Novel di-tertiary-butyl phenylhydrazones as dual cyclooxygenase-2/5-lipoxygenase inhibitors: Synthesis,COX/LOX inhibition,molecular modeling,and insights into their cytotoxicities

Although dual inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) enzymes is highly effective than targeting COX or LOX alone, there are only a few reports of examining such compounds in case of colorectal cancers (CRC). In the present work we report that the novel di-tert-butyl phenol-based dual inhibitors DTPSAL, DTPBHZ, DTPINH, and DTPNHZ exhibit significant cytotoxicity against human CRC cell lines. Molecular docking studies revealed a good fit of these compounds in the COX-2 and 5-LOX protein cavities. The inhibitors show significant inhibition of COX-2 and 5-LOX activities and are effective against a panel of human colon cancer cell lines including HCA-7, HT-29, SW480 and intestinal Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressing colon cancer cells, through inhibition of the Hyaluronan/CD44v6 cell survival pathway. Western blot analysis and qRT-PCR analyses indicated that the di-tert-butyl phenol-based dual inhibitors reduce the expression of COX-2, 5-LOX, and CD44v6 in human colon cancer HCA-7 cells, while the combination of CD44v6shRNA and DTPSAL has an additional inhibitory effect on CD44v6 mRNA expression. The synergistic inhibitory effect of Celecoxib and Licofelone on CD44v6 mRNA expression suggests that the present dual inhibitors down-regulate cyclooxygenase and lipoxygenase enzymes through CD44v6. The compounds also exhibited enhanced antiproliferative potency compared to standard dual COX/LOX inhibitor, viz. Licofelone. Importantly, the HA/CD44v6 antagonist CD44v6shRNA in combination with synthetic compounds had a sensitizing effect on the cancer cells which enhanced their antiproliferative potency, a finding which is crucial for the anti-proliferative potency of the novel synthetic di-tert-butyl phenol based dual COX–LOX inhibitors in colon cancer cells.     

Lipoxygenase inhibitors abolish proliferation of human pancreatic cancer cells.

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism, but their role in pancreatic cancer growth is unknown. The expression of 5-LOX and 12-LOX as well as their effects on cell proliferation was investigated in four human pancreatic cancer cell lines (PANC-1, MiaPaca2, Capan2, and ASPC-1). Expression of 5-LOX and 12-LOX mRNA was measured by nested RT-PCR. Effects of LOX inhibitors and specific LOX antisense oligonucleotides on pancreatic cancer cell proliferation were measured by (3)H-thymidine incorporation. Our results showed that (1) 5-LOX and 12-LOX were expressed in all pancreatic cancer cell lines tested, while they were not detectable in normal human pancreatic ductal cells; (2) both LOX inhibitors and LOX antisense markedly inhibited cell proliferation in a concentration-dependent and time-dependent manner; (3) the 5-LOX and 12-LOX metabolites 5-HETE and 12-HETE as well as arachidonic and linoleic acids directly stimulated pancreatic cancer cell proliferation; (4) LOX inhibitor-induced growth inhibition was reversed by 5-HETE and 12-HETE. The current studies indicate that both 5-LOX and 12-LOX expression is upregulated in human pancreatic cancer cells and LOX plays a critical role in pancreatic cancer cell proliferation. LOX inhibitors may be valuable for the treatment of pancreatic cancer.     

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.     

Apigenin up-regulates transgelin and inhibits invasion and migration of colorectal cancer through decreased phosphorylation of AKT

