PGE2-induced colon cancer growth is mediated by mTORC1

The inflammatory prostaglandin E₂ (PGE₂) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE₂ directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE₂-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE₂ increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE₂ EP₄ receptor was responsible for transducing the signal to mTORC1. Moreover, PGE₂ increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE₂-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE₂ increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE₂ mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.     

A colonic tissue architecture assay applied to human colon carcinoma cells

Summary A two-component tissue architecture assay system has been devised that tests the ability of human colon carcinoma cells to conform to the specific three-dimensional cell-cell and cell-substratum interactions characteristic of normal colonic tissues. Dissociated fetal rat colonic cells (FRCC) were allowed to reaggregate in suspension with or without the addition of different proportions (0.1%, 1%, and 10% of the total cells) of the human colon carcinoma cell lines, SW-1222 and LS-174T. Cellular aggregates obtained after 36 hours’ incubation exhibited cell sorting by the formation of recognizable epithelial colonic crypt-like structures with glandular lumens in a mesenchyme-like background. Carcinoembryonic antigen (CEA)-positive SW-1222 cells in 10% mixed aggregates were organized into numerous well-formed glandular structures with a polarized apical distribution of CEA. LS-174T cells, on the other hand, were self-sorted but structurally disorganized with a continuous cell surface CEA distribution. Pure FRCC and mixed aggregates were implanted under the kidney capsules of Swiss nu/nu (nude) or CD-1 nu/nu mice and allowed to grow for a period of 7–10 days. Whereas the normal FRCC readily formed colonic tissue, the SW-1222 cells exhibited a capacity for differentiation into colonic crypts which became progressively less normal and more tumor-like as the proportion of carcinoma cells in the aggregates was increased. The LS-174T cells demonstrated poor differentiation at all concentrations. Cell surface levels of CEA and the CEA family member nonspecific crossreacting antigen (NCA), both overexpressed in colon cancer, were higher in LS-174T than in SW-1222 cells, whereas family member biliary glycoprotein (BGP), downregulated in colon carcinoma was higher in the SW-1222 cells. These results thus support the suggestion that deregulated expression of CEA family members can be involved in the ability of colonocytes to differentiate and conform to normal tissue architecture as assessed by the assay. The assay is therefore amenable to genetic analysis of normal and perturbed architectural phenotypes. This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. C. I. is a recipient of a studentship from “le Fonds pour la Formation de Chercheurs et l’Aide à la Recherche.”     

Silencing of Jagged1 inhibits cell growth and invasion in colorectal cancer

Dysregulated Notch signaling has a critical role in the tumorigenesis. Jagged1, a Notch ligand, is overexpressed in various human cancers. Recent studies revealed the involvement of Jagged1 in colorectal cancer (CRC) development. These basic studies provide a promising potential for inhibition of the Notch pathway for the treatment of CRC. Herein, we aimed to investigate the consequences of targeting Jagged1 using shRNA on CRC both in vitro and in vivo to test their potential to inhibit this key element for CRC treatment. We found that downregulation of Jagged1 with lentiviral Jagged1-shRNA resulted in decreased colon cancer cell viability in vitro, most likely mediated through reduced cell proliferation. Importantly, Jagged1 knockdown induced G₀/G₁ phase cell cycle arrest, with reduced Cyclin D1, Cyclin E and c-Myc expression. Silencing of Jagged1 reduced the migration and invasive capacity of the colon cancer cells in vitro. Furthermore, colon cancer cells with knockdown of Jagged1 had much slower growth rate than control cells in a xenograft mouse model in vivo, with a marked downregulation of cell proliferation markers (PCNA, Ki-67, and c-Myc) and metastasis markers (MMP-2 and MMP-9). These findings rationalize a mechanistic approach to CRC treatment based on Jagged1-targeted therapeutic development.     

