4-(N,N-dipropylamino)benzaldehyde inhibits the oxidation of all-trans retinal to all-trans retinoic acid by ALDH1A1, but not the differentiation of HL-60 promyelocytic leukemia cells exposed to all-trans retinal

BACKGROUND: The signal transduction pathways mediated by retinoic acid play a critical role in the regulation of cell growth and differentiation during embryogenesis and hematopoiesis as well as in a variety of tumor cell lines in culture. Following the reports that two members of the superfamily of aldehyde dehydrogenase (ALDH) enzymes, ALDH1A1 and ALDH1A2, were capable of catalyzing the oxidation of all-trans retinal to all-trans retinoic acid with submicromolar K(m) values, we initiated an investigation of the ability of 4-(N,N-dipropylamino)benzaldehyde (DPAB) to inhibit the oxidation of retinal by purified mouse and human ALDH1A1. RESULTS: Our results show that DPAB potently inhibits retinal oxidation, with IC(50) values of 0.11 and 0.13 μM for purified mouse and human ALDH1A1, respectively. Since the HL-60 human myeloid leukemic cell line has been used extensively to study the retinoic acid induced differentiation of HL-60 cells to granulocytes, and ALDH1A1 activity had previously been reported in HL-60 cells, we investigated the ability of DPAB to block differentiation of HL-60 promyelocytic leukemia cells exposed to retinal in culture. In HL-60 cells coincubated with 1 μM retinal and 50 μM DPAB for 144 hours, cell differentiation was inhibited only 30%. Furthermore, the NAD-dependent oxidation of propanal or retinal was less than 0.05 nmoles NADH formed/min-10(7) cells in spectrophotometric assays using HL-60 cell extracts. CONCLUSION: Although ALDH1A1 may be the major catalytic activity for retinal oxidation in some retinoid-dependent mouse and Xenopus embryonic tissues and in adult human and mouse hematopoietic stem cells, another catalytic activity appears to synthesize the retinoic acid ligand necessary to stimulate the differentiation of HL-60 cells to end stage granulocytes

Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery.

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and αvβ3 integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer

The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells

MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors

BACKGROUND MicroRNA (miRNA) expression profiles have been described in pancreatic ductal adenocarcinoma (PDAC), but these have not been compared with pre-malignant pancreatic tumors. We wished to compare the miRNA expression signatures in pancreatic benign cystic tumors (BCT) of low and high malignant potential with PDAC, in order to identify miRNAs deregulated during PDAC development. The mechanistic consequences of miRNA dysregulation were further evaluated. METHODS Tissue samples were obtained at a tertiary pancreatic unit from individuals with BCT and PDAC. MiRNA profiling was performed using a custom microarray and results were validated using RT-qPCR prior to evaluation of miRNA targets. RESULTS Widespread miRNA down-regulation was observed in PDAC compared to low malignant potential BCT. We show that amongst those miRNAs down-regulated, miR-16, miR-126 and let-7d regulate known PDAC oncogenes (targeting BCL2, CRK and KRAS respectively). Notably, miR-126 also directly targets the KRAS transcript at a "seedless" binding site within its 3'UTR. In clinical specimens, miR-126 was strongly down-regulated in PDAC tissues, with an associated elevation in KRAS and CRK proteins. Furthermore, miR-21, a known oncogenic miRNA in pancreatic and other cancers, was not elevated in PDAC compared to serous microcystic adenoma (SMCA), but in both groups it was up-regulated compared to normal pancreas, implicating early up-regulation during malignant change. CONCLUSIONS Expression profiling revealed 21 miRNAs down-regulated in PDAC compared to SMCA, the most benign lesion that rarely progresses to invasive carcinoma. It appears that miR-21 up-regulation is an early event in the transformation from normal pancreatic tissue. MiRNA expression has the potential to distinguish PDAC from normal pancreas and BCT. Mechanistically the down-regulation of miR-16, miR-126 and let-7d promotes PDAC transformation by post-transcriptional up-regulation of crucial PDAC oncogenes. We show that miR-126 is able to directly target KRAS; re-expression has the potential as a therapeutic strategy against PDAC and other KRAS-driven cancers

EGFR-Mediated Carcinoma Cell Metastasis Mediated by Integrin αvβ5 Depends on Activation of c-Src and Cleavage of MUC1

Receptor tyrosine kinases and integrins play an essential role in tumor cell invasion and metastasis. We previously showed that EGF and other growth factors induce human carcinoma cell invasion and metastasis mediated by integrin αvβ5 that is prevented by Src blockade [1]. MUC1, a transmembrane glycoprotein, is expressed in most epithelial tumors as a heterodimer consisting of an extracellular and a transmembrane subunit. The MUC1 cytoplasmic domain of the transmembrane subunit (MUC1.CD) translocates to the nucleus where it promotes the transcription of a metastatic gene signature associated with epithelial to mesenchymal transition. Here, we demonstrate a requirement for MUC1 in carcinoma cell metastasis dependent on EGFR and Src without affecting primary tumor growth. EGF stimulates Src-dependent MUC1 cleavage and nuclear localization leading to the expression of genes linked to metastasis. Moreover, expression of MUC1.CD results in its nuclear localization and is sufficient for transcription of the metastatic gene signature and tumor cell metastasis. These results demonstrate that EGFR and Src activity contribute to carcinoma cell invasion and metastasis mediated by integrin αvβ5 in part by promoting proteolytic cleavage of MUC1 and highlight the ability of MUC1.CD to promote metastasis in a context-dependent manner. Our findings may have implications for the use and future design of targeted therapies in cancers known to express EGFR, Src, or MUC1

Colorectal Cancer Metastases Settle in the Hepatic Microenvironment Through \u3b15\u3b21 Integrin

Colorectal cancer (CRC) metastasis dissemination to secondary sites represents the critical point for the patient0s survival. The microenvironment is crucial to cancer progression, influencing tumour cell behaviour by modulating the expression and activation of molecules such as integrins, the cell-extracellular matrix interacting proteins participating in different steps of the tumour metastatic process. In this work, we investigated the role of a5b1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver. The culture medium conditioned by the IHH hepatic cell line, and the extracellular matrix (ECM) proteins, modulate the activation of a5b1 integrin in the colon cancer cell line HCT-116, and drives FAK phosphorylation during the process of cell adhesion to fibronectin, one of the main components of liver ECM. In these conditions, a5b1 modulates the expression/activity of another integrin, a2b1, involved in the cell adhesion to collagen I. These results suggest that a5b1 integrin holds a leading role in HCT-116 colorectal cancer cells adhesion to the ECM through the modulation of the intracellular focal adhesion kinase FAK and the a2b1 integrin activity. The driving role of the tumour microenvironment on CRC dissemination, here detected, and described, strengthens and adds new value to the concept that a5b1 integrin can be an appropriate and relevant therapeutic target for the control of CRC metastases