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. Apigenin is a flavonoid that possesses various clinically relevant properties such as anti-tumour, anti-platelet and anti-inflammatory activities. Our results showed that apigenin has anti-proliferation, anti-invasion and anti-migration effects in three kinds of colorectal adenocarcinoma cell lines, namely SW480, DLD-1 and LS174T. Proteomic analysis with SW480 indicated that apigenin up-regulated the expression of transgelin (TAGLN) in mitochondria to exert its anti-tumour growth and anti-metastasis effects. Real-time quantitative polymerase chain reaction (RQ-PCR) and western blot confirm the up-regulation in all the three colorectal adenocarcinoma cells. An inverse correlation was observed between TAGLN expression and CRC metastasis in tissue microarray staining. TAGLN siRNA increased the viability of SW480. Apigenin decreased the expression of MMP-9 in a dose-dependent manner. Transfection of three truncated forms of TAGLN and wild type has identified TAGLN as a repressor of MMP-9 expression. A synergetic effect was observed in overexpression of TAGLN wild type and apigenin treatment which manifested as lowered phosphorylation of AKT Ser473 and ATK Thr308. In an orthotopic CRC model, apigenin inhibited tumour growth and metastasis to liver and lung. In conclusion, our research provided direct evidence that apigenin inhibited tumour growth and metastasis both in vitro and in vivo. Apigenin up-regulated TAGLN and hence down-regulated MMP-9 expression through decreasing phosphorylation of Akt at Ser473 and in particular Thr308 to prevent cell proliferation and migration.     

MIF/SCL3A2 depletion inhibits the proliferation and metastasis of colorectal cancer cells via the AKT/GSK‐3β pathway and cell iron death

This study investigated the mechanisms of migration inhibitory factor (MIF) and solute carrier family 3 member 2 (SLC3A2) in colorectal cancer progression. The levels of MIF and SLC3A2 expression in cells were measured by RT‐qPCR. SW480 and SW620 cells were transfected with sh‐MIF and sh‐SLC3A2, respectively. MIF, SLC3A2, GPX4, E‐cadherin and N‐cadherin expression were detected by immunofluorescence (IF). CCK8 and Transwell assays were performed to detect cell proliferation and migration. Co‐immunoprecipitation (CoIP) was used to measure the binding activity of MIF and SLC3A2. Finally, a nude mouse tumorigenicity assay was used to confirm the functions of MIF and SLC3A2 in colorectal cancer. Results showed that the levels of MIF and SLC3A2 expression were up‐regulated in colorectal cancer cells. Inhibition of MIF or SLC3A2 expression prevented cell proliferation, migration, epithelial‐mesenchymal transition (EMT) and invasion. In addition, knockdown of MIF and SLC3A2 promoted iron death in SW480 and SW620 cells. CoIP results showed that MIF and SLC3A2 directly interact with each other. Knockdown of both MIF and SLC3A2 inhibited tumour growth and metastasis via the AKT/GSK‐3β pathway in vivo. The Akt/GSK‐3β pathway was found to participate in regulating MIF and SLC3A2 both in vivo and in vitro. MIF and SLC3A2 might be potential biomarkers for monitoring the treatment of colorectal cancer.     

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.     

Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism. We previously reported that 5-LOX and 12-LOX are upregulated in human pancreatic cancer cells and that blockade of these enzymes abolishes pancreatic cancer cell growth. The present study was aimed at evaluating the effect of LOX inhibition on differentiation and apoptosis in pancreatic cancer cells in parallel with growth inhibition. Four human pancreatic cancer cell lines, PANC-1, MiaPaca2, Capan2, and HPAF, were used. Apoptosis was evaluated by three separate methods, including DNA propidium iodide staining, DNA fragmentation, and the TUNEL assay. Morphological changes and carbonic anhydrase activity were used to determine the effect of LOX inhibitors on differentiation. The general LOX inhibitor NDGA, the 5-LOX inhibitor Rev5901, and the 12-LOX inhibitor baicalein all induced apoptosis in all four pancreatic cancer cell lines, as confirmed by all three methods, suggesting that both the 5-LOX and 12-LOX pathways are important for survival of these cells. Furthermore, NDGA, Rev5901, or baicalein resulted in marked cellular morphological changes in parallel with increased intracellular carbonic anhydrase activity, indicating that LOX blockade induced a more differentiated phenotype in human pancreatic cancer cells. Together with our previous findings, this study suggests that perturbations of LOX activity affect pancreatic cancer cell proliferation and survival. Blockade of LOX enzymes may be valuable for the treatment of human pancreatic cancer.     

