Gene modulation by the cyclooxygenase inhibitor,sulindac sulfide,in human colorectal carcinoma cells: possible link to apoptosis

The mechanisms underlying the anti-tumorigenic properties of cyclooxygenase inhibitors are not well understood. One novel hypothesis is alterations in gene expression. To test this hypothesis sulindac sulfide, which is used to treat familial adenomatous polyposis, was selected to detect gene modulation in human colorectal cells at physiological concentrations with microarray analysis. At micromolar concentrations, sulindac sulfide stimulated apoptosis and inhibited the growth of colorectal cancer cells on soft agar. Sulindac sulfide (10 microm) altered the expression of 65 genes in SW-480 colorectal cancer cells, which express cyclooxygenase-1 but little cyclooxygenase-2. A more detailed study of 11 genes revealed that their expression was altered in a time- and dose-dependent manner as measured by real-time RT-PCR. Northern analysis confirmed the expression of 9 of these genes, and Western analysis supported the conclusion that sulindac sulfide altered the expression of these proteins. Cyclooxygenase-deficient HCT-116 cells were more responsive to sulindac sulfide-induced gene expression than SW-480 cells. However, this response was diminished in HCT-116 cells overexpressing cyclooxygenase-1 compared with normal HCT-116 cells suggesting the presence of cyclooxygenase attenuates this response. However, prostaglandin E2, the main product of cyclooxygenase, only suppressed the sulindac sulfide-induced expression of two genes, with little known biological function while it modulated the expression of two more. The most likely explanation for this finding is the metabolism of sulindac sulfide to inactive metabolites by the peroxidase activity of cyclooxygenase. In conclusion, this is the first report showing sulindac sulfide, independent of cyclooxygenase, altered the expression of several genes possibly linked to its anti-tumorigenic and pro-apoptotic activity.     

雷公藤甲素对结肠癌细胞SW480 的基因表达谱的影响

目的:探讨雷公藤甲素对结肠癌SW480细胞的基因表达谱的影响。方法:雷公藤甲素处理结肠癌SW480细胞24h后,分别提取给药组和空白对照组SW480细胞总RNA,纯化并逆转录成用Cy3和Cy5标记的cDNA探针,经全基因芯片杂交,洗涤,通过生物信息学方法分析雷公藤甲素处理组和空白对照组SW480细胞基因表达谱的差异。结果:与空白对照组比较,共发现了902个差异基因,雷公藤甲素处理组有196个基因上调,706个基因下调。上调基因主要涉及细胞代谢。下调基因主要涉及wnt通路、细胞周期通路、Toll样受体通路以及MAPK等通路。结论:雷公藤甲素能导致结肠癌细胞基因表达谱的改变,这些基因改变可能参与了细胞增殖、分化、凋亡等过程。这些信息可能为探讨雷公藤抗结肠癌作用机制提供线索。     

Proteomics identification of ITGB3 as a key regulator in reactive oxygen species-induced migration and invasion of colorectal cancer cells

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and second in females worldwide. Unfortunately 40-50% of patients already have metastatic disease at presentation when prognosis is poor with a 5-year survival of <10%. Reactive oxygen species (ROS) have been proposed to play a crucial role in tumor metastasis. We now show that higher levels of ROS accumulation are found in a colorectal cancer-derived metastatic cell line (SW620) compared with a cell line (SW480) derived from the primary lesion from the same patient. In addition, ROS accumulation can affect both the migratory and invasive capacity of SW480 and SW620 cells. To explore the molecular mechanism underlying ROS-induced migration and invasion in CRC, we have compared protein expression patterns between SW480 and SW620 cells using a two-dimensional electrophoresis-based proteomics strategy. A total of 63 altered proteins were identified from tandem MS analysis. Cluster analysis revealed dysregulated expression of multiple redox regulative or ROS responsive proteins, implicating their functional roles in colorectal cancer metastasis. Molecular and pathological validation demonstrated that altered expression of PGAM1, GRB2, DJ-1, ITGB3, SOD-1, and STMN1 was closely correlated with the metastatic potential of CRC. Functional studies showed that ROS markedly up-regulated expression of ITGB3, which in turn promoted an aggressive phenotype in SW480 cells, with concomitant up-regulated expression of STMN1. In contrast, knockdown of ITGB3 expression could mitigate the migratory and invasive potential of SW620 or H(2)O(2)-treated SW480 cells, accompanied by down-regulated expression of STMN1. The function of ITGB3 was dependent on the surface expression of integrin αvβ3 heterodimer. Furthermore, STMN1 expression and the PI3K-Akt-mTOR pathway were found to be involved in ROS-induced and ITGB3-mediated migration and invasion of colorectal cancer cells. Taken together, these studies suggest that ITGB3 plays an important role in ROS-induced migration and invasion in CRC.     

