-jun were up-regulated in AEGl-overexpressing cells. Both PDTC and curoumin reduced COX-2 expression in HepG2 cells with AEG1 overexpression. Conclusion： AEG1- overexpressing and -knockdown HepG2 cells are established successfully. AEG1 could induce COX-2 expression though activating NF-KB and AP-1 in human hepatoma HepG2 cells.

Protective effect of notoginsenoside and tanshinone IIA on inflammation-related colorectal cancer mice and the inhibition effect on COX-2 expression

Objective To explore the preventive effects and possible mechanisms of action of notoginsenoside(NGS)and tanshinone IIA(TSN)in inflammation-related colorectal cancer(IRCC)in mice.Methods Eighty-eight male C57BL/6 mice were randomly assigned to 11 groups(n=8 each group).Azomethane oxide+dextran sulfate(AOM+DSS)model control(model),NGS lowdose(l-NGS),NGS medium-dose(m-NGS),NGS high-dose(h-NGS),TSN low-dose(l-TSN),TSN medium-dose(m-TSN),TSN high-dose(h-TSN),(NGS+TSN)low-dose[l-(NGS+TSN)],(NGS+TSN)medium-dose[m-(NGS+TSN)],(NGS+TSN)high-dose[h-(NGS+TSN)],and blank groups were established.The first 10 groups were intraperitoneally injected with AOM to induce inflammatory colon cancer,whereas the blank group was intraperitoneally injected with 0.9%NaCl solution.The first 10 groups drank a 2.5%sodium DSS aqueous solution continuously from day 5 for three cycles(one cycle:five days,every three weeks),and the blank group was allowed free access to water.Drug groups were administered NGS(low,medium,or high dose),TSN(low,medium,or high dose),or NGS+TSN(low,medium,or high dose),and the model and blank groups were administered saline by lavage until the end of the experiment.The general activity,body weight,and survival rate of and incidence of adenocarcinoma in mice were detected and the expression of cyclooxygenase 2(COX-2)was detected by immunohistochemistry.Results(1)The survival rate of mice with IRCC in the h-NGS,m-TSN,h-TSN,m-(NGS+TSN),and h-(NGS+TSN)groups was significantly increased than that in other groups(P<0.05).(2)The incidence of tumors in the h-(NGS+TSN),m-TSN,and l-NGS groups was significantly lower than that in the model group(P<0.05).(3)The expression level of COX-2 in tumor tissues of mice in the m-(NGS+TSN)and h-(NGS+TSN)groups was significantly lower than that in the model group(P<0.05).Conclusion Tumor formation was inhibited by m-TSN and h-(NGS+TSN)treatments in mice with IRCC,and h-(NGS+TSN)treatment inhibited the COX-2 pathway.

The Prognostic Importance of EGFR and COX-2 Expression in Cervix Cancer Stages IIb-IVa

Objectives. Locally advanced cervix cancer represents a therapeutic challenge with a delicate balance between effect and toxicity. Consequently, there is an obvious need for new prognostic parameters with a perspective of a more individualized treatment. The aim of the present study was to evaluate the possible prognostic importance of epidermal growth factor receptor and cyclooxygenase-2 expression in locally advanced cervix cancer. Methods. The study included 91 patients with cervix cancer, FIGO stages IIb-IVa, and were treated with curative intent according to the Nordic Cervix Cancer protocol (NOCECA). The median observation time was 7 years. Epidermal growth factor receptor and cyclooxygenase-2 expression was evaluated by immunohistochemistry using commercially available antibodies. The tumor marker expression was evaluated according to a semi-quantitative scoring system with a positive score when more than 10% of the tumor cells were moderately or strongly stained. Results. Epidermal growth factor receptor and cyclooxygenase-2 over-expression was found in 22% and 18% of the patients, respectively. The survival differed according to epidermal growth factor receptor and cyclooxygenase-2 over-expression as calculated by Kaplan-Meier plots and log-rank test (p = 0.0002 and p = 0.0084 respectively). A multivariate Cox Regression analysis identified each tumor marker as an independent prognostic factor. Conclusion. The results clearly indicate epidermal growth factor receptor and cyclooxygenase-2 hold important prognostic information, markedly appearing with a long-term observation. We found that both parameters were independent prognostic factors, and to further clarify this matter our retrospective analysis should be confirmed in a prospective study with more patients.

