ABSTRACT
Our previous study found that splicing factor polypyrimidine tract-binding protein 1 (PTBP1) had a role in tumorigenesis but the underlying mechanism remained unclear. In this study, we observed that knockdown of PTBP1 inhibited filopodia formation. Subsequently, we found that PTBP1 regulated the alternative splicing of CDC42, a major regulator of filopodia formation. Two CDC42 variants, CDC42-v1 and CDC42-v2, can be generated through alternative splicing. Knockdown of PTBP1 increased the expression of CDC42-v2. Ectopic expression of individual variants showed that CDC42-v2 suppressed filopodia formation, opposite to the effect of CDC42-v1. Quantitative RT-PCR revealed that CDC42-v2 was expressed at lower levels in ovarian cancer cell lines and ovarian tumor tissues than in normal control cells and tissues. Further, CDC42-v2 was observed to have inhibitory effects on ovarian tumor cell growth, colony formation in soft agar and invasiveness. In contrast, these inhibitory effects were not found with CDC42-v1. Taken together, above results suggest that the role of PTBP1 in tumorigenesis may be partly mediated by its regulation of CDC42 alternative splicing and CDC42-v2 might function as a tumor suppressor.
Subject(s)
Alternative Splicing , Gene Expression Regulation, Neoplastic , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Polypyrimidine Tract-Binding Protein/genetics , cdc42 GTP-Binding Protein/genetics , Animals , Base Sequence , Blotting, Western , Cell Line, Tumor , Cell Proliferation/genetics , Female , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Polypyrimidine Tract-Binding Protein/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Pseudopodia/genetics , Pseudopodia/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Tumor Stem Cell Assay , cdc42 GTP-Binding Protein/metabolismABSTRACT
Extract from the cultured freshwater cf. Oscillatoria sp. UIC 10045 showed antiproliferative activity against HT-29 cell line. Bioassay-guided fractionation led to the isolation of two new cyclic lipopeptides, named trichormamides C (1) and D (2). The planar structures were determined by combined analyses of HRESIMS, Q-TOF ESIMS/MS, and 1D and 2D NMR spectra. The absolute configurations of the amino acid residues were assigned by advanced Marfey's analysis after partial and complete acid hydrolysis. Trichormamides C (1) is a cyclic undecapeptide and D (2) is a cyclic dodecapeptide, both containing a lipophilic ß-aminodecanoic acid residue. Trichormamide C (1) displayed antiproliferative activities against HT-29 and MDA-MB-435 cancer cell lines with IC50 values of 1.7 and 1.0µM, respectively, as well as anti-Mycobacterium tuberculosis activity with MIC value of 23.8µg/mL (17.3µM). Trichormamide D (2) was found to be less potent against both HT-29 and MDA-MB-435 cancer cell lines with IC50 values of 11.5 and 11.7µM, respectively.
Subject(s)
Anti-Bacterial Agents/chemistry , Antineoplastic Agents/chemistry , Cyanobacteria/chemistry , Lipopeptides/chemistry , Peptides, Cyclic/chemistry , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Drug Design , Drug Screening Assays, Antitumor , HT29 Cells , Humans , Inhibitory Concentration 50 , Lipopeptides/isolation & purification , Lipopeptides/pharmacology , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/growth & development , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Structure-Activity RelationshipABSTRACT
Bioactivity-directed fractionation of the organic extracts of two filamentous fungi of the Bionectriaceae, strains MSX 64546 and MSX 59553 from the Mycosynthetix library, led to the isolation of a new dimeric epipolythiodioxopiperazine alkaloid, verticillin H (1), along with six related analogs, Sch 52900 (2), verticillin A (3), gliocladicillin C (4), Sch 52901 (5), 11'-deoxyverticillin A (6) and gliocladicillin A (7). The structures of compounds 1-7 were determined by extensive NMR and HRMS analyses, as well as by comparisons to the literature. All compounds (1-7) were evaluated for cytotoxicity against a panel of human cancer cell lines, displaying IC(50) values ranging from 1.2 µM to 10 nM. Compounds 1-5 were examined for activity in the NF-κB assay, where compounds 2 and 3 revealed activity in the sub-micromolar range. Additionally, compounds 1, 3 and 4 were tested for EGFR inhibition using an enzymatic assay, while compound 3 was examined against an overexpressing EGFR(+ve) cancer cell line.
Subject(s)
Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Disulfides/isolation & purification , Disulfides/pharmacology , Hypocreales/chemistry , Indole Alkaloids/isolation & purification , Indole Alkaloids/pharmacology , Piperazines/isolation & purification , Piperazines/pharmacology , Terphenyl Compounds/isolation & purification , Terphenyl Compounds/pharmacology , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/isolation & purification , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Disulfides/chemistry , Drug Screening Assays, Antitumor , ErbB Receptors/antagonists & inhibitors , Humans , Indole Alkaloids/chemistry , Indoles/chemistry , Indoles/isolation & purification , Indoles/pharmacology , Magnetic Resonance Spectroscopy , Molecular Structure , NF-kappa B/antagonists & inhibitors , Piperazines/chemistry , Terphenyl Compounds/chemistryABSTRACT
Dolichols are long-chain unsaturated polyisoprenoids with multiple cellular functions, such as serving as lipid carriers of sugars used for protein glycosylation, which affects protein trafficking in the endoplasmic reticulum. The biological functions of dolichols in plants are largely unknown. We isolated an Arabidopsis thaliana mutant, lew1 (for leaf wilting1), that showed a leaf-wilting phenotype under normal growth conditions. LEW1 encoded a cis-prenyltransferase, which when expressed in Escherichia coli catalyzed the formation of dolichol with a chain length around C(80) in an in vitro assay. The lew1 mutation reduced the total plant content of main dolichols by approximately 85% and caused protein glycosylation defects. The mutation also impaired plasma membrane integrity, causing electrolyte leakage, lower turgor, reduced stomatal conductance, and increased drought resistance. Interestingly, drought stress in the lew1 mutant induced higher expression of the unfolded protein response pathway genes BINDING PROTEIN and BASIC DOMAIN/LEUCINE ZIPPER60 as well as earlier expression of the stress-responsive genes RD29A and COR47. The lew1 mutant was more sensitive to dark treatment, but this dark sensitivity was suppressed by drought treatment. Our data suggest that LEW1 catalyzes dolichol biosynthesis and that dolichol is important for plant responses to endoplasmic reticulum stress, drought, and dark-induced senescence in Arabidopsis.
