RESUMO
Background: The differentiation of high-grade glioma and brain tumors of an extracranial origin is eminent for the decision on subsequent treatment regimens. While in high-grade glioma, a surgical resection of the tumor mass is a fundamental part of current standard regimens, in brain metastasis, the burden of the primary tumor must be considered. However, without a cancer history, the differentiation remains challenging in the imaging. Hence, biopsies are common that may help to identify the tumor origin. An additional tool to support the differentiation may be of great help. For this purpose, we aimed to identify a biomarker panel based on the expression analysis of a small sample of tissue to support the pathological analysis of surgery resection specimens. Given that an aberrant glutamate signaling was identified to drive glioblastoma progression, we focused on glutamate receptors and key players of glutamate homeostasis. Methods: Based on surgically resected samples from 55 brain tumors, the expression of ionotropic and metabotropic glutamate receptors and key players of glutamate homeostasis were analyzed by RT-PCR. Subsequently, a receiver operating characteristic (ROC) analysis was performed to identify genes whose expression levels may be associated with either glioblastoma or brain metastasis. Results: Out of a total of 29 glutamatergic genes analyzed, nine genes presented a significantly different expression level between high-grade gliomas and brain metastases. Of those, seven were identified as potential biomarker candidates including genes encoding for AMPA receptors GRIA1, GRIA2, kainate receptors GRIK1 and GRIK4, metabotropic receptor GRM3, transaminase BCAT1 and the glutamine synthetase (encoded by GLUL). Overall, the biomarker panel achieved an accuracy of 88% (95% CI: 87.1, 90.8) in predicting the tumor entity. Gene expression data, however, could not discriminate between patients with seizures from those without. Conclusion: We have identified a panel of seven genes whose expression may serve as a biomarker panel to discriminate glioblastomas and brain metastases at the molecular level. After further validation, our biomarker signatures could be of great use in the decision making on subsequent treatment regimens after diagnosis.
RESUMO
Several cues for a directional migration of colorectal cancer cells were identified as being crucial in tumor progression. However, galvanotaxis, the directional migration in direct-current electrical fields, has not been investigated so far. Therefore, we asked whether direct-current electrical fields could be used to mobilize colorectal cancer cells along field vectors. For this purpose, five patient-derived low-passage cell lines were exposed to field strengths of 150-250 V/m in vitro, and migration along the field vectors was investigated. To further study the role of voltage-gated calcium channels on galvanotaxis and intracellular signaling pathways that are associated with migration of colorectal cancer cells, the cultures were exposed to selective inhibitors. In three out of five colorectal cancer cell lines, we found a preferred cathodal migration. The cellular integrity of the cells was not impaired by exposure of the cells to the selected field strengths. Galvanotaxis was sensitive to inhibition of voltage-gated calcium channels. Furthermore, signaling pathways such as AKT and MEK, but not STAT3, were also found to contribute to galvanotaxis in our in vitro model system. Overall, we identify electrical fields as an important contributor to the directional migration of colorectal cancer cells.
RESUMO
Glioblastoma is the most common and aggressive primary brain tumor. Multiple genetic and epigenetic alterations in several major signaling pathways-including the phosphoinositide 3-kinases (PI3K)/AKT/mTOR and the Raf/MEK/ERK pathway-could be found. We therefore aimed to investigate the biological and molecular effects of small-molecule kinase inhibitors that may interfere with those pathways. For this purpose, patient-derived glioblastoma cells were challenged with dactolisib, ipatasertib, MK-2206, regorafenib, or trametinib. To determine the effects of the small-molecule kinase inhibitors, assays of cell proliferation and apoptosis and immunoblot analyses were performed. To further investigate the effects of ipatasertib on organotypic brain slices harboring glioblastoma cells, the tumor growth was estimated. In addition, the network activity in brain slices was assessed by electrophysiological field potential recordings. Multi-kinase inhibitor regorafenib and both MK-2206 and dactolisib were very effective in all preclinical tumor models, while with respect to trametinib, two cell lines were found to be highly resistant. Only in HROG05 cells, ipatasertib showed anti-tumoral effects in vitro and in organotypic brain slices. Additionally, ipatasertib diminished synchronous network activity in organotypic brain slices. Overall, our data suggest that ipatasertib was only effective in selected tumor models, while especially regorafenib and MK-2206 presented a uniform response pattern.