Correlation of VEGF and COX-2 Expression with VM in Malignant Melanomas

OBJECTIVE To investigate the relationship between vascular epithelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in melanomas and the expressive difference of VEGF and COX-2 between melanomas with and without vasculogenic mimicry(VM).METHODS Sixty cases of malignant melanomas emoeaaea In paraffin were studied. The tumors were divided into a high-grade malignant group and a low-grade malignant group based on their tumor type, atypia and survival time of the patient. Then tissue microarrays were produced from these paraffin-embedded tumor tissues which were stained for VEGF, COX-2 and PAS. The difference in expression between VEGF and COX-2 in the malignant melanomas was compared using a grid-count. In addition, the tumors were also divided into mimicry and non-mimicry groups based on their PAS staining. Then the differences between the PAS positive and negative areas of the 2 groups were compared.RESULTS In malignant melanomas with VM, VEGF and COX-2 expression was less in tumors in which VM was absent, but VEGF, COX-2 expression in high-grade malignant melanomas was higher than that in low-grade grade malignant melanomas. Expression of VEGF was correlated with COX-2 expression.CONCLUSION VM exists in some high-grade malignant melanomas. The differences and relations between VEGF and COX-2 showed that some high-grade malignant melanomas possess a unique molecular-mechanism of tumor metastasis and blood supply.

Gene expression analysis of cytokines and MMPs in melatonin and rhBMP-2 enhanced bone remodeling

BACKGROUND In the medical and dental fields,there is a need for studies of new therapeutic approaches for the treatment of bone defects that cause extensive bone loss.Melatonin may be an important endogenous biological factor for bone remodeling,and growth factors may enhance the repair process.AIM To evaluate the gene expression of cytokines(IL-1β,IL-6,IL-10 and TNF-α),markers of osteoclastogenesis(RANK,RANKL and OPG)and MMPs(MMP-1,MMP-2,MMP-8 and MMP-13)from the treatment of melatonin associated with an osteogenic membrane and rhBMP-2 on the recovery of a bone injury.METHODS Sixty-four rats were used and divided into 9 experimental groups and were formed according to the treatment carried out in the region of the bone lesion,which varied between the combination of 1,10 and 100μmol/L of melatonin.Gene Expression analysis was performed using real time-PCR by reading the concentration of total RNA and reverse transcription.RESULTS There were differences between groups when compared with clot or scaffold control,and improvement with a higher concentration of melatonin or rhBMP-2.The combination melatonin(1μg)with 5μg of rhBMP-2,using the guided bone regeneration technique,demonstrated some effects,albeit mild,on bone repair of critical bone defects.CONCLUSION This indicates that the approach for administering these substances needs to be reassessed,with the goal of ensuring their direct application to the affected area.Therefore,future research must be carried out,seeking to produce materials with these ideal characteristics.