Effects of siRNA Targeting c‐Myc and VEGF on Human Colorectal Cancer Volo Cells

c‐Myc and vascular endothelial growth factor (VEGF) genes are frequently deregulated and overexpressed in this malignancy, and strategies designed to inhibit c‐Myc and VEGF expression in cancer cells may have considerable therapeutic value. In the present study, we design and use short interfering RNA (siRNA) to inhibit c‐Myc and VEGF expression in colorectal cancer Volo cells and validate their effects on cell proliferation, cell cycle, apoptosis, and cell metastasis. Upon transient transfection with plasmid‐encoding siRNA, it was found that expression of c‐Myc and VEGF was significantly downregulated in siRNA‐transfected cells and the downregulation of c‐Myc and VEGF inhibited cell growth and induced apoptosis and metastasis of Volo cells. c‐Myc and VEGF downregulation also increased cell population in the G0–G1 phase. In conclusion, the specific siRNA efficiently silenced the expression of c‐Myc and VEGF, further suppressed the cell proliferation, triggered cell apoptosis, and inhibited cell invasiveness of colorectal cancer Volo cells. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:499‐505, 2012;Viewthis article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21455     

Effects of iodonium-class flavin dehydrogenase inhibitors on growth,reactive oxygen production,cell cycle progression,NADPH oxidase 1 levels,and gene expression in human colon cancer cells and xenografts

Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174 T colon cancer cells at concentrations (10–250 nM for DPI, 0.5–2.5 μM for DTI, and 155 nM to 10 μM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H₂O₂ production and diminished intracellular ROS levels, lasting up to 24 h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G₁/S interface in both LS-174 T and HT-29 cells exposed to either DPI or DTI; and the G₁ block was produced, for LS-174 T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H₂O₂. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174 T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.     

AS2O3抑制人结肠癌生长的体内外实验研究

目的：通过研究三氧化二砷（AS2O3）对人结肠癌细胞株LS-174T在体内外实验中增殖情况的影响,探讨As2O3治疗结肠癌的机制。方法：1、试验分组：体外细胞培养及体内动物实验均分三组：对照组,As2O3低剂量组,As2O3高剂量组。2、MTT法测定细胞增殖数的情况。3、丫啶橙/溴乙啶（AO/EB）双荧光染色检测细胞凋亡率。4、免疫组织化学法测定结肠癌细胞和裸鼠实体肿瘤中HIF-1α和VEGF的表达。5、测量各组裸鼠实体肿瘤的重量及体积。结果：体外试验中,由MTT法可以看出,As2O3治疗组细胞生存率明显低于对照组（P〈0.01）,其中AS2O3高剂量组明显低于低剂量组。双荧光染色结果As2O3高剂量组细胞凋亡百分率明显高于低剂量组和对照组（P〈0.01）。免疫组化结果显示结肠癌细胞HIF-1α和VEGF在对照组的阳性表达率明显高于As2O3治疗组（P〈0.01）,低剂量组高于高剂量组（P〈0.05）。体内实验中,治疗组平均瘤重和瘤体积均较对照组明显减低,（P〈0.01）。HIF-1α及VEGF在裸鼠肿瘤组织中均有阳性表达,其中对照组较AS2O3治疗组高（P〈0.01）。结论：As2O3能够抑制体内外人结肠癌细胞的生长,并能明显抑制HIF-1α和VEGF的表达,从而抑制肿瘤的生长。     

Expression of developmentally regulated endothelial cell locus 1 was induced by tumor-derived factors including VEGF

Developmentally regulated endothelial cell locus 1 (Del1) is a new angiogenic molecules expressed specifically in early embryonic endothelial cells. We investigated the relationship between Del1 and tumor cell-derived vascular endothelial growth factor (VEGF). Dunn osteosarcoma cells and high- and low-metastatic murine sarcoma cells did not express Del1. However, the expression of Del1 was observed in these primary tumor tissues and the pulmonary metastatic tissues after subcutaneous inoculation in vivo. Every tumor cell-conditioned medium containing VEGF induced the expression of Del1 in murine lung microvascular endothelial (MLE) cells, although control MLE cells did not express Del1. The anti-mouse VEGF monoclonal antibody inhibited the induction of the Del1 expression. In addition, mouse recombinant interleukin-1alpha and tumor necrosis factor-alpha also induced Del1 in MLE cells. Del1 may play an important role in tumor angiogenesis through the effects of tumor-derived factors including VEGF.     