RESUMO
Microbeam radiotherapy could help to cure malignant tumours which are currently still considered therapy-resistant. With an irradiation target in the thoracic cavity, the heart would be one of the most important organs at risk. To assess the acute adverse effects of microbeam irradiation in the heart, a powerful ex vivo tool was created by combining the Langendorff model of the isolated beating mammalian heart with X-Tream dosimetry. In a first pilot experiment conducted at the Biomedical and Imaging Beamline of the Australian Synchrotron, the system was tested at a microbeam peak dose approximately ten times higher than the anticipated future microbeam irradiation treatment doses. The entire heart was irradiated with a dose of 4000â Gy at a dose rate of >6000â Gyâ s-1, using an array of 50â µm-wide microbeams spaced at a centre-to-centre distance of 400â µm. Although temporary arrhythmias were seen, they reverted spontaneously to a stable rhythm and no cardiac arrest occurred. This amazing preservation of cardiac function is promising for future therapeutic approaches.
Assuntos
Radiometria , Síncrotrons , Animais , Austrália , Mamíferos , Radiometria/métodosRESUMO
PURPOSE: Microbeam radiation therapy (MRT) has shown several advantages compared with conventional broad-beam radiation therapy in small animal models, including a better preservation of normal tissue function and improved drug delivery based on a rapidly increased vascular permeability in the target region. Normal tissue tolerance is the limiting factor in clinical radiation therapy. Knowledge of the normal tissue tolerance of organs at risk is therefore a prerequisite in evaluating any new radiation therapy approach. With an irradiation target in the thoracic cavity, the heart would be the most important organ at risk. METHODS AND MATERIALS: We used the ex vivo beating rodent heart in the Langendorff perfusion system at the synchrotron to administer microbeam irradiation (MBI) with a peak dose of 40 or 400 Gy. By continuously recording the electrocardiogram, the left ventricular pressure, and the aortic pressure before, during and after MBI, we were able to assess acute and subacute effects of MBI on electrophysiological and mechanical cardiac function. In addition, we analyzed histologic and ultrastructural sequelae caused by MBI. RESULTS: There were no significant changes in heart rate, heart rate variability, systolic increase of left ventricular pressure or aortic pressure. Moreover, the changes of heart rate, left ventricular pressure and aortic pressure by adding 10-5 mol/L norepinephrine to the perfusate, were also not significant between MBI and sham experiments. However, the rate-pressure product as a surrogate marker for maximum workload after MBI was significantly lower compared with sham-irradiated controls. On the structural level, no severe membranous, sarcomeric, mitochondrial or nuclear changes caused by MBI were detected by desmin immunohistochemistry and electron microscopy. CONCLUSIONS: With respect to acute and subacute toxicity, an MBI peak dose up to 400 Gy did not result in severe changes in cardiac electrophysiology or mechanics.
Assuntos
Roedores , Síncrotrons , Animais , Coração , Imuno-Histoquímica , Modelos AnimaisRESUMO
Galvanotaxis, the migration along direct current electrical fields, may contribute to the invasion of brain cancer cells in the tumor-surrounding tissue. We hypothesized that pharmacological perturbation of the epidermal growth factor (EGF) receptor and downstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway prevent galvanotactic migration. In our study, patient-derived glioblastoma and brain metastases cells were exposed to direct current electrical field conditions. Velocity and direction of migration were estimated. To determine the effects of EGF receptor antagonist afatinib and AKT inhibitor capivasertib, assays of cell proliferation, apoptosis and immunoblot analyses were performed. Both inhibitors attenuated cell proliferation in a dose-dependent manner and induced apoptosis. We found that most of the glioblastoma cells migrated preferentially in an anodal direction, while brain metastases cells were unaffected by direct current stimulations. Afatinib presented only a mild attenuation of galvanotaxis. In contrast, capivasertib abolished the migration of glioblastoma cells without genetic alterations in the PI3K/AKT pathway, but not in cells harboring PTEN mutation. In these cells, an increase in the activation of ERK1/2 may in part substitute the inhibition of the AKT pathway. Overall, our data demonstrate that glioblastoma cells migrate in the electrical field and the PI3K/AKT pathway was found to be highly involved in galvanotaxis.
