Bile acid-induced colon cancer cell proliferation

胆汁酸诱导结肠癌细胞增殖

• 批准号: 7422304
• 负责人: JEAN-PIERRE RAUFMAN
• 金额: $ 21.39万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-28 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7422304
• 关键词: Address; Animals; Antibodies; Antisense Oligonucleotides; Bile Acids; Binding; Biological Assay; CREB1 gene; Cell Proliferation; Cell membrane; Cells; Cholesterol Homeostasis; Cholinergic Agonists; Cholinergic Receptors; Colon Carcinoma; Complementary DNA; DTR gene; Data; Dependence; Dominant-Negative Mutation; Enzymes; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; FOS gene; Genes; Genetic Transcription; Human; Immune Sera; Immunoblotting; In Situ Hybridization; JUN gene; Ligands; Lipids; MAPK14 gene; MAPK8 gene; Mediating; Metalloproteases; Molecular; Muscarinic Acetylcholine Receptor; Muscarinic M3 Receptor; Muscarinics; NF-kappa B; Northern Blotting; Nuclear Receptors; Pathway interactions; Phospholipase C; Phosphorylation; Protein Kinase C; Proteins; Receptor Activation; Receptor Signaling; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Testing; Transactivation; Transduction Gene; absorption; antibody inhibitor; cancer cell; cancer risk; citrate carrier; diphtheria toxin receptor; heparin-binding EGF-like growth factor; inhibitor/antagonist; knock-down; programs; radioligand; receptor; transcription factor

DESCRIPTION (provided by applicant): The central hypothesis to be tested in this proposal is that bile acids induce colon cancer cell proliferation by interaction with M3 muscarinic receptors (M3R), thereby causing transactivation of epidermal growth factor receptors (EGFR). To elucidate post-receptor signaling that results in bile acid-induced colon cancer cell proliferation and to determine the requirement for, and mechanism of, transactivation of EGFR the following Specific Aims are proposed: 1. To elucidate further post-receptor signal transduction pathways which mediate cholinergic agonist-induced transactivation of EGFR and stimulate colon cancer cell proliferation. 1a. For use in the proposed studies, in addition to cells that naturally express M3R and EGFR, colon cancer cells will be transfected with cDNA clones for M3R and/or EGFR. 1b. The mechanism of cholinergic agonist-induced cell proliferation will be studied using immunoblotting to probe for activated proteins in the p44/42 MARK, p38 MARK and JNK pathways, and by examining the roles of phospholipase C and protein kinase C activation and Ca2+ mobilization.1c. The requirement for cholinergic agonist-induced transactivation of EGFR will be confirmed by examining EGFR phosphorylation and by using EGFR inhibitors, antisense oligonucleotides, and dominant negative mutants.2. To determine the requirement for co-expression of M3R and EGFR and delineate the molecular mechanisms whereby bile acids regulate colon cancer cell proliferation. 2a. Bile acid-induced post-receptor signaling and the requirement for co-expression of M3R and EGFR will be determined using radioligand binding assays, immunoblotting for activated p44/42 MARK, p38 MARK and JNK cascade proteins, and by using EGFR inhibitors, antisense oligonucleotides, and dominant negative mutants. 2b. Bile acid-induced activation and expression of transcription factors (p90RSK, p38 MARK and JNK) and genes (CREB, NF-kappaB, c-Fos and c-Jun) related to colon cancer proliferation will be elucidated. 3. To determine the molecular mechanism in human colon cancer cells of bile acid-induced transactivation of EGFR. 3a) Expression and release of EGFR ligands, including HB-EGF, will be determined using immunoblots, northern blots, and RT-PCR, and the dependence of bile acid-induced EGFR transactivation on release of EGFR ligands will be determined using EGFR antibodies, metalloproteinase inhibitors, and specific antibodies and inhibitors for EGFR. 3b) Colon cancer cell metalloproteases will be identified by immunoblotting and in situ hybridization and the role of these enzymes in mediating bile acid-induced HB-EGF release will be determined by using metalloprotease inhibitors and antisera, and by knocking down metalloprotease expression. 3c) The mechanism whereby bile acids activate metalloproteases will be determined by exploring the roles of PKC, Ca2+, and Src in bile acid-induced HB-EGF release.

描述（由申请人提供）：该提案中要检验的中心假设是胆汁酸通过与M3毒蕈碱受体（M3R）相互作用诱导结肠癌细胞增殖，从而导致表皮生长因子受体（EGFR）的反式激活。为了阐明受感染后的信号传导，导致胆汁酸诱导的结肠癌细胞增殖并确定EGFR反式激活的需求和机制，提出了以下特定目的：1。阐明了进一步的后促进剂促进的EGFR和刺激性刺激性刺激性刺激性刺激性刺激性刺激性刺激性刺激性刺激性和刺激性刺激性刺激性刺激性的促进后信号转导途径。 1a。为了用于拟议的研究，除了自然表达M3R和EGFR的细胞外，结肠癌细胞还将用M3R和/或EGFR转染结肠癌细胞。 1B。将使用免疫印迹来研究胆碱能激动剂诱导的细胞增殖的机制，以探测p44/42 Mark，p38 mark和JNK途径中激活的蛋白质的活化蛋白，并检查磷脂酶C和蛋白质激酶C活化和CA2+ Motilization的机制。通过检查EGFR磷酸化和使用EGFR抑制剂，反义寡核苷酸和显性阴性突变体，可以证实胆碱能激动剂诱导的EGFR反式活化的需求。2。为了确定M3R和EGFR共表达的需求，并描绘了胆汁酸调节结肠癌细胞增殖的分子机制。 2a。胆汁酸诱导的受损伤后信号传导以及M3R和EGFR共表达的需求将使用放射性结合测定法确定，用于激活的P44/42 MARK，p38 MARK和JNK CASCADE CASCADE蛋白的免疫印迹，并使用EGFR抑制剂，抗浓度寡核酸固醇和抗氧化甲基核酸固醇和替代剂。 2b。胆汁酸诱导的转录因子的激活和表达（P90RSK，P38 MARK和JNK）以及与结肠癌增殖有关的基因（CREB，NF-KAPPAB，C-FOS和C-JUN）将被阐明。 3。确定胆汁酸诱导的EGFR反式激活的人类结肠癌细胞中的分子机制。 3A）将使用免疫印迹，北印迹和RT-PCR确定EGFR配体的表达和释放，包括HB-EGF，以及使用EGFR抗体，金属蛋白酶抑制剂和特定的抗生素来确定胆汁酸诱导的EGFR反式激活对EGFR配体释放的依赖性。 3B）结肠癌细胞金属蛋白酶将通过免疫印迹和原位杂交来鉴定，并且这些酶在介导胆汁酸诱导的HB-EGF释放中的作用将通过使用金属蛋白酶抑制剂和抗血液来确定，并通过使用金属蛋白酶抑制蛋白抑制剂来确定。 3C）通过探索PKC，Ca2+和SRC在胆汁酸诱导的HB-EGF释放中的作用来确定胆汁酸激活金属蛋白酶的机制。