Expression of COX-2 and HER-2 in colorectal cancer and their correlation

AIM: To detect the expression of COX-2 and HER-2 in colorectal cancer and to analyze their correlation and clinical significance.METHODS: A total of 1026 colorectal cancer surgical specimens were collected from patients treated fromDecember 2002 to December 2007 at the First Affiliated Hospital of Anhui Medical University. All specimens were made into 4-μm slices. The expression of COX-2 and HER-2 were detected by immunohistochemistry using the streptavidin-biotin-peroxidase method. The correlations between COX-2 and HER-2 expression and colorectal cancer clinical features were analyzed.RESULTS: The positive rates of COX-2 and HER-2 expression in colorectal cancer were 77.97%(800/1026) and 46.20%(474/1026), respectively. There was a significant correlation between COX-2 and HER-2 expression in colorectal cancer(P < 0.05). In patients with tumor size ≥ 5 cm, the positive rates of COX-2 and HER-2 expression were 81.48%(308/378) and 57.94%(219/378), respectively. In patients with serosal invasion, the positive COX-2 and HER-2 expression rates were 80.53%(612/760) and 49.21%(374/760), respectively. In patients with lymph node metastasis, the positive expression rates were 85.04%(506/595) and 54.62%(325/595), respectively, and the positive expression rates differed significantly between patients with lymph node metastasis and those without(P < 0.05). In patients with Duke's C and D colorectal cancer, the positive COX-2 and HER-2 expression rates were 82.80%(443/535) and 57.94%(310/535), respectively. In patients with poorly differentiated colorectal cancer, the positive expression rates were 74.49%(210/282) and 52.84%(149/282), respectively(P < 0.05). In patients with distant metastasis, the positive expression rates were 82.27%(116/141) and 53.90%(76/141), respectively(P < 0.05). These findings suggest that COX-2 and HER-2 have synergistic effects in colorectal cancer. COX-2 and HER-2 expression had no significant correlation with sex, age, or tumor location. CONCLUSION: COX-2 and HER-2 are important markers for invasion and metastasis of colorectal cancer, and they act together to regulate the invasion and metastasis of colorectal cancer.

Relationship between COX-2 expression and clinicopathological features of colorectal cancers

Background Cyclo-oxgenase 2 (COX-2) is involved in prostaglandin synthesis in central nervous system, and it also plays a role in human carcinogenesis Our purpose of this study is to investigate the COX-2 expression in different development stages of colorectal cancer, and to discuss the relationship between the gene expression and clinicopathological features of the cancer Methods COX-2 expression was examined by immunohistochemical staining in 76 surgical specimens of colorectal cancer (44 of advanced stage and 32 of early stage), thirty-three adenomas and 18 normal colonic mucosal tissues taken by endoscopic biopsy Kaplan-Meier survival curves and Cox proportional hazards regression were used to evaluate the relation of COX-2 to prognosis Results COX-2 expression, divided into 4 grades from “-” to“+++”, is respectively 83 3%, 16 7%, 0% and 0% in normal colonic mucosal tissues; 12 1%, 42 4%, 36 4% and 9 1% in adenomas; 6 3%, 28 1%, 46 9% and 18 7% in early colorectal cancers (ECCs), and 6 8%, 20 5%, 18 2% and 54 5% in advanced colorectal cancers (CRCs) The differences in COX-2 expression between advanced CRCs and early colorectal cancers (ECCs) as well as between the advanced CRCs and adenomas were statistically significant ( P <0 01); but there was no significant difference between ECCs and adenomas Kaplan-Meier survival analysis showed a significant difference in the survival curves between low high COX-2 groups ( P <0 05) Cox proportional hazards regression showed that COX-2 expression was related to poorer long-term outcome with a hazard ratio of 2 665 unadjusted for other variables ( P <0 05), and COX-2 expression was an independent risk factor of poor prognosis KH*2/5DConclusions COX-2 expression is gradually up-regulated in the development from normal epithelium to adenomas and from ECCs to advanced CRCs Alhough the COX-2 protein can not be regarded as a tumor marker to diagnose CRCs early, COX-2 expression can be regarded as an independent risk factor of poor prognosis for postoperative patients with advanced CRCs

Expression of COX-2,iNOS,p53 and Ki-67 in gastric mucosa-associated lymphoid tissue lymphoma

