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1.
Neurooncol Adv ; 3(1): vdab063, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34131650

RESUMO

BACKGROUND: Genetically susceptible individuals can develop malignancies after irradiation of normal tissues. In the context of therapeutic irradiation, it is not known whether irradiating benign neoplasms in susceptible individuals promotes neoplastic transformation and worse clinical outcomes. Individuals with Neurofibromatosis 1 (NF1) are susceptible to both radiation-induced second malignancies and spontaneous progression of plexiform neurofibromas (PNs) to malignant peripheral nerve sheath tumors (MPNSTs). The role of radiotherapy in the treatment of benign neoplasms such as PNs is unclear. METHODS: To test whether radiotherapy promotes neoplastic progression of PNs and reduces overall survival, we administered spinal irradiation (SI) to conditional knockout mouse models of NF1-associated PNs in 2 germline contexts: Nf1 fllfl ; PostnCre + and Nf1 fl/- ; PostnCre + . Both genotypes develop extensive Nf1 null spinal PNs, modeling PNs in NF1 patients. A total of 101 mice were randomized to 0 Gy, 15 Gy (3 Gy × 5), or 30 Gy (3 Gy × 10) of spine-focused, fractionated SI and aged until signs of illness. RESULTS: SI decreased survival in both Nf1 fllfl mice and Nf1 fl/- mice, with the worst overall survival occurring in Nf1 fl/- mice receiving 30 Gy. SI was also associated with increasing worrisome histologic features along the PN-MPNST continuum in PNs irradiated to higher radiation doses. CONCLUSIONS: This preclinical study provides experimental evidence that irradiation of pre-existing PNs reduces survival and may shift PNs to higher grade neoplasms.

2.
Radiat Res ; 190(1): 22-27, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29813005

RESUMO

Circulating tumor DNA (ctDNA) analysis has been shown to aid in both the detection of cancer and evaluation of somatic mutations in tumors. CtDNA concentration in plasma increases in proportion to tumor volume and/or metabolic activity and growth; however, this principle has yet to be applied to cell culture. We hypothesized that cell line-specific cell-free DNA (cfDNA) can be used to measure cell viability and cell survival in cell culture. Clonogenic assays on non-small cell lung cancer (NSCLC) cell lines H322, A549 and H322 were exposed to radiation doses of 0, 4 and 8 Gy. Prior to colony fixation and counting, cfDNA was extracted and quantified from cell culture media. The correlation between cell line-specific cfDNA and number of colonies grown on culture plates was examined. An H1299:A549 coculture model was used to evaluate the differential release of cell line-specific cfDNA. The results of this work indicate a strong correlation between CfDNA quantification from cell culture media and clonogenic survival at all radiation doses and in all cell lines tested (R2 range = 0.77-0.99). Cell survival curves derived from cfDNA were virtually indistinguishable from matched traditional clonogenic survival data ( P > 0.05; no significant difference exists between clonogenic curves). CfDNA quantification also accurately estimates colony count in a two-cell-line coculture model. In conclusion, cell-free DNA quantification from cell culture media can be used to measure cell survival, and appears suitable for development in a high-throughput clonogenic assay and radiosensitizer screening platform.


Assuntos
Sobrevivência Celular/efeitos da radiação , Ácidos Nucleicos Livres/metabolismo , Técnicas Citológicas/métodos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Neoplasias Pulmonares/patologia
3.
Free Radic Biol Med ; 112: 318-326, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28782644

RESUMO

Constitutive activation of the pro-survival transcription factor NF-κB has been associated with resistance to both chemotherapy and radiation therapy in many human cancers, including prostate cancer. Our lab and others have demonstrated that the natural product parthenolide can inhibit NF-κB activity and sensitize PC-3 prostate cancers cells to X-rays in vitro; however, parthenolide has poor bioavailability in vivo and therefore has little clinical utility in this regard. We show here that treatment of PC-3 and DU145 human prostate cancer cells with dimethylaminoparthenolide (DMAPT), a parthenolide derivative with increased bioavailability, inhibits constitutive and radiation-induced NF-κB binding activity and slows prostate cancer cell growth. We also show that DMAPT increases single and fractionated X-ray-induced killing of prostate cancer cells through inhibition of DNA double strand break repair and also that DMAPT-induced radiosensitization is, at least partially, dependent upon the alteration of intracellular thiol reduction-oxidation chemistry. Finally, we demonstrate that the treatment of PC-3 prostate tumor xenografts with oral DMAPT in addition to radiation therapy significantly decreases tumor growth and results in significantly smaller tumor volumes compared to xenografts treated with either DMAPT or radiation therapy alone, suggesting that DMAPT might have a potential clinical role as a radiosensitizing agent in the treatment of prostate cancer.


Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , NF-kappa B/antagonistas & inibidores , Neoplasias da Próstata/terapia , Radiossensibilizantes/farmacologia , Sesquiterpenos/farmacologia , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Próstata/efeitos da radiação , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Transdução de Sinais , Raios X , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
4.
Int J Radiat Biol ; 92(8): 427-33, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27258472

RESUMO

Purpose To investigate whether irradiated human endothelial progenitor cells (hEPC) could induce bystander killing in the A549 non-small cell lung cancer (NSCLC) cells and help explain the improved radiation-induced tumor cures observed in A549 tumor xenografts co-injected with hEPC. Materials and methods We investigated whether co-injection of CBM3 hEPC with A549 NSCLC cells would alter tumor xenograft growth rate or tumor cure after a single dose of 0 or 5 Gy of X-rays. We then utilized dual chamber Transwell dishes, to test whether medium from irradiated CBM3 and CBM4 hEPC would induce bystander cell killing in A549 cells, and as an additional control, in human pancreatic cancer MIA PaCa-2 cells. The CBM3 and CBM4 hEPC were plated into the upper Transwell chamber and the A549 or MIA PaCa-2 cells were plated in the lower Transwell chamber. The top inserts with the CBM3 or CBM4 hEPC cells were subsequently removed, irradiated, and then placed back into the Transwell dish for 3 h to allow for diffusion of any potential bystander factors from the irradiated hEPC in the upper chamber through the permeable membrane to the unirradiated cancer cells in the lower chamber. After the 3 h incubation, the cancer cells were re-plated for clonogenic survival. Results We found that co-injection of CBM3 hEPC with A549 NSCLC cells significantly increased the tumor growth rate compared to A549 cells alone, but paradoxically also increased A549 tumor cure after a single dose of 5 Gy of X-rays (p < 0.05). We hypothesized that irradiated hEPC may be inducing bystander killing in the A549 NSCLC cells in tumor xenografts, thus improving tumor cure. Bystander studies clearly showed that exposure to the medium from irradiated CBM3 and CBM4 hEPC induced significant bystander killing and decreased the surviving fraction of A549 and MIA PaCa-2 cells to 0.46 (46%) ± 0.22 and 0.74 ± 0.07 (74%) respectively (p < 0.005, p < 0.0001). In addition, antibody depletion studies demonstrated that the bystander killing induced in both A549 and MIA PaCa-2 cells was mediated by the cytokines TNF-α and TGF-ß (p < 0.05). Conclusions These data provide evidence that irradiated hEPC can induce strong bystander killing in A549 and MIA PaCa-2 human cancer cells and that this bystander killing is mediated by the cytokines TNF-α and TGF-ß.


Assuntos
Efeito Espectador/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/efeitos da radiação , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Células A549 , Linhagem Celular Tumoral , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Células Progenitoras Endoteliais/patologia , Humanos , Doses de Radiação
5.
Oncotarget ; 7(15): 20788-800, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-26959112

RESUMO

Pro-oxidative stressors can suppress host immunity due to their ability to generate oxidized lipid agonists of the platelet-activating factor-receptor (PAF-R). As radiation therapy also induces reactive oxygen species, the present studies were designed to define whether ionizing radiation could generate PAF-R agonists and if these lipids could subvert host immunity. We demonstrate that radiation exposure of multiple tumor cell lines in-vitro, tumors in-vivo, and human subjects undergoing radiation therapy for skin tumors all generate PAF-R agonists. Structural characterization of radiation-induced PAF-R agonistic activity revealed PAF and multiple oxidized glycerophosphocholines that are produced non-enzymatically. In a murine melanoma tumor model, irradiation of one tumor augmented the growth of the other (non-treated) tumor in a PAF-R-dependent process blocked by a cyclooxygenase-2 inhibitor. These results indicate a novel pathway by which PAF-R agonists produced as a byproduct of radiation therapy could result in tumor treatment failure, and offer important insights into potential therapeutic strategies that could improve the overall antitumor effectiveness of radiation therapy regimens.


