RESUMO
Solid tumors with areas of low oxygen tension (hypoxia) have a poor prognosis, as cells in this environment often survive radiation and chemotherapy. In this report we describe how this hypoxic environment can be used to activate heterologous gene expression driven by a hypoxia-responsive element (HRE), which interacts with the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Our results demonstrate that the HIF-1/HRE system of gene regulation is active in hypoxic tumor cells and show the potential of exploiting tumor-specific conditions for the targeted expression of diagnostic or therapeutic genes in cancer therapy.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fibrossarcoma/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Nucleares/fisiologia , Oxigênio/farmacologia , Fosfoglicerato Quinase/genética , Fatores de Transcrição , Animais , Antimetabólitos Antineoplásicos/farmacologia , Hipóxia Celular , Citosina Desaminase , Fibrossarcoma/metabolismo , Flucitosina/farmacologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos , Camundongos Nus , Misonidazol/análogos & derivados , Misonidazol/farmacologia , Transplante de Neoplasias , Nucleosídeo Desaminases/genética , Pró-Fármacos/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/biossíntese , Células Tumorais CultivadasRESUMO
BACKGROUND: Metastases cause most cancer-related deaths. We investigated the use of hypoxia-selective cytotoxins as adjuvants to radiotherapy in the control of metastatic tumour growth. METHODS: The NLCQ-1, RB6145 and tirapazamine were assessed against the spontaneously metastasising KHT model. Subcutaneous KHT tumours (250 mm(3)) were irradiated with 25 Gy (single fraction) to control primary growth. Equitoxic drug treatments (NLCQ-1 (10 mg kg(-1)) once daily; RB6145 (75 mg kg(-1)) and tirapazamine (13 mg kg(-1)) twice daily) were administered 3-6 days post-radiotherapy when hypoxic cells were evident in lung micrometastases. Mice were culled when 50% of controls exhibited detrimental signs of lung metastases. RESULTS: In total, 95% of control mice presented with lung disease. This was significantly reduced by NLCQ-1 (33%; P=0.0002) and RB6145 (60%; P=0.02). Semi-quantitative grading of lung disease revealed a significant improvement with all treatments, with NLCQ-1 proving most efficacious (median grades: control, 4; NLCQ, 0 (P<0.0001); RB6145, 1 (P<0.001), tirapazamine, 3 (P=0.007)). Positron emission tomography (PET) was evaluated as a non-invasive means of assessing metastatic development. Primary and metastatic KHT tumours showed robust uptake of [(18)F]fluorodeoxyglucose ([(18)F]FDG). Metastatic burden discernable by [(18)F]FDG PET correlated well with macroscopic and histological lung analysis. CONCLUSION: The hypoxia-selective cytotoxin NLCQ-1 controls metastatic disease and may be a successful adjuvant to radiotherapy in the clinical setting.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Hipóxia Celular/efeitos dos fármacos , Imidazóis/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Quinolinas/administração & dosagem , Sarcoma/tratamento farmacológico , Sarcoma/secundário , Animais , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Terapia Combinada , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Camundongos , Camundongos Endogâmicos C3H , Metástase Neoplásica , Nitroimidazóis/administração & dosagem , Tirapazamina , Triazinas/administração & dosagemRESUMO
Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.
Assuntos
Experimentação Animal/normas , Bem-Estar do Animal/normas , Neoplasias/patologia , Neoplasias/terapia , Guias de Prática Clínica como Assunto , Algoritmos , Experimentação Animal/ética , Bem-Estar do Animal/ética , Bem-Estar do Animal/organização & administração , Animais , Biomarcadores Farmacológicos/análise , Pesquisa Biomédica/ética , Pesquisa Biomédica/legislação & jurisprudência , Pesquisa Biomédica/organização & administração , Pesquisa Biomédica/normas , Linhagem Celular Transformada , Diagnóstico por Imagem , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Transplante de Neoplasias/métodos , Transplante de Neoplasias/patologia , Transplante de Neoplasias/normas , Neoplasias/diagnóstico , Neoplasias/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The purpose of the work was to identify novel inhibitors of the enzyme NQO2. Using computational molecular modelling, a QSAR (R(2)=0.88) was established, relating inhibitory potency with calculated binding affinity. From this, the imidazoacridin-6-one, NSC660841, was identified as the most potent inhibitor of NQO2 yet reported (IC(50)=6 nM).