MTA1 promotes the invasion and migration of pancreatic cancer cells potentially through the HIF-α/VEGF pathway

Abstract

The metastasis-associated gene 1 (MTA1) has previously been recognized as an oncogene, and abnormal MTA1 expression has been related to progression of numerous cancer types to the metastasis stage. However, the function of MTA1 in the regulation of pancreatic cancer progression and metastasis remains unclear. Western blot analysis was adopted to determine the expression of MTA1 in pancreatic cancer tissues and corresponding near normal tissues. Steady clone with MTA1-overexpression and MTA1-inhibitionweregenerated via lentivirus technology in BxPc-3 cells. Transwell assay was carried out for detecting the invasion of pancreatic cancer cells. The migration activity was assessed using the wound scratch assay. The effect of MTA1 in pancreatic cancer was evaluated in the mice xenografts. Western blot analysis was employed to determine the expression of hypoxia inducible factor-α (HIF-α) and vascular endothelial growth factor (VEGF) in vitro and in vivo. We observed that MTA1 overexpression enhanced migration and invasion ability of pancreatic cancer cells in vitro and increased HIF-α and VEGF protein levels in vitro and in vivo. MTA1 inhibition had the opposite effects. MTA1 protein level was positively related to HIF-α and VEGF protein levels. These results indicated that MTA1 potentially promoted pancreatic cancer metastasis via HIF-α/VEGF pathway. This research supplies a new molecular mechanism for MTA1 in the pancreatic cancer progression and metastasis. MTA1 may be an effective therapy target in pancreatic cancer.     

Knockdown of YKL-40 inhibits angiogenesis through regulation of VEGF/VEGFR2 and ERK1/2 signaling in endometrial cancer

Studies have demonstrated that small interfering RNA (siRNA) targeting YKL-40 (siYKL-40) inhibits the proliferation, migration, invasion, and induces antiapoptotic abilities of endometrial cancer (EC) HEC-1A cells. However, its effect on angiogenesis is unclear. The present study aimed to investigate the role of YKL-40 in endometrial cancer and the related molecular mechanisms. YKL-40 was knocked down by transfection with siYKL-40 and the effects on angiogenesis, cell viability, and signaling pathways were investigated. The results showed that siYKL-40 inhibited VEGFA levels and tube formation in endothelial cells. Additionally, inhibition of YKL-40 decreased the expression levels of vascular endothelial growth factor (VEGF), phosphorylated vascular endothelial growth factor receptor 2 (pVEGFR2), and phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2). Furthermore, a nude mice xenograft model of EC showed that siYKL-40 inhibited tumor growth. Inhibition of YKL-40 led to suppression of angiogenesis and reduction of microvessel density through VEGF/VEGFR2 and ERK1/2 signaling in endometrial cancer cells. Taken together, this study demonstrated novel molecular mechanisms for role of YKL-40 in EC.     

Apatinib inhibits the growth of small cell lung cancer via a mechanism mediated by VEGF,PI3K/Akt and Ki-67/CD31