AIM: To assess the expression of cyclooxygenase-2 (COX-2),nitric oxide synthase (iNOS), p53 and Ki-67 in gastric mucosaassociated lymphoid tissue (MALT) lymphoma and clarify the relationship between COX-2 expression and iNOS or p53 expression in these patients.METHODS: The expressions of COX-2, iNOS, p53 and Ki-67 were detected in 32 gastric MALT lymphoma specimens and 10 adjacent mucosal specimens by immunohistochemical Envision method.RESULTS: COX-2 and iNOS expressions were significantly higher in gastric MALT lymphoma tissues than those in adjacent normal tissues. The expression of COX-2 was observed in 22 of 32 cases of MALT lymphoma tissues (68.8%). A positive cytoplasmic immunoreactivity for iNOS was detected in 17 of 31 cases (53.1%). COX-2 expression in gastric MALT lymphoma tissues was positively correlated with iNOS expression (r=-0.448, P=0.010) and cell proliferative activity analyzed by Ki-67 labeling index (r=0.410, P=0.020).The expression of COX-2 protein did not correlate with age,sex, stage of disease, lymph node metastasis or differentiation.The accumulation of p53 nuclear phosphoprotein was detected in 19(59.4%) of tumors, p53 protein was expressed in 11 of 23 assessed LG tumors and in 8 of 9 assessed HG tumors.The difference of p53 positivity was found statistically significant between LG and HG cases (P=0.0302). The p53 accumulation correlated with advanced clinical stage (stage Ⅲ+Ⅳ vs stage Ⅰ+Ⅱ, P=0.017). There was a significant positive correlation between COX-2 expression and p53 accumulation status (r=0.403,/=0.022). The mean PI of Ki-67 in each grade group were 36.0±7.73% in HG and 27.4±9.21% in LG. High-proliferation rate correlated with HG tumors (r=0.419, P=0.017). The correlation coefficient showed a significant positive correlation between PI and COX-2 expression in MALT lymphoma patients (r=-0.410,P=0.020).CONCLUSION: COX-2 expresses in the majority of gastric MALT lymphoma tissues and correlates with cellular proliferation and iNOS expression. COX-2 overexpression is closely associated with p53 accumulation status, iNOS and COX-2 may play a synergistic role in the pathogenesis of gastric MALT lymphoma.

COX-2, MMP-7 expression in oral lichen planus and oral squamous cell carcinoma

Objective: To observe cyclooxygenase (COX)-2 expression in normal oral mucosa (NOM), oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC) and explore its significance in the incidence of oral cancer. Methods: The immunohistochemical method and RT-PCR method were applied to detect the expression of COX-2 and MMP-7 in 10 cases with NOM, 33 cases of with OLP and 38 cases with OSCC. Results: The expression of COX-2 mRNA in OSCC tissues (68.4%, 26/38) was significantly higher than in the OLP (24.2%, 8/33) and NOM (0.0%, 0/10) ( P<0.01). The expression of MMP-7 mRNA in OSCC tissues (65.8%, 25/38) was significantly higher than in the OLP (30.3%, 10/33) and NOM (0.0%, 0/10) ( P<0.01). The expression of MMP-7 in OLP was significantly higher than in the NOM ( P<0.05). There was no significant expression of COX-2 protein in NOM, and the positive rate was 42.4% (14/33) and 89.5% (34/38) in OLP and OSCC group, respectively. The COX-2 expression in cancer tissues was significantly higher than in NOM and OLP ( P<0.05). The MMP-7 protein expression in cancer tissues (84.2%, 32/38) was significantly higher than in NOM (10.0%, 1/10) and in OLP (42.4%, 14/33), and the positive rate in OLP was significantly higher than in NOM ( P<0.01). The COX-2 expression was associated with clinical stage ( P<0.05), the MMP-7 expression was associated with clinical stage and lymph node metastasis ( P<0.05). The expressions of COX-2 and MMP-7 mRNA were positively correlated with OSCC. Conclusions: The abnormal expressions of COX-2 and MMP-7 are closely related to the biological behavior of OSCC, the MMP-7 may be induced by COX-2, and further lead to the invasion and metastasis of OSCC.

