COX-2调节HIF-1α和VEGF在人胰腺癌中的表达

目的 检测环氧合酶-2、低氧诱导因子-1α和血管内皮生长因子在胰腺癌组织中的表达情况.并进一步研究选择性环氧合酶-2抑制剂尼美舒利对胰腺癌BxPC-3细胞株中这3种蛋白表达的影响.探讨环氧合酶-2在胰腺恶性肿瘤血管生成过程中的作用及意义.方法 应用Western blot-ting检测12例胰腺癌及相应癌旁组织中环氧合酶-2、低氧诱导因子-1α和血管内皮生长因子蛋白的表达情况,应用不同浓度(分别为DMSO组.50 μmol/L,100 μmol/L组和200 μmol/L组)尼美舒利作用于BxPC-3细胞24 h后.提取细胞蛋白行Western blotting.观察3种蛋白表达水平的变化.结果 12例胰腺癌组织标本中均检测到有环氧合酶-2蛋白的高表达.相比之下,癌旁组织中则相对低表达甚至未表达.低氧诱导因子-1α和血管内皮生长因子蛋白也存在相同情况,即在癌组织中的表达要明显高于癌旁组织.而在尼美舒利不同浓度作用于胰腺癌BxPC-3细胞后,可明显抑制3种蛋白的表达水平(P<0.01),并呈剂量依赖性.结论 胰腺癌组织中环氧合酶-2、低氧诱导因子-1α和血管内皮生长因子呈过量表达.提示环氧合酶-2可能与胰腺肿瘤血管生成有关,而选择性环氧合酶-2抑制剂尼美舒利可抑制这3种蛋白的表达水平,这可能为其抗肿瘤新生血管形成的机制之一.     

COX-2特异性抑制剂NS-398对胰腺癌生长及肿瘤血管 生成影响

目的:探讨COX-2特异性抑制剂NS-398对胰腺癌生长的影响及其机制。方法:分别用q RT-PCR与Western blot检测不同人胰腺癌细胞株(Bx PC-3、SWl990、Capan-2、Aspc-1、PANC-1)中COX-2及VEGF表达,并用MTT法检测NS-398在体外对人胰腺癌细胞增殖抑制作用;用体外实验最敏感细胞株建立裸鼠胰腺癌原位移植瘤模型,并随机将荷瘤鼠分为实验组和对照组,分别用NS-398与生理盐水处理,比较两组移植瘤的生长情况,并检测肿瘤组织中COX-2、VEGF蛋白表达及肿瘤微血管密度(MVD)。结果:各胰腺癌细胞中均有COX-2及VEGF表达,NS-398呈时间与浓度依赖性抑制各胰腺癌细胞的体外增殖,其中Bxpc-3细胞COX-2与VEGF表达量最高,且对NS-398最敏感。用Bxpc-3细胞建立原位移植瘤的实验组与对照组裸鼠比较,平均肿瘤体积明显减小(20.215 2 mm~3 vs.204.444 4 mm~3),瘤组织中COX-2与VEGF表达及MVD均明显降低(均P0.05)。结论:NS-398对胰腺癌的生长有抑制作用,其机制可能是通过COX-2途径降低VEGF基因表达从而抑制肿瘤血管生成有关。     

尼美舒利对结肠腺癌细胞MMP-2表达的影响

目的 探讨选择性环氧合酶-2（COX-2）抑制剂——尼美舒利对体外培养的结肠癌细胞的生长及对基质金属蛋白酶-2（MMP-2）表达的影响。方法实验分为尼美舒利组、二甲亚砜（DMSO）对照组及不接种细胞的空白对照组，采用MTT法检测不同浓度的尼美舒利对人结肠癌细胞株HT-29（COX-2高表达）及HCT-116（COX-2低表达）增殖的抑制效应；采用改良明胶酶谱分析法检测MMP-2的表达。结果尼美舒利对人结肠癌细胞株HT-29和HCT-116生长的抑制作用呈时间、剂量依赖性，且对HT-29细胞的抑制作用强于HCT-116细胞；尼美舒利抑制了HT-29细胞的MMP-2表达，而对HCT-116细胞MMP-2的表达的抑制作用不明显。结论尼美舒利可明显抑制COX-2高表达的结肠癌细胞株HT-29的增殖和生长，提示尼美舒利可能通过抑制COX-2活性而降低细胞MMP-2的表达。     

