EFFECTS OF p53 GENE THERAPY COMBINED WITH CYCLOOXYGENASE-2 INHIBITOR ON CYCLOOXYGENASE-2 GENE EXPRESSION AND GROWTH INHIBITION OF HUMAN LUNG CANCER CELLS

1 INTRODUCTIONCOXis the key enzyme involved in the synthesis of prostanoids,a collective termfor the PGs andthromboxanes.Of the two major isoforms of the COXenzyme,COX-1 is ubiquitous and constitutively ex-pressedin virtually all normal tissues.In contras…     

Cyclooxygenase-2 dependent and independent antitumor effects induced by celecoxib in urinary bladder cancer cells

Transitional cell carcinoma of the urinary bladder is the second most common genitourinary malignancy in people in the United States. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer. COX-2 inhibitors have had antitumor activity against bladder cancer, but the mechanisms of action are unclear. Clinically relevant concentrations of COX-2 inhibitors fail to inhibit proliferation in standard in vitro assays. In pilot experiments, different culture conditions [standard monolayer, modified monolayer, soft agar, collagen, and poly(2-hydroxyethyl methacrylate)-coated plates] were assessed to determine conditions suitable for the study of COX inhibitor growth-inhibitory effects. This was followed by studies of the effects of clinically relevant concentrations of a selective COX-2 inhibitor (celecoxib) on urinary bladder cancer cell lines (HT1376, TCCSUP, and UMUC3). Celecoxib (相似文献   

Selective inhibition of cyclooxygenase-2 suppresses the growth of pancreatic cancer cells in vitro and in vivo

Cyclooxygenase-2 (COX-2), a prostaglandin synthetase, is involved in development of certain tumors. We therefore analyzed COX-2 expression in pancreatic cancer tissues (53 samples) and Panc-1 human pancreatic cancer cells by immunohistochemistry, RT-PCR and western-blotting analyses. Also, immunohistochemistry of proliferating cell nuclear antigen (PCNA) was performed. We found expression of COX-2 was dramatically upregulated in 36 of 53 cases (67.9%) and the expression of COX-2 was associated with the diameter (> 3 cm) of the tumors (p < 0.05), but not with the age, gender, tumor location, differentiation, lymph-node metastases and TNM stage. The positivity rate of PCNA expression in the pancreatic cancer cells of the COX-2 positive group (32.88 +/- 13.26%) was significantly higher than that in the COX-2 negative group (24.56 +/- 11.51%) (p < 0.05). Then we investigated the effect of selective inhibitors of COX-2 (NS398 and celecoxib) on proliferation of Panc-1 cells by 3-(4,5 dimethyl-2-thiazolyl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) assay. Either NS398 or celecoxib suppressed proliferation of Panc-1 cells dose-dependently in vitro. Furthermore, Panc-1 cells were implanted into nude mice, and celecoxib was administrated orally with feed. The volume of the tumor xenografted into nude mice was decreased by 51.6% in the celecoxib group (p < 0.01). In conclusion, the increased expression of COX-2 may be responsible for rapid proliferation of pancreatic cancer, and specific inhibition of COX-2 suppresses proliferation of Panc-1 cells in vitro and in nude mice. The selective inhibitor of COX-2 may be an effectual agent for pancreatic cancer chemoprevention.     

Acetaminophen selectively suppresses peripheral prostaglandin E2 release and increases COX-2 gene expression in a clinical model of acute inflammation

Acetaminophen is widely used for pain management as an alternative to NSAIDs and selective COX-2 inhibitors, but its action at a molecular level is still unclear. We evaluated acetaminophen’s effect on PG release and the expression patterns of genes related to PG production in a clinical model of tissue injury and acute inflammation. Subjects (119 outpatients) received either 1000 mg acetaminophen, 50 mg rofecoxib (a selective COX-2 inhibitor), 30 mg ketorolac (a dual COX-1/COX-2 inhibitor), or placebo before the surgical removal of two impacted mandibular third molars. Microdialysis was used to collect inflammatory transudate from the surgical site for measurement of PGE₂ and TXB₂ levels at the site of injury. Biopsies were collected to investigate the expression patterns of genes related to PG production at baseline prior to surgery and at 3 or 24 h following surgery. PGE₂ release was suppressed by ketorolac, rofecoxib and acetaminophen compared to placebo at 3 h coincident with increased COX-2 gene expression in biopsies collected from the surgical site. TXB₂ release was suppressed only by ketorolac. COX-2 gene expression remained elevated at 24 h with continued ketorolac and acetaminophen treatment. COX-1 gene expression was significantly down-regulated at 24 h by ketorolac, rofecoxib and acetaminophen. Acetaminophen suppression of PGE₂ without inhibiting TXB₂ release, when COX-2 gene expression is up-regulated, suggests that acetaminophen is a selective COX-2 inhibitor in vivo. The up-regulation of COX-2 gene and down-regulation of COX-1 gene expression suggests that acetaminophen may result in changes in COX-derived prostanoids with repeated doses.     