A diterpenoid derivative 15-oxospiramilactone inhibits Wnt/β-catenin signaling and colon cancer cell tumorigenesis

The Wnt/β-catenin signaling pathway is a highly conserved pathway in organism evolution and regulates many biological processes. Aberrant activation of the Wnt/β-catenin signaling pathway is closely related to tumorigenesis. In order to identify potent small molecules to treat the over-activated Wnt signaling-mediated cancer, such as colon cancer, we established a mammalian cell line-based reporter gene screening system. The screen revealed a diterpenoid derivative, 15-oxospiramilactone (NC043) that inhibits Wnt3a or LiCl-stimulated Top-flash reporter activity in HEK293T cells and growth of colon cancer cells, SW480 and Caco-2. Treatment of SW480 cells with NC043 led to decreases in the mRNA and/or protein expression of Wnt target genes Axin2, Cyclin D1 and Survivin , as well as decreases in the protein levels of Cdc25c and Cdc2. NC043 did not affect the cytosol-nuclear distribution and protein level of soluble β-catenin, but decreased β-catenin/TCF4 association in SW480 cells. Moreover, NC043 inhibited anchorage-independent growth and xenograft tumorigenesis of SW480 cells. Collectively these results demonstrate that NC043 is a novel small molecule that inhibits canonical Wnt signaling downstream of β-catenin stability and may be a potential compound for treating colorectal cancer.     

Nonsteroidal anti-inflammatory drugs and oxidative stress in cancer cells

Nonsteroidal antiinflammatory drugs (NSAIDs) induce apoptosis in a variety of cancer cells, including those of colon, prostate, breast and leukemia. In addition, the classical NSAIDs sulindac and aspirin are promising chemopreventive agents against colon cancer. NSAIDs inhibit cyclooxygenases (COX) preventing the formation of prostaglandins, prostacyclin and thromboxane. NSAIDs also exert other biological effects, including generation of reactive oxygen species (ROS) and inhibition of NF-kappaB-mediated signals. Despite many suggested mechanisms for their anticancer effects, it remains uncertain how they induce cell cycle arrest and apoptosis in cancer cells. Furthermore, there is little information on the selectivity of NSAIDs-mediated anticancer effects, although this is one of the most important issues in cancer therapy. Increased understanding of the biological basis for the anticancer activity of NSAIDs and their selectivity is essential for future therapeutic advances. In this paper, we propose that increased ROS generation is one of the key mechanisms for NSAIDs-mediated anticancer effects on various cancer cells.     

Up-regulation of GADD45α expression by NSAIDs leads to apoptotic and necrotic colon cancer cell deaths

Growth arrest and DNA damage inducible 45 alpha (GADD45α) is a central player in mediating apoptosis induced by a variety of stress stimuli and genotoxic agents. Regular usage of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and sulindac is associated with reduced risk for various cancers, including colon cancer. The role of GADD45α in NSAID-induced colon cancer cell cytotoxicity is unknown. In this study, we report that indomethacin and sulindac sulfide treatments up-regulate GADD45α mRNA expression and protein levels in colon cancer HT-29, RKO and Caco-2 cells. This up-regulation of GADD45α is accompanied by necrotic cell death and apoptosis. Anti-sense suppression of GADD45α expression inhibited indomethacin and sulindac sulfide-induced necrotic cell death and apoptosis. These findings confirm a role for GADD45α in NSAID-induced cytotoxicity, a mechanism for the anti-neoplastic effect of NSAIDs in colon tumorigenesis and cancer growth.     