Assuntos
Acridinas/química , Acridonas/química , Inibidores Enzimáticos/química , Imidazóis/química , Quinona Redutases/antagonistas & inibidores , Acridinas/farmacologia , Acridonas/farmacologia , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Bases de Dados Factuais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Relação Quantitativa Estrutura-Atividade , Quinona Redutases/metabolismo , TermodinâmicaRESUMO
BACKGROUND: Hypoxia is as an indicator of poor treatment outcome. Consistently, hypoxic HCT116 colorectal cancer cells are resistant to oxaliplatin, although the mechanistic basis is unclear. This study sought to investigate the relative contribution of HIF-1 (hypoxia-inducible factor-1)-mediated gene expression and drug penetrance to oxaliplatin resistance using three-dimensional spheroids. METHODS: Hypoxia-inducible factor-1alpha function was suppressed by the stable expression of a dominant-negative form in HCT116 cells (DN). Cells were drug exposed as monolayer or multicellular spheroid cultures. Cells residing at differing oxygenation status were isolated from Hoechst 33342-treated spheroids using flow cytometry. Sub-populations were subjected to clonogenic survival assays and to Inductively-Coupled Plasma Mass Spectroscopy to determine oxaliplatin uptake. RESULTS: In spheroids, a sensitivity gradient (hypoxicAssuntos
Antineoplásicos/farmacologia
, Neoplasias Colorretais/tratamento farmacológico
, Fator 1 Induzível por Hipóxia/fisiologia
, Compostos Organoplatínicos/farmacologia
, Hipóxia Celular
, Sobrevivência Celular/efeitos dos fármacos
, Neoplasias Colorretais/metabolismo
, Neoplasias Colorretais/patologia
, Resistencia a Medicamentos Antineoplásicos
, Células HCT116
, Humanos
, Compostos Organoplatínicos/farmacocinética
, Oxaliplatina
, Esferoides Celulares
RESUMO
BACKGROUND: The DREAMtherapy (Dual REctal Angiogenesis MEK inhibition radiotherapy) trial is a novel intertwined design whereby two tyrosine kinase inhibitors (cediranib and selumetinib) were independently evaluated with rectal chemoradiotherapy (CRT) in an efficient manner to limit the extended follow-up period often required for radiotherapy studies. PATIENTS AND METHODS: Cediranib or selumetinib was commenced 10 days before and then continued with RT (45 Gy/25#/5 wks) and capecitabine (825 mg/m2 twice a day (BID)). When three patients in the cediranib 15-mg once daily (OD) cohort were in the surveillance period, recruitment to the selumetinib cohort commenced. This alternating schedule was followed throughout. Three cediranib (15, 20 and 30 mg OD) and two selumetinib cohorts (50 and 75 mg BID) were planned. Circulating and imaging biomarkers of inflammation/angiogenesis were evaluated. RESULTS: In case of cediranib, dose-limiting diarrhoea, fatigue and skin reactions were seen in the 30-mg OD cohort, and therefore, 20 mg OD was defined as the maximum tolerated dose. Forty-one percent patients achieved a clinical or pathological complete response (7/17), and 53% (9/17) had an excellent clinical or pathological response (ECPR). Significantly lower level of pre-treatment plasma tumour necrosis factor alpha (TNFα) was found in patients who had an ECPR. In case of selumetinib, the 50-mg BID cohort was poorly tolerated (fatigue and diarrhoea); a reduced dose cohort of 75-mg OD was opened which was also poorly tolerated, and further recruitment was abandoned. Of the 12 patients treated, two attained an ECPR (17%). CONCLUSIONS: This novel intertwined trial design is an effective way to independently investigate multiple agents with radiotherapy. The combination of cediranib with CRT was well tolerated with encouraging efficacy. TNFα emerged as a potential predictive biomarker of response and warrants further evaluation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiorradioterapia/métodos , Neoplasias Retais/terapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Benzimidazóis/administração & dosagem , Biomarcadores Tumorais , Estudos de Coortes , Feminino , Seguimentos , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Prognóstico , Quinazolinas/administração & dosagem , Neoplasias Retais/patologia , Distribuição TecidualRESUMO
Hypoxia inducible factor 1 (HIF-1) represses the transcription of pro-apoptotic bid in colorectal cancer cells in vitro. To assess the clinical relevance of this observation, HIF-1alpha and Bid were assessed in serial sections of 39 human colorectal adenocarcinomas by immunohistochemistry. In high HIF-1alpha nuclear-positive cell subpopulations, there was a significant reduction in Bid expression (ANOVA, P=0.04). Given the role of Bid in drug-induced apoptosis, these data add impetus to strategies targeting HIF-1 for therapeutic gain.
