RESUMO
The cucurbits (prebiotics) were investigated as novel agents for radio-modification against gastrointestinal injury. The cell-cycle fractions and DNA damage were monitored in HCT-15 cells. A cucurbit extract was added to culture medium 2 h before irradiation (6 Gy) and was substituted by fresh medium at 4 h post-irradiation. The whole extract of the fruits of Lagenaria siceraria, Luffa cylindrica, or Cucurbita pepo extract enhanced G2 fractions (42%, 34%, and 37%, respectively) as compared with control (20%) and irradiated control (31%). With cucurbits, the comet tail length remained shorter (L. siceraria, 28 µm; L. cylindrica, 34.2 µm; C. pepo, 36.75 µm) than irradiated control (41.75 µm). For in vivo studies, L. siceraria extract (2 mg/kg body weight) was administered orally to mice at 2 h before and 4 and 24 h after whole-body irradiation (10 Gy). L. siceraria treatment restored the glutathione contents to 48.8 µmol/gm as compared with control (27.6 µmol/gm) and irradiated control (19.6 µmol/gm). Irradiation reduced the villi height from 379 to 350 µm and width from 54 to 27 µm. L. siceraria administration countered the radiation effects (length, 366 µm; width, 30 µm, respectively) and improved the villi morphology and tight junction integrity. This study reveals the therapeutic potential of cucurbits against radiation-induced gastrointestinal injury.
Assuntos
Frutas/química , Gastroenteropatias/prevenção & controle , Lagenidium/química , Extratos Vegetais/uso terapêutico , Prebióticos , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Animais , Linhagem Celular Tumoral , Cucurbita/química , Dano ao DNA , Frutas/economia , Fase G2/efeitos da radiação , Gastroenteropatias/dietoterapia , Gastroenteropatias/metabolismo , Gastroenteropatias/patologia , Glutationa/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos da radiação , Mucosa Intestinal/ultraestrutura , Luffa/química , Masculino , Camundongos , Microvilosidades/metabolismo , Microvilosidades/patologia , Microvilosidades/efeitos da radiação , Microvilosidades/ultraestrutura , Extratos Vegetais/metabolismo , Efeitos da Radiação , Lesões Experimentais por Radiação/dietoterapia , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Protetores contra Radiação/metabolismo , Distribuição Aleatória , Análise de Sobrevida , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Junções Íntimas/efeitos da radiação , Junções Íntimas/ultraestruturaRESUMO
High expression of vascular endothelial growth factor (VEGF) in patients with breast cancer has been associated with a poor prognosis, indicating that VEGF could be linked to the efficacy of chemotherapy and radiotherapy. It has also been suggested that radiation resistance is partly due to tumour cell production of angiogenic cytokines, particularly VEGF receptor (VEGFR). This evidence indicates that inhibition of VEGFR might enhance the radiation response. Sorafenib tosylate (Bay 54-9085) is an oral, small-molecule multikinase inhibitor of several targets including RAF/MEK/ERK MAP kinase signalling, VEGFR-2, VEGFR-3 and platelet-derived growth factor receptor-beta. Sorafenib has shown clinical efficacy in treating solid tumours such as renal cell and hepatocellular carcinomas. However, strategies are yet to be identified to prolong and maximize the anticancer effect of this multikinase inhibitor. The objective of this study was to determine whether a combination of Sorafenib and radiation will enhance the treatment response in vitro and in vivo. Radio-modulating effect of Sorafenib was assessed by performing clonogenic assays. In addition, cell cycle analyses as well as annexin-V apoptosis assays were performed 24 and 48 h after treatment, respectively. To confirm our in-vitro results, tumour growth delay assays were performed. Our results showed a strong and supra-additive antitumour effect of radiation combined with Sorafenib in vitro (dose enhancement factor of 1.76). The combined therapy demonstrated a strong and significant G2/M cell cycle arrest (combined treatment vs. irradiated alone: P<0.0008). Moreover, annexin-V staining showed a significant increase in the level of apoptosis (combined treatment vs. irradiated alone: P<0.0004). Study of the syngeneic model demonstrated the superior potency of the Sorafenib combined with radiotherapy. Our results demonstrate that higher antitumour activity can be achieved when radiation and Sorafenib are combined.