Assuntos
Antioxidantes/farmacologia , Melanoma/terapia , Fator de Ativação de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/agonistas , Receptores Acoplados a Proteínas G/agonistas , Neoplasias Cutâneas/terapia , Raios Ultravioleta , Animais , Feminino , Humanos , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Estresse Oxidativo , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/secundário , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
DNA Repair (Amst) ; 40: 35-46, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26991853

RESUMO

Non-small cell lung cancers (NSCLC) are commonly treated with a platinum-based chemotherapy such as cisplatin (CDDP) in combination with ionizing radiation (IR). Although clinical trials have demonstrated that the combination of CDDP and IR appear to be synergistic in terms of therapeutic efficacy, the mechanism of synergism remains largely uncharacterized. We investigated the role of the DNA damage response (DDR) in CDDP radiosensitization using two NSCLC cell lines. Using clonogenic survival assays, we determined that the cooperative cytotoxicity of CDDP and IR treatment is sequence dependent, requiring administration of CDDP prior to IR (CDDP-IR). We identified and interrogated the unique time and agent-dependent activation of the DDR in NSCLC cells treated with cisplatin-IR combination therapy. Compared to treatment with CDDP or IR alone, CDDP-IR combination treatment led to persistence of γH2Ax foci, a marker of DNA double-strand breaks (DSB), for up to 24h after treatment. Interestingly, pharmacologic inhibition of DDR sensor kinases revealed the persistence of γ-H2Ax foci in CDDP-IR treated cells is independent of kinase activation. Taken together, our data suggest that delayed repair of DSBs in NSCLC cells treated with CDDP-IR contributes to CDDP radiosensitization and that alterations of the DDR pathways by inhibition of specific DDR kinases can augment CDDP-IR cytotoxicity by a complementary mechanism.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Cisplatino/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Reparo de DNA por Recombinação , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Tolerância a Radiação/genética , Raios X
7.
Free Radic Biol Med ; 89: 263-73, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26393423

RESUMO

We investigated whether altering Warburg metabolism (aerobic glycolysis) by treatment with the metabolic agent dichloroacetate (DCA) could increase the X-ray-induced cell killing of the radiation-resistant human non-small-cell lung cancer (NSCLC) cell lines A549 and H1299. Treatment with 50mM DCA decreased lactate production and glucose consumption in both A549 and H1299, clear indications of attenuated aerobic glycolysis. In addition, we found that DCA treatment also slowed cell growth, increased population-doubling time, and altered cell cycle distribution. Furthermore, we report that treatment with 50mM DCA significantly increased single and fractionated X-ray-induced cell killing of A549 and H1299 cells. Assay of DNA double-strand break repair by neutral comet assays demonstrated that DCA inhibited both the fast and the slow kinetics of X-ray-induced DSB repair in both A549 and H1299 NSCL cancer cells. Taken together the data suggest a correlation between an attenuated aerobic glycolysis and enhanced cytotoxicity and radiation-induced cell killing in radiation-resistant NSCLC cells.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Ácido Dicloroacético/farmacologia , Glicólise/fisiologia , Neoplasias Pulmonares/radioterapia , Tolerância a Radiação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Ensaio Cometa , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Glicólise/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células Tumorais Cultivadas , Raios X
8.
J Bone Miner Res ; 30(7): 1268-79, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25588731