Expression and Significance of NF-κB, IL-1β and COX-2 in the Murine Model of Estrogen-dependent Experimental Vulvovaginal Candidiasis

Objective To investigate the possible role of estrogen in the pathogenesis of vulvovaginal candidiasis（VVC）. Methods Estrogen-dependent experimental murine model of C. albicans vaginal infection was established by injecting subcutaneously with estradiol benzoate and then 5 × 10^6 stationary-phase C. albicans blastoconidia was inoculated intravaginally to mice （group El）, and other 3 groups were set up.＂ estrogen-treated but not infected （group E）; estrogen-untreated but infected （group I）; normal control （group C）. The dynamic change of colony-forming unit （CFU） of cervivovaginal lavage fluid was observed. Vaginal tissues at different time points （d 2, d 4, d 7 and d 14） after inoculation of C. albicans were obtained. In situ hybridization staining was used to detect expression of cyclooxygenase-2 （COX-2） mRNA and expression of nuclear factor-κB （NF-κB） was examined by immuohistochemistry. ELISA was applied to determine the interlenkin-1β （IL-1β） level. Results The constitutional high level expression of COX-2 mRNA in the vaginal tissue of group E was significantly higher than that in group C on d 4 and d 7 （P〈0. 01）, and the optical density （OD） in group EI was higher than that in the other 3 groups （P〈0.05）. There were higher IL-1β levels in vaginal tissues from d 4 to d 7 postinoculation in group EI and group I than group C （P〈0.01）. Furthermore, IL-1β in group EI was markedly elevated on d 4 and d 7 compared with group I （P〈0.01）. Compared with group C, the expression of NF-κB in group E was increased obviously on d 4 （P〈0.01）, and there was significant difference between group E1 and group I on d 4 and d 7 （P〈0.01）. Conclusions In the murine model of estrogen-dependent experimental WC, estrogen promotes the infection establishment by up-regulating expression of COX-2 via activating NF-κB signal pathway, and the high expression of COX-2 promoted by the interaction of IL-1β and NF-κB after infection formation was involved in persistence of infection.

Correlation and Expression of COX-2 and P53 Protein in Basal Cell Carcinoma of Eyelid

The correlation between the expression of COX-2 and p53 protein in basal cell carcinoma （BCC） of eyelid and apoptosis was investigated. Specimens of BCC were collected from 40 cases （aged 28-68 y） at the Department of Pathology, Renmin Hospital of Wuhan University, and Department of Pathology, Zhongnan Hospital of Wuhan University during from 1999 to 2006. Five specimens of paracancerous tissues served as control group. Immunohistochemical staining was performed to detect the expression of COX-2 and p53 in the tissues. The average absorbance （A） and the average positive area rate of COX-2 and p53 protein were measured by image analysis. The positive area rate of COX-2 and p53 protein was analyzed by linear correlation analysis. It was found that COX-2 and p53 proteins were highly expressed in BCC of eyelid, and weakly expressed in paracancerous tissues. Image analysis revealed that the expression of COX-2 and p53 proteins in BCC of eyelid was sig- nificantly higher than that in paracancerous tissues （P〈0.01）. Spearman rank correlation analysis demonstrated a positive correlation between the expression of COX-2 and p53 （r=0.113, P=0.421）. It was concluded that COX-2 can increase the expression of p53 protein, therefore suppressing apoptosis.

The effects of interfering in COX-2 gene expression on malignant proliferation of the human lung adenocarcinoma A2 cell in vitro