沉默FOXQ1可逆转SW480细胞的上皮-间质转化

FOXQ1是FOX家族的的重要成员之一,其参与了多种人类肿瘤的上皮间质转化(epithelial- mesenchymal transition,EMT).本研究设计合成了FOXQ1基因的shRNA（short hairpin RNA）,用此转染SW480细胞,通过显微镜观察细胞形态,Transwell小室、细胞黏附试验检测转移能力及黏附能力,以探索FOXQ1与结直肠癌细胞EMT的关系.结果显示,沉默FOXQ1后,SW480细胞顶底极性及细胞间紧密连接增加,侵袭、迁移的细胞数目减少,同种黏附能力增加,异种黏附能力降低.进一步的机制研究表明,沉默FOXQ1基因可以导致SW480细胞的上皮标志因子E-cadherin表达显著增高,而间质细胞标志因子N-cadherin、Vimentin及MMP2表达均降低.以上结果表明,沉默FOXQ1基因可以逆转SW480细胞EMT,其机制可能与E-cadherin的上调和N cadherin、Vimentin、MMP2的下调有关,这为进一步研究FOXQ1在结直肠癌发生发展中的作用提供实验基础.     

Cyclooxygenase-independent effects of aspirin on HT-29 human colon cancer cells, revealed by oligonucleotide microarrays

Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation of human colon cancer cells in vitro. Transmission electron microscope detected morphological features of apoptosis in the aspirin-treated (5 mM, 72 h) HT-29 cells in which cyclooxygenoase-2 is catalytically inactive. We investigated aspirin-induced genome-wide expression changes in HT-29 cells and further studied the time- and concentration-dependent expression changes in 374 apoptosis-related genes, which is the first to show stimulation of genome-wide expression of HT-29 cells by aspirin. The most marked effects of aspirin are on ribosome assembly and rRNA metabolism, which could explain why the quasi-apoptotic morphological changes are not accompanied by a classical DNA ladder. These findings demonstrate that aspirin induces apoptosis in HT-29 cells, bolstering the hypothesis that apoptosis may be a mechanism by which NSAIDs inhibit colon carcinogenesis.     

PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs.

PPARB was identified as a target of APC through the analysis of global gene expression profiles in human colorectal cancer (CRC) cells. PPARdelta expression was elevated in CRCs and repressed by APC in CRC cells. This repression was mediated by beta-catenin/Tcf-4-responsive elements in the PPARdelta promotor. The ability of PPARs to bind eicosanoids suggested that PPARdelta might be a target of chemopreventive non-steroidal anti-inflammatory drugs (NSAIDs). Reporters containing PPARdelta-responsive elements were repressed by the NSAID sulindac. Furthermore, sulindac was able to disrupt the ability of PPARdelta to bind its recognition sequences. These findings suggest that NSAIDs inhibit tumorigenesis through inhibition of PPARdelta, the gene for which is normally regulated by APC.     

Effects of silk sericin on the proliferation and apoptosis of colon cancer cells

Sericin is a silk protein woven from silkworm cocoons (Bombyx mori). In animal model, sericin has been reported to have anti-tumoral action against colon cancer. The mechanisms underlying the activity of sericin against cancer cells are not fully understood. The present study investigated the effects of sericin on human colorectal cancer SW480 cells compared to normal colonic mucosal FHC cells. Since the size of the sericin protein may be important for its activity, two ranges of molecular weight were tested. Sericin was found to decrease SW480 and FHC cell viability. The small sericin had higher anti-proliferative effects than that of the large sericin in both cell types. Increased apoptosis of SW480 cells is associated with increased caspase-3 activity and decreased Bcl-2 expression. The anti-proliferative effect of sericin was accompanied by cell cycle arrest at the S phase. Thus, sericin reduced SW480 cell viability by inducing cell apoptosis via caspase-3 activation and down-regulation of Bcl-2 expression. The present study provides scientific data that support the protective effect of silk sericin against cancer cells of the colon and suggests that this protein may have significant health benefits and could potentially be developed as a dietary supplement for colon cancer prevention.     

