phenylmercuric-acetate and Neoplasms

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

Topics: AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Humans; Lipogenesis; Neoplasms; Oxidative Stress; Pentose Phosphate Pathway; Phosphogluconate Dehydrogenase; Protein Serine-Threonine Kinases; Ribulosephosphates; Signal Transduction

α1(IV)NC1 inhibits angiogenesis by regulating MAPK activation, this biological function was partly attributed α1(IV)NC1 binding to α1β1-integrin. However, its potent antiangiogenic activity and the molecular targets of α1(IV)NC1 has not been investigated. In the present study, the regulation of MMP-2 activation by α1(IV)NC1 was evaluated. α1β1-integrin which is required for inhibition of angiogenesis is not playing a role in cellular invasion and inhibition of MMP-2 activation by α1(IV)NC1. We found that α1(IV)NC1 binds the CBD of MMP-2 and forming a stable complex that prevents activation of MMP-2. The antiangiogenic activity of α1(IV)NC1 is mediated, in part, by this binding activity. In addition, up-regulation of TIMP-2 by α1(IV)NC1 led to saturation of MT1-MMP binding sites, which in turn led to inhibition of MMP-2 activation. In-vivo studies using α1-integrin null-mice treated with higher doses of α1(IV)NC1 showed integrin independent inhibition of tumor growth and active-MMP-2, without affecting MMP-9, MMP-7 and angiostatin.

Topics: Angiostatins; Animals; Collagen Type IV; Enzyme Activation; Human Umbilical Vein Endothelial Cells; Humans; Integrin alpha1; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; Phenylmercuric Acetate; Protein Interaction Domains and Motifs; Recombinant Proteins; Vascular Endothelial Growth Factor A

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7x10(5) receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-alpha). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.

Topics: Amino Acid Sequence; Animals; Cattle; Cell Line, Tumor; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Sequence Data; Neoplasms; Peptides; Phenylmercuric Acetate; Protease Inhibitors; Protein Isoforms; Protein Kinase C; Protein Structure, Tertiary; Sequence Alignment; Spectrometry, Mass, Electrospray Ionization; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Vanadates

Clinical studies demonstrate a positive correlation between the extent of matrix metalloproteinase (MMP) activation and malignant progression of precancerous lesions. Therefore, identification of effective, well-tolerated MMP inhibitors represents a rational chemopreventive strategy. A variety of agents, including proteinases and thiol-oxidizing compounds, activate MMPs by initiating release of the propeptide's cysteine sulfur "blockage" of the MMP active site. Despite the importance of the propeptide's cysteine thiol in preserving MMP latency, limited studies have evaluated the effects of reduced thiols on MMP function. This study investigated the effects of two naturally occurring nonprotein thiols, i.e., glutathione (GSH) and N-acetylcysteine (NAC), on activation, function, and cellular-extracellular matrix interactions of the basement-membrane-degrading gelatinase, MMP-9. Our results reveal that NAC and GSH employ protein S-thiolation to inhibit organomercurial activation of pro-MMP-9. Gelatinase activity assays showed that GSH and NAC significantly inhibited MMP-9 but not MMP-2 function, implying isoform structural specificity. Immunoblot analyses, which suggested GSH interacts with MMP-9's active-site Zn, were corroborated by computational molecular modeling. Cell invasion assays revealed that NAC enhanced endostatin's ability to inhibit human cancer cell invasion. Collectively, these data demonstrate that nonprotein thiols suppress MMP-9 activation and function and introduce the prospect for their use in chemopreventive applications.

Topics: Acetylcysteine; Anticarcinogenic Agents; Catalytic Domain; Cell Line, Tumor; Chemoprevention; Enzyme Activation; Glutathione; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Models, Molecular; Neoplasms; Oxidation-Reduction; Phenylmercuric Acetate; Protein Conformation; Zinc