Objective:We explored the interfering effect of COX-2 gene expression and the influence on the malignant proliferation of A2 cells by RNAi after quenching COX-2 in vitro. Methods:COX-2 was selected as the subject. Three COX-2 siRNA expression vectors with human U6 promoter were constructed and three vectors and the vacant vector (pEGFP) were transfected into A2 cells with lipofectamine respectively. The cell strains transfected were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A2 cells after silencing COX-2 were studied by cell growth curve and clonogenic assay in vitro. Results: The three siRNA and U6 promoter cloned into pEGFP plasmid were validated by PCR, restriction endonucleases identification, DNA sequencing and BLAST alignment. The cell strains transfected were named as A2-3, A2-7, A2-10 and A2-p respectively. Green fluorescence was seen in A2-p cells and not in A2-3, A2-7 and A2-10 cells in 24, 48 and 72 h after transfected. The results of RT-PCR and Western blot showed the three siRNA expression vectors produced effects and the expression of COX-2 was inhibited in different extent. In contrast to A2 cells, the levels of COX-2 mRNA of A2-3, A2-7 and A2-10 cells reduced 15.6%, 20.4% and 64.2% respectively; the levels of COX-2 protein of A2-3, A2-7 and A2-10 cells reduced 23.7%, 36.7% and 60.2% respectively. The results of cell growth curve and clonogenic assay showed the growth of A2-10 cell slowed and the colonial formation rate reduced but the growth of A2-3 and A2-7 cells had not obvious changes in contrast to the controls (A2 and A2-p). Conclusion: Silencing the COX-2 gene in vitro by RNAi technique can significantly inhibit the malignant proliferation of A2 cells.

Prognostic Value of Combined;Cox-2, Cyclin D1 and P21 Expression in Colorectal Cancer (CRC) Patients: An Immunohistochemical Study

Background: Cyclooxygenase (COX) is a rate limiting enzyme in synthesis of prostanoids pathway and it has 2 isoforms (COX-1 and COX-2). It has many biological roles in inflammation and oncogenesis. Cox-2 was incriminated in performing disturbances in the cell cycle control in CRC, but its role of in CRC needs clarification of the mechanisms by which Cox-2 might affect the process of colorectal carcinogenesis. Cyclin D1 is an oncogene that regulates G1 phase progression to S phase of the cell cycle. Its stimulatory role on cell cycle is antagonized by Cyclin D1-dependent kinase (CDK) inhibitors like p21. P21 plays an essential role in cell cycle regulation;it may have a pro-apoptotic or an antiapoptotic role in cancer. P21 was found to have many roles in cancer;invasion metastases, cellular senescence and stem cells aging. The roles of combined expression of Cox-2, Cyclin D1 and P21 in CRC tissues and their role of prognosis and patients survival are not sufficiently clarified. Aim of the Study: To evaluate tissue expression of Cox-2, Cyclin D1 and P21 in CRC and to correlate such expression with pathological parameters, clinical and prognostic data of the patients. Methods: Cox-2, Cyclin D1 and P21 are evaluated in colon cancer tissues. Correlations between their level of expressions pathological parameters, clinical and prognostic data of patients were analyzed. Results: Cox-2, Cyclin D1 over-expression was associated with higher grade, higher incidence of occurrence of lymph node & distant metastasis and advanced stage (P = 0.000). Cox-2 was related to higher tumor recurrence rate (P = 0.04) and decreased overall patients survival rate (p = 0.002). (r correlation coefficient = +0.987). Conclusion: Cox-2 and Cyclin D1 are markers of poor prognosis colon cancer patients.

The effects of the same target on malignant proliferation of human lung cancer cells with different expression levels of COX-2 protein

