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Results for “RNA, Small Interfering”

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PubMed2003

PACSIN3 binds ADAM12/meltrin alpha and up-regulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor.

A disintegrin and metalloprotease 12 (ADAM12/meltrin alpha) is a key enzyme implicated in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor (proHB-EGF)-dependent epidermal growth factor receptor (EGFR) transactivation. However, the activation mechanisms of ADAM12 are obscure. To determine how ADAM12 is activated, we screened proteins that bind to the cytoplasmic domain of ADAM12 using a yeast two-hybrid system and identified a protein called PACSIN3 that contains a Src homology 3 domain. An analysis of interactions between ADAM12 and PACSIN3 using glutathione S-transferase fusion protein revealed that a proline-rich region (amino acid residues 829-840) of ADAM12 was required to bind PACSIN3. Furthermore, co-immunoprecipitation and co-localization analyses of ADAM12 and PACSIN3 proteins also revealed their interaction in mammalian cells expressing both of them. The overexpression of PACSIN3 in HT1080 cells enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced proHB-EGF shedding. Furthermore, knockdown of endogenous PACSIN3 by small interfering RNA in HT1080 cells significantly attenuated the shedding of proHB-EGF induced by TPA and angiotensin II. Our data indicate that PACSIN3 has a novel function as an up-regulator in the signaling of proHB-EGF shedding induced by TPA and angiotensin II.

ADAM Proteins↗

PubMed2005

Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells.

Hyaluronan is enriched in many types of human cancers, and manipulations of hyaluronan expression or interactions have a major influence on tumor progression in animal models. Increased ErbB2 activity is characteristic of several cancers and is responsible for many aspects of malignant cell behavior in these cancers. In this study we show that constitutively high levels of active, i.e. autophosphorylated, ErbB2 in HCT116 colon carcinoma cells and TA3/St mammary carcinoma cells are dependent on endogenous hyaluronan-CD44 interaction. Dependence on hyaluronan-CD44 interaction was demonstrated by the administration of hyaluronan oligomers, experimentally induced expression of soluble CD44, and small interfering RNA knockdown of CD44 expression. On the other hand, increasing hyaluronan production by overexpression of hyaluronan synthase 2 or emmprin causes elevated ErbB2 phosphorylation in MCF-7 mammary carcinoma cells, which normally exhibit low levels of ErbB2 activity. Furthermore, in HCT116 and TA3/St cells, inhibition of endogenous hyaluronan-CD44 interaction causes disassembly of a constitutive, lipid raft-associated, signaling complex containing phosphorylated ErbB2, CD44, ezrin, phosphoinositide 3-kinase, and the chaperone molecules, Hsp90 and cdc37. Stimulation of hyaluronan production in MCF-7 cells induces assembly of this complex. We conclude that hyaluronan regulates ErbB2 activity and its interactions with other signaling factors in carcinoma cells.

Breast Neoplasms↗

PubMed2005

Hypoxia-driven proliferation of human pulmonary artery fibroblasts: cross-talk between HIF-1alpha and an autocrine angiotensin system.

Pulmonary artery adventitial fibroblasts (FBPA) may play a central role in lung vascular remodeling under conditions of hypoxia and inflammation, the result being pulmonary hypertension and cor pulmonale. In cultured human FBPA, both angiotensin II (Ang II) and hypoxia promoted cell cycle progression and cell proliferation and suppressed apoptosis. These effects were further enhanced when both stimuli were applied simultaneously. Hypoxia elevated the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased the expression of genes regulated by the hypoxia-responsive element (HRE). Up-regulation of both angiotensin-converting enzyme (ACE) and Ang II receptor type 1 (AT1) was also observed. Exogenous Ang II further increased HIF/HRE-dependent signaling in FBPA, whereas suppression of the autocrine ACE-Ang II-AT1 loop with inhibitors of ACE, AT1, and phosphatidylinositol 3-kinase (PI3K) reduced the proliferative response to both hypoxia and exogenous Ang II. Overexpression of HIF-1alpha by transient transfection caused the same proliferative effect and up-regulation of AT1 expression that were observed under hypoxic conditions. In contrast, small interfering RNA targeting HIF-1alpha inhibited hypoxia-induced ACE and AT1 expression. Our studies indicate that the ACE-Ang II-AT1 system serves as a positive feedback loop and fosters FBPA proliferation under hypoxic conditions, with the PI3K-HIF-HRE axis as the central effector pathway. This pathway may thus facilitate vascular remodeling under hypoxic conditions.

Angiotensin II↗

PubMed2006

Downregulation of protein disulfide isomerase inhibits infection by the mouse polyomavirus.

