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1.
Radiat Res ; 200(1): 21-31, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37212749

RESUMO

This study conducted fundamental research to develop a more effective BNCT targeting cancer stem cells. We constructed plasmids that induced the overexpression of L-type amino acid transporter 1 (LAT1) tagged with tdTomato on the cytoplasmic membranes of CD133 expressing cancer cells. After transfection of the plasmids into a glioblastoma cell line (T98G), several clones overexpressing LAT1-tdTomato in the hypoxic microenvironment of the spheroids formed from each clone were obtained. Confocal laser microscopic observation confirmed that signals from LAT1-tdTomato overlapped with immunofluorescence signals from the second antibody binding to CD133 in the hypoxic microenvironment of the spheroids. As CD133-positive cells in the hypoxic microenvironment of T98G spheroids have cancer stem cell characteristics, LAT1 seems to be selectively overexpressed in cancer stem cell-like cells. An RI tracer method showed that cells overexpressing LAT1-tdTomato in the hypoxic microenvironment of spheroids incorporate 14C-BPA much more than cells that do not overexpress LAT1-tdTomato. Neutron radiation experiments showed a more significant regression in spheroids formed with clones than in spheroids formed with parental cells when spheroids were treated with 10BPA. These results suggest that BNCT combined with gene therapy targeting cancer stem cells is more effective in glioblastoma therapy.


Assuntos
Terapia por Captura de Nêutron de Boro , Glioblastoma , Humanos , Glioblastoma/radioterapia , Linhagem Celular Tumoral , Terapia por Captura de Nêutron de Boro/métodos , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Células-Tronco Neoplásicas/metabolismo , Microambiente Tumoral
2.
Oncol Lett ; 23(3): 102, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35154433

RESUMO

The present study examined the radiosensitization induced by a heat shock protein 90 inhibitor, N-vinylpyrrolidone (NVP)-AUY922, in CD133-positive cells in a hypoxic area of T98G spheroids. CD133-positive cells that are induced in the hypoxic microenvironment of spheroids have previously been reported to exhibit cancer stem cell-like properties. The present study used CD133-positive cells from a glioblastoma cell line (T98G) as cancer stem cell-like cells. CD133-positive and negative cells were sorted from T98G spheroids using fluorescence-activated cell sorting and used for colony formation assay. Colony formation assay results indicated that NVP-AUY922 enhanced radiosensitivity more strongly in CD133-positive cells compared with CD133-negative cells. This result showed that NVP-AUY922 was a preferential radiosensitization candidate targeting glioblastoma cancer stem cells. The mechanisms underlying radiosensitization by NVP-AUY922 are discussed in relation to the properties of cancer stem cells. Overall, HIF-1α inhibition by NVP-AUY922 may induce higher sensitization of cancer stem cells to radiation.

3.
Radiat Res ; 196(1): 17-22, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33956158

RESUMO

In this study, we examined whether the cancer cell-killing effects of boron neutron capture therapy (BNCT) are enhanced by manipulating the expression levels of l-type amino acid transporter 1 (LAT1) of human cancer cells, which transports boronophenylalanine into cells. We transfected pCMV/LAT1-GFP plasmids into a T98G glioblastoma cell line and selected several clones. Confocal laser microscopic observation was performed to confirm the stable overexpression of LAT1 in the plasma membranes of the clones. Western blot was used to analyze the cellular accumulation of LAT1 protein in the clones. Relative intracellular uptake of boronophenylalanine (BPA) was determined by measuring the radioactivity of 14C-BPA using a radioactive iodine (RI) tracer method. Sensitivity to neutron and gamma (γ)-ray fluences generated by a research reactor facility at Kyoto University was assayed using colony formation assay. Green fluorescent protein (GFP)-tagged LAT1 was observed in the plasma membranes of the LAT1-overexpressing clones and the cellular accumulation of GFP-tagged LAT1 was largely increased in these clones. Intracellular uptake of BPA was 1.5-5.0 times greater among the clones than that in a control clone. The LAT1-overexpressing clones and transiently LAT1-lipofected T98G cells showed clearly enhanced sensitivity to neutron and γ-ray fluences compared to the control clone when they were treated with 10BPA. The sensitivity of cancer cells to the fluences was well correlated with the expression level of LAT1 in the cells and the level of BPA uptake. These results suggest that overexpression of LAT1 in cancer cells results in enhanced anticancer effects of BNCT, and BNCT combined with gene therapy is beneficial for tumors with low LAT1 expression.