Assuntos
Antineoplásicos/uso terapêutico , Benzenossulfonatos/uso terapêutico , Ciclo Celular , Raios gama/uso terapêutico , Neoplasias Mamárias Experimentais , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Benzenossulfonatos/administração & dosagem , Benzenossulfonatos/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Citometria de Fluxo , Fase G2/efeitos dos fármacos , Fase G2/efeitos da radiação , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/radioterapia , Camundongos , Camundongos Endogâmicos BALB C , Niacinamida/análogos & derivados , Compostos de Fenilureia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacologia , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Colorectal cancer is the second leading cause of death in developed countries. Tumor therapies should on the one hand aim to stop the proliferation of tumor cells and to kill them, and on the other hand stimulate a specific immune response against residual cancer cells. Dying cells are modulators of the immune system contributing to anti-inflammatory or pro-inflammatory responses, depending on the respective cell death form. The positive therapeutic effects of temperature-controlled hyperthermia (HT), when combined with ionizing irradiation (X-ray), were the origin to examine whether combinations of X-ray with HT can induce immune activating tumor cell death forms, also characterized by the release of the danger signal HMGB1. Human colorectal tumor cells with differing radiosensitivities were treated with combinations of HT (41.5 degrees C for 1h) and X-ray (5 or 10Gy). Necrotic cell death was prominent after X-ray and could be further increased by HT. Apoptosis remained quite low in HCT 15 and SW480 cells. X-ray and combinations with HT arrested the tumor cells in the radiosensitive G2 cell cycle phase. The amount of released HMGB1 protein was significantly enhanced after combinatorial treatments in comparison to single ones. We conclude that combining X-ray with HT may induce anti-tumor immunity as a result of the predominant induction of inflammatory necrotic tumor cells and the release of HMGB1.
Assuntos
Neoplasias Colorretais/terapia , Proteína HMGB1/metabolismo , Hipertermia Induzida , Apoptose/imunologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Terapia Combinada , Fase G2/efeitos da radiação , Humanos , Sistema Imunitário/efeitos da radiação , Necrose/imunologia , Tolerância a RadiaçãoRESUMO
Arsenic compounds have been used as anti-cancer agents in traditional Chinese medicine. Ionizing radiation (IR) is one of the most effective tools in the clinical treatment of cancer. The induction of apoptotic cell death is a significant mechanism of tumor cells under the influence of radio-/chemotherapy, and resistance to these treatments has been linked to some cancer cell lines with a low propensity for apoptosis. A combination of different anti-tumoral treatment modalities is advantageous in limiting non-specific toxicity often observed by an exceedingly high dose of single regimen. The present study aimed at investigating the enhanced effects and mechanisms in cell cycle distribution and apoptosis of U937 cells, a human pre-monocytic leukemia cell line lacking functional p53 protein, after combination treatment with irradiation and As(2)O(3). Our results indicated that combined treatment led to activation of cdc-2, which is related to the expression of cyclin B. In addition, combined treatment increased apoptotic cell death in U937 cells, which is correlated with the induction of mitotic arrest, the increase in intracellular reactive oxygen species (ROS) generation, the decrease in B-cell leukemia/lymphoma 2 (Bcl-2) and B-cell leukemia/lymphoma XL (Bcl-XL) levels, the loss of mitochondria membrane potential, and the activation of caspase-3. We found that combining radiation and As(2)O(3) may be an effective strategy against p53-deficient leukemia cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Arsenicais/farmacologia , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Óxidos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Trióxido de Arsênio , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Ciclina A/biossíntese , Ciclina A/efeitos dos fármacos , Ciclina A/efeitos da radiação , Ciclina B/biossíntese , Ciclina B/efeitos dos fármacos , Ciclina B/efeitos da radiação , Citocromos c/efeitos dos fármacos , Citocromos c/metabolismo , Citocromos c/efeitos da radiação , Ensaios de Seleção de Medicamentos Antitumorais , Fase G2/efeitos dos fármacos , Fase G2/efeitos da radiação , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/efeitos da radiação , Radiação Ionizante , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos da radiação , Células U937RESUMO
G(2) checkpoint inhibitors can force cells arrested in G(2) phase by DNA damage to enter mitosis. In this manner, several G(2) checkpoint inhibitors can enhance killing of cancer cells by ionizing radiation and DNA-damaging chemotherapeutic agents, particularly in cells lacking p53 function. All G(2) checkpoint inhibitors identified to date target protein phosphorylation by inhibiting checkpoint kinases or phosphatases. Using a phenotypic cell-based assay for G(2) checkpoint inhibitors, we have screened a large collection of plant extracts and identified Z-Cryptofolione and Cryptomoscatone D2 as highly efficacious inhibitors of the G(2) checkpoint. These compounds and related pyrones also inhibit nuclear export. Leptomycin B, a potent inhibitor of Crm1-mediated nuclear export, is also a very potent G(2) checkpoint inhibitor. These compounds possess a reactive Michael acceptor site and do not appear promising as a radiosensitizing agents because they are toxic to unirradiated cells at checkpoint inhibitory concentrations. Nevertheless, the results show that inhibition of nuclear export is an alternative to checkpoint kinase inhibition for abrogating the G(2) checkpoint and they should stimulate the search for less toxic nuclear export inhibitors.
Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Cryptocarya/metabolismo , Cryptocarya/efeitos da radiação , Fase G2/efeitos da radiação , Pironas/metabolismo , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Ácidos Graxos Insaturados/farmacologia , Raios gama , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Humanos , Carioferinas/fisiologia , Extratos Vegetais/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteína Exportina 1RESUMO
OBJECTIVE: We have previously shown that all-trans retinoic acid (ATRA) enhanced the maintenance of early human hematopoietic progenitor cells (HPCs) in the presence of an irradiated stromal cell line AFT024. In this study, we examined the effects of ATRA on the stromal cell component with particular reference to cellular proliferation and gene expression. METHODS: Irradiated AFT024 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum and were incubated with ATRA at 1 mumol/L up to 21 days. The cells were examined in terms of immunostaining for proliferative cell nuclear antigen (PCNA) and BrdU incorporation, apoptosis assay, cell cycle analysis, and gene expression using semiquantitative reverse-transcriptase polymerase chain reaction. RESULTS: In the control experiments, AFT024 cells lost their confluence in culture after 15-Gy irradiation and were arrested in G2/M phase on days 7 and 21. ATRA restored the cellular confluence with an increase in proliferation on day 21 (BrdU incorporation: 20.6-fold; PCNA staining: 51.7-fold) with reversal of cell cycle arrest (S phase: 2.7-fold increase; G2/M phase: 2.0-fold decrease). There was no effect on apoptosis as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. ATRA significantly upregulated the expression of cell cycle genes for checkpoint transition, including cyclin A2, B2, and aurora kinase B, as well as genes associated with a putative role in HPC maintenance, including osteopontin, HoxA5, enhancer of zeste homolog 2, and peroxisome proliferator-activated receptor gamma. CONCLUSION: We concluded that ATRA induced cellular proliferation of irradiated AFT024 cells and expression of a number of genes whose relevance to HPC homeostasis would have to be further examined.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estromais/citologia , Tretinoína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Fase G2/efeitos dos fármacos , Fase G2/efeitos da radiação , Regulação da Expressão Gênica/genética , Genes cdc/efeitos dos fármacos , Camundongos , Células-Tronco/citologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Similar to what has been observed after irradiation, the fraction of G(2)-phase cells increases as a consequence of heat treatment. On the basis of cell cycle distributions alone, however, it is difficult to say whether the two results are related. In particular, comparison is complicated by the fact that the accompanying changes in the S-phase transition are different. These changes play a minor role after irradiation but constitute by far the most important cell cycle effect after heat treatment. Two-parameter flow cytometry was used here to study the proliferation of human melanoma cells in vitro. Cultures were pulse-labeled with BrdU after irradiation and/or heat treatment and were fixed either immediately or after a delay of up to 36 h. DNA-synthesizing cells were identified with the help of an FITC-conjugated antibody against BrdU; DNA was quantified after staining with propidium iodide. In this way, the cell cycle distribution could be determined and the progression through the cell cycle could be analyzed. From the movement of labeled cells through the cycle, in particular the appearance of labeled cells in the G(1) compartment (after they had gone through mitosis), the delay in G(2) phase could be determined. The duration of the G(2)/M phase in control cells was about 6 h. This was increased to 12, 13 and 16 h after irradiation (4 Gy X rays), heat treatment (1 h at 43 degrees C), and a combination of the two, respectively. In all these cases, the G(2)-phase block was completely overcome within 48 h after treatment, whereas changes in the S phase were still observable at this time. As expected, the radiation-induced G(2)-phase block was almost completely removed by incubating the cells with 5 or 10 mM caffeine. In the case of hyperthermia alone or in combination with radiation, however, caffeine was somewhat less effective. This does not mean, however, that the mechanisms involved are necessarily different. It can also be seen as a result of the differences in the time course of events. The long delay in S phase after heat treatment may lead to a loss of susceptibility to caffeine by the time the cells move into the G(2) phase.