RESUMO
BACKGROUND: High-dose-rate radiotherapy has shown promising results with respect to normal tissue preservation. We developed an ex vivo model to study the physiological effects of experimental radiotherapy in the rodent esophageal smooth muscle. METHODS: We assessed the physiological parameters of the esophageal function in ex vivo preparations of the proximal, middle, and distal segments in the organ bath. High-dose-rate synchrotron irradiation was conducted using both the microbeam irradiation (MBI) technique with peak doses greater than 200 Gy and broadbeam irradiation (BBI) with doses ranging between 3.5-4 Gy. RESULTS: Neither MBI nor BBI affected the function of the contractile apparatus. While peak latency and maximal force change were not affected in the BBI group, and no changes were seen in the proximal esophagus segments after MBI, a significant increase in peak latency and a decrease in maximal force change was observed in the middle and distal esophageal segments. CONCLUSION: No severe changes in physiological parameters of esophageal contraction were determined after high-dose-rate radiotherapy in our model, but our results indicate a delayed esophageal function. From the clinical perspective, the observed increase in peak latency and decreased maximal force change may indicate delayed esophageal transit.
Assuntos
Esôfago , Roedores , Animais , Contração Muscular/fisiologia , Músculo LisoRESUMO
The progression of glioblastomas is associated with a variety of neurological impairments, such as tumor-related epileptic seizures. Seizures are not only a common comorbidity of glioblastoma but often an initial clinical symptom of this cancer entity. Both, glioblastoma and tumor-associated epilepsy are closely linked to one another through several pathophysiological mechanisms, with the neurotransmitter glutamate playing a key role. Glutamate interacts with its ionotropic and metabotropic receptors to promote both tumor progression and excitotoxicity. In this review, based on its physiological functions, our current understanding of glutamate receptors and glutamatergic signaling will be discussed in detail. Furthermore, preclinical models to study glutamatergic interactions between glioma cells and the tumor-surrounding microenvironment will be presented. Finally, current studies addressing glutamate receptors in glioma and tumor-related epilepsy will be highlighted and future approaches to interfere with the glutamatergic network are discussed.
Assuntos
Neoplasias Encefálicas/complicações , Encéfalo/metabolismo , Epilepsia/etiologia , Glioblastoma/complicações , Ácido Glutâmico/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Anticonvulsivantes/uso terapêutico , Antineoplásicos/uso terapêutico , Encéfalo/patologia , Encéfalo/fisiopatologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Ondas Encefálicas , Progressão da Doença , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Transdução de SinaisRESUMO
Gliomas are the most common primary brain tumors and often become apparent through symptomatic epileptic seizures. Glial cells express the inwardly rectifying K+ channel Kir4.1 playing a major role in K+ buffering, and are presumably involved in facilitating epileptic hyperexcitability. We therefore aimed to investigate the molecular and functional expression of Kir4.1 channels in cultured rat and human glioma cells. Quantitative PCR showed reduced expression of Kir4.1 in rat C6 and F98 cells as compared to control. In human U-87MG cells and in patient-derived low-passage glioblastoma cultures, Kir4.1 expression was also reduced as compared to autopsy controls. Testing Kir4.1 function using whole-cell patch-clamp experiments on rat C6 and two human low-passage glioblastoma cell lines (HROG38 and HROG05), we found a significantly depolarized resting membrane potential (RMP) in HROG05 (-29 ± 2 mV, n = 11) compared to C6 (-71 ± 1 mV, n = 12, P < 0.05) and HROG38 (-60 ± 2 mV, n = 12, P < 0.05). Sustained K+ inward or outward currents were sensitive to Ba2+ added to the bath solution in HROG38 and C6 cells, but not in HROG05 cells, consistent with RMP depolarization. While immunocytochemistry confirmed Kir4.1 in all three cell lines including HROG05, we found that aquaporin-4 and Kir5.1 were also significantly reduced suggesting that the Ba2+-sensitive K+ current is generally impaired in glioma tissue. In summary, we demonstrated that glioma cells differentially express functional inwardly rectifying K+ channels suggesting that impaired K+ buffering in cells lacking functional Ba2+-sensitive K+ currents may be a risk factor for increased excitability and thereby contribute to the differential epileptogenicity of gliomas.