Early stages of infection by the mouse polyomavirus have been studied using HeLa cells stably expressing small interfering RNA to protein disulfide isomerase (PDI). Infectibility measured by nuclear T antigen expression was reduced commensurately with the degree of PDI downregulation. Infectibility was restored by transfection with a plasmid expressing PDI but not with a control expressing catalytically inactive enzyme. Deconvolution microscopy using fluorescently labeled virus and cellular markers showed that virus reaches the endoplasmic reticulum (ER) normally in cells with reduced PDI but subsequently fails to exit the ER. Simian virus 40 infection was not inhibited in PDI-downregulated cells. The results are discussed in terms of structural differences between the two viruses and current knowledge of virus disassembly in the ER.

Animals↗

PubMed2005

Varicella-zoster virus activates inflammatory cytokines in human monocytes and macrophages via Toll-like receptor 2.

The pattern recognition receptor Toll-like receptor 2 (TLR2) has been implicated in the response to several human viruses, including herpes simplex viruses (types 1 and 2) and cytomegalovirus. We demonstrated that varicella-zoster virus (VZV) activates inflammatory cytokine responses via TLR2. VZV specifically induced interleukin-6 (IL-6) in human monocytes via TLR2-dependent activation of NF-kappaB, and small interfering RNA designed to suppress TLR2 mRNA reduced the IL-6 response to VZV in human monocyte-derived macrophages. Unlike other herpesviruses, the cytokine response to VZV was species specific. VZV did not induce cytokines in murine embryonic fibroblasts or in a mouse cell line, although VZV did activate NF-kappaB in a human cell line expressing a murine TLR2 construct. Together, these results suggest that TLR2 may play a role in the inflammatory response to VZV infection.

Animals↗

PubMed2005

PIAS1 activates the expression of smooth muscle cell differentiation marker genes by interacting with serum response factor and class I basic helix-loop-helix proteins.

Although a critical component of vascular disease is modulation of the differentiated state of vascular smooth muscle cells (SMC), the mechanisms governing SMC differentiation are relatively poorly understood. We have previously shown that E-boxes and the ubiquitously expressed class I basic helix-loop-helix (bHLH) proteins, including E2-2 and E12, are important in regulation of the SMC differentiation marker gene, the SM alpha-actin gene. The aim of the present study was to identify proteins that bind to class I bHLH proteins in SMC and modulate transcriptional regulation of SMC differentiation marker genes. Herein we report that members of the protein inhibitor of activated STAT (PIAS) family interact with class I bHLH factors as well as serum response factor (SRF). PIAS1 interacted with E2-2 and E12 based on yeast two-hybrid screens, mammalian two-hybrid assays, and/or coimmunoprecipitation assays. Overexpression of PIAS1 significantly activated the SM alpha-actin promoter and mRNA expression, as well as SM myosin heavy chain and SM22alpha, whereas a small interfering RNA for PIAS1 decreased activity of these promoters, as well as endogenous mRNA expression, and SRF binding to SM alpha-actin promoter within intact chromatin in cultured SMC. Of significance, PIAS1 bound to SRF and activated SM alpha-actin promoter expression in wild-type but not SRF(-/-) embryonic stem cells. These results provide novel evidence that PIAS1 modulates transcriptional activation of SMC marker genes through cooperative interactions with both SRF and class I bHLH proteins.

Actins↗

PubMed2004

MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

Adult↗

PubMed2006

Müllerian inhibiting substance regulates its receptor/SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Müllerian duct regression.

Examination of Müllerian inhibiting substance (MIS) signaling in the rat in vivo and in vitro revealed novel developmental stage- and tissue-specific events that contributed to a window of MIS responsiveness in Müllerian duct regression. The MIS type II receptor (MISRII)-expressing cells are initially present in the coelomic epithelium of both male and female urogenital ridges, and then migrate into the mesenchyme surrounding the male Müllerian duct under the influence of MIS. Expression of the genes encoding MIS type I receptors, Alk2 and Alk3, is also spatiotemporally controlled; Alk2 expression appears earlier and increases predominantly in the coelomic epithelium, whereas Alk3 expression appears later and is restricted to the mesenchyme, suggesting sequential roles in Müllerian duct regression. MIS induces expression of Alk2, Alk3 and Smad8, but downregulates Smad5 in the urogenital ridge. Alk2-specific small interfering RNA (siRNA) blocks both the transition of MISRII expression from the coelomic epithelium to the mesenchyme and Müllerian duct regression in organ culture. Müllerian duct regression can also be inhibited or accelerated by siRNA targeting Smad8 and Smad5, respectively. Thus, the early action of MIS is to initiate an epithelial-to-mesenchymal transition of MISRII-expressing cells and to specify the components of the receptor/SMAD signaling pathway by differentially regulating their expression.