Assuntos
Apoptose/efeitos da radiação , Terapia por Captura de Nêutron de Boro , Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/patologia , Proteínas de Fluorescência Verde/genética , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/genética
4.
Biochem Biophys Res Commun ; 546: 150-154, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33582558

RESUMO

In this study, we examined the phenotypes of CD133-positive cells that were induced in a hypoxic microenvironment of spheroids formed using a glioblastoma cell line (T98G). Colony-formation assay showed that spheroid CD133-positive cells (SCPCs) were more resistant to X-rays and Temozolomide (TMZ) than spheroid CD133-negative cells (SCNCs) sorted from T98G spheroids. In contrast, the sensitivity to X-rays and TMZ was not different between hypoxic cells and normoxic cells of T98G spheroids in a colony-formation assay using green fluorescent protein (GFP) reporter-transfectants to monitor hypoxia. This result suggests that the difference in the sensitivity to X-rays and TMZ between SCPCs and SCNCs did not result from hypoxia. Transwell membrane assay indicated that the migration and inversion ability of SCPCs was higher than that of SCNCs. These results, including the findings obtained previously regarding nestin positivity in SCPCs, strongly suggest that SCPCs are cancer stem cell (CSC)-like cells. Additionally, based on experiments of monolayer culture of T98G cells, it was shown that hypoxia or low pH culture condition is not sufficient for the induction of SCPCs. The three-dimensional cell structure might be a critical factor for SCPC induction.


Assuntos
Hipóxia Celular , Glioblastoma/patologia , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Microambiente Tumoral , Antígeno AC133/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Citometria de Fluxo , Genes Reporter , Humanos , Concentração de Íons de Hidrogênio , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/efeitos da radiação , Temozolomida/farmacologia , Raios X
5.
J Radiat Res ; 61(2): 237-242, 2020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-31904079

RESUMO

The aim of this study was to determine whether membrane lipid peroxidation in mammalian cells is enhanced by X-ray irradiation at the K-shell resonance absorption peak of phosphorus. A549 and wild-type p53-transfected H1299 (H1299/wtp53) cell lines derived from human lung carcinoma were irradiated with monoenergetic X-rays at 2.153 keV, the phosphorus K-shell resonance absorption peak, or those at 2.147 or 2.160 keV, which are off peaks. Immunofluorescence staining for 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, was used as marker for protein modification. In both cell lines, the HNE production was significantly enhanced after irradiation at 2.153 keV compared to sham-irradiation. The enhancement (E) was calculated as the ratio of the fluorescence intensity of irradiated cells to that of sham-irradiated cells. In both the cell lines, E2.153 was significantly larger than E2.147 and no significant difference between E2.147 and E2.160 was observed. The extra enhancement at 2.153 keV was possibly caused by energy transition within the phosphorus K-shell resonance absorption. Our results indicate that membrane lipid peroxidation in cells is enhanced by the Auger effect after irradiation at the K-shell resonance absorption peak of phosphorus rather than by the photoelectric effect of the constituent atoms in the membrane lipid at 2.147 keV.