环氧合酶2抑制剂塞来昔布对结肠癌多药耐药逆转作用的研究

目的 探讨环氧合酶2(COX-2)抑制剂塞来昔布对结肠癌多药耐药(MDR)的逆转作用.方法 在结肠癌多药耐药细胞株colo320/5-FU中,应用四甲基偶氮唑蓝(MTT)方法检测塞来昔布时5-氟尿嘧啶(5-FU)的耐药逆转倍数;反转录-聚合酶链反应(RT-PCR)方法检测耐药基因的变化;免疫组织化学法检测细胞膜跨膜蛋白p-170(P-gP)的表达.结果 MTT结果显示塞来昔布对结肠癌细胞的耐药产生明显的逆转作用(F=2285.660,P＜0.01),其逆转倍数分别为1.70,3.80,8.48,且呈浓度依赖性.RT-PCR结果显示塞来昔布明显抑制多药耐药基因1(MDR1)mRNA的表达(F=986.776,P＜0.01),且呈浓度依赖性减低,各浓度组之间差异有统计学意义(P＜0.01).免疫荧光结果显示,细胞膜P-gp荧光强度随着塞来昔布浓度的逐渐增高而减弱.结论 选择性COX-2抑制剂塞来昔布对多药耐药细胞株colo320/5-FU具有明显逆转作用,在非细胞毒剂量范围内呈现剂量依赖性,初步机制可能涉及干预多种转录因子的表达而下调MDR1,继而阻滞细胞周期及增加细胞凋亡.     

构建人 COX-2反义核酸真核表达载体对膀胱癌 T24细胞株COX-2表达的作用

【目的】构建人环氧化酶-2（COX-2）反义核酸真核表达载体,探讨其抑制膀胱癌 T24细胞COX-2表达的作用。【方法】经PCR法从含人COX-2基因的质粒中扩增其cDNA ,通过限制性内切酶克隆于 T载体EcoR Ⅰ和Xba Ⅰ位点之间,并将其反向插入pcDNA3．1中获得重组质粒。应用脂质体介导将其导入T24膀胱癌细胞中,经过G418筛后进行流式细胞术、间接免疫荧光染色和RT-PCR鉴定。【结果】核酸测序和限制性内切酶消化证实重组COX-2反义核酸真核表达质粒 pcDNA3．1/COX-2 as是正确的,经鉴定转导 pcD-NA3/COX-2 as的T24细胞中环氧化酶-2的表达明显抑制。【结论】成功构建人COX-2反义核酸真核表达载体并获得环氧化酶-2基因表达持续下调的膀胱癌细胞株T24-AS。     

The cancer pain related factors affected by celecoxib together with cetuximab in head and neck squamous cell carcinoma

Pain is the most disruptive influence on the quality of prognosis among head and neck squamous cell carcinoma (HNSCC) patients. The development of pain is closely associated with tumor growth and inflammation in the cancer patients. Notably, cyclooxygenase-2 (COX-2) is an important mediator during inflammation. Celecoxib, a selective inhibitor of COX-2, was hailed as a promising chemopreventive agent for HNSCC. Dose-dependent cardiac toxicity limits long-term use of celecoxib. However, the toxicity can be diminished by lowering the dosage. In this study, we hypothesized that a combinatory strategy to reduce cancer pain via two distinct pathways, tumor grown inhibition and inflammation blockade, which would enhance analgesia effect induced by HNSCC. We found that treatment of cetuximab (C225), a monoclonal anti-epidermal growth factor receptor (EGFR) antibody, with low-dose celecoxib results in a more pronounced anticancer effect in HNSCC than either agent alone. More noticeably, the combination could downregulate the phosphorylation of constitutively active extracellular signal regulated kinase (ERK) in CAL-27 and Fadu cells. Furthermore, combination therapy enhancing S phase arrest and downregulating cyclin D1 was observed in Fadu cells. The COX-2 expression was significantly blocked by celecoxib combined with C225, and other cancer pain related factors, such as ET-1 and NGF, was also downregulated by combination treatment. Taken together, these results strongly suggest that combination of celecoxib with C225 holds potential as a new therapy strategy in developing cancer pain treatment in HNSCC.     

