RESUMEN
Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Permeabilidad Capilar/fisiología , Microvasos/metabolismo , Animales , Encéfalo/irrigación sanguínea , Caveolas/metabolismo , Femenino , Proteínas del Choque Térmico HSC70/metabolismo , Immunoblotting , Inmunoprecipitación , Ratones , Proteína Disulfuro Isomerasas/metabolismo , Transporte de Proteínas , Proteómica , Ratas , Ratas Sprague-DawleyRESUMEN
Characteristics of cancer cells include a more oxidized redox environment, metabolic reprogramming and apoptosis resistance. Our studies with a lymphoma model have explored connections between the cellular redox environment and cancer cell phenotypes. Alterations seen in lymphoma cells made resistant to oxidative stress include: a more oxidized redox environment despite increased expression of antioxidant enzymes, enhanced net tumour growth, metabolic changes involving the mitochondria and resistance to the mitochondrial pathway to apoptosis. Of particular importance, the cells show cross-resistance to multiple chemotherapeutic agents used to treat aggressive lymphomas. Analyses of clinical and tumour data reveal the worst prognosis when patients' lymphomas have gene expression patterns consistent with the most oxidized redox environment. Lymphomas from patients with the worst survival outcomes express increased levels of proteins involved in oxidative phosphorylation, including cytochrome c. This is consistent with these cells functioning as metabolic opportunists. Using lymphoma cell models and primary lymphoma cultures, we observed enhanced killing using genetic and drug approaches which further oxidize the cellular redox environment. These approaches include increased expression of SOD2 (superoxide dismutase 2), treatment with a manganoporphyrin that oxidizes the glutathione redox couple, or treatment with a copper chelator that inhibits SOD1 and leads to peroxynitrite-dependent cell death. The latter approach effectively kills lymphoma cells that overexpress the anti-apoptotic protein Bcl-2. Given the central role of mitochondria in redox homoeostasis, metabolism and the intrinsic pathway to apoptosis, our studies support the development of new anti-cancer drugs to target this organelle.
Asunto(s)
Mitocondrias/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Linfoma/metabolismo , Mitocondrias/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacosRESUMEN
Since the approval of brentuximab vedotin (BV), assessment of CD30 status by immunohistochemistry gained increasing importance in the clinical management of patients diagnosed with CD30-expressing lymphomas, including classical Hodgkin lymphoma (CHL). Paradoxically, patients with low or no CD30 expression respond to BV. This discrepancy may be due to lack of standardization in CD30 staining methods. In this study, we examined 29 cases of CHL and 4 cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) for CD30 expression using a staining protocol that was designed to detect low CD30 expression levels, and an evaluation system similar to the Allred scoring system used for breast cancer evaluation. For CHL, 10% of cases had low scores and 3% were CD30 negative, with 3 cases in which the majority of tumor cells showed very weak staining. Unexpectedly, one of four cases of NLPHL was positive. We demonstrate intra-patient heterogeneity in CD30 expression levels and staining patterns in tumor cells. Three CHL cases with weak staining may have been missed without the use of control tissue for low expression. Thus, standardization of CD30 immunohistochemical staining with use of known low-expressing controls may aid in proper CD30 assessment and subsequent therapeutic stratification of patients.
Asunto(s)
Enfermedad de Hodgkin , Humanos , Brentuximab Vedotina/uso terapéutico , Diagnóstico Diferencial , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/patología , Inmunohistoquímica , Coloración y EtiquetadoRESUMEN
Acquired resistance to drugs commonly used for lymphoma treatment poses a significant barrier to improving lymphoma patient survival. Previous work with a lymphoma tissue culture model indicates that selection for resistance to oxidative stress confers resistance to chemotherapy-induced apoptosis. This suggests that adaptation to chronic oxidative stress can contribute to chemoresistance seen in lymphoma patients. Oxidative stress-resistant WEHI7.2 cell variants in a lymphoma tissue culture model exhibit a range of apoptosis sensitivities. We exploited this phenotype to test for mitochondrial changes affecting sensitivity to apoptosis in cells made resistant to oxidative stress. We identified impaired release of cytochrome c, and the intermembrane proteins adenylate kinase 2 and Smac/DIABLO, indicating inhibition of the pathway leading to permeabilization of the outer mitochondrial membrane. Blunting of a glucocorticoid-induced signal and intrinsic mitochondrial resistance to cytochrome c release contributed to both points of resistance. The level of Bcl-2 family members or a difference in Bim induction were not contributing factors. The extent of cardiolipin oxidation following dexamethasone treatment, however, did correlate with apoptosis resistance. The differences found in the variants were all proportionate to the degree of resistance to glucocorticoid treatment. We conclude that tolerance to oxidative stress leads to mitochondrial changes that confer resistance to apoptosis.