Activin Receptors, Type I↗

PubMed2004

Down-regulation of sphingosine kinase-1 by DNA damage: dependence on proteases and p53.

Sphingosine kinase 1 (SK1), a key enzyme in sphingosine 1-phosphate (S1P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of sphingosine kinase (SK1). Act D caused a reduction in the protein levels of SK1, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was post-transcriptional, because the mRNA levels of SK1 remained unchanged. Similar decreases in SK1 protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and gamma-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK1, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK1. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK1 loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK1 decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK1 decrease. When p53 up-regulation after Act D treatment was inhibited, SK1 down-regulation was rescued, demonstrating p53 dependence of SK1 modulation. Treatment of cells with S1P, the product of SK1, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK1 may be in part necessary for cell death to occur. Furthermore, the knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth.

Apoptosis↗

PubMed2005

P2Y2 receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation.

Neurotrophins are essential for neuronal differentiation, but the onset and the intensity of neurotrophin signaling within the neuronal microenvironment are poorly understood. We tested the hypothesis that extracellular nucleotides and their cognate receptors regulate neurotrophin-mediated differentiation. We found that 5'-O-(3-thio)triphosphate (ATPgammaS) activation of the G protein-coupled receptor P2Y(2) in the presence of nerve growth factor leads to the colocalization and association of tyrosine receptor kinase A and P2Y(2) receptors and is required for enhanced neuronal differentiation. Consistent with these effects, ATPgammaS promotes phosphorylation of tyrosine receptor kinase A, early response kinase 1/2, and p38, thereby enhancing sensitivity to nerve growth factor and accelerating neurite formation in both PC12 cells and dorsal root ganglion neurons. Genetic or small interfering RNA depletion of P2Y(2) receptors abolished the ATPgammaS-mediated increase in neuronal differentiation. Moreover, in vivo injection of ATPgammaS into the sciatic nerve increased growth-associated protein-43 (GAP-43), a marker for axonal growth, in wild-type but not P2Y(2)(-/-) mice. The interactions of tyrosine kinase- and P2Y(2)-signaling pathways provide a paradigm for the regulation of neuronal differentiation and suggest a role for P2Y(2) as a morphogen receptor that potentiates neurotrophin signaling in neuronal development and regeneration.

Adenosine Triphosphate↗

PubMed2006

The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair.

Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

ATP-Binding Cassette Transporters↗

PubMed2006

Cofilin plays a critical role in IL-8-dependent chemotaxis of neutrophilic HL-60 cells through changes in phosphorylation.

Cofilin is a ubiquitous, actin-binding protein. Only unphosphorylated cofilin binds actin and severs or depolymerizes filamentous actin (F-actin), and the inactive form of cofilin is phosphorylated at Ser 3. We reported recently that cofilin plays a regulatory role in superoxide production and phagocytosis by leukocytes, and in the present study, we investigated the role of cofilin in the chemotaxis of neutrophilic HL-60 cells. IL-8 is a potent, physiological chemokine, and it triggers a rapid, transient increase in F-actin beneath the plasma membrane and rapid dephosphorylation and subsequent rephosphorylation of cofilin. In this study, cofilin phosphorylation was found to be inhibited by S3-R peptide, which consists of a peptide corresponding to part of the phosphorylation site of cofilin and a membrane-permeable arginine polymer. When S3-R peptide was introduced into the neutrophilic cells, their chemotactic activity was enhanced, whereas a control peptide that contained an inverted sequence of the phosphorylation site of cofilin had no enhancing effect. Cofilin small interfering RNA (siRNA) decreased cofilin expression by about half and inhibited chemotaxis. In IL-8-stimulated cells, unphosphorylated cofilin accumulated around F-actin, and colocalization of F-actin and phosphorylated cofilin was observed, but these changes in cofilin localization were less prominent in cofilin siRNA-treated cells. The inhibitors of PI-3K wortmannin and LY294002 inhibited the chemotaxis and suppressed IL-8-evoked dephosphorylation and rephosphorylation of cofilin. These results suggested that unphosphorylated cofilin plays a critical role in leukocyte chemotaxis and that PI-3K is involved in the control of the phosphorylation/dephosphorylation cycle of cofilin.

Actin Depolymerizing Factors↗

PubMed2006

Class II phosphoinositide 3-kinase alpha-isoform regulates Rho, myosin phosphatase and contraction in vascular smooth muscle.