敲低HSP60 对结肠癌SW480 细胞增殖能力的影响及其机制探究

目的:研究热休克蛋白60(HSP60)敲低对结肠癌SW480细胞增殖的影响,并进一步探究其作用机制。方法:通过含HSP60sh RNA载体的慢病毒感染加上流式细胞仪无菌分选的方法构建结肠SW480 HSP60基因稳定RNA干扰(RNAi)单克隆细胞系,利用Western blot和q-PCR验证结肠癌细胞中HSP60的敲低效率;使用CCK-8试剂检测结肠癌细胞增殖能力,并用流式细胞仪检测其HSP60敲低对细胞周期的影响。结果:Western blot和q-PCR结果验证了HSP60在结肠癌细胞中的敲低效率,与对照组细胞相比,实验组细胞HSP60的m RNA水平和蛋白水平均降低了60%以上。CCK-8实验结果表明,敲低HSP60后SW480细胞的增殖能力下降了约70%;流式细胞周期实验显示敲低HSP60后SW480细胞中G0/G1期、S期、G2/M期的分布比例变化不大。结论:敲低HSP60能够显著抑制SW480细胞的增殖能力,而SW480细胞周期并没有发生明显变化,推测HSP60的敲低引起的线粒体损伤导致细胞生长速度变慢。     

Cyclooxygenase inhibitors induce apoptosis in oral cavity cancer cells by increased expression of nonsteroidal anti-inflammatory drug-activated gene

We have investigated whether NAG-1 is induced in oral cavity cancer cells by various NSAIDs and if apoptosis induced by NSAIDs can be linked directly with the induction of NAG-1. NAG-1 expression was increased by diclofenac, aceclofenac, indomethacin, ibuprofen, and sulindac sulfide, in the order of NAG-1 induction, but not by acetaminophen, piroxicam or NS-398. Diclofenac was the most effective NAG-1 inducer. Incubation with diclofenac inhibited cell proliferation and induced apoptosis. The expression of NAG-1 was observed in advance of the induction of apoptosis. Conditioned medium from NAG-1-overexpressing Drosophila cells inhibited SCC 1483 cells proliferation and induced apoptosis. In summary, some NSAIDs induce NAG-1 expression in oral cavity cancer cells and the induced NAG-1 protein appears to mediate apoptosis. Therefore, NSAIDs may be considered as a possible chemopreventive agent against oral cavity cancer.     

microRNA-211 promotes invasion and migration of colorectal cancer cells by targeting FABP4 via PPARγ

Fatty acid binding protein 4 (FABP4) is a novel tumor regulator that is abnormally expressed in many human cancers. In our study, upregulated microRNA-211 (miR-211) and reduced FABP4 expression were detected in colorectal cancer (CRC) patients and CRC cells. Mimic miR-211 or anti-miR-211 were transfected to investigate the effects of miR-211 on SW480 cells. The results showed that miR-211 promoted but anti-miR-211 inhibited cell migration, invasion, and epithelial–mesenchymal transition (EMT) of SW480 cells. Luciferase activity was decreased after cotransfection with miR-211 and WT-FABP4-UTR in SW480 cells. And reduced FABP4 protein expression by miR-211 indicated that FABP4 was the targeted gene of miR-211. miR-211 inhibited the activation of peroxisome proliferator-activated receptor (PPAR) γ, whereas overexpression of FABP4 reversed that effect. Finally, FABP4 inhibited the migration, invasion, and EMT of SW480 cells, whereas PPARγ agonist reversed the effects of FABP4. Thus, the miR-211/FABP4/PPARγ axis may be a novel target for CRC therapy.