Assuntos
Adenocarcinoma/metabolismo , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Neoplasias Colorretais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Adenocarcinoma/patologia , Análise de Variância , Neoplasias Colorretais/patologia , Humanos , Imuno-HistoquímicaAssuntos
Ensaios Clínicos como Assunto/normas , Avaliação Pré-Clínica de Medicamentos/normas , Drogas em Investigação/uso terapêutico , Neoplasias/radioterapia , Guias de Prática Clínica como Assunto , Radiossensibilizantes/uso terapêutico , Algoritmos , Ensaios Clínicos como Assunto/métodos , Drogas em Investigação/farmacologia , Humanos , Modelos Biológicos , Radiossensibilizantes/farmacologiaRESUMO
Fifteen nitroaromatic and nitroheterocyclic compounds that can act as "radiosensitizers" were tested for their cytotoxicity toward hypoxic Chinese hamster V79 cells in vitro. The cytotoxicity increased markedly as the electron affinity, measured as a one-electron reduction potential, increased. Non-nitro-containing compounds of similar electron affinities (such as quinones) that also act as radiosensitizers did not exhibit this specific toxicity toward hypoxic cells. The implications of the presence of the nitro group as a prerequisite for the hypoxic cell toxicity were discussed, and the mechanism of the cytotoxicity was compared with that of hypoxic cell radiosensitization.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Hipóxia , Nitrocompostos/toxicidade , Animais , Células Cultivadas , Cricetinae , Oxirredução , Oxigênio , Radiossensibilizantes/toxicidade , Relação Estrutura-AtividadeRESUMO
The inhibition of de novo thymidine (dThd) synthesis by the novel folate-based thymidylate synthase (TS) inhibitor ZD1694 (Tomudex) can achieve tumor cell-specific cytotoxicity in vivo. However, nucleosides in the surrounding microenvironment of tumors may be used by the salvage pathway to regenerate any depleted pools, thus providing an efficient mechanism through which to circumvent the ZD1694-dependent toxicity. Anabolism of dThd to dTMP by dThd kinase (TK) is the first committed step in the dThd salvage pathway. However, dThd phosphorylase (dThdPase) can compete with TK by catalyzing the reversible phosphorolytic cleavage of dThd to thymine and deoxyribose 1-phosphate and rendering the salvaged dThd metabolically unavailable. Both TK and dThdPase are up-regulated in some tumors, and their relative importance is not fully defined. We have studied the influence of dThdPase expression on the capacity of exogenous dThd to reverse ZD1694-dependent growth inhibition and have shown that both intra- and extracellular dThdPase activity can effectively moderate dThd-rescue. This suggests that tumor levels of dThdPase may be an important factor in the outcome of ZD1694 therapy.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Quinazolinas/farmacologia , Tiofenos/farmacologia , Timidina Fosforilase/fisiologia , Timidina/farmacologia , Timidilato Sintase/antagonistas & inibidores , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Meios de Cultivo Condicionados/química , Relação Dose-Resposta a Droga , Feminino , Humanos , Timidina/administração & dosagem , Timidina/metabolismo , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismo , Timidilato Sintase/análise , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
We report that hypoxia regulates and influences the level of the angiogenic enzyme platelet-derived endothelial cell growth factor (PD-ECGF), also called thymidine phosphorylase, in vitro and in vivo. Levels of PD-ECGF protein increased 6-fold in the breast cancer cell line MDA 231 after 16 h of growth in 0.3% oxygen. A simultaneous increase in enzyme activity was observed. Immunohistochemical staining of MDA 231 tumors grown in nu/nu mice showed increased expression of PD-ECGF in those parts of the tumor that are proximal to the areas of necrosis. In addition, increased and widespread staining for PD-ECGF protein was obtained when the tumor vascular supply was occluded for 2 h by clamping. Lowering the media pH to 6.3-6.7 in vitro also resulted in an increase in PD-ECGF protein levels. This study demonstrates that tumor microenvironmental factors can result in the specific up-regulation of an angiogenic enzyme that can also activate 5-fluorouracil prodrugs and hence is exploitable therapeutically.