We demonstrated previously that membrane depolarization and excitatory receptor agonists such as noradrenaline induce Ca2+-dependent Rho activation in VSM (vascular smooth muscle), resulting in MP (myosin phosphatase) inhibition through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit MYPT1. In the present study, we show in de-endothelialized VSM strips that the PI3K (phosphoinositide 3-kinase) inhibitors LY294002 and wortmannin inhibited KCl membrane depolarization- and noradrenaline-induced Rho activation and MYPT1 phosphorylation, with concomitant inhibition of MLC (20-kDa myosin light chain) phosphorylation and contraction. LY294002 also augmented de-phosphorylation of MLC and resultantly relaxation in KCl-contracted VSM, whereas LY294002 was much less effective or ineffective under the conditions in which MP was inhibited by either a phosphatase inhibitor or a phorbol ester in Rho-independent manners. VSM express at least four PI3K isoforms, including the class I enzymes p110alpha and p110beta and the class II enzymes PI3K-C2alpha and -C2beta. The dose-response relationships of PI3K-inhibitor-induced inhibition of Rho, MLC phosphorylation and contraction were similar to that of PI3K-C2alpha inhibition, but not to that of the class I PI3K inhibition. Moreover, KCl and noradrenaline induced stimulation of PI3K-C2alpha in a Ca2+-dependent manner, but not of p110alpha or p110beta. Down-regulation of PI3K-C2alpha expression by siRNA (small interfering RNA) inhibited contraction and phosphorylation of MYPT1 and MLC in VSM cells. Finally, intravenous wortmannin infusion induced sustained hypotension in rats, with inhibition of PI3K-C2alpha activity, GTP-loading of Rho and MYPT1 phosphorylation in the artery. These results indicate the novel role of PI3K-C2alpha in Ca2+-dependent Rho-mediated negative control of MP and thus VSM contraction.

Animals↗

PubMed2004

Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors.

Bcr-Abl kinase is known to reverse apoptosis of cytokine-dependent cells due to cytokine deprivation, although it has been controversial whether chronic myeloid leukemia (CML) progenitors have the potential to survive under conditions in which there are limited amounts of cytokines. Here we demonstrate that early hematopoietic progenitors (Sca-1(+) c-Kit(+) Lin(-)) isolated from normal mice rapidly undergo apoptosis in the absence of cytokines. In these cells, the expression of Bim, a proapoptotic relative of Bcl-2 which plays a key role in the cytokine-mediated survival system, is induced. In contrast, those cells isolated from our previously established CML model mice resist apoptosis in cytokine-free medium without the induction of Bim expression, and these effects are reversed by the Abl-specific kinase inhibitor imatinib mesylate. In addition, the expression levels of Bim are uniformly low in cell lines established from patients in the blast crisis phase of CML, and imatinib induced Bim in these cells. Moreover, small interfering RNA that reduces the expression level of Bim effectively rescues CML cells from apoptosis caused by imatinib. These findings suggest that Bim plays an important role in the apoptosis of early hematopoietic progenitors and that Bcr-Abl supports cell survival in part through downregulation of this cell death activator.

Animals↗

PubMed2004

Developmentally essential protein flightless I is a nuclear receptor coactivator with actin binding activity.

Hormone-activated nuclear receptors (NR) activate transcription by recruiting multiple coactivator complexes to the promoters of target genes. One important coactivator complex includes a p160 coactivator (e.g., GRIP1, SRC-1, or ACTR) that binds directly to activated NR, the histone acetyltransferase p300 or CBP, and the arginine-specific histone methyltransferase CARM1. We previously demonstrated that the coactivator function of CARM1 depends both on the methyltransferase activity and on additional unknown proteins that bind to CARM1. In this study a yeast two-hybrid screen for proteins that bind CARM1 identified the protein Flightless I (Fli-I), which has essential roles in Drosophila and mouse development. Fli-I bound to CARM1, GRIP1, and NRs and cooperated synergistically with CARM1 and GRIP1 to enhance NR function. Fli-I bound poorly to and did not cooperate with PRMT1, a CARM1-related protein arginine methyltransferase that also functions as an NR coactivator. The synergy between GRIP1, CARM1, and Fli-I required the methyltransferase activity of CARM1. The C-terminal AD1 (binding site for p300/CBP) and AD2 (binding site for CARM1) activation domains of GRIP1 contributed to the synergy but were less stringently required than the N-terminal region of GRIP1, which is the binding site for Fli-I. Endogenous Fli-I was recruited to the estrogen-regulated pS2 gene promoter of MCF-7 cells in response to the hormone, and reduction of endogenous Fli-I levels by small interfering RNA reduced hormone-stimulated gene expression by the endogenous estrogen receptor. A fragment of Fli-I that is related to the actin binding protein gelsolin enhanced estrogen receptor activity, and mutations that reduced actin binding also reduced the coactivator function of this Fli-I fragment. These data suggest that Fli-I may facilitate interaction of the p160 coactivator complex with other coactivators or coactivator complexes containing actin or actin-like proteins.