Synergistic anticancer effects of combined γ-tocotrienol and celecoxib treatment are associated with suppression in Akt and NFκB signaling

The selective cyclooxygenase (COX)-2 inhibitor, celecoxib, and the vitamin E isoform, γ-tocotrienol, both display potent anticancer activity. However, high dose clinical use of selective COX-2 inhibitors has been limited by gastrointestinal and cardiovascular toxicity, whereas limited absorption and transport of γ-tocotrienol by the body has made it difficult to obtain and sustain therapeutic levels in the blood and target tissues. Studies were conducted to characterize the synergistic anticancer antiproliferative effects of combined low dose celecoxib and γ-tocotrienol treatment on mammary tumor cells in culture. The highly malignant mouse +SA mammary epithelial cells were maintained in culture on serum-free defined control or treatment media. Treatment effects on COX-1, COX-2, Akt, NFκB and prostaglandin E₂ (PGE₂) synthesis were assessed following a 3- or 4-day culture period. Treatment with 3–4 μM γ-tocotrienol or 7.5–10 μM celecoxib alone significantly inhibited +SA cell growth in a dose-responsive manner. However, combined treatment with subeffective doses of γ-tocotrienol (0.25 μM) and celecoxib (2.5 μM) resulted in a synergistic antiproliferative effect, as determined by isobologram analysis, and this growth inhibitory effect was associated with a reduction in PGE₂ synthesis, and decrease in COX-2, phospho-Akt (active), and phospho-NFκB (active) levels. These results demonstrate that the synergistic anticancer effects of combined celecoxib and γ-tocotrienol therapy are mediated by COX-2 dependent and independent mechanisms. These findings also suggest that combination therapy with these agents may provide enhanced therapeutic response in breast cancer patients, while avoiding the toxicity associated with high-dose COX-2 inhibitor monotherapy.     

吲哚美辛对喉癌细胞Hep-2增殖与凋亡及COX-2蛋白表达的影响

目的探索吲哚美辛对喉癌细胞增殖与凋亡的作用以及对细胞COX-2蛋白表达的影响。方法Hep-2细胞暴露于含吲哚美辛（125，250，500μM）的培养基中，培养24小时，用MTT方法检测各组药物与溶剂对照组相比对细胞增殖的影响；用台盼蓝染色法检测吲哚美辛对细胞活力的影响；吖啶橙染色法观察吲哚美辛（250μM）对细胞形态的影响；流式细胞仪分析法检测吲哚美辛（250μM）对细胞周期和细胞凋亡的影响，Western blot检测吲哚美辛对细胞COX-2蛋白表达的影响。结果吲哚美辛使Hep-2细胞增殖及细胞活力明显降低，细胞形态发生明显变化，细胞质萎缩而细胞核相对较大，S期细胞分数明显增加，细胞出现凋亡峰，而COX-2蛋白表达量增加。结论吲哚美辛强烈抑制Hep-2细胞增殖与细胞活力，明显改变细胞初始形态，阻滞细胞从S期向G2／M期转化，并诱导其凋亡，吲哚美辛对Hep-2细胞的上述作用机制与COX-2的特异性抑制途径无关。     

膀胱癌中COX-2、Survivin的表达及临床意义

目的检测COX-2和Survivin在膀胱癌中的表达,分析它们与不同临床病理因素及预后的关系。方法应用免疫组织化学方法对手术切除的56例膀胱癌COX-2,Survivin蛋白表达进行检测。结果膀胱癌中COX-2、Survivin蛋白阳性表达率分别为55.3%,62.5%,随着肿瘤分级、分期的升高,COX-2、Survivin表达阳性率增加(P<0.05),差别有显著性(P<0.05)。经Spearman等级相关性分析表明,COX-2表达水平与Survivin表达水平之间成正相关关系(rs=0.324,P=0.001)。Kaplan-Meier生存分析表明COX-2,Survivin表达阳性组的生存时间明显低于阴性组,二者差异显著。结论COX-2和Survivin可作为膀胱癌早期诊断、治疗和判断预后的新靶点。     