Assuntos
Membrana Celular/metabolismo , Peroxidação de Lipídeos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fósforo/química , Aldeídos/química , Linhagem Celular Tumoral , Fluorescência , Humanos , Doses de Radiação , Raios X
6.
World J Oncol ; 10(3): 132-141, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31312280

RESUMO

BACKGROUND: The aim of the study was to examine the dependency of p53 status and the usefulness of mild hyperthermia (MHT) as an inhibitor of recovery from radiation-induced damage, referring to the response of quiescent (Q) tumor cell population. METHODS: Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector (SAS/neo) were injected subcutaneously into left hind legs of nude mice. Tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells. They received high dose-rate γ-ray irradiation (HDR) immediately followed by localized MHT (40 °C for 2 h), or caffeine or wortmannin administration, or low dose-rate γ-ray irradiation simultaneously with localized MHT or caffeine or wortmannin administration. Nine hours after the start of irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. RESULTS: SAS/neo tumor cells, especially intratumor Q cell populations, showed a marked reduction in sensitivity due to the recovery from radiation-induced damage, compared with the total or Q tumor cells within SAS/mp53 tumors that showed little recovery capacity. The recovery from radiation-induced damage was thought to be a p53-dependent event. In both total and Q tumor cells within SAS/neo tumors, especially the latter, MHT efficiently suppressed the reduction in sensitivity caused by leaving an interval between HDR irradiation and the assay and decreasing the irradiation dose-rate, as well as the combination with wortmannin administration. CONCLUSIONS: From the viewpoint of solid tumor control as a whole, including intratumor Q-cell control, non-toxic MHT is useful for suppressing the recovery from radiation-induced damage, as well as wortmannin treatment combined with γ-ray irradiation.

7.
Int J Radiat Biol ; 93(3): 286-294, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27707083

RESUMO

PURPOSE: To examine the enhancing effects of syringetin on the radiosensitivity of normal and cancer cells, and the related mechanism. MATERIALS AND METHODS: We used normal human lung and mouse fibroblasts as well as human lung and mouse cancer cells derived from the above normal fibroblasts. Cell radiosensitivity was measured using a colony formation assay. Apoptosis was analyzed with DAPI staining and Western blots. DNA lesions were analyzed with γH2AX immunofluorescent staining. RESULTS: The colony formation assay showed that syringetin enhanced radiosensitivity more effectively in cancer cells (H1299 and C3H/MCA clone 15) compared with normal cells (HFL-III and C3H/10T1/2). The radiosensitizing effect of syringetin was observed in mutated p53 and wild-type p53-transfected H1299 cells regardless of p53 status. Apoptosis was more frequently observed in X-ray-irradiated H1299 cells combined with syringetin compared with X-ray-only-treated cells. Enhanced apoptosis by syringetin was not observed in HFL-III cells. Western blot analysis showed that X-ray-induced Caspase-3 activation was enhanced by syringetin in H1299 cells. The number of X-ray-induced DNA double-strand breaks (DSB) measured by quantitative analysis of γH2AX foci was the same for H1299 cells treated with X-rays with or without syringetin. CONCLUSIONS: This study supports the hypothesis that syringetin enhances radiosensitivity more effectively in cancer cells than in normal cells through enhancement of the Caspase-3-mediated apoptosis pathway. Syringetin could be useful in the development of novel efficacious radiosensitizers.


Assuntos
Apoptose/efeitos dos fármacos , Flavonoides/administração & dosagem , Neoplasias Experimentais/patologia , Neoplasias Experimentais/radioterapia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/administração & dosagem , Animais , Linhagem Celular Tumoral , Dano ao DNA , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Estudos de Viabilidade , Camundongos , Neoplasias Experimentais/genética , Dosagem Radioterapêutica
8.
Biochem Biophys Res Commun ; 457(4): 706-11, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-25619133

