RESUMO
Three types of transmembrane protein, IRE1α/IRE1ß, PERK, and ATF6α/ATF6ß, are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1α inhibitors. IRE1α, IRE1ß, ATF6α and ATF6ß are ubiquitously expressed in medaka. We found that IRE1α/ATF6α-double knockout is lethal, similarly to IRE1α/IRE1ß- and ATF6α/ATF6ß-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6α-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1α in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1α inhibitors in vivo.Key words: endoplasmic reticulum, inhibitor screening, mRNA splicing, phenotypic assay, unfolded protein response.
Assuntos
Endonucleases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Endonucleases/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Oryzias , Proteínas Serina-Treonina Quinases/genética , Fatores de TempoRESUMO
Antiestrogen agents are commonly used to treat patients with estrogen receptor (ER)-positive breast cancer. Tamoxifen has been the mainstay of endocrine treatment for patients with early and advanced breast cancer for many years. Following tamoxifen treatment failure, however, there are still limited options for subsequent hormonal therapy. We discovered a novel compound, NK150460, that inhibits 17ß-estradiol (E2)-dependent transcription without affecting binding of E2 to ER. Against our expectations, NK150460 inhibited growth of not only most ER-positive, but also some ER-negative breast cancer cell lines, while never inhibiting growth of non-breast cancer cell lines. Cell-based screening using a random shRNA library, identified aryl hydrocarbon receptor nuclear translocator (ARNT) as a key gene involved in NK150460's antitumor mechanism. siRNAs against not only ARNT but also its counterpart aryl hydrocarbon receptor (AhR) and their target protein, CYP1A1, dramatically abrogated NK150460's growth-inhibitory activity. This suggests that the molecular cascade of AhR/ARNT plays an essential role in NK150460's antitumor mechanism. Expression of ERα was decreased by NK150460 treatment, and this was inhibited by an AhR antagonist. Unlike two other AhR agonists now undergoing clinical developmental stage, NK150460 did not induce histone H2AX phosphorylation or p53 expression, suggesting that it did not induce a DNA damage response in treated cells. Cell lines expressing epithelial markers were more sensitive to NK150460 than mesenchymal marker-expressing cells. These data indicate that NK150460 is a novel AhR agonist with selective antitumor activity against breast cancer cell lines, and its features differ from those of the other two AhR agonists.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Quinolinas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citocromo P-450 CYP1A1/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , Estrogênios/farmacologia , Feminino , Compostos Heterocíclicos com 2 Anéis/química , Humanos , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Camundongos Nus , Quinolinas/química , Ratos Nus , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Topoisomerase II (Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2alpha is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2beta is nonessential for growth and has recently been implicated in treatment-associated secondary malignancies, highlighting the validity of a Top2alpha-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c]phenanthridine alkaloid, targets Top2alpha and not Top2beta in vivo. Unlike other Top2 inhibitors, NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an alpha isoform-specific manner. Heterozygous disruption of the human TOP2alpha gene confers increased NK314 resistance, whereas TOP2beta homozygous knock-out cells display increased NK314 sensitivity, indicating that the alpha isoform is the cellular target. We further show that the absence of Top2beta does not alleviate NK314 hypersensitivity of cells deficient in non-homologous end-joining, a critical pathway for repairing Top2-mediated DSBs. Our results indicate that NK314 acts as a Top2alpha-specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock-out cell lines are useful in assessing DNA damage and repair induced by potential topoisomerase-targeting agents.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Fenantrenos/farmacologia , Inibidores da Topoisomerase II , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/uso terapêutico , Deleção de Genes , Células HeLa , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , Fenantrenos/uso terapêutico , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that shows strong antitumor activity. It inhibited topoisomerase II activity and stabilized topoisomerase II-DNA cleavable complexes. The DNA breaks occurred within 1h after treatment with NK314 even without digestion of topoisomerase II by proteinase K, whereas etoposide required digestion of the enzyme protein in cleavable complex to detect DNA breaks. Pretreatment with topoisomerase II catalytic inhibitors, ICRF-193 and suramin, reduced both cleavable complex-mediated DNA breaks and proteinase K-independent DNA breaks, but protease inhibitors and nuclease inhibitors only decreased the latter. These results indicate that NK314 might affect topoisomerase II in the different manner from cleavable complex formation and activate intracellular proteinase and nuclease to produce DNA fragmentation. As a result of this unique mechanism of DNA breakage, NK314 showed substantial growth inhibition of topoisomerase II inhibitor-resistant tumors.