Mitochondrial DNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells

We investigated the potential role of mitochondrial DNA (mtDNA) in colorectal carcinogenesis by constructing a eukaryotic expression vector of the mitochondrial D-loop gene from colorectal cancer cell SW480 and transfected NIH3T3 cells. The NIH3T3/SW480 cells exhibited a significantly increased growth rate and colony formation rate, and also had a decreased apoptotic rate. Polyploidy and aberrant chromosomes were detected in the NIH3T3/SW480 cells by chromosome karyotype analysis. Our results suggested that mtDNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells. Further study of the biological functions of NIH3T3/SW480 cells might be helpful in understanding the role of mtDNA in colorectal carcinogenesis.     

NOP14 regulates the growth,migration, and invasion of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide. Recently, nucleolar complex protein 14 (NOP14) has been discovered to play a critical role in cancer development and progression, but the mechanisms of action of NOP14 in colorectal cancer remain to be elucidated. In this study, we used collected colorectal cancer tissues and cultured colorectal cancer cell lines (SW480, HT29, HCT116, DLD1, Lovo), and measured the mRNA and protein expression levels of NOP14 in colorectal cancer cells using qPCR and Western blotting. GFP-NOP14 was constructed and siRNA fragments against NOP14 were synthesized to investigate the importance of NOP14 for the development of colorectal cells. Transwell migration assays were used to measure cell invasion and migration, CCK-8 kits were used to measure cell activity, and flow cytometry was applied to the observation of apoptosis. We found that both the mRNA and protein levels of NOP14 were significantly upregulated in CRC tissues and cell lines. Overexpression of GFP-NOP14 markedly promoted the growth, migration, and invasion of the CRC cells HT19 and SW480, while genetic knockdown of NOP14 inhibited these behaviors. Overexpression of NOP14 promoted the expression of NRIP1 and phosphorylated inactivation of GSK-3β, leading to the upregulation of β-catenin. Genetic knockdown of NOP14 had the opposite effect on NRIP1/GSK-3/β-catenin signals. NOP14 therefore appears to be overexpressed in clinical samples and cell lines of colorectal cancer, and promotes the proliferation, growth, and metastasis of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway.Key words: Colorectal cancer, NOP14, proliferation, migration, invasion     

Suppression of mucin 2 enhances the proliferation and invasion of LS174T human colorectal cancer cells

Altered expression of MUC2 (mucin 2) is related to tumour development in colorectal cancer. Colorectal mucinous carcinomas are positive for MUC2 expression, whereas MUC2 is down-regulated in non-mucinous adenocarcinomas. In the present study, we down-regulated MUC2 expression by RNAi (RNA interference) and investigated the in vitro and in vivo effects on the proliferation and invasion/migration potential of the LS174T human colorectal cancer cells. The LS174T cell line is a goblet-cell-like colorectal cancer cell line that continuously produces high levels of MUC2. Inhibition of MUC2 expression in vitro by transfection of LS174T cells with the recombinant plasmid pcDNA6.2-GW/EmGFP-miR-MUC2 led to the production of a stably transfected MUC2-RNAi LS174T cell line. The proliferation and invasion/migration of MUC2-RNAi cells in vitro were significantly higher than those in control cells, as assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], colony formation and transwell assays. Subcutaneous injection of MUC2-RNAi LS174T cells into nude mice resulted in the development of subcutaneous tumours visible to the naked eye after 1 week. The growth rate of tumours derived from MUC2-RNAi LS174T cells was greater than that of tumours derived from control cells. Ki67 and matrix metalloproteinase-9 proteins were detected by immunohistochemistry in the xenografts. The expression levels of these proteins were higher in the MUC2-RNAi-derived xenografts than in xenografts derived from control cells. Although the role of MUC2 in colorectal tumorigenesis is not fully understood, these results strongly suggest a relationship between the proliferation and invasion of LS174T cells and the expression of MUC2.     