RESUMO

While an increase in progression free survival time is seen when an angiogenesis inhibitor is used in the treatment of high-relapse rate ovarian cancer, it has little effect on overall survival. A possible cause of treatment-resistance to angiogenesis inhibitors is the growth of stem cells in a hypoxic microenvironment built inside the tumor tissue by angiogenesis inhibition. In this study, we examined the possible suppression of stem cell and cancer stem cell (CSC) expansion by hypoxic cytotoxin, TX-402. TX-402, an analogue of tirapazamine, has been developed as a hypoxia selective prodrug with inhibitory effects of HIF-1 and angiogenesis. We considered TX-402 as a possible molecular-target drug candidate for ovarian cancer due to its inhibition of CSC expansion. In this study, we found that the expressions of HIF-1α and HIF-2α were increased under hypoxia in serous ovarian cancer cell lines. The expressions of HIF-1α and HIF-2α induced under hypoxia were repressed by TX-402 in a dose-dependent manner. Next, we investigated the effects of hypoxia on the expression levels of stem cell factors, Oct4, Nanog, Sox2 and Lin28, and showed that their expressions were induced by hypoxia. It was also observed that the expressions of putative ovarian cancer stem cell markers, CD133 and CD44 were induced under hypoxia. Furthermore, TX-402 was found to dose-dependently inhibit the expressions of CSC markers and stem cell factors. Oct4 expression was repressed by HIF-2α silencing, but not by HIF-1α silencing, indicating that TX-402 may repress the expression of Oct4 by inhibiting HIF-2α. We constructed CaOV3 spheroids as a 3-dimensional hypoxia model, in which the internal hypoxic region contained CSC-like cells expressing Oct4. The internal hypoxic region, which contained Oct4 expressing cells, disappeared following TX-402 treatment. In conclusion, hypoxia promoted the expansion of CSCs expressing CD133 and CD44 accompanied by an increase of stem cell factors. Its inhibition of hypoxia-induced CSC expansion makes TX-402 promising agent usable in combination for ovarian cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Quinoxalinas/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia
9.
Int J Oncol ; 45(2): 581-6, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24897999

RESUMO

Recent studies showed that the stemness of cancer stem cells is maintained under a hypoxic microenvironment. However, the relationship of the hypoxic microenvironment in a three-dimensional cell mass and the induction of cancer stem cell-like phenotype is not well known. We examined the relationship between CD133 expression and the hypoxic microenvironment using glioblastoma spheroids formed with the T98G cell line. CD133(AC133)- and HIF-1α-positive cells were observed in the marginal region of the central hypoxic area positive for HIF-1α 10 days after plating T98G cells. CD133(AC133)-positive cells were positive for nestin. Quantitative PCR analysis showed that the CD133 expression level is not different in spheroids during the tested period after spheroid formation, indicating that post-translational regulation of the CD133 protein mediates positivity to CD133(AC133). When spheroids were trypsinized and the dissociated cells were cultured under the adherent monolayer conditions, the CD133(AC133)-positive cells gradually disappeared. These results show that CD133(AC133)-positive cells, which may incline toward undifferentiated cells because of nestin positivity, are plastically induced under the different culture conditions, spheroid and monolayer. In this plasticity, HIF-1α is involved in the induction and maintenance of CD133(AC133)-positive cells. Spheroids as an in vitro tumor model are useful to study the dynamic changes in the tumor cell phenotype in the different cell microenvironments.


Assuntos
Antígenos CD/metabolismo , Glioblastoma/patologia , Glicoproteínas/metabolismo , Células-Tronco Neoplásicas/patologia , Peptídeos/metabolismo , Esferoides Celulares/patologia , Microambiente Tumoral/fisiologia , Antígeno AC133 , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Imunofluorescência , Glioblastoma/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Esferoides Celulares/metabolismo , Transfecção
10.
Case Rep Ophthalmol ; 5(1): 60-5, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24707274

RESUMO

PURPOSE: To report a case of orbital capillary hemangioma in an adult who was successfully treated with topical timolol maleate 0.5% solution. METHODS: Case report. RESULTS: A 43-year-old female presented both superficial and deep orbital capillary hemangioma. Topical timolol maleate was applied twice daily. The superficial lesions have nearly disappeared after 1 year of treatment. The deeper lesions have also been reduced in size according to MRI. CONCLUSION: We report an adult patient with a relatively large orbital capillary hemangioma who was successfully treated with a topical ß-blocker solution. This treatment might be applicable for orbital capillary hemangiomas, regardless of the patient's age, because of its effectiveness and safety.