Prediction of poor survival by cyclooxygenase-2 in patients with T4 nasopharyngeal cancer treated by radiation therapy: clinical and in vitro studies

BACKGROUND: This study was undertaken to determine the status of cyclooxygenase-2 (COX-2) in nasopharyngeal cancer (NPC) in Taiwanese patients and its relationship to survival after radiotherapy (RT). In addition, the effect of NS-398, a potent selective COX-2 inhibitor, was tested in vitro alone and in combination with radiation on NPC-BM1 human NPC cells as a prelude to using this drug along with RT in the treatment of patients with NPC. METHODS: Thirty-seven patients diagnosed with T4N0-3M0 NPC were enrolled into this study. COX-2 expression was determined by immunohistochemical staining of formalin-fixed, paraffin-embedded tumor tissue. Patient survival was the clinical end point. The effects of COX-2 expression on cell survival and radioresistance was tested in vitro using the selective COX-2 inhibitor NS-398 in conjunction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) and clonogenic assays. RESULTS: COX-2 immunoreactivity was detected in 62% of NPC tumors, and expression levels were high in 43%. Survival analysis showed the 5-year overall survival rates for patients who had high COX-2 expression was 27% compared with 60% for those with low/absent expression (p = .047). Pattern of failure analysis showed no significant difference between high and low COX-2 expression in locoregional failure (27% vs 25%, p = .91). However, patients with N0 to N1 disease and high COX-2 expression had a significantly higher incidence of distant metastasis compared with patients with stage N0 to N1 disease and low COX-2 expression (83% vs 15%, p = .004). This difference was not observed in patients with N2 to N3 disease. This difference contributed to worse survival of patients whose tumors had high COX-2 expression levels. The selective COX-2 inhibitor NS-398 was directly cytotoxic to NPC-BM1 cells in vitro, as judged in an MTT assay (viable cells decreased from 92% to 76%, 52%, and 22%, with increases of NS-398 from 20 to 40, 60, and 80 microM, respectively). Radiation-induced cell death was also increased by treatment with NS-398. At a 10% survival level, 40 microM NS-398 increased radiation cytotoxicity by a factor of 1.37, whereas 60 microM increased it by a factor of 4.9. CONCLUSIONS: COX-2 overexpression is a predictor for poor survival for advanced stage NPC. In vitro, NS-398 radiosensitizes the NPC-BM1 cell line, providing a basis for testing the combination of COX-2 inhibitors with radiation in the treatment of patients with NPC.     

Transforming growth factor beta1, urokinase-type plasminogen activator and plasminogen activator inhibitor-1 mRNA expression in head and neck squamous carcinoma and normal adjacent mucosa