COX-2和HER-2在非小细胞肺癌中的表达及临床意义

目的研究COX-2和HER-2在非小细胞肺癌(NSCLC)组织中的表达及其临床意义。方法采用免疫组织化学法检测了68例非小细胞肺癌组织中COX-2和HER-2的表达水平,分析两者与临床病理参数之间的关系以及分析两者之间的相互关系。结果非小细胞肺癌中COX-2阳性表达率为54/68(79.41%),COX-2的高表达与病理类型及TNM分期相关(P<0.05);HER-2阳性表达率为26/68(38.24%),HER-2的表达与年龄相关(P<0.05)。HER-2与COX-2的表达强度之间呈显著正相关(r=0.2486,P=0.041)。HER-2阳性/COX-2高表达的NSCLC临床分期晚、淋巴结转移率高。结论COX-2在NSCLC中高水平表达并与HER-2密切相关,检测COX-2与HER-2的表达对NSCLC预后的判断可能具有重要价值。     

TPX2 siRNA regulates growth and invasion of esophageal cancer cells

Purpose

Observe how specific small RNA interference (siRNA) aimed at TPX2 gene suppresses TPX2 gene expression in esophageal cancer EC9706 cells and the effect on esophageal cancer cell growth and invasion ability.

Methods

Transfect TPX2 siRNA into EC9706 cells via lipofectamin 2000. The experiments were divided into three groups, a negative control, a blank control and an siRNA interference group (24 h, 48 h, 72 h, 96 h). We examined RNA and protein level alteration of the TPX2 gene after TPX2 siRNA transfection by RT-PCR and Western blot analysis. Detection of how TPX2 siRNA influences EC9706 cell proliferation was done by MTT, cell apoptosis monitored through Tunel assay, in vitro invasion ability via Boyden chamber and cell cycle change by flow cytometry.

Results

After effective siRNA transfection, TPX2 mRNA and protein expression level in siRNA interference group were (0.31 ± 0.08, 0.39 ± 0.12),72 h after transfection, significantly lower than blank control group (1.00 ± 0.01) and negative control group (0.98 ± 0.11), (F = 71.182, t1 = 8.17, t2 = 7.90, P < 0.05); MTT results demonstrated that cell growth and proliferation were inhibited and the inhibition rate was up to 35.4% (P < 0.05) compared with the control group. TUNEL results indicated that cell apoptosis index in siRNA interference group was 18.28 ± 0.35, higher than that in blank control group (4.07 ± 0.26)and negative control group (4.13 ± 0.22), (F = 244.5, t1 = 60.61, t2 = 53.32, P < 0.01). Boyden chamber results showed that the transmembrane cell number was 45.30 ± 8.08 in siRNA interference group, less than blank control group (121.90 ± 7.83), (F = 122.46, t1 = 11.81, t2 = 10.47, P < 0.01); besides, in siRNA interference group cell invasion inhibition rate was 71.42 ± 9.12, higher than negative control group (5.65 ± 3.55), (t = 14.256, P < 0.01). Flow cytometry results illustrated that more EC9706 cells went into apoptosis and cell cycle arrested in S phase. Similar results were obtained by in vivo transplantation, as TPX2 siRNA transfection significantly reduced tumor growth of the xenograft in nude mice.

Conclusion

siRNA could effectively inhibit the invasion and metastasis of EC9706 cells, promote the apoptosis of tumor cells and may become a new approach for treatment of esophageal carcinoma.     

Survivin蛋白在卵巢上皮性癌中的表达及其与COX-2相关性的研究

目的探讨卵巢上皮性癌组织中Survivin的表达与环氧合酶2(COX-2)表达的相关性。方法采用免疫组化Elivision染色法检测Survivin、COX-2蛋白在60例卵巢上皮性癌组织、20例正常卵巢、20例良性卵巢上皮性肿瘤及20例交界性卵巢上皮性肿瘤中的表达。结果Survivin蛋白在正常卵巢组织、良性卵巢上皮性肿瘤组织、交界性卵巢上皮性肿瘤组织及卵巢上皮性癌组织中的阳性率分别为0%、20.0%、65.0%、71.7%,差异均有显著性(P<0.05)。COX-2蛋白在正常卵巢组织、良性卵巢上皮性肿瘤组织、交界性卵巢上皮性肿瘤组织及卵巢上皮性癌组织中的阳性率分别为0%、0%、55.0%、65.0%,差异均有显著性(P<0.05)。卵巢上皮性癌中,Survivin蛋白表达与COX-2蛋白表达呈显著正相关(P<0.001),列联系数0.978,两者有密切相关。结论Survivin、COX-2蛋白均在卵巢上皮性癌中高表达,与卵巢上皮性癌的发生、发展密切相关,Survivin蛋白表达与COX-2蛋白表达有密切相关。两者可能存在共同的激活机制,构成抑制卵巢上皮性癌细胞凋亡的多种途径。     