11.
Int J Mol Med ; 33(3): 559-64, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24366006

RESUMO

The molecular chaperone heat shock protein 90 (Hsp90) is involved in the maturation and stabilization of a wide range of oncogenic client proteins for oncogenesis and malignant cell proliferation, which renders this protein a promising target in the development of cancer therapeutics. PU-H71 is a purine-scaffold Hsp90 inhibitor with less toxicity in normal cells than in cancer cells. In this study, we examined the in vitro radiosensitizing activity and molecular mechanisms of action of PU-H71 in human lung cancer cell lines. PU-H71 enhanced the sensitivity of the SQ-5 and A549 cancer cells to radiation. When the cancer cells were pre-treated with PU-H71, the repair of DNA double-strand breaks (DSBs) was markedly inhibited after irradiation compared with the cells that were not pre-treated with PU-H71, as evaluated by counting the foci of phosphorylated histone H2AX (γ-H2AX). We further demonstrated that post-irradiation, PU-H71 inhibited Rad51 foci formation, a critical protein for the homologous recombination pathway of DNA DSB repair. These data indicate that targeting Hsp90 with PU-H71 may be novel therapeutic strategy for radioresistant carcinomas.


Assuntos
Benzodioxóis/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Purinas/administração & dosagem , Tolerância a Radiação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/biossíntese , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Purinas/metabolismo
12.
Int J Mol Med ; 28(6): 1043-7, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21833466

RESUMO

Isothiocyanates are a class of naturally occurring chemopreventive agents known to suppress proliferation of cancer cells in culture. The present study was undertaken in order to examine the effects of benzyl isothiocyanate (BITC), one of the common dietary isothiocyanates, on the radiosensitivity of human pancreatic cancer cells and to gain insights into the underlying molecular mechanism of BITC-induced radiosensitization. Two human pancreatic cancer cell lines, PANC-1 and MIAPaCa-2, were treated with BITC and irradiated with X-rays. Radiation sensitivity, apoptosis, and protein levels were determined by a clonogenic assay, fluorescence microscopic analysis with DAPI staining and Western blotting, respectively. MIAPaCa-2 cells were relatively more sensitive to BITC treatment compared with PANC-1 cells. Radiosensitization was observed in both PANC-1 and MIAPaCa-2 cells incubated with BITC at 5 to 10 µM and 2.5 to 5 µM for 24 h, respectively. The combination treatments with BITC and X-rays also revealed an increased percentage of apoptotic cells. In addition, treatment with BITC and X-rays resulted in a decrease in the protein levels of the X-linked inhibitor of apoptosis (XIAP), inhibitor of apoptosis (IAP) family protein, and in a marked increase in the apoptosis protease activating factor-1 (Apaf-1), essential for activation of caspase-9 in stress-induced apoptosis. BITC may be a useful radiosensitizer for radiotherapy of pancreatic cancers.


Assuntos
Antineoplásicos/farmacologia , Terapia Combinada/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isotiocianatos/farmacologia , Neoplasias Pancreáticas , Tolerância a Radiação , Radiossensibilizantes/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Western Blotting , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Isotiocianatos/uso terapêutico , Microscopia de Fluorescência , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiação , Radiossensibilizantes/uso terapêutico , Regulação para Cima , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Raios X
13.
Int J Hyperthermia ; 27(3): 297-304, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21501031

RESUMO

Nijmegen breakage syndrome 1 (NBS1) plays an important role as a key protein in the repair of radiation-induced DNA double strand breaks (DSBs), and the work described here was designed to examine the effect of NBS1 on heat sensitivity for human anaplastic thyroid carcinoma 8305c cells. Cellular heat sensitivity was evaluated with colony formation assays. Apoptosis was detected and quantified with terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay and Hoechst33342 staining assay. Heat-induced DSBs were measured with flow cytometry using γH2AX antibodies. The transfection of NBS1-siRNA into cells specifically inhibited the expression of NBS1, and enhanced heat sensitivity and the frequency of apoptosis through caspase pathway. In addition, more frequent γH2AX foci were observed in the NBS1-siRNA transfected cells than in control cells transfected with scrambled siRNA at 24 h after heat treatment with a pan-caspase inhibitor. These results suggest that heat sensitisation might result from NBS1-siRNA mediated suppression of heat-induced DSB repair, indicating that NBS1-siRNA could potentially function as a heat sensitiser for cancer patients.