RESUMO

Increased fracture risk is commonly reported in cancer patients receiving radiotherapy, particularly at sites within the field of treatment. The direct and systemic effects of ionizing radiation on bone at a therapeutic dose are not well-characterized in clinically relevant animal models. Using 20-week-old male C57Bl/6 mice, effects of irradiation (right hindlimb; 2 Gy) on bone volume and microarchitecture were evaluated prospectively by microcomputed tomography and histomorphometry and compared to contralateral-shielded bone (left hindlimb) and non-irradiated control bone. One week postirradiation, trabecular bone volume declined in irradiated tibias (-22%; p < 0.0001) and femurs (-14%; p = 0.0586) and microarchitectural parameters were compromised. Trabecular bone volume declined in contralateral tibias (-17%; p = 0.003), and no loss was detected at the femur. Osteoclast number, apoptotic osteocyte number, and marrow adiposity were increased in irradiated bone relative to contralateral and non-irradiated bone, whereas osteoblast number was unchanged. Despite no change in osteoblast number 1 week postirradiation, dynamic bone formation indices revealed a reduction in mineralized bone surface and a concomitant increase in unmineralized osteoid surface area in irradiated bone relative to contralateral and non-irradiated control bone. Further, dose-dependent and time-dependent calvarial culture and in vitro assays confirmed that calvarial osteoblasts and osteoblast-like MC3T3 cells were relatively radioresistant, whereas calvarial osteocyte and osteocyte-like MLO-Y4 cell apoptosis was induced as early as 48 hours postirradiation (4 Gy). In osteoclastogenesis assays, radiation exposure (8 Gy) stimulated murine macrophage RAW264.7 cell differentiation, and coculture of irradiated RAW264.7 cells with MLO-Y4 or murine bone marrow cells enhanced this effect. These studies highlight the multifaceted nature of radiation-induced bone loss by demonstrating direct and systemic effects on bone and its many cell types using clinically relevant doses; they have important implications for bone health in patients treated with radiation therapy.


Assuntos
Reabsorção Óssea/patologia , Osso e Ossos/patologia , Osso e Ossos/efeitos da radiação , Membro Posterior/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Composição Corporal , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Osteogênese/efeitos da radiação , Células RAW 264.7 , Crânio/patologia , Crânio/efeitos da radiação , Fatores de Tempo , Raios X
9.
Int J Radiat Oncol Biol Phys ; 88(2): 412-8, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24411612

RESUMO

PURPOSE: To image the intratumor vascular physiological status of pancreatic tumors xenografts and their response to anti-angiogenic therapy using dynamic contrast-enhanced computed tomography (DCE-CT), and to identify parameters of vascular physiology associated with tumor x-ray sensitivity after anti-angiogenic therapy. METHODS AND MATERIALS: Nude mice bearing human BxPC-3 pancreatic tumor xenografts were treated with 5 Gy of radiation therapy (RT), either a low dose (40 mg/kg) or a high dose (150 mg/kg) of DC101, the anti-VEGF receptor-2 anti-angiogenesis antibody, or with combination of low or high dose DC101 and 5 Gy RT (DC101-plus-RT). DCE-CT scans were longitudinally acquired over a 3-week period post-DC101 treatment. Parametric maps of tumor perfusion and fractional plasma volume (Fp) were calculated and their averaged values and histogram distributions evaluated and compared to controls, from which a more homogeneous physiological window was observed 1-week post-DC101. Mice receiving a combination of DC101-plus-RT(5 Gy) were imaged baseline before receiving DC101 and 1 week after DC101 (before RT). Changes in perfusion and Fp were compared with alternation in tumor growth delay for RT and DC101-plus-RT (5 Gy)-treated tumors. RESULTS: Pretreatment with low or high doses of DC101 before RT significantly delayed tumor growth by an average 7.9 days compared to RT alone (P ≤ .01). The increase in tumor growth delay for the DC101-plus-RT-treated tumors was strongly associated with changes in tumor perfusion (ΔP>-15%) compared to RT treated tumors alone (P=.01). In addition, further analysis revealed a trend linking the tumor's increased growth delay to its tumor volume-to-DC101 dose ratio. CONCLUSIONS: DCE-CT is capable of monitoring changes in intratumor physiological parameter of tumor perfusion in response to anti-angiogenic therapy of a pancreatic human tumor xenograft that was associated with enhanced radiation response.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias Pancreáticas/terapia , Tolerância a Radiação/efeitos dos fármacos , Tomografia Computadorizada por Raios X/métodos , Animais , Proliferação de Células , Terapia Combinada/métodos , Meios de Contraste , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Fluxo Sanguíneo Regional/efeitos da radiação , Resultado do Tratamento , Carga Tumoral
10.
Mol Cancer Ther ; 12(6): 1038-48, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23468532