Assuntos
Biomarcadores Tumorais/metabolismo , Hipóxia Celular/fisiologia , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Timidina Fosforilase/metabolismo , Animais , Neoplasias da Mama/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Nus , Células Tumorais Cultivadas/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Mitomycin C (MMC) is regarded as the prototype bioreductive alkylating agent in clinical use. To elucidate the biochemical basis of MMC resistance, we isolated a drug resistant derivative (designated CHO-MMC) of a Chinese hamster ovary cell line (CHO-K1) by exposure to progressively higher concentrations of MMC. CHO-MMC cells exhibited a 17-fold increase in resistance to MMC and were 33-fold cross-resistant to the monofunctional derivative, decarbamoyl mitomycin C. In contrast, CHO-MMC cells showed only a 2-fold level of resistance to BMY 25282, a more easily activated analogue of MMC, and exhibited parental sensitivity to MMC under radiobiologically hypoxic conditions. CHO-MMC cells showed no increased resistance to a range of DNA damaging agents including several other alkylating agents (e.g., melphalan and methyl methanesulfonate). Cross-resistance to drugs associated with the multidrug resistant phenotype (e.g., Adriamycin and vincristine) was present only at very low levels. Using a specific high performance liquid chromatography technique, we examined the rates of reduction of MMC and BMY 25282 in cell extracts from CHO-K1 and CHO-MMC cells under both aerobic (air) and hypoxic (N2) conditions. Reduction rates for both drugs were at least 30-fold faster under nitrogen than in air. Metabolism of MMC was undetectable in air but was readily detectable under nitrogen and was 2- 3-fold slower in CHO-MMC cell extracts than in CHO-K1 cell extracts. Although BMY 25282 was more readily reduced under nitrogen, no difference was detected between extracts from CHO-K1 or CHO-MMC cells in the rate of reduction of BMY 25282 under either air or nitrogen. The activity of NADPH:cytochrome P-450 (cytochrome c) reductase, an enzyme implicated in the bioreductive activation of MMC, was 3-4-fold lower in CHO-MMC cells than in the parental line. These findings suggest that the resistance of CHO-MMC cells to MMC under aerobic conditions may be due to impaired metabolic activation of the drug as a result of a decrease in NADPH:cytochrome P-450 reductase activity. This supports the view that decreased bioreductive enzyme activity may be a significant mechanism for acquired resistance to MMC in tumor cells in vivo and that more readily activated analogues may be potentially useful in overcoming this specific form of resistance.
Assuntos
Alquilantes/farmacologia , Mitomicinas/farmacologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Aerobiose , Anaerobiose , Animais , Biotransformação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Resistência a Medicamentos , Feminino , Hipóxia/metabolismo , Mitomicina , Mitomicinas/metabolismo , Ovário , Raios UltravioletaRESUMO
The nitric oxide synthase inhibitor NG-nitro-L-arginine (NOARG) was examined for its ability to alter energy metabolism in three murine tumors using 31P magnetic resonance spectroscopy. NOARG (10 mg/kg, i.v.) increased the inorganic phosphate:total phosphate ratio (Pi:total) 2-3-fold in the KHT, RIF-1, and SCCVII/Ha intradermal back tumors from 30 min to 6 h after injection, but the 31P magnetic resonance spectrum from normal tissue on the mouse back was unchanged after this treatment. NOARG (10 mg/kg, i.v.) injected 30 min before X-rays increased tumor cell survival 3-5-fold in SCCVII/Ha and 50-200-fold in RIF-1, measured using an in vivo/in vitro clonogenic assay. These effects were equivalent to those obtained from clamped tumors, indicating full radiobiological hypoxia. In KHT, only a 2-fold increase in radioresistance was observed after NOARG, which was less than the response of clamped tumors. In RIF-1 tumors, NOARG induced full radiobiological hypoxia when given from 30 min to 6 h prior to X-rays, consistent with the time course for the increase in Pi:total, measured by 31P magnetic resonance spectroscopy. Pi:total after NOARG doses of 0.1-10 mg/kg, i.v., increased in a dose-dependent manner in this tumor. Increased RIF-1 tumor radioresistance was similarly dependent on NOARG dose. The combination of the bioreductive agent RB6145 (300 mg/kg, i.p.) 15 min prior to NOARG (10 mg/kg, i.v.) produced greater than 5 decades of KHT tumor cell killing at 24 h after treatment. This combination also increased Pi:total 4.5-fold over the control value at 24 h in the KHT tumor. Histological examination of tumors at this time indicated extensive necrosis.