BACKGROUND: A balance between urokinase-type plasminogen activator (uPA) and its main inhibitor type-1 (PAI-1) appears to be important for cancer invasive behavior. Since uPA/PAI-1 system seems to be regulated by transforming growth factor beta1 (TGFbeta1) in different cell types, our aim was to investigate the relationship between the expression of the three genes and lymph node status in head and neck squamous cell carcinomas (HNSCC) at specific sites. MATERIALS AND METHODS: uPA, PAI-1, and TGFbeta1 mRNAs were determined by Northern analysis in tumor, and paired normal mucosa samples were obtained from 91 operable HNSCC patients. RESULTS: In oral cavity, excluding tongue, TGFbeta1, PAI-1, and uPA mRNAs values were consistently lower in the normal tissues than in tumors. In larynx tumors, TGFbeta1 expression was increased, but no statistically significant differences were found for uPA or PAI-1 mRNAs as compared with normal tissues. Tongue tumors overexpressed only uPA mRNA, and uPA levels showed significant parallel variations with TGFbeta1 and PAI-1 mRNAs mainly in pN+ tumors. In oral cavity tumors, an inverse correlation between TGFbeta1 and uPA was observed in pN0 subgroup, elevated uPA mRNA was counterbalanced by high PAI-1 mRNA TGFbeta1, and PAI-1 were not coordinately expressed. Correlations between the three markers were not found in larynx. Hypopharynx tumors, all staged as pN+, expressed the lowest TGFbeta1 mRNA mean values. CONCLUSIONS: Combined information about TGFbeta1, uPA, and PAI-1 mRNAs may add some clues to the understanding of the pathophysiological role of uPA system in head and neck squamous cell carcinoma.     

尼美舒利对二甲基苯并蒽诱导的乳腺癌的影响及机制的实验研究

目的探讨选择性环氧化酶-2(COX-2)抑制剂尼美舒利(NIM)对二甲基苯并蒽(DMBA)诱导的大鼠乳腺癌的影响及其作用机制。方法将76只大鼠随机分为致癌组(30只)、NIM组(30只)、饮食对照组(8只)及NIM药物对照组(8只),观察乳腺肿瘤诱发率。通过逆转录聚合酶链反应方法、蛋白印迹法测定每组肿瘤组织中COX-2mRNA含量及其蛋白表达水平;放射免疫法测定各组大鼠血浆、肿瘤组织中前列腺素E2(PGE2)含量;TUNEL法、增殖细胞核抗原分别检测凋亡和增殖指数。结果NIM组(40.3%)大鼠乳腺肿瘤发生率明显低于致癌组(69.2%);NIM组COX-2mRNA、蛋白水平较致癌组明显下调[A值:(0.21±0.05)vs.(0.46±0.12),P<0.05;(30.26±8.75)vs.(58.13±10.02),P<0.05],血浆、肿瘤组织中PGE2的含量显著降低[(233±59)pg/mlvs.(452±82)pg/ml,P<0.01;(167±42)pg/mg蛋白vs.(250±67)pg/mg蛋白,P<0.05];NIM组肿瘤细胞的增殖指数与致癌组相比下降[(20±5)vs.(36±5),P<0.01],凋亡指数明显增高[(43±13)vs.(18±7),P<0.01],差异有统计学意义。结论NIM通过下调COX-2的表达,抑制前列腺素的合成,抑制肿瘤细胞的增殖、诱导凋亡,降低DMBA诱发大鼠乳腺癌的发生率。     

Inverse correlation between serum PGE2 and T classification in head and neck cancer

BACKGROUND: Prostaglandin E2 (PGE2) serum levels have been shown previously to be increased in tumor bearing mice as well as in patients with solid tumors; however, the impact on the course or stage of disease has not been shown. We hypothesized that PGE2 is strictly required for aggressive and especially early-stage tumors of the head and neck to provoke invasion and angiogenesis. METHODS: We analyzed the serum PGE2 levels of 100 patients with head and neck squamous cell carcinoma of different stages before and 1 year after treatment and compared the results with the serum levels of 40 healthy donors and the secretion profile of 8 different squamous cell carcinoma cell lines. RESULTS: Our investigations showed a statistically significant inverse correlation between PGE2 serum levels and tumor stage. Furthermore, this effect has been reflected by the results of our cell culture analyses, which showed an inversely regulated PGE2 secretion into the medium during the process of proliferation. Interestingly, the serum levels of PGE2 were significantly downregulated 1 year after successful treatment. CONCLUSIONS: We conclude that PGE2 serum level as an indicator for early-stage cancer of the head and neck may function as a tumor marker during the follow-up period.     