RESUMO

Pancreatic cancer often presents in advanced stages and is unresponsive to conventional treatments. Thus, the need to develop novel treatment strategies for pancreatic cancer has never been greater. Here, we report that combination of focal irradiation with hedgehog (Hh) signaling inhibition exerts better than additive effects on reducing metastases. In an orthotopic model, we found that focal irradiation alone effectively reduced primary tumor growth but did not significantly affect metastasis. We hypothesized that cancer stem cells (CSC) of pancreatic cancer are responsible for the residual tumors following irradiation, which may be regulated by Hh signaling. To test our hypothesis, we showed that tumor metastasis in our model was accompanied by increased expression of CSC cell surface markers as well as Hh target genes. We generated tumor spheres from orthotopic pancreatic and metastatic tumors, which have elevated levels of CSC markers relative to the parental cells and elevated expression of Hh target genes. Irradiation of tumor spheres further elevated CSC cell surface markers and increased Hh target gene expression. Combination of Hh signaling inhibition with radiation had more than additive effects on tumor sphere regeneration in vitro. This phenotype was observed in two independent cell lines. In our orthotopic animal model, focal radiation plus Hh inhibition had more than additive effects on reducing lymph node metastasis. We identified several potential molecules in mediating Hh signaling effects. Taken together, our data provide a rationale for combined use of Hh inhibition with irradiation for clinical treatment of patients with pancreatic cancer.


Assuntos
Carcinogênese/genética , Proteínas Hedgehog/biossíntese , Metástase Neoplásica/genética , Neoplasias Pancreáticas/genética , Alcaloides de Veratrum/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Carcinogênese/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Terapia de Alvo Molecular , Metástase Neoplásica/patologia , Metástase Neoplásica/terapia , Estadiamento de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Análise de Célula Única , Raios X
11.
Free Radic Biol Med ; 51(12): 2249-58, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22019440

RESUMO

We investigated the efficacy and mechanism of dimethylaminoparthenolide (DMAPT), an NF-κB inhibitor, to sensitize human lung cancer cells to X-ray killing in vitro and in vivo. We tested whether DMAPT increased the effectiveness of single and fractionated X-ray treatment through inhibition of NF-κB and/or DNA double-strand break (DSB) repair. Treatment with DMAPT decreased plating efficiency, inhibited constitutive and radiation-induced NF-κB binding activity, and enhanced radiation-induced cell killing by dose modification factors of 1.8 and 1.4 in vitro. X-ray fractionation demonstrated that DMAPT inhibited split-dose recovery/repair, and neutral DNA comet assays confirmed that DMAPT altered the fast and slow components of X-ray-induced DNA DSB repair. Knockdown of the NF-κB family member p65 by siRNA increased radiation sensitivity and completely inhibited split-dose recovery in a manner very similar to DMAPT treatment. The data suggest a link between inhibition of NF-κB and inhibition of DSB repair by DMAPT that leads to enhancement of X-ray-induced cell killing in vitro in non-small-cell lung cancer cells. Studies of A549 tumor xenografts in nude mice demonstrated that DMAPT enhanced X-ray-induced tumor growth delay in vivo.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Neoplasias Pulmonares/terapia , NF-kappa B/antagonistas & inibidores , Sesquiterpenos/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Nus , Relação Estrutura-Atividade , Raios X
12.
Radiat Res ; 176(2): 208-16, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21663393