Inhibitory effects of anti-CXCR4 antibodies on human colon cancer cells

Background: CXCR4, the chemokine receptor for CXCL12, has recently been involved in the metastatic process of several neoplasms. Materials and methods: The expression of CXCR4 was evaluated by immunohistochemistry of colorectal tissue samples and by flow cytometry on Caco2, GEO, SW480, SW48, Lovo and SW620 human colon carcinoma cell lines. Correlations with pathological characteristics of the specimens were analysed with chi-square test. To verify the functional status of CXCR4, cell lines were tested in adhesion, migration, and proliferation assays. Results: We studied the expression of CXCR4 in 88 human colorectal tissues and we found that CXCR4 was expressed in >10% of epithelial cells in 50% of normal mucosae (7/14), in 55% of polyps (29/53), in all of carcinomas (16/16) and hepatic metastasis (5/5). Notably, CXCR4 was significantly over-expressed in cancerous lesions (carcinomas and metastasis) compared to non-cancerous lesions (normal mucosa and polyps) (P=0.003) and in adenomatous polyps versus hyperplastic polyps (P=0.009). The diameter of a polyp was also significantly associated with CXCR4 expression (P=0.031). SW480, SW48 and SW620 cell lines showed the highest levels of CXCR4 (60–80% of positive cells). Adhesion, migration, and proliferation increased in response to the CXCL12 chemokine. These effects were abrogated by the addition of anti-CXCR4 antibodies. Further, CXCL12 activated ERK1/2 in SW480 cells. Conclusions: These data suggest that CXCR4 might play a role in colon cancer cell properties and that anti-CXCR4 antibodies could have therapeutic effects against colorectal cancer.Partially presented at the 94th Annual Meeting of the American Association for Cancer Research, Washington, DC, 11–14 July 2003 (abstract LB-132).     

microRNA‐577 suppresses tumor growth and enhances chemosensitivity in colorectal cancer

In this work, we aimed to determine the expression and biological functions of microRNA (miR)‐577 in colorectal cancer (CRC). The results showed that miR‐577 was downregulated in CRC specimens and cell lines. Restoration of miR‐577 significantly suppressed the proliferation and colony formation and induced a G0/G1 cell cycle arrest in CRC cells. 5‐Fluorouracil (5‐FU)‐resistant SW480 cells (SW480/5‐FU) were found to have elevated levels of miR‐577. Ectopic expression of miR‐577 enhanced 5‐FU sensitivity in SW480/5‐FU cells. Heat shock protein 27 (HSP27) was identified as a target gene of miR‐577. Enforced expression of HSP27 reversed the effects of miR‐577 on CRC cell growth and 5‐FU sensitivity. Xenograft tumors derived from miR‐577‐overexpressing SW480 cells exhibited significantly slower growth than control tumors. In conclusion, our results support that miR‐577 acts as a tumor suppressor in CRC likely through targeting HSP27. Therefore, miR‐577 may have therapeutic potential in the treatment of CRC.     

STAT3-siRNA对人结直肠癌细胞的干涉作用

目的:研究STAT3-siRNA对STAT3基因表达阳性的结直肠癌细胞凋亡的影响。方法:应用脂质体转染试剂将STAT3-siRNA表达盒(STAT3-siRNA expression cassettes,STAT3-SECs)体外转染至人结直肠癌SW480细胞及人成纤维细胞中,同时分别设立人成纤维对照组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组。于48h后收集细胞,先经荧光染色方法观察细胞表象变化,再通过流式细胞仪检测人结直肠癌SW480细胞凋亡情况,后分别提取细胞总RNA,用RT-PCR测定STAT3基因在mRNA水平的表达。结果:SW480STAT3-SECs组的细胞可见凋亡小体,出现明显的凋亡现象,而人成纤维对照组、人成纤维STAT3-SECs组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组未出现明显的凋亡现象。SW480STAT3-SECs组细胞的凋亡比率较SW480对照组、SW480错配链-SECs组和SW480空转染试剂组有明显的增高。RT-PCR所得数据经统计学处理得出:SW480STAT3-SECs组细胞的STAT3基因表达在mRNA水平上显著低于SW480对照组(P0.01);而人成纤维对照组与人成纤维STAT3-SECs组,SW480细胞对照组与SW480错配链-SECs组、SW480空转染试剂组之间无明显差异(P0.05)。结论:应用RNAi技术沉默STAT3基因可以降低人结直肠癌SW480细胞中STAT3的表达,诱导细胞的凋亡。     

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.