曲列格酮对Hep G2肝癌细胞增殖、COX-2表达和前列腺素E2水平的影响

目的　探讨曲列格酮 (TGZ)对HepG2肝癌细胞增殖的影响。方法　TGZ处理HepG2细胞后 ,用MTT、[3 H]-胸苷摄入、Northern杂交、RT -PCR、Western印迹等方法分别测定细胞增殖、COX - 2表达及PGE2水平。结果　TGZ以剂量依赖方式抑制细胞增殖 ,并下凋COX - 2表达及抑制PGE2产生。结论　TGZ抑制HepG2肝癌细胞增殖 ,这种抑制作用可能与COX - 2表达减少有关。     

The cyclooxygenase-2 inhibitor celecoxib blocks phosphorylation of Akt and induces apoptosis in human cholangiocarcinoma cells

The expression of cyclooxygenase (COX)-2 is increased in human cancers including cholangiocarcinoma. This study was designed to evaluate the effect and mechanisms of the selective COX-2 inhibitor celecoxib in the growth control of human cholangiocarcinoma cells. Immunohistochemical analysis using human cholangiocarcinoma tissues showed increased levels of COX-2 as well as phospho-Akt (Thr (308)), a protein kinase activated by COX-2-mediated prostaglandins, in human cholangiocarcinoma cells. Treatment of cultured human cholangiocarcinoma cells (HuCCT1, SG231, and CCLP1) with celecoxib resulted in a dose- and time-dependent reduction of cell viability. Fluorescence microscopy, Western blot, and caspase activity assays demonstrated that celecoxib induced morphological features of apoptosis, activation of caspase-9 and caspase-3, and release of cytochrome c. The celecoxib-induced cell death was significantly blocked by N-benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone, a wide-spectrum caspase inhibitor. Furthermore, cholangiocarcinoma cells treated with celecoxib showed significant reduction of Akt phosphorylation, whereas the levels of Bcl-2 and Bax were not altered. Inhibition of Akt activation by LY294002 significantly decreased the viability of human cholangiocarcinoma cells. These findings suggest that celecoxib inhibits cholangiocarcinoma growth partly through induction of apoptosis and inhibition of Akt phosphorylation.     

肺癌患者血清环氧化酶-2的水平及临床意义

目的探讨环氧化酶-2（COX-2）在肺癌患者血清中的表达情况以及病理类型、临床分期、预后关系。方法用ELISA方法检测56例肺癌患者血清COX-2水平,比较不同患者血清COX-2水平差异。结果血清COX-2水平在非小细胞肺癌患者明显高于小细胞肺癌患者,Ⅲ＋Ⅳ期患者高于Ⅰ＋Ⅱ期患者,有淋巴结转移者高于无淋巴结转移者。结论血清COX-2水平可用于非小细胞肺癌病理类型、分期、转移、预后的评估。     

乳腺癌环氧合酶-2和基质金属蛋白酶-2及其抑制因子的表达

目的 研究环氧合酶(COX)-2、基质金属蛋白酶(MMP)-2及其抑制因子(TIMP-2)在乳腺癌组织中的蛋白表达及其相互关系.方法 建立组织芯片平台,应用免疫组织化学S-P法检测127例乳腺癌组织COX-2、MMP-2和TIMP-2蛋白的表达情况.结果 乳腺癌COX-2、MMP-2和TIMP-2阳性率分别为81.1%(103/127)、96.9%(123/127)和60.6%(77/127);COX-2的表达与乳腺癌腋淋巴结转移和TNM分期均呈正相关(P<0.01,P<0.05),与孕激素受体表达呈负相关(P<0.05);MMP-2蛋白表达与COX-2表达呈显著正相关(r=0.290,P<0.01).结论 乳腺癌COX-2表达状况与肿瘤侵袭转移有密切关系,COX-2可能通过调控MMP-2表达来促进肿瘤侵袭转移.