RESUMO

Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells that circulate at low concentration in human umbilical cord and adult peripheral blood and are largely resident in blood vessels. ECFCs not only appear to be critical for normal vascular homeostasis and repair but may also contribute to tumor angiogenesis and response to therapy. To begin to characterize the potential role of ECFCs during the treatment of tumors in children and adults with radiation, we characterized the X-ray sensitivity of cord and adult blood-derived ECFCs. We found both cord blood and adult ECFCs to be highly radiation sensitive (3 Gy resulted in >90% killing without induction of apoptosis). The X-ray survival curves suggested reduced potential for repair capacity, but X-ray fractionation studies demonstrated that all the ECFCs exhibited repair when the radiation was fractionated. Finally, the mechanisms of X-ray-induced cell death for cord blood and adult ECFCs were different at low and high dose. At low dose, all ECFCs appear to die by mitotic death/catastrophe. However, at high radiation doses (≥ 10 Gy) cord blood ECFCs underwent p53 stabilization and Bax-dependent apoptosis as well as p21-dependent G1 and G2/M cell cycle checkpoints. By contrast, after 10 Gy adult ECFCs undergo only large-scale radiation-induced senescence, which is a cellular phenotype linked to premature development of atherosclerosis and vasculopathies. These data demonstrate that the ECFC response to radiation is dose-dependent and developmentally regulated and may provide potential mechanistic insight into their role in tumor and normal tissue response after ionizing radiation treatment.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos da radiação , Apoptose/efeitos da radiação , Células Endoteliais/citologia , Sangue Fetal/citologia , Adulto , Animais , Ciclo Celular/efeitos da radiação , Separação Celular , Relação Dose-Resposta a Droga , Humanos , Camundongos , Tolerância a Radiação/efeitos da radiação , Fatores de Tempo , Raios X/efeitos adversos
13.
Radiat Res ; 171(4): 389-96, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19397439

RESUMO

We have shown that parthenolide, a sesquiterpene lactone, is a radiation sensitizer for human CGL1 hybrid cells that have constitutively activated NF-kappaB and wild-type p53. Since many malignant cells have nonfunctional p53, we investigated whether parthenolide could alter the X-ray sensitivity of PC-3 prostate cancer cells, a p53 null cell line with constitutively activated NF-kappaB. The addition of 5 microM parthenolide induced non-apoptotic cell death, inhibited PC-3 proliferation, and increased the population doubling time from 23+/-1 h to 49+/-4 h. Parthenolide also inhibited constitutive and radiation-induced NF-kappaB binding activity and enhanced the X-ray sensitivity of these p53 null PC-3 cells by a dose modification factor of 1.7. Cell cycle analysis of PC-3 cells treated with parthenolide showed only small alterations in G1 and G2/M cells, and these appeared to be insufficient to explain the observed radiosensitization. Split-dose studies using clinically relevant 2- and 4-Gy fractions demonstrated that parthenolide completely inhibited split-dose repair in PC-3 cells. We hypothesized that inhibition of NF-kappaB activity by parthenolide was responsible for the observed X-ray sensitization and inhibition of split-dose repair. To test this hypothesis, we knocked down the expression of NF-kappaB p65 protein, an active component of NF-kappaB in both PC-3 and CGL1 cells, by siRNA. Inhibition of NF-kappaB activity by knockdown of p65 increased radiation sensitivity and completely inhibited split-dose repair in both cell lines in a nearly identical manner as parthenolide treatment alone. Treating p65-depleted PC-3 cells with 5 microM parthenolide did not further increase their radiation sensitivity or the inhibition of split-dose repair. We propose that the suppression of radiation-induced NF-kappaB activity by parthenolide leads to X-ray sensitization through inhibition of split-dose repair in p53 null PC-3 prostate cancer cells.


Assuntos
NF-kappa B/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Sesquiterpenos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Humanos , Masculino , Modelos Biológicos , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação , Fator de Transcrição RelA/metabolismo , Raios X
14.
Cancer Chemother Pharmacol ; 63(4): 723-30, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18622615

RESUMO

PURPOSE: Clinical drug resistance to platinum-based chemotherapy is considered a major impediment in the treatment of human ovarian cancer. Multiple pathways associated with drug resistance have been suggested by many previous studies. Over expression of several key proteins involved in DNA repair, drug transport, redox regulation, and apoptosis has been recently reported by our group using a global quantitative proteomic profiling approach. Superoxide dismutase 1 (SOD1) is one of these proteins consistently over-expressed in cisplatin-resistant ovarian cancer cells as compared to their sensitive counterparts, but its precise role in drug resistance is yet to be defined. METHOD: In the current study, we examined the role of SOD1 in drug resistance by inhibiting its redox activity in cisplatin-resistant ovarian cancer cells using a small-molecule inhibitor, triethylenetetramine (TETA). The effect of TETA was determined by the cell proliferation assay, clonogenic cell survival assay, and SOD1 activity assay. RESULTS: The inhibition of the SOD1 activity enhanced the cisplatin sensitivity in the resistant cells supporting the hypothesis that SOD1 is a key determinant of cisplatin resistance and is an exploitable target to overcome cisplatin drug resistance. CONCLUSION: SOD1 plays an important role in cisplatin resistance and modulation of its activity may overcome this resistance and ultimately lead to improved clinical outcomes.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Superóxido Dismutase/antagonistas & inibidores , Bromodesoxiuridina , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quelantes/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Trientina/farmacologia , Ensaio Tumoral de Célula-Tronco
15.
DNA Repair (Amst) ; 7(2): 177-86, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17974506