Objective: The aim of the study was to explore the effects of the same target (si-10) on lung cancer cells with different expression levels of cyclooxygenase-2 (COX-2) protein by RNAi and malignant proliferation of these cells. Methods: COX-2 was selected as the target and one siRNA expression vector with the best effect was selected and thought as the subject from three COX-2 siRNA expression vectors with human U6 promoter. The siRNA expression vector (psi-10) and the vacant vector (pEGFP) were transfected into these cells with different COX-2 expression states (801D, A549 and LTEP-A2) with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve and clonogenic assay. Results: The siRNA and U6 promoter were validated by PCR, restriction endonucleases identification and DNA sequencing and BLAST alignment and cloned into the pEGFP vector. The cell strains transfected that 801D was used as maternal line were named as 801D-p and 801D-10 respectively. The cell strains transfected that A549 was used as maternal line were named as A549-p and A549-10 respectively. The cell strains transfected that LTEP-A2 was used as maternal line were named as LTEP-A2-p and LTEP-A2-10 respectively. These cells transfected pEGFP (801D-p, A549-p and LTEP-A2-p) had the expression of GFP and 801D-10, A549-10 and LTEP-A2-10 cells had not in 24, 48 and 72 hours after transfected. The results of RT-PCR and Western blot showed the siRNA expression vector produced marked effects in two cells (A549 and LTEP-A2) expressing COX-2 and the expression of COX-2 was inhibited. But the inhibited effects were differ- ent and the expression of COX-2 was more inhibited obviously in LTEP-A2 cells than in A549 cells though the expression of COX-2 was also inhibited obviously in A549 cells. In contract to their maternal line, the levels of COX-2 mRNA of LTEP-A2-10 and A549-10 cells reduced 64.2% and 61.2% respectively; the levels of COX-2 protein reduced 60.2% and 56.2% respectively. But the levels of COX-2 mRNA and protein had not change in 801D cells not expressing COX-2. The results of cell growth curve and clonogenic assay showed the growth of LTEP-A2-10 cells slowed and the clonal formation rate reduced and the size of the colonies became small; the growth of A549-10 cells showed slow and more obviously in the cell growth curve especially. But the growth of 801D-10 cells had not obvious change. Conclusion: The si-10 target of COX-2 has different inhibition effects on lung cancer cells with different COX-2 expression levels and the different inhibition effects have different effects on cells malignant proliferation.

次苷酸查尔酮对LPS诱导的RAW264.7细胞iNOS和COX-2表达的影响

补骨脂为豆科植物补骨脂(Psoraleacorylifolia L.)果实,具有温肾助阳、温脾止泻的功效[1],临床上被用于治疗皮肤病、肾炎、骨折等[2-3]。次苷酸查尔酮(corylifol A,CYA)是中药补骨脂的活性成分之一,是一种异黄酮类化合物,具有抗炎、抗菌、抗氧化、抗骨质疏松的特性[4-5]。有研究表明,CYA能明显抑制破骨细胞的分化,且能明显降低LPS诱导的巨噬细胞一氧化氮(NO)的生成[6-7]。

Effects of Light and Temperature on the Expression of the Lhcb2 Gene in Pea

An approximately 800 bp cDNA ( Lhcb 2) encoding light_harvesting chlorophyll a/b_binding protein complex (type Ⅱ) was cloned from the seedling of pea ( Pisum sativum L.) with RT_PCR method. Southern blotting using special probe demonstrated that there existed one copy of Lhcb 2 in pea genome. RT_PCR and Northern blotting revealed the expression of Lhcb 2 which was regulated by light in a time_dependent expression manner. The Lhcb 2 gene didn't express untill 2 h after irradiated with white light. Low temperature (4 ℃) also affected the Lhcb 2 gene by decreasing half of its expression under 25 ℃.

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

The Role of Predominant Expression of Th2 Type Cytokines Gene in the Genesis and Development of Human Gliomas

Objective: To explore the expression of Th1/Th2 cytokines gene in human gliomas and its role in the genesis and development of human gliomas.Methods: Using IL-2 and IFNγ as Th1 type cytokines, IL-4, IL-6 and IL-10 as Th2 type cytokines, the biological activity of cytokines in the supernatant of glioma cell lines was assayed by ELISA method, and the gene expression of Th1/Th2 cytokines in human glioma cells, glioma infiltrating lymphocytes and glioma cell lines were detected by RT-PCR.Results: There was predominant expression of Th2 type cytokines in human glioma cells, glioma infiltrating lymphocytes and glioma cell lines, but there was no such expression in normal brain tissues.Conclusion: It suggested that there is a relationship between the Th2 type cytokines expression in human gliomas and the immunosupressive status of human glioma patients. The predominant expression of Th2 type cytokines may play an important role in the genesis and development of human gliomas. Key words glioma - Th1/Th2 - gene expression - RT-PCR This project was supported by a grant from National Natural Sciences foundation of China (No. 30271335).