This study aimed to investigate the anti-tumour effect of apatinib on extensive-stage small cell lung cancer (SCLC) and elucidate the associated mechanisms. NCI-H345 cells were selected as model cells because of high expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and phosphorylated-VEGFR2 (pVEGFR2). Cells were exposed to recombinant human VEGF (rhVEGF) and apatinib. Cells were then divided into eight groups, namely, control, rhVEGF, apatinib, rhVEGF+apatinib, serum-free medium (SM), SM+rhVEGF, SM+apatinib and SM+rhVEGF+apatinib. In comparison with the control group, cell proliferation in vitro in apatinib, SM, SM+apatinib and SM+rhVEGF+apatinib groups was inhibited, particularly in SM+apatinib group. The effect of apatinib on tumour growth in vivo was investigated using a mouse xenograft tumour model. In comparison with the control group, tumour sizes were reduced in apatinib-treated group on days 34 and 37. Immunohistochemical and immunofluorescence staining revealed that VEGF, pVEGFR2, PI3K, AKT, p-ERK1/2, Ki-67 and CD31 in the tumour cells of apatinib-treated group were downregulated compared with control group. Haematoxylin and eosin staining revealed that apatinib promoted the necrosis of SCLC cells in vivo. In conclusion, apatinib inhibited the growth of SCLC cells by downregulating the expression of VEGF, pVEGFR2, p-PI3K, p-AKT, p-ERK1/2, Ki-67 and CD31.     

Colon cancer cell adhesion to endothelial E-selectin inhibits detachment of endothelial cells through activation of beta(1)-integrin

Previous studies have implicated a role for E-selectin in carcinoma cell adhesion to vascular endothelium. We examined the role of colon cancer cell adhesion to vascular endothelium via E-selectin using adenoviral vector-mediated transfection in human umbilical vein endothelial cells (HUVECs). We found that the amount of HUVEC detachment from the gelatin matrix 24 h after LS-180 cell adhesion was inhibited only when the HUVECs were transduced with wild-type E-selectin, but not with a cytoplasmic domain truncated mutant E-selectin or the control Lac-Z vector. We also found that the adhesion of LS-180 cells to wild-type E-selectin transduced HUVEC-induced activation of beta(1)-integrin receptors without affecting MMP activity. These results indicate that colon cancer cell adhesion via E-selectin inhibits HUVEC detachment from the monolayer, at least in part by modulating beta(1)-integrin activity in HUVECs. In addition, they indicate the importance of the cytoplasmic domain of E-selectin with this phenomenon.     

Aberrant,ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy.     

血管内皮生长因子与肿瘤

血管内皮生长因子是新近确定的一种具有旁分泌机制的生长因子,能特异作用于血管内皮细胞,促进其增殖及新生血管的形成,同时还有增加血管通透性的作用．由于其生物学活性与实体瘤的生长密切相关,因此对它的研究倍受关注,进展非常迅速．     

Resveratrol derivative,trans‐3,5,4′‐trimethoxystilbene,exerts antiangiogenic and vascular‐disrupting effects in zebrafish through the downregulation of VEGFR2 and cell‐cycle modulation

Angiogenesis plays an important role in the development of neoplastic diseases such as cancer. Resveratrol and its derivatives exert antiangiogenic effects, but the mechanisms of their actions remain unclear. The aim of this study was to evaluate the antiangiogenic activity of resveratrol and its derivative trans‐3,5,4′‐trimethoxystilbene in vitro using human umbilical vein endothelial cells (HUVECs) and in vivo using transgenic zebrafish, and to clarify their mechanisms of action in zebrafish by gene expression analysis of the vascular endothelial growth factor (VEGF) receptor (VEGFR2/KDR) and cell‐cycle analysis. trans‐3,5,4′‐Trimethoxystilbene showed significantly more potent antiangiogenic activity than that of resveratrol in both assays. In zebrafish, trans‐3,5,4′‐trimethoxystilbene caused intersegmental vessel regression and downregulated VEGFR2 mRNA expression. Trans‐3,5,4′‐trimethoxystilbene also induced G2/M cell‐cycle arrest, most specifically in endothelial cells of zebrafish embryos. We propose that the antiangiogenic and vascular‐targeting activities of trans‐3,5,4′‐trimethoxystilbene result from the downregulation of VEGFR2 expression and cell‐cycle arrest at G2/M phase. J. Cell. Biochem. 109: 339–346, 2010. © 2009 Wiley‐Liss, Inc.