RESUMO

Apurinic endonuclease 1/redox effector factor-1 (Ape1/Ref-1 or Ape1) is an essential protein with two distinct functions. It is a DNA repair enzyme in the base excision repair (BER) pathway and a reduction-oxidation (redox) signaling factor maintaining transcription factors in an active reduced state. Our laboratory previously demonstrated that Ape1 is overexpressed in ovarian cancer and potentially contributes to resistance. Therefore, we utilized siRNA technology to knockdown protein levels of Ape1 in ovarian cancer cell line, SKOV-3x. Knocking Ape1 down had dramatic effects on cell growth in vitro but was not due to an increase in apoptosis and at least partially due to an extension in transit time through S-phase. Similarly, human ovarian tumor xenografts with reduced levels of Ape1 protein demonstrated a dramatic reduction in tumor volume (p<0.01) and also statistically significant (p=0.02) differences in (18)F-fluorodeoxyglucose (FDG) uptake indicating reduced glucose metabolism and cellular proliferation. Ape1's role in DNA repair and redox signaling is important to our basic understanding of ovarian cancer cell growth and these findings strongly support Ape1 as a therapeutic target.


Assuntos
Ciclo Celular/fisiologia , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Neoplasias Ovarianas/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Oxirredução , Tomografia por Emissão de Pósitrons , Interferência de RNA , RNA Interferente Pequeno/genética , Transfecção
16.
Radiat Res ; 168(6): 689-97, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18088190

RESUMO

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-kappaB-activated CGL1 cells. Treatment with 5 microM parthenolide for 48 to 72 h inhibited constitutive NF-kappaB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21 waf1/cip1 (CDKN1A) and the onset of G1/S and G2/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , NF-kappa B/metabolismo , Sesquiterpenos/farmacologia , Raios X , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/classificação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tubulina (Proteína)/metabolismo , Proteína Supressora de Tumor p53/metabolismo
17.
Radiat Res ; 163(6): 614-22, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15913393

RESUMO

Exposing CGL1 (HeLa x fibroblast) hybrid cells to 7 Gy of X rays results in the onset of a delayed apoptosis in the progeny of the cells 10 to 12 cell divisions postirradiation that correlates with the emergence of neoplastically transformed foci. The delayed apoptosis begins around day 8 postirradiation and lasts for 11 days. We now demonstrate that the delayed apoptosis is also characterized by the appearance of approximately 50-kb apoptotic DNA fragments and caspase 3 activation postirradiation. In addition, we confirm that stabilization of TP53 and transactivation of pro-apoptosis BAX also occurs during the delayed apoptosis and show that anti-apoptosis BCL-X(L) is down-regulated. To test whether the delayed apoptosis was due to a nonfunctional acute TP53 damage response in CGL1 cells, studies of acute apoptosis were completed. After irradiation, CGL1 cells underwent an acute wave of apoptosis that involves TP53 stabilization, transactivation of BAX gene expression, and a rapid caspase activation that ends by 96 h postirradiation. In addition, the acute onset of apoptosis correlates with transactivation of a standard wild-type TP53-responsive reporter (pG13-CAT) in CGL1 cells after radiation exposure. We propose that the onset of the delayed apoptosis is not the result of a nonfunctional acute TP53 damage response pathway but rather is a consequence of X-ray-induced genomic instability arising in the distant progeny of the irradiated cells.


Assuntos
Apoptose/efeitos da radiação , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/efeitos da radiação , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Transformação Celular Neoplásica/patologia , Relação Dose-Resposta à Radiação , Fibroblastos/patologia , Humanos , Células Híbridas/metabolismo , Células Híbridas/patologia , Células Híbridas/efeitos da radiação , Doses de Radiação , Proteína X Associada a bcl-2
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