Assuntos
Arginina/análogos & derivados , Carcinoma/tratamento farmacológico , Metabolismo Energético/efeitos dos fármacos , Sarcoma Experimental/tratamento farmacológico , Animais , Arginina/farmacologia , Carcinoma/metabolismo , Carcinoma/radioterapia , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Nitroarginina , Nitroimidazóis/farmacologia , Fosfatos/metabolismo , Pró-Fármacos/farmacologia , Sarcoma Experimental/metabolismo , Sarcoma Experimental/radioterapiaRESUMO
Nitric oxide synthases (NOSs) play a crucial role in the control of blood flow, memory formation, and the immune response. These proteins can be structurally divided into oxygenase and reductase domains. The reductase domain shares a high degree of sequence homology with P450 reductase, which is thought to be the major enzyme responsible for the one-electron reduction of foreign compounds, including bioreductive antitumor agents currently undergoing clinical trials. In view of the structural similarities between NOS and P450 reductase, we investigated the capacity of NOS to reduce the hypoxic cytotoxin tirapazamine, the antitumor agent doxorubicin, and also the redox cycling compound menadione. All three isoforms exhibited high levels of activity toward these compounds. In the case of doxorubicin and menadione, the activity of NOS II was 5-10-fold higher than the other enzymes, whereas with tirapazamine, the activities were broadly similar. NOS-mediated metabolism of tirapazamine resulted in a large increase in plasmid DNA strand breaks, demonstrating that the reduction was a bioactivation process. In addition, tirapazamine inhibited NOS activity. Because nitric oxide is implicated in maintaining tumor vascular homeostasis, it is conceivable that tirapazamine could potentiate its own toxicity by increasing the degree of hypoxia. This study suggests that the NOSs could play a key role in the therapeutic effects of tirapazamine, particularly because NOS activity is markedly increased in several human tumors. In addition, the presence of NOS in the heart indicates that these enzymes may contribute to the cardiotoxicity of redox cycling drugs, such as doxorubicin.
Assuntos
Antineoplásicos/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Antineoplásicos/farmacologia , Catálise , Bovinos , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Humanos , Camundongos , NADH NADPH Oxirredutases/química , NADPH-Ferri-Hemoproteína Redutase , Óxido Nítrico Sintase/efeitos dos fármacos , Oxirredução , Oxigênio/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Tirapazamina , Triazinas/metabolismo , Triazinas/farmacologia , Vitamina K/metabolismo , Vitamina K/farmacologiaRESUMO
There is increasing evidence that hypoxia-regulated gene expression influences tumor aggressiveness, contributing to the poorer outcome of patients with hypoxic tumors. The role of the transcriptional complex hypoxia-inducible factor-1 as an important mediator of hypoxia-regulated gene expression is one of the best documented pathways. Recently, it has emerged that certain tumor-associated carbonic anhydrases (CAs) can be added to the list of known hypoxia-inducible factor-responsive genes. Here we show that the immunohistochemical expression of the tumor-associated CA IX is correlated with the level of hypoxia in human cervical tumors. We performed a prospective study in 68 patients where needle electrodes were used to make direct measurements of tumor oxygenation levels. CA IX expression was evaluated immunohistochemically in pretreatment tumor biopsies. There was a significant positive correlation between the level of tumor hypoxia (HP5) and the extent of CA IX expression. A retrospective study of 130 squamous cell cervical carcinomas demonstrated that a semiquantitative immunohistochemical analysis of CA IX expression in tumor biopsies is a significant and independent prognostic indicator of overall survival and metastasis-free survival after radiation therapy. These studies provide clinical evidence that CA IX expression is up-regulated in hypoxic human cervical tumors and is associated with a poor prognosis. CA IX may act as an intrinsic marker of tumor hypoxia and poor outcome after radiation therapy. The level of CA IX expression may be used to aid in the selection of patients who would benefit most from hypoxia-modification therapies or bio-reductive drugs.