Assuntos
Proteínas de Ciclo Celular/genética , Temperatura Alta , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Apoptose , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Marcação In Situ das Extremidades Cortadas , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide/patologia
14.
Biochem Biophys Res Commun ; 404(1): 206-10, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21111709

RESUMO

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA(-/-), FANCC(-/-) and FANCA(-/-)C(-/-) cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2(-/-) cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.


Assuntos
Dano ao DNA , Reparo do DNA/genética , DNA Recombinante , Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Animais , Proteína BRCA2/fisiologia , Células CHO , Cricetinae , Cricetulus , Proteína do Grupo de Complementação A da Anemia de Fanconi/fisiologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/fisiologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Formaldeído/toxicidade , Histonas/metabolismo , Camundongos
15.
Int J Radiat Biol ; 87(3): 293-301, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21142704

RESUMO

PURPOSE: To clarify the role of p53 in boron neutron capture therapy (BNCT) for oral squamous cell carcinoma (SCC), the effect of BNCT on oral SCC xenografts with either wild-type or mutant-type p53 was examined. MATERIALS AND METHODS: Oral SCC cells expressing either wild-type (SAS/neo) or mutant-type p53 (SAS/mp53) were used to produce nude mouse tumours. Tumour-bearing mice received boronophenylalanine (BPA) at a dose of 250 mg/kg and tumours were exposed to neutron irradiation. RESULTS: After BNCT, the growth of SAS/neo and SAS/mp53 tumours was suppressed remarkably and all tumours became undetectable within two weeks. However, three of six SAS/mp53 tumours showed regrowth in two months. Histological examination of BNCT-treated tumours revealed chromosomal condensation, micronucleation, nuclear segmentation and intra- and intercelluar vacuolation. Notably, multinucleated giant cells appeared in SAS/mp53 tumours early after BNCT, suggesting mitotic catastrophe. In SAS/mp53 tumours treated with BNCT, a rapid decrease in phosphorylated cell division cycle 2 (cdc2) and a high level of cyclin B1, required for premature mitosis, were observed. CONCLUSION: These results indicate that BNCT suppressed oral SCC xenografts in nude mice efficiently, but cells survived in mutant-type p53 tumours. BNCT induces multinucleation which represents prestage of apoptosis or necrosis in oral SCC with mutant-type p53, but it may be also associated with the recurrence of BNCT-treated tumours.


Assuntos
Terapia por Captura de Nêutron de Boro/efeitos adversos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Genes p53 , Neoplasias Bucais/genética , Neoplasias Bucais/radioterapia , Mutação , Animais , Compostos de Boro/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitose , Transplante de Neoplasias , Fenótipo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Radiossensibilizantes/farmacologia
16.
Int J Oncol ; 37(4): 1001-10, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20811722

RESUMO

Activation to a large extent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and mutations in the p53 gene are involved in lung cancer therapeutic resistance. The mammalian target of rapamycin (mTOR) acts as a downstream effector for Akt. Activation of the Akt/mTOR signal is a contributing factor to decreased radiation sensitivity. The purpose of this study was to examine whether the effect of rapamycin on radiation sensitivity is affected by cellular p53 gene status. Cellular radiation sensitivity was evaluated by using two human non-small cell lung cancer (NSCLC) cell lines with the same genetic background except for their p53 gene status (H1299/wtp53 and H1299/mp53). The cells were treated with rapamycin and/or radiation. Cell viability, cell proliferation, apoptosis, cell cycle and Akt/mTOR signaling activity were explored. Rapamycin synergistically enhanced the cytotoxicity of radiation, promoting the induction of apoptosis. Moreover, the combined treatment augmented the cytostatic effects of radiation regardless of cellular p53 gene status. Rapamycin in combination with radiation increased G1 arrest and suppressed progression to S phase in both cell lines. Furthermore, the combined treatment conduced to a prominent p53-independent down-regulation of the mTOR signal and pro-survival molecule, cyclin D1. Rapamycin can enhance the effect of radiation through the repression of pro-survival signals and the reduction in the apoptotic threshold. Taken together, inhibition of the mTOR signal may be a promising strategy for radiosensitization with no relevance to p53 gene status from the aspects of cell lethality and cell growth depression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ciclina D1/metabolismo , Relação Dose-Resposta à Radiação , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/genética
17.
Cancer Sci ; 101(8): 1881-5, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20487264