Treatment of lung cancer using clinically relevant oral doses of the cyclooxygenase-2 inhibitor rofecoxib: potential value as adjuvant therapy after surgery

OBJECTIVE: To investigate the uses and limitations of cyclooxygenase- (COX) 2 inhibition using clinically relevant doses of oral rofecoxib in the treatment of murine models of non-small-cell lung cancer (NSCLC). SUMMARY BACKGROUND DATA: Overexpression of COX-2 has been reported in lung cancer. Several studies have demonstrated that high doses of COX-2 inhibitors could inhibit the growth of rodent and human lung cancer cell lines. The potential uses and limitations of COX-2 inhibition at doses equivalent to those currently approved for use in humans have not been well studied. METHODS: Three murine NSCLC cell lines were injected into the flanks of mice to establish tumor xenografts. Mice were treated orally with low doses of a COX-2 inhibitor (rofecoxib chow, 0.0075%). Mechanisms were evaluated by analysis of tumor-infiltrating lymphocytes. To study rofecoxib as adjuvant therapy, large established tumors (14-18 days after tumor inoculation) were surgically debulked and animals were treated with rofecoxib starting 3 days before surgery. Recurrence of the tumor after debulking was monitored. RESULTS: Rofecoxib significantly slowed the growth of small (0-120 mm) tumors (P < 0.01-0.05) in all 3 cell lines, with higher efficacy in the more immunogenic tumors. Minimal responses were noted in larger tumors. Rofecoxib appeared to augment CD8 T cell infiltration in immunogenic tumors. Rofecoxib significantly reduced the recurrence rate after debulking (P < 0.01). CONCLUSIONS: Clinically relevant doses of the COX-2 inhibitor rofecoxib given orally were effective in inhibiting the growth of small (but not large) tumors in 3 murine NSCLC cell lines tested and in preventing recurrences after surgical debulking. Depending on the immunogenicity of human tumors, COX-2 inhibition might be useful as adjuvant therapy for surgically resectable NSCLC.     

C-erb-B2 (HER2/neu) expression in synovial sarcoma of the head and neck

BACKGROUND: Synovial sarcoma is a malignant mesenchymal tumor composed of varying proportions of spindle and epithelial cell components. Because of the histologic and immunohistochemical similarity of synovial sarcoma to epithelial carcinomas, we hypothesized that the human epithelial growth factor receptor 2 (C-erb-B2, also termed HER2/neu) may contribute to the tumor phenotype and provide a new therapeutic target for this soft tissue tumor. METHODS: Three head and neck, one chest wall, and seven extremity synovial sarcomas were evaluated for C-erb-B2 (HER2/neu) expression by immunohistochemistry, Western immunoblotting, and fluorescence in situ hybridization (FISH). RESULTS: The head and neck cases demonstrated immunohistochemically strong positive staining, whereas tumors from other anatomic locations showed neither positive nor cytoplasmic restricted staining. Antigen-targeted antibody therapy (trastuzumab) was initiated in two patients. CONCLUSIONS: These results demonstrate that C-erb-B2 (HER2/neu) may play a role in the tumorigenesis of synovial sarcoma; and, therefore, antigrowth factor therapies may provide a previously unrecognized pharmaceutical approach to soft tissue tumors. The data also suggest that although synovial sarcoma of the head and neck and synovial sarcoma of the extremities have similar morphologic features, they may be clinically and mechanistically distinct entities.     