Assuntos
Antígenos de Neoplasias , Biomarcadores Tumorais/biossíntese , Anidrases Carbônicas , Carcinoma de Células Escamosas/enzimologia , Proteínas de Neoplasias/biossíntese , Oxigênio/metabolismo , Neoplasias do Colo do Útero/enzimologia , Anidrase Carbônica IX , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Hipóxia Celular/fisiologia , Eletrodos , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Estudos Prospectivos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Resultado do Tratamento , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/radioterapiaRESUMO
Tumour hypoxia confers poor prognosis in a wide range of solid tumours, due to an increased malignancy, increased likelihood of metastasis and treatment resistance. Poorly oxygenated tumours are resistant to both radiation therapy and chemotherapy. However, although the link between radiation therapy and hypoxia is well established in a range of clinical studies, evidence of its influence on chemotherapy response is lacking. In this study, a panel of human tumour-derived xenografts that have been characterised previously for in vivo response to a large series of anti-cancer agents, and have been found to show chemosensitivities that correlate strongly with the parent tumour, were used to address this issue. Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded sections of xenograft samples to detect expression of the intrinsic hypoxia marker Glut-1 and adducts of the bioreductive hypoxia marker pimonidazole. Glut-1 scores correlated significantly with T/C values for CCNU sensitivity (r = 0.439, P = 0.036, n = 23) and showed a borderline significant correlation with dacarbazine T/C (r = 0.405, P = 0.076, n = 20). However, there was no correlation between both Glut-1 and pimonidazole scores and T/C obtained for the bioreductive drug mitomycin C. The use of human tumour-derived xenografts offers a potentially useful way of using archival material to determine the influence of hypoxia and other tumour-microenvironmental factors on chemosensitivity without the direct use of human subjects.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Proteínas de Transporte de Monossacarídeos/fisiologia , Neoplasias Experimentais/tratamento farmacológico , Animais , Biomarcadores , Hipóxia Celular , Chaperona BiP do Retículo Endoplasmático , Transportador de Glucose Tipo 1 , Proteínas de Choque Térmico/fisiologia , Humanos , Camundongos , Chaperonas Moleculares/fisiologia , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Transplante HeterólogoRESUMO
NO synthases are unique among eukaryotic enzymes in being dimeric, calmodulin-dependent or calmodulin-containing cytochrome P-450-like haemoproteins that combine reductase and oxygenase catalytic domains in one monomer. They catalyse the formation of NO from L-arginine in the presence of NADPH and molecular oxygen. There are, broadly, three distinctive forms of NO synthase, of which two are constitutively expressed in a variety of cells and are calcium dependent. Of these, the endothelial cell-specific form (eNOS) can play an important role in vascular development, maintenance of vascular tone and tumour growth. A third, inducible, calcium-independent form (iNOS), is important in the immunogenic and cytotoxic response of T-lymphocytes and macrophages. NO acts as an intracellular secondary messenger and provides an efficient system for cellular regulation, interaction and defence, while striking a very fine balance in its role in tumour growth and--under some circumstances--appearing to promote tumour growth, whereas other evidence suggests its production can be growth inhibitory. Nevertheless, tumour cells do express both the constitutive and inducible forms of NO synthase, albeit at widely different levels, and their presence in some human cancers correlates positively with tumour grade. Its role is strictly dependent upon its chemical reactivity with oxygen and metals, e.g. in haem-containing proteins, rather than specific structural interactions with physiological targets. Conflicting evidence still surrounds the effects of expressing high levels of iNOS activity and consequent production of NO on tumour growth. Similar conflicting results have been obtained by applying various NO donors and NO synthase inhibitors. Overall, NO may be acting as part of a signalling cascade for neovascularization in vivo, whereas in vitro cytotoxic properties contribute to the 'apparent' slowing of the growth of cells. It is our contention that low concentrations of NO can be pro-angiogenic and pro-tumour growth, whereas higher NO concentrations can have the opposite effect. Like many other areas of therapeutics, the concept of dose-response is very important. Modification of NO synthase activity in tumours, and hence NO biosynthesis, may be regarded as a promising means for selective tumour blood flow modification and provides a novel approach for reducing tumour oxygenation aimed at enhancing the efficiency of hypoxia-mediated, bioreductively activated anti-cancer drugs.