Actins↗

PubMed2005

Decreased growth of Vhl-/- fibrosarcomas is associated with elevated levels of cyclin kinase inhibitors p21 and p27.

Inactivating mutations within the von Hippel-Lindau (VHL) tumor suppressor gene predispose patients to develop a variety of highly vascularized tumors. pVHL targets alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), a critical regulator of energy metabolism, angiogenesis, hematopoiesis, and oxygen (O(2)) delivery, for ubiquitin-mediated degradation in an O(2)-dependent manner. To investigate the role of Vhl in cellular proliferation and tumorigenesis, we utilized mouse embryonic fibroblasts (MEFs), a common tool for analyzing cell cycle regulation, and generated Vhl(-)(/)(-) MEF-derived fibrosarcomas. Surprisingly, growth of both Vhl(-)(/)(-) MEFs and fibrosarcomas was impaired, although tumor vascularity was increased. Decreased proliferation of Vhl(-)(/)(-) MEFs was correlated with an overexpression of cyclin kinase inhibitors (CKIs) p21 and p27. The transcription of p21 and p27 is inhibited by c-Myc; therefore, the induction of CKIs was attributed to the ability of HIF to antagonize c-Myc activity. Indeed, p21 mRNA levels were elevated under normoxia in Vhl(-)(/)(-) MEFs, while c-Myc transcriptional activity was markedly reduced. Gene silencing of HIF-1alpha by small interfering RNA reduced p21 and p27 protein and mRNA levels in Vhl(-)(/)(-) MEFs. The induction of p21 and p27, mediated by constitutive activation of the HIF pathway, provides a mechanism for the decreased proliferation rates of Vhl(-)(/)(-) MEFs and fibrosarcomas. These results demonstrate that a loss of pVHL can induce growth arrest in certain cells types, which suggests that additional genetic mutations are necessary for VHL-associated tumorigenesis.

Animals↗

PubMed2006

Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells.

Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes, where it is often referred to as an "insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types, agents that facilitate signaling through these receptors would be expected to enhance proliferation. We show here that metformin acts as a growth inhibitor rather than an insulin sensitizer for epithelial cells. Breast cancer cells can be protected against metformin-induced growth inhibition by small interfering RNA against AMP kinase. This shows that AMP kinase pathway activation by metformin, recently shown to be necessary for metformin inhibition of gluconeogenesis in hepatocytes, is also involved in metformin-induced growth inhibition of epithelial cells. The growth inhibition was associated with decreased mammalian target of rapamycin and S6 kinase activation and a general decrease in mRNA translation. These results provide evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent population studies and justify further work to explore potential roles for activators of AMP kinase in cancer prevention and treatment.

Adenylate Kinase↗

PubMed2005

The methyltransferase inhibitor 5-aza-2-deoxycytidine induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells.

DNA methylation by DNA methyltransferases in CpG-rich promoter regions of genes is a well-described component of epigenetic silencing in human cells. Dysregulation of this process in cancer cells may lead to hypermethylation of promoter CpG islands, thus disabling transcription initiation of certain genes, such as tumor suppressor genes. Reversing epigenetic silencing and up-regulating genes involved in preventing or reversing the malignant phenotype has become a new, important targeted approach for cancer prevention and treatment. Therefore, methyltransferase inhibitors (MTI) have emerged recently as promising chemotherapeutic or preventive agents. The potent MTI 5-aza-2-deoxycytidine (5-Azadc) causes growth arrest, differentiation, and/or apoptosis of many tumor types in vitro and in vivo. The present study shows that low micromolar concentrations of 5-Azadc induce the expression of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer cells. The expression of 15-LOX-1 correlates with 5-Azadc-induced increases in 13-S-hydroxyoctadecadienoic acid levels, growth inhibition, and apoptosis in these cells. Furthermore, specific inhibition of 15-LOX-1 by pharmacologic means or small interfering RNA significantly reduced the 5-Azadc-induced effects. These novel findings are the first demonstration of a mechanistic link between the induction of 15-LOX-1 by a MTI and apoptosis in cancer cells. This result has important implications for the study of 5-Azadc and other MTIs in the prevention and therapy of colorectal cancer and supports future investigations of the mechanisms by which MTIs up-regulate 15-LOX-1.

Antimetabolites, Antineoplastic↗