胃癌组织中E26转录因子-1表达与血管生成的相关性研究

目的研究胃癌组织中E26转录因子-1(Ets-1)表达与血管生成的关系。方法应用免疫组织化学方法(SP法)检测86例胃癌标本中Ets-1的表达及微血管密度(MVD);建立裸鼠胃癌皮下种植瘤模型,分别应用Ets-1反义,正义寡核苷酸与PBS行瘤内注射,应用逆转录聚合酶链式反应检测瘤组织Ets-1mRNA的变化,并检测瘤组织Ets-1蛋白及微血管密度的差异。结果胃癌组织中Ets-1标记指数(LI)和MVD呈正相关(γ=0.495,P<0.05);经Ets-1反义寡核苷酸治疗后,裸鼠瘤组织Ets-1mRNA及Ets-1蛋白表达降低,反义组MVD显著低于正义组与PBS组(10.4±3.1VS24.5±8.4,20.6±7.5,P<0.01)。结论胃癌组织中Ets-1在促进血管生成中起重要作用。     

Involvement of IL-8 in COX-2-mediated bone metastases from breast cancer

BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression by a primary tumor correlates with poor prognosis in breast cancer, including early spread to bone. Interleukin-8 (IL-8) stimulates osteoclastogenesis and resorption of bone, and elevated IL-8 levels predict early metastatic spread of breast cancer. The purpose of this study was to test our hypothesis that tumors that overexpress COX-2 induce IL-8 production. MATERIALS AND METHODS: We cotransfected MCF-10A (nonmalignant breast epithelial) cells, as well as MDA-231 (highly metastatic human breast cancer) cell lines with a pSG5-COX-2 vector and pEF1a-Luc-IRES-Neo vector (luciferase reporter). COX-2 overexpression was confirmed by Western blot and PGE2 (a product of the COX-2 pathway) immunoassay. IL-8 production was measured by immunoassay. In vivo testing used a nude mouse model to measure COX-2 and IL-8 production from breast cancer cells that had metastasized to bone (bone-seeking clones (BSCs)). Long bone metastases were localized and quantified by luciferase imaging (Xenogen IVIS system) and X-ray. BSCs were isolated and cultured and then tested for the production of PGE2 and IL-8. RESULTS: COX-2 overexpression caused a 4- to 5-fold increase in IL-8 production in both MCF-10A and MDA-231 cells in vitro. In vivo, we observed that the MDA-231-BSC (metastatic cells isolated from bone metastases) produced significantly greater levels of both PGE2 and IL-8 compared to the parental MDA-231 cells (P < 0.01). In contrast to the results obtained with these estrogen receptor-negative cell lines, COX-2 expression failed to induce IL-8 in the MCF-7 estrogen receptor-positive breast cancer cell line. Treatment with the COX-2 inhibitor NS-398 at a low 1-mu[scap]M dose reduced the production of IL-8 in COX-2-transfected MDA-231 cells by 30%, thus confirming the involvement of COX-2 in IL-8 induction. CONCLUSION: COX-2 expression induced formation of PGE2 and IL-8 in breast cancer cells. Since PGE2 and IL-8 stimulate osteoclasts to resorb bone, COX-2 inhibition is a potential target for treatment to prevent bone metastases.     

环氧合酶-2对乳腺癌淋巴管形成的影响

目的探讨环氧合酶-2（COX-2）对人乳腺癌淋巴管形成的影响。方法1998年11月至2002年3月行手术治疗并经病理证实的乳腺癌患者94例,随访83例,失访11例。免疫组织化学方法检测COX-2、血管内皮生长因子-C（VEGF—C）及D2-40在乳腺癌中的表达。逆转录聚合酶链式反应（RT-PCR）和Western印迹法分别检测加入不同浓度COX-2抑制剂尼美舒利后VEGF-CmRNA及蛋白的变化。Western印迹法检测加入外源性前列腺素E2（PGE2）和曲妥珠单抗后VEGF—C蛋白的变化。结果46．8％的乳腺癌组织呈COX-2高表达,51．1％呈VEGF—C高表达。COX-2与VEGF—C（P〈0．01）、淋巴管密度（P=0．032）和淋巴结转移（P＝0．035）呈正相关,与乳腺癌患者的无病生存率（P=0．010）和总生存率（P＝0．040）呈负相关。尼美舒利以剂量依赖性方式下调VEGF-CmRNA及蛋白表达,而PGE2上调其蛋白表达。曲妥珠单抗可显著降低VEGF—C的蛋白表达。结论COX-2可通过上调VEGF—C的表达,促进乳腺癌的淋巴管形成和淋巴结转移,并影响乳腺癌患者的预后。     