Assuntos
Neoplasias/metabolismo , Óxido Nítrico/fisiologia , Animais , Divisão Celular , Fatores de Crescimento Endotelial/metabolismo , Estrogênios/farmacologia , Estrogênios/fisiologia , Humanos , Hipóxia/metabolismo , Linfocinas/metabolismo , Neoplasias/tratamento farmacológico , Neovascularização Fisiológica , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Hypoxic tumors are known to be more malignant, to be more likely to metastasize, and to have a poor prognosis. They are also radio- and chemoresistant. For this reason, it is desirable that a clinically useful marker of hypoxia is found, so that treatment with radiotherapy and bioreductive chemotherapy can be rationally applied to individual patients. Glut-1 is a facilitative glucose transporter that is ubiquitously expressed in normal tissue and expressed at higher levels in a number of tumors. Its potential as an intrinsic hypoxia marker arises from its dual control in hypoxic conditions by reduced oxidative phosphorylation and the hypoxia-inducible factor (HIF-1) oxygen-sensing pathway. Eppendorf histography, by virtue of its proven predictive qualities, is a suitable gold standard used in our laboratory to validate new hypoxia markers. Using this technique, pretreatment pO(2) measurements were performed on 54 patients with locally advanced cervical carcinoma. Then, immunohistochemical staining was used to detect Glut-1 protein in individual tumor biopsy sections. Both measurements were made before initiation of treatment. By using a low-tech scoring system, pO(2) was found to correlate weakly with Glut-1 score (r = 0.28; P = 0.04). To extrapolate this correlation to the known adverse effects of tumor hypoxia on outcome, we examined the prognostic significance of Glut-1 staining in a retrospective series of 121 patients. An absence of Glut-1 significantly increased the likelihood of metastasis-free survival (P = 0.022) but did not significantly effect disease-free or recurrence-free survival. These findings suggest that Glut-1 be an intrinsic marker of hypoxia that can easily be applied in a clinical setting.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Transporte de Monossacarídeos/biossíntese , Neoplasias do Colo do Útero/metabolismo , Intervalo Livre de Doença , Feminino , Transportador de Glucose Tipo 1 , Humanos , Hipóxia/diagnóstico , Hipóxia/metabolismo , Imuno-Histoquímica , Pessoa de Meia-Idade , Oxigênio/metabolismo , Prognóstico , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologiaRESUMO
Hypoxia is a feature that exists in most, if not all, solid tumours and hypoxia has been shown to exist in a variety of other diseases. Bioreductive prodrugs have been developed to preferentially target the hypoxic cells in tumours. They are prodrugs, that are reductively activated (catalysed by reductive enzymes) to afford their active (toxic) species. More recently, bioreductive delivery agents that "release" a therapeutic entity preferentially under hypoxic conditions have also been developed to target hypoxia, not only in tumours, but also in a host of other diseases. This new technology platform is described in this review. In addition, we discuss the potential of utilising hypoxia to deliver selective gene therapy based upon the transcription factor HIF-1 and the use of unique genetic sequences termed HRE's (hypoxia responsive elements) that specifically control gene expression under hypoxic conditions. Finally, we describe how these drugs and gene-based therapeutic approaches can be combined to potentially deliver a highly selective form of therapy for cancer and other diseases where hypoxia plays a major pathophysiological role.
Assuntos
Terapia Genética/métodos , Hipóxia/terapia , Animais , Humanos , Oxirredução , QuinonasRESUMO
The effect of hydralazine treatment on 3 murine tumours (RIF-1, KHT and 16/C) was monitored using 31P-NMR. Changes in the 31P-NMR spectrum are compared with measurements of radiobiological hypoxic fraction (RHF) in the RIF-1 and KHT. Hydralazine is known to reduce temporarily blood flow in experimental tumours, and thus cause a transient increase in the RHF to 100% (in RIF-1 and KHT). This correlates with a decline in energy status as measured by 31P-NMR (i.e. there was an increase in Pi in all three tumours). Time-course data from the RIF-1 and KHT tumours show that maintenance of anaesthesia prolongs the hypoxia induced by hydralazine.