RESUMO

Nimustine (ACNU) is a chloroethylating agent which was the most active chemotherapy agent used for patients with high-grade gliomas until the introduction of temozolomide, which became the standard of care for patients with newly diagnosed glioblastomas in Japan. Since temozolomide was established as the standard first-line therapy for glioblastoma multiforme (GBM), ACNU has been employed as a salvage chemotherapy agent for recurrent GBM in combination with other drugs. The acting molecular mechanism in ACNU has yet to be elucidated. ACNU is a cross-linking agent which induces DNA double-strand breaks (DSBs). The work described here was intended to clarify details in repair pathways which are active in the repair of DNA DSBs induced by ACNU. DSBs are repaired through the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. Cultured mouse embryonic fibroblasts were used which have deficiencies in DNA DSB repair genes which are involved in HR repair (X-ray repair cross-complementing group 2 [XRCC2] and radiation sensitive mutant 54 [Rad54]), and in NHEJ repair (DNA ligase IV [Lig4]). Cellular sensitivity to ACNU treatment was evaluated with colony forming assays. The most effective molecular target which correlated with ACNU cell sensitivity was Lig4. In addition, it was found that Lig4 small-interference RNA (siRNA) efficiently enhanced cell lethality which was induced by ACNU in human glioblastoma A172 cells. These findings suggest that the down-regulation of Lig4 might provide a useful tool which can be used to increase cell sensitivity in response to ACNU chemotherapy.


Assuntos
Antineoplásicos/farmacologia , DNA Ligases/antagonistas & inibidores , Nimustina/farmacologia , Animais , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Helicases , DNA Ligase Dependente de ATP , DNA Ligases/fisiologia , Reparo do DNA , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Camundongos , Proteínas Nucleares/fisiologia
18.
J Radiat Res ; 51(4): 417-22, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20448412

RESUMO

Heating induces histone H2AX phosphorylation at serine 139 (gammaH2AX). Phosphorylated H2AX subsequently forms foci in numerous mammalian cell lines. The aim of this study was to clarify details in the mechanisms involved in the phosphorylation of H2AX after heating. The cell lines used were DNA-PKcs knockout cells, ATM knockout cells, and their parental cell lines. To elucidate mechanisms of induction of phosphorylation of H2AX after heating, ATM/ATR inhibitor (CGK733) and DNA-PK inhibitor (NU7026) were used. The intensity of gammaH2AX signals was assayed with flow cytometry. The thermal dose-response curve for the fluorescence intensity of gammaH2AX appearance in DNA-PKcs-/- cells during the heating period was similar to that observed in DNA-PKcs+/+ cells. On the other hand, the slope of thermal dose-response curve for them in ATM-/- cells was lower than that in ATM+/+ cells. Phosphorylation of H2AX after heating was suppressed by a combination of CGK733 and NU7026 in the culture medium in DNA-PKcs-/- cells, ATM-/- cells and in their parental cells. Although the phosphorylation of H2AX after heating was not suppressed by NU7026 in their parental cells, such phosphorylation was suppressed by CGK733 in their parental cells. These results indicate that ATM is the predominant protein which is active in the phosphorylation of histone H2AX after heating.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Benzenoacetamidas/farmacologia , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromonas/farmacologia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/deficiência , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Histonas/química , Temperatura Alta , Humanos , Camundongos , Morfolinas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos da radiação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Serina/química , Tioureia/análogos & derivados , Tioureia/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética
19.
Int J Radiat Oncol Biol Phys ; 77(2): 559-65, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20381264

