RESUMO
Premature senescence may play an important role as an acute, drug-, or ionizing radiation (IR)-inducible growth arrest program along with interphase apoptosis and mitotic catastrophe. The aim of the study was to evaluate whether IR can induce senescence-like phenotype (SLP) associated with terminal growth arrest in the thyroid cells, and if so, to evaluate impact of terminal growth arrest associated with SLP in intrinsic radiosensitivity of various thyroid carcinomas. The induction of SLP in thyroid cells were identified by: (1) senescence associated beta-galactosidase (SA-beta-Gal) staining method, (2) dual-flow cytometric analysis of cell proliferation and side light scatter using vital staining with PKH-2 fluorescent dye, (3) double labeling for 5-bromodeoxyuridine and SA- beta-Gal, (4) Staining for SA-beta-Gal with consequent antithyroglobulin immunohistochemistry. IR induced SLP associated with terminal growth arrest in four thyroid cancer cells lines and in primary thyrocytes in time- and dose-dependent manner. Analysis of relationship between induction of SLP and radiosensitivity revealed a trend in which more radioresistant cell lines strongly tended to show lower specific SLP yields (r = -0.93, p = 0.068). We find out that SA-beta-Gal staining is detectable in irradiated ARO xenotransplants, but not in control tumors. We, therefore, conclude that induction of SLP with terminal growth arrest contribute to the elimination of clonogenic populations after IR.
Assuntos
Senescência Celular/efeitos da radiação , Glândula Tireoide/efeitos da radiação , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitose , Tolerância a Radiação , beta-Galactosidase/análiseRESUMO
PURPOSE: The objective of the study was to determine the effects of a novel selective nuclear factor kappaB (NF-kappaB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in thyroid carcinoma cells in vitro and in vivo and to additionally elucidate the molecular mechanisms underlying the action of this chemotherapeutic agent. EXPERIMENTAL DESIGN: In the in vitro experiments, the induction of apoptosis by DHMEQ in various human thyroid carcinoma cell types was determined by flow cytometry analysis of annexin-V binding and the caspase activation by Western blotting. For the in vivo study, female nu/nu mice were xenografted with s.c. FRO thyroid tumors. DHMEQ solution was injected i.p. at a dose of 8 mg/kg/day for two weeks. Tumor dimensions were monitored twice weekly, and apoptosis in tumor specimens was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. RESULTS: Treatment with DHMEQ substantially inhibited the translocation of p65 and p50 NF-kappaB subunits to the nucleus, the DNA-binding activity of the RelA/p65, NF-kappaB-dependent expression of the inhibitor of apoptosis (IAP)-family proteins, cIAP-1, cIAP-2, and XIAP, and the de novo synthesis of inhibitor of nuclear factor kappaB alpha. At concentration levels ranging from 0.1 to 5 microg/ml, DHMEQ induced a caspase-mediated apoptotic response that could be abrogated by the c-Jun NH(2)-terminal kinase inhibitor SP600125 but not by either mitogen-activated protein/extracellular signal-regulated kinase kinase or p38 inhibitors. In contrast, normal human thyrocytes were resistant to DHMEQ-induced apoptosis. At higher doses of DHMEQ we observed the necrotic-like killing of both normal and malignant thyrocytes, which was resistant to mitogen-activated protein kinase inhibitors. In nude mice DHMEQ substantially inhibited tumor growth without observable side effects, and increased numbers of apoptotic cells were observed in the histologic sections of tumors treated with DHMEQ. CONCLUSIONS: Our results show the potential usefulness of the novel NF-kappaB inhibitor, DHMEQ, in future therapeutic strategies for the treatment of thyroid cancers that do not respond to conventional approaches.
Assuntos
Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Carcinoma/patologia , Cicloexanonas/farmacologia , Neoplasias da Glândula Tireoide/patologia , Humanos , NF-kappa B/antagonistas & inibidores , NF-kappa B/farmacologia , Células Tumorais CultivadasRESUMO
Nuclear transcription factor-kappaB (NF-kappaB) is a transcriptional complex that is rapidly activated in the course of an immediate early response of cells after exposure to different stresses including ionizing radiation (IR). To overcome the limitation of radiation therapy for thyroid cancers, we studied the response of the NF-kappaB cascade to IR in cultured normal human thyroid cells and various thyroid cancer cell lines. Exposure to IR resulted in a dose-dependent increase of DNA-binding activity of p65 and p50 subunits in all types of thyroid cells. Specific inhibitors of NF-kappaB or phosphorylation deficient mutant inhibitory protein IkappaBalpha reduced thyroid cancer cell survival after exposure to IR and enhanced IR-induced cell death in a model undifferentiated thyroid cancer cell line. Tumors harboring mutant IkappaBalpha implanted into nude mice exhibited delayed growth rate and increased radiosensitivity. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and annexinV-propidium iodide staining revealed the increase of radiation-induced apoptosis in the cells with inhibited NF-kappaB signaling. Our results indicate that radiosensitivity of transformed thyroid cells is due in part to elevated basal activity and rapid induction of the active form of NF-kappaB. We therefore suggest that inhibition of NF-kappaB could be an effective modality for radiation therapy of advanced human thyroid cancers.