Assuntos
Bário/administração & dosagem , Neoplasias Encefálicas/fisiopatologia , Glioma/fisiopatologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Glioma/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos WistarRESUMO
An abnormal glutamate signaling of glioblastoma may contribute to both tumor progression and the generation of glioma-associated epileptic seizures. We hypothesized that the AMPA receptor antagonist perampanel (PER) could attenuate tumor growth and epileptic events. F98 glioma cells, grown orthotopically in Fischer rats, were employed as a model of glioma to investigate the therapeutic efficiency of PER (15 mg/kg) as adjuvant to standard radiochemotherapy (RCT). The epileptiform phenotype was investigated by video-EEG analysis and field potential recordings. Effects on glioma progression were estimated by tumor size quantification, survival analysis and immunohistological staining. Our data revealed that orthotopically-growing F98 glioma promote an epileptiform phenotype in rats. RCT reduced the tumor size and prolonged the survival of the animals. The adjuvant administration of PER had no effect on tumor progression. The tumor-associated epileptic events were abolished by PER application or RCT respectively, to initial baseline levels. Remarkably, PER preserved the glutamatergic network activity on healthy peritumoral tissue in RCT-treated animals. F98 tumors are not only a robust model to investigate glioma progression, but also a viable model to simulate a glioma-associated epileptiform phenotype. Furthermore, our data indicate that PER acts as a potent anticonvulsant and may protect the tumor-surrounding tissue as adjuvant to RCT, but failed to attenuate tumor growth or promote animal survival.
RESUMO
INTRODUCTION: Ageing-related processes such as cellular senescence are believed to underlie the accumulation of diseases in time, causing (co)morbidity, including cancer, thromboembolism and stroke. Interfering with these processes may delay, stop or reverse morbidity. The aim of this study is to investigate the link between (co)morbidity and ageing by exploring biomarkers and molecular mechanisms of disease-triggered deterioration in patients with pancreatic ductal adenocarcinoma (PDAC) and (thromboembolic) ischaemic stroke (IS). METHODS AND ANALYSIS: We will recruit 50 patients with PDAC, 50 patients with (thromboembolic) IS and 50 controls at Rostock University Medical Center, Germany. We will gather routine blood data, clinical performance measurements and patient-reported outcomes at up to seven points in time, alongside in-depth transcriptomics and proteomics at two of the early time points. Aiming for clinically relevant biomarkers, the primary outcome is a composite of probable sarcopenia, clinical performance (described by ECOG Performance Status for patients with PDAC and the Modified Rankin Scale for patients with stroke) and quality of life. Further outcomes cover other aspects of morbidity such as cognitive decline and of comorbidity such as vascular or cancerous events. The data analysis is comprehensive in that it includes biostatistics and machine learning, both following standard role models and additional explorative approaches. Prognostic and predictive biomarkers for interventions addressing senescence may become available if the biomarkers that we find are specifically related to ageing/cellular senescence. Similarly, diagnostic biomarkers will be explored. Our findings will require validation in independent studies, and our dataset shall be useful to validate the findings of other studies. In some of the explorative analyses, we shall include insights from systems biology modelling as well as insights from preclinical animal models. We anticipate that our detailed study protocol and data analysis plan may also guide other biomarker exploration trials. ETHICS AND DISSEMINATION: The study was approved by the local ethics committee (Ethikkommission an der Medizinischen Fakultät der Universität Rostock, A2019-0174), registered at the German Clinical Trials Register (DRKS00021184), and results will be published following standard guidelines.
Assuntos
Adenocarcinoma , Isquemia Encefálica , AVC Isquêmico , Neoplasias Pancreáticas , Acidente Vascular Cerebral , Adenocarcinoma/epidemiologia , Envelhecimento , COVID-19 , Senescência Celular , Estudos de Coortes , Comorbidade , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Neoplasias Pancreáticas/epidemiologia , Estudos Prospectivos , Qualidade de Vida , SARS-CoV-2 , Acidente Vascular Cerebral/epidemiologiaRESUMO
Epileptic seizures are frequent in patients with glioma, and anticonvulsive treatment is often indicated. Glioma cells release glutamate via the Xc- antiporter system, which appears to be a major pathomechanism of glioma-associated seizures and excitotoxicity. In addition, the proliferation and survival of the tumor cells are promoted. Therefore, anticonvulsants that attenuate glutamate-mediated receptor activation could be especially effective. In this study, we investigated the effects of AMPA receptor antagonist perampanel in rat C6 glioma model. In first pilot experiments, perampanel reduced glucose uptake but had no impact of extracellular glutamate level in vitro. To analyze the effects of perampanel in vivo, we injected C6 cells orthotopically into the neocortex of Wistar rats in order to establish a model of glioma-associated epilepsy. Spontaneous recurrent discharges in brain slices were abolished upon perfusion with the AMPA receptor blocker perampanel, supporting the major role of glutamatergic excitation. With respect to the tumor progression, no effect of perampanel on survival of the animals or on glioma size was determined. Our data demonstrate that perampanel inhibit epileptiform discharges in organotypic brain slices of glioma, but failed to attenuate tumor growth or promote animal survival.
Assuntos
Glioma/patologia , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Piridonas/farmacologia , Convulsões/prevenção & controle , Animais , Linhagem Celular Tumoral , Glioma/complicações , Masculino , Nitrilas , Fenótipo , Ratos , Convulsões/complicações , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epileptic seizures are frequent in patients with glioblastoma, and anticonvulsive treatment is often necessary. While clinical guidelines recommend all approved anticonvulsants, so far it is still unclear which of the available drugs is the best therapeutic option for treating glioma-associated seizures, also in view of possible anti-tumorigenic effects. In our study, we employed four patient-derived low-passage cell lines of glioblastoma and three cell lines of brain metastases, and challenged these cultures with four anticonvulsants with different mechanisms of action: levetiracetam, valproic acid, carbamazepine and perampanel. Cell proliferation was determined by bromodeoxyuridine incorporation. To further analyze the effects of perampanel, apoptosis induction was measured by caspase 3/7 activation. Glutamate release was quantified and glucose uptake was determined using 18F-fluorodeoxyglucose. Real-time polymerase chain reaction was employed to assess the expression of genes associated with glutamate release and uptake in brain tumor cells. Of the four anticonvulsants, only perampanel showed systematic inhibitory effects on cell proliferation, whereas all other anticonvulsants failed to inhibit glioma and metastasis cell growth in vitro. Metastasis cells were much more resistant to perampanel than glioblastoma cell lines. Glucose uptake was attenuated in all glioblastoma cells after perampanel exposure, whereas cell death via apoptosis was not induced. Extracellular glutamate levels were found to be significantly higher in glioblastoma cell lines as compared to metastasis cell lines, but could be reduced by perampanel exposure. Incubation with perampanel up-regulated glutamine synthetase expression in glioblastoma cells, whereas treatment with valproic acid and levetiracetam downregulated excitatory amino acid transporter-2 expression. Overall, our data suggest that perampanel acts as an anticonvulsive drug and additionally mediated anti-tumorigenic effects.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Ácido Glutâmico/metabolismo , Piridonas/farmacologia , Receptores de AMPA/antagonistas & inibidores , Anticonvulsivantes/farmacologia , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Glioblastoma/metabolismo , Humanos , Nitrilas , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Regulação para Cima/efeitos dos fármacos , Ácido Valproico/farmacologiaRESUMO
AIM: Mitochondrial DNA (mtDNA) mutations can negatively influence lifespan and organ function. More than 250 pathogenic mtDNA mutations are known, often involving neurological symptoms. Major neurodegenerative diseases share key etiopathogenetic components ie mtDNA mutations, mitochondrial dysfunction and oxidative stress. METHODS: Here, we characterized a conplastic mouse strain (C57BL/6 J-mtNOD) carrying an electron transport chain complex IV mutation that leads to an altered cytochrome c oxidase subunit III. Since this mouse also harbours adenine insertions in the mitochondrial tRNA for arginine, we chose the C57BL/6 J-mtMRL as control strain which also carries a heteroplasmic stretch of adenine repetitions in this tRNA isoform. RESULTS: Using MitoSOX fluorescence, we observed an elevated mitochondrial superoxide production and a reduced gene expression of superoxide dismutase 2 in the 24-month-old mtNOD mouse as compared to control. Together with the decreased expression of the fission-relevant gene Fis1, these data confirmed that the ageing mtNOD mouse had a mitochondrial dysfunctional phenotype. On the functional level, we could not detect significant differences in synaptic long-term potentiation, but found a markedly poor physical constitution to perform the Morris water maze task at the age of 24 months. Moreover, the median lifespan of mtNOD mice was significantly shorter than of control animals. CONCLUSION: Our findings demonstrate that a complex IV mutation leads to mitochondrial dysfunction that translates into survival.
Assuntos
Deficiência de Citocromo-c Oxidase/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Longevidade/genética , Espécies Reativas de Oxigênio/metabolismo , Animais , Encéfalo/metabolismo , Deficiência de Citocromo-c Oxidase/genética , Proteína Glial Fibrilar Ácida/metabolismo , Técnicas In Vitro , Memória/fisiologia , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxidos/metabolismoRESUMO
AIM: To establish patient-individual tumor models of rectal cancer for analyses of novel biomarkers, individual response prediction and individual therapy regimens. METHODS: Establishment of cell lines was conducted by direct in vitro culturing and in vivo xenografting with subsequent in vitro culturing. Cell lines were in-depth characterized concerning morphological features, invasive and migratory behavior, phenotype, molecular profile including mutational analysis, protein expression, and confirmation of origin by DNA fingerprint. Assessment of chemosensitivity towards an extensive range of current chemotherapeutic drugs and of radiosensitivity was performed including analysis of a combined radio- and chemotherapeutic treatment. In addition, glucose metabolism was assessed with 18F-fluorodeoxyglucose (FDG) and proliferation with 18F-fluorothymidine. RESULTS: We describe the establishment of ultra-low passage rectal cancer cell lines of three patients suffering from rectal cancer. Two cell lines (HROC126, HROC284Met) were established directly from tumor specimens while HROC239 T0 M1 was established subsequent to xenografting of the tumor. Molecular analysis classified all three cell lines as CIMP-0/ non-MSI-H (sporadic standard) type. Mutational analysis revealed following mutational profiles: HROC126: APCwt , TP53wt , KRASwt , BRAFwt , PTENwt ; HROC239 T0 M1: APCmut , P53wt , KRASmut , BRAFwt , PTENmut and HROC284Met: APCwt , P53mut , KRASmut , BRAFwt , PTENmut . All cell lines could be characterized as epithelial (EpCAM+) tumor cells with equivalent morphologic features and comparable growth kinetics. The cell lines displayed a heterogeneous response toward chemotherapy, radiotherapy and their combined application. HROC126 showed a highly radio-resistant phenotype and HROC284Met was more susceptible to a combined radiochemotherapy than HROC126 and HROC239 T0 M1. Analysis of 18F-FDG uptake displayed a markedly reduced FDG uptake of all three cell lines after combined radiochemotherapy. CONCLUSION: These newly established and in-depth characterized ultra-low passage rectal cancer cell lines provide a useful instrument for analysis of biological characteristics of rectal cancer.
Assuntos
Biomarcadores Tumorais/análise , Células Epiteliais/patologia , Neoplasias Retais/patologia , Reto/citologia , Idoso , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Análise Mutacional de DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Feminino , Fluordesoxiglucose F18/administração & dosagem , Glucose/metabolismo , Humanos , Fígado/citologia , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mutação , Tolerância a Radiação/efeitos da radiação , Neoplasias Retais/genética , Neoplasias Retais/metabolismo , Neoplasias Retais/terapia , Reto/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: To study the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells (PSCs). METHODS: Quiescent PSCs were isolated from mouse pancreas and activated in vitro by seeding on plastic surfaces. The cells were exposed to D-vitamins as primary cultures (early-activated PSCs) and upon re-culturing (fully-activated cells). Exhibition of vitamin A-containing lipid droplets was visualized by oil-red staining. Expression of α-smooth muscle actin (α-SMA), a marker of PSC activation, was monitored by immunofluorescence and immunoblot analysis. The rate of DNA synthesis was quantified by 5-bromo-2'-deoxyuridine (BrdU) incorporation assays. Real-time PCR was employed to monitor gene expression, and protein levels of interleukin-6 (IL-6) were measured by ELISA. Uptake of proline was determined using 18F-proline. RESULTS: Sustained culture of originally quiescent PSCs induced cell proliferation, loss of lipid droplets and exhibition of stress fibers, indicating cell activation. When added to PSCs in primary culture, all three D-vitamins diminished expression of α-SMA (to 32%-39% of the level of control cells; P < 0.05) and increased the storage of lipids (scores from 1.97-2.15 on a scale from 0-3; controls: 1.49; P < 0.05). No such effects were observed when Dvitamins were added to fully-activated cells, while incorporation of BrdU remained unaffected under both experimental conditions. Treatment of re-cultured PSCs with Dvitamins was associated with lower expression of IL-6 (-42% to -49%; P < 0.05; also confirmed at the protein level) and increased expression of the vitamin D receptor gene (209%-321% vs controls; P < 0.05). There was no effect of Dvitamins on the expression of transforming growth factor-ß1 and collagen type 1 (chain α1). The lowest uptake of proline, a main component of collagen, was observed in calcipotriol-treated PSCs. CONCLUSION: The three D-vitamins inhibit, with similar efficiencies, activation of PSCs in vitro, but cannot reverse the phenotype once the cells are fully activated.
Assuntos
Calcitriol/análogos & derivados , Colecalciferol/farmacologia , Ergocalciferóis/farmacologia , Miofibroblastos/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Pancreatopatias/prevenção & controle , Células Estreladas do Pâncreas/efeitos dos fármacos , Actinas/metabolismo , Animais , Calcitriol/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Fibrose , Interleucina-6/genética , Interleucina-6/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatopatias/metabolismo , Pancreatopatias/patologia , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/patologia , Fenótipo , Prolina/metabolismoRESUMO
OBJECTIVES: Patient-derived tumor cell lines are a powerful tool to analyze the sensitivity of individual tumors to specific therapies in mice. An essential prerequisite for such an approach are reliable quantitative techniques to monitor tumor progression in vivo. METHODS: We have employed HROC24 cells, grown heterotopically in NMRI Foxn1nu mice, as a model of microsatellite instable colorectal cancer to investigate the therapeutic efficiencies of 5'-fluorouracil (5'-FU) and the mutant BRAF inhibitor PLX4720, a vemurafenib analogue, by three independent methods: external measurement by caliper, magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) with 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG). RESULTS: Repeated measure ANOVA by a general linear model revealed that time-dependent changes of anatomic tumor volumes measured by MRI differed significantly from those of anatomic volumes assessed by caliper and metabolic volumes determined by PET/CT. Over the investigation period of three weeks, neither 5'-FU, PLX4720 nor a combination of both drugs affected the tumor volumes. Also, there was no drug effect on the apparent diffusion constant (ADC) value as detected by MRI. Interestingly, however, PET/CT imaging showed that PLX4720-containing therapies transiently reduced the standardized uptake value (SUV), indicating a temporary response to treatment. CONCLUSIONS: 5'-FU and PLX4720 were largely ineffective with respect to HROC24 tumor growth. Tumoral uptake of 18F-FDG, as expressed by the SUV, proved as a sensitive indicator of small therapeutic effects. Metabolic imaging by 18F-FDG PET/CT is a suitable approach to detect effects of tumor-directed therapies early and even in the absence of morphological changes.
RESUMO
BACKGROUND: Aging is broadly considered to be a dynamic process that accumulates unfavourable structural and functional changes in a time dependent fashion, leading to a progressive loss of physiological integrity of an organism, which eventually leads to age-related diseases and finally to death. SCOPE OF REVIEW: The majority of aging-related studies are based on reductionist approaches, focusing on single genes/proteins or on individual pathways without considering possible interactions between them. Over the last few decades, several such genes/proteins were independently analysed and linked to a role that is affecting the longevity of an organism. However, an isolated analysis on genes and proteins largely fails to explain the mechanistic insight of a complex phenotype due to the involvement and integration of multiple factors. MAJOR CONCLUSIONS: Technological advance makes it possible to generate high-throughput temporal and spatial data that provide an opportunity to use computer-based methods. These techniques allow us to go beyond reductionist approaches to analyse large-scale networks that provide deeper understanding of the processes that drive aging. GENERAL SIGNIFICANCE: In this review, we focus on systems biology approaches, based on network inference methods to understand the dynamics of hallmark processes leading to aging phenotypes. We also describe computational methods for the interpretation and identification of important molecular hubs involved in the mechanistic linkage between aging related processes. This article is part of a Special Issue entitled "System Genetics" Guest Editor: Dr. Yudong Cai and Dr. Tao Huang.
Assuntos
Envelhecimento/genética , Redes Reguladoras de Genes/genética , Animais , Humanos , Longevidade/genética , Fenótipo , Biologia de SistemasRESUMO
Uncoupling protein (UCP) 2 is a mitochondrial transporter protein that plays various roles in cellular metabolism, including the glucose and lipid metabolism. Polymorphisms in UCP2 are associated with longevity in humans. In line with this, mice carrying the UCP2 transgene under the control of hypocretin promoter were reported to have an extended lifespan, while, conversely, mice deficient in Ucp2 demonstrated a significantly shorter lifespan. In this study, we examined the phenotype of aging in a large colony of Ucp2-deficient (Ucp2(-/-)) mice on the molecular level. We have found that the significantly shorter lives of Ucp2(-/-) mice is the result of an accelerated aging process throughout their entire lifespan. Thus, Ucp2(-/-) mice not only earlier gained sexual maturity, but also earlier progressed into an aging phenotype, reflected by a decrease in body weight, increased neutrophil numbers, and earlier emergence of spontaneous ulcerative dermatitis. Intriguingly, on the molecular level this acceleration in aging predominantly driven by increased levels of circulating IGF-1 in Ucp2(-/-) mice, hinting at a crosstalk between UCP2 and the classical Insulin/IGF-1 signaling aging pathway.
Assuntos
Envelhecimento , Proteína Desacopladora 2/metabolismo , Animais , Estudos Transversais , Feminino , Fator de Crescimento Insulin-Like I/análise , Estudos Longitudinais , Masculino , Camundongos , Camundongos Knockout , Soro/química , Proteína Desacopladora 2/deficiênciaRESUMO
Large-scale, heteroplasmic and generally pathogenic mtDNA defects (as induced by defective mitochondrial DNA polymerase, clonal mutations or DNA deletions) are known to negatively impact on life span and can result in apoptosis and tissue loss in, e.g., skeletal muscle or reduce learning abilities. The functional impact of homoplasmic specific mtDNA point mutations, e.g., in genes coding for the electron transport chain, however, remains a matter of debate. The present study contributes to this discussion and provides evidence that a single point mutation in complex I of the respiratory chain is associated with impairment of spatial navigation in adolescent (6-month-old) mice, i.e., reduced performance in the Morris Water Maze, which goes along with increased production of reactive oxygen species (ROS) in juvenile mice (3 months) but not at the age of phenotype expression. A point mutation in complex III goes along with only a mild and non-significant negative effect on cognitive performance and no significant changes in ROS production. These findings suggest to also consider the ontogenetic development of phenotypes when studying mtDNA mutations and highlights a possible impact of complex I dysfunction on the emergence of neurological deficits.