Combined evaluation of expression of telomerase, survivin, and anti-apoptotic Bcl-2 family members in relation to loss of differentiation and apoptosis in human head and neck cancers

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers, and it accounts for 5% of all adult cancers worldwide. Loss of growth control and a marked resistance to apoptosis are considered major mechanisms driving tumor progression. Little is known about the distribution of inhibitors of apoptosis in HNSCC or how they correlate with other biomarkers of malignancy, such as telomerase, an enzyme that plays a critical role in cellular immortalization. The objective of this study was to assess the protein expression of anti-apoptotic members of Bcl-2 family and survivin and correlate them with telomerase activity. METHODS: We compared anti-apoptotic protein expression in tumor tissue sections of 50 HNSCC patients and 19 histopathologically normal tissues by immunohistochemistry and Western blotting. Apoptotic index was studied by TUNEL assay. Telomerase activity was determined by polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA). RESULTS: Bcl-2, Bcl-XL, and survivin were found to be significantly upregulated in tumor tissues as compared with the normal tissue. Protein expression of Bcl-2 and survivin was significantly associated with the loss of differentiation in tumors and that of Bcl-XL with nodal metastasis. Telomerase activity was found to be upregulated in tumors as compared with the normal tissue (p <.001) and was found to be significantly associated with the loss of differentiation in tumors. CONCLUSIONS: Mechanisms that promote both cell proliferation (telomerase activity) and cell survival (expression of inhibitors of apoptosis protein [IAPs]) appear to be activated in HNSCC. This is the first study of its kind to look into the correlation of the apoptotic pathway and proliferation promoting enzyme activity simultaneously in relation to loss of apoptosis and differentiation in HNSCCs. Telomerase activity in these tumors was found to be correlated with Bcl-2, Bcl-XL, and survivin overexpression and with reduced apoptosis, thereby suggesting that novel strategies can be developed to control cancer cell growth by co-targeting telomerase and apoptotic pathways.     

乳腺癌细胞凋亡及侵袭转移与诱导型一氧化氮合酶、抑癌基因PTEN及环氧合酶-2表达的关系

目的 探讨乳腺癌组织中诱导型一氧化氮合酶(iNOS)、抑癌基因PTEN和环氧合酶-2(COX-2)的表达与肿瘤细胞凋亡、侵袭转移的关系.方法 收集乳腺浸润性导管癌组织90例和乳腺良性病变组织30例,分别应用原位杂交法和免疫组织化学法检测组织中iNOS、PTEN和COX-2的mRNA及蛋白表达,用TdT介导的dUTP缺口末端标记(TUNEL)法检测细胞凋亡.结果 在90例乳腺癌中iNOS、PTEN、COX-2的mRNA和蛋白表达率分别为66.7%、68.9%、51.1%和70.0%、65.6%、48.9%,凋亡指数为(4.98±0.57)%,与良性对照组比较差异均有统计学意义(P<0.01).iNOS表达与乳腺癌肿瘤大小、分期呈正相关,与凋亡指数负相关(P<0.05);PTEN表达与淋巴结转移、分期呈负相关,与凋亡指数正相关(P<0.05);COX-2表达与肿瘤分级、淋巴结转移、分期呈正相关(P<0.05).PTEN表达与iNOS表达呈负相关(P<0.05),与COX-2表达差异无统计学意义(P>0.05).结论 iNOS、PTEN、COX-2的异常表达在乳腺癌的凋亡和侵袭转移中具有重要作用,PTEN的转录表达是iNOS促进乳腺癌进展的拮抗因素.