RESUMO

PURPOSE: The aim of this study was to clarify the effects of nitric oxide (NO) on radiation-induced cell killing and chromosome aberrations in two human lung cancer cell lines with a different p53 gene status. METHODS AND MATERIALS: We used wild-type (wt) p53 and mutated (m) p53 cell lines that were derived from the human lung cancer H1299 cell line, which is p53 null. The wtp53 and mp53 cell lines were generated by transfection of the appropriate p53 constructs into the parental cells. Cells were pretreated with different concentrations of isosorbide dinitrate (ISDN) (an NO donor) and/or 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) (an NO scavenger) and then exposed to X-rays. Cell survival, apoptosis, and chromosome aberrations were scored by use of a colony-forming assay, Hoechst 33342 staining assay and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP [deoxyuridine triphosphate] nick end labeling) assay, and chromosomal banding techniques, respectively. RESULTS: In wtp53 cells the induction of radioresistance and the inhibition of apoptosis and chromosome aberrations were observed in the presence of ISDN at low 2- to 10-mumol/L concentrations before X-irradiation. The addition of c-PTIO and ISDN into the culture medium 6 h before irradiation almost completely suppressed these effects. However, at high concentrations of ISDN (100-500 mumol/L), clear evidence of radiosensitization, enhancement of apoptosis, and chromosome aberrations was detected. However, these phenomena were not observed in mp53 cells at either concentration range with ISDN. CONCLUSIONS: These results indicate that low and high concentrations of NO radicals can choreograph inverse radiosensitivity, apoptosis, and chromosome aberrations in human lung cancer cells and that NO radicals can affect the fate of wtp53 cells.


Assuntos
Apoptose/efeitos dos fármacos , Aberrações Cromossômicas/efeitos dos fármacos , Genes p53/genética , Neoplasias Pulmonares/radioterapia , Doadores de Óxido Nítrico/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Apoptose/efeitos da radiação , Benzoatos/administração & dosagem , Benzoatos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular , Bandeamento Cromossômico/métodos , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Marcação In Situ das Extremidades Cortadas , Dinitrato de Isossorbida/administração & dosagem , Dinitrato de Isossorbida/farmacologia , Neoplasias Pulmonares/genética , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/administração & dosagem , Tolerância a Radiação/genética , Ensaio Tumoral de Célula-Tronco/métodos
20.
J Radiat Res ; 51(1): 27-35, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19801892

RESUMO

The usefulness of hexamethylenetetramine as an adjuvant to radiation and cisplatin in the treatment of solid tumors and its dependency on the p53 status of tumor cells were examined. Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53), or with neo vector as a control (SAS/neo), were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. The tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. Then, they received hexamethylenetetramine (HMTA), intraperitoneally or continuously, combined with or without gamma-ray irradiation or cisplatin treatment. Immediately after treatment following HMTA, the response of quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (= P + Q) tumor cells was determined from the BrdU non-treated tumors. A higher toxicity of HMTA to Q cells than total cells, especially in SAS/neo, was made less clear by continuous administration. There was no apparent difference in the radio- and cisplatin-sensitivity enhancing effects by HMTA combination between SAS/neo and SAS/mp53 tumors, with a slightly greater effect in SAS/mp53. In both SAS/neo and SAS/mp53 tumors, continuous HMTA administration produced higher radio- and cisplatin-sensitivity enhancing effects than intraperitoneal single administration. Therefore, the use of HMTA as an adjuvant to radiation or cisplatin might be promising in curing solid tumors with large fraction of hypoxic cells and also with frequent loss-of-function in p53.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Cisplatino/administração & dosagem , Metenamina/administração & dosagem , Radioterapia Conformacional/métodos , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/administração & dosagem , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimioterapia Adjuvante/métodos , Humanos , Camundongos , Camundongos Nus , Resultado do Tratamento
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