Asunto(s)
Adaptación Fisiológica , Apoptosis , Linfoma/patología , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo , Neoplasias del Timo/patología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Cardiolipinas/metabolismo , Citocromos c/metabolismo , Immunoblotting , Linfoma/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Neoplasias del Timo/metabolismo , Células Tumorales CultivadasRESUMEN
Barrett's esophagus (BE) is a premalignant condition, where normal squamous epithelium is replaced by intestinal epithelium. BE is associated with an increased risk of developing esophageal adenocarcinoma (EAC). However, the BE cell of origin is not clear. We hypothesize that BE tissue originates from esophageal squamous cells, which can differentiate to columnar cells as a result of repeated exposure to gastric acid and bile acids, two components of refluxate implicated in BE pathology. To test this hypothesis, we repeatedly exposed squamous esophageal HET1A cells to 0.2 mM bile acid (BA) cocktail at pH 5.5 and developed an HET1AR-resistant cell line. These cells are able to survive and proliferate after repeated 2-h treatments with BA at pH 5.5. HET1AR cells are resistant to acidification and express markers of columnar differentiation, villin, CDX2, and cytokeratin 8/18. HET1AR cells have increased amounts of reactive oxygen species, concomitant with a decreased level and activity of manganese superoxide dismutase compared with parental cells. Furthermore, HET1AR cells express proteins and activate signaling pathways associated with inflammation, cell survival, and tumorigenesis that are thought to contribute to BE and EAC development. These include STAT3, NF-κB, epidermal growth factor receptor (EGFR), cyclooxygenase-2, interleukin-6, phosphorylated mammalian target of rapamycin (p-mTOR), and Mcl-1. The expression of prosurvival and inflammatory proteins and resistance to cell death could be partially modified by inhibition of STAT3 signaling. In summary, our study shows that long-term exposure of squamous cells to BA at acidic pH causes the cells to display the same characteristics and markers as BE.
Asunto(s)
Esófago de Barrett/etiología , Ácidos y Sales Biliares/farmacología , Esófago/efectos de los fármacos , Ácidos/farmacología , Adenocarcinoma/metabolismo , Biomarcadores/metabolismo , Línea Celular Transformada , Regulación hacia Abajo , Resistencia a Medicamentos , Epitelio/metabolismo , Neoplasias Esofágicas/metabolismo , Esófago/citología , Esófago/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Interleucina-6/metabolismo , Metaplasia/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Superóxido Dismutasa/metabolismo , Regulación hacia ArribaRESUMEN
Superoxide dismutases play an important role in human health and disease. Three decades of effort have gone into synthesizing SOD mimics for clinical use. The result is the Mn porphyrins which have SOD-like activity. Several clinical trials are underway to test the efficacy of these compounds in patients, particularly as radioprotectors of normal tissue during cancer treatment. However, aqueous chemistry data indicate that the Mn porphyrins react equally well with multiple redox active species in cells including H2O2, O2â¢-, ONOO-, thiols, and ascorbate among others. The redox potential of the Mn porphyrins is midway between the potentials for the oxidation and reduction of O2â¢-. This positions them to react equally well as oxidants and reductants in cells. The result of this unique chemistry is that: 1) the species the Mn porphyrins react with in vivo will depend on the relative concentrations of the reactive species and Mn porphyrins in the cell of interest, and 2) the Mn porphyrins will act as catalytic (redox cycling) agents in vivo. The ability of the Mn porphyrins to catalyze protein S-glutathionylation means that Mn porphyrins have the potential to globally modulate cellular redox regulatory signaling networks. The purpose of this review is to summarize the data that indicate the Mn porphyrins have diverse reactions in vivo that are the basis of the observed biological effects. The ability to catalyze multiple reactions in vivo expands the potential therapeutic use of the Mn porphyrins to disease models that are not SOD based.
Asunto(s)
Manganeso/farmacología , Porfirinas/farmacología , Compuestos de Sulfhidrilo/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Humanos , Oxidación-Reducción , TermodinámicaRESUMEN
P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Dolor Agudo/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Microvasos/metabolismo , Dolor/metabolismo , Animales , Caveolina 1/metabolismo , Femenino , Transporte de Proteínas/fisiología , RatasRESUMEN
Cortical spreading depression (CSD) in the CNS is suggested as a common mechanism contributing to headache. Despite strong evidence for CNS involvement in headache disorders, drug development for headache disorders remains focused on peripheral targets. Difficulty in delivering drugs across the blood-brain barrier (BBB) may partially account for this disparity. It is known, however, that BBB permeability is increased during several CNS pathologies. In this study, we investigated BBB changes in response to KCl-induced CSD events and subsequent allodynia in rats. Cortical KCl injection in awake, freely moving rats produced facial allodynia with peak intensity between 1.5 and 3 h and CSD induction within 0.5-2 h postinjection. Brain perfusion of 14C-sucrose as a marker of BBB paracellular permeability revealed increased leak in the cortex, but not brainstem, beginning 0.5 h post-KCl injection and resolving within 6 h; no changes in tight junction (TJ) proteins occludin or claudin-5 expression were observed. Acute pretreatment with topiramate to inhibit CSD did not prevent the increased BBB paracellular permeability. CNS delivery of the abortive anti-migraine agent sumatriptan was increased in the cortex 1.5 h post-KCl injection. Surprisingly, sumatriptan uptake was also increased in the brainstem following CSD induction, suggesting regulation of active transport mechanisms at the BBB. Together, these results demonstrate the ability of CSD events to produce transient, time-dependent changes in BBB permeability when allodynia is present and to mediate access of clinically relevant therapeutics (i.e., sumatriptan) to the CNS.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Tronco Encefálico/efectos de los fármacos , Fármacos del Sistema Nervioso Central/farmacología , Corteza Cerebral/efectos de los fármacos , Depresión de Propagación Cortical/fisiología , Cefalea/tratamiento farmacológico , Hiperalgesia/fisiopatología , Sumatriptán/farmacología , Animales , Fármacos del Sistema Nervioso Central/farmacocinética , Corteza Cerebral/fisiopatología , Modelos Animales de Enfermedad , Femenino , Cefalea/fisiopatología , Hiperalgesia/inducido químicamente , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Sumatriptán/farmacocinética , Topiramato/farmacologíaRESUMEN
The rates of opioid prescription and use have continued to increase over the last few decades resulting in a greater number of opioid tolerant patients. Treatment of acute pain from surgery and injury is a clinical challenge for these patients. Several pain management strategies including prescribing increased opioids are used clinically with limited success; all currently available strategies have significant limitations. Many opioids are a substrate for p-glycoprotein (p-gp), an efflux transporter at the blood-brain barrier (BBB). Increased p-gp is associated with a decreased central nervous system uptake and analgesic efficacy of morphine. Our laboratory previously found that acute peripheral inflammatory pain (PIP) induces p-gp trafficking from the nucleus to the luminal surface of endothelial cells making up the BBB concomitant with increased p-gp activity and decreased morphine analgesic efficacy. In the current study, we tested whether PIP-induced p-gp trafficking could contribute to decreased opioid efficacy in morphine tolerant rats. A 6-day continuous dosing of morphine from osmotic minipumps was used to establish morphine tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase in morphine tolerant rats. This observation suggests that p-gp trafficking contributes to the decreased morphine analgesic effects in morphine tolerant rats experiencing an acute pain stimulus. Attenuating p-gp trafficking during an acute pain stimulus could improve pain management by increasing the amount of opioid that could reach CNS analgesic targets and decrease the need for the dose escalation that is a serious challenge in pain management.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Encéfalo/irrigación sanguínea , Núcleo Celular/metabolismo , Microvasos/metabolismo , Morfina/administración & dosificación , Animales , Carragenina/toxicidad , Femenino , Dolor/inducido químicamente , Transporte de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS: We aim here to demonstrate that radiation (RT) enhances tumor sensitization by only those Mn complexes that are redox active and cycle with ascorbate (Asc), thereby producing H2O2 and utilizing it subsequently in protein S-glutathionylation in a glutathione peroxidase (GPx)-like manner. In turn, such compounds affect cellular redox environment, described by glutathione disulfide (GSSG)/glutathione (GSH) ratio, and tumor growth. To achieve our goal, we tested several Mn complexes of different chemical and physical properties in cellular and animal flank models of 4T1 breast cancer cell. Four other cancer cell lines were used to substantiate key findings. RESULTS: Joint administration of cationic Mn porphyrin (MnP)-based redox active compounds, MnTE-2-PyP5+ or MnTnBuOE-2-PyP5+ with RT and Asc contributes to high H2O2 production in cancer cells and tumor, which along with high MnP accumulation in cancer cells and tumor induces the largest suppression of cell viability and tumor growth, while increasing GSSG/GSH ratio and levels of total S-glutathionylated proteins. Redox-inert MnP, MnTBAP3- and two other different types of redox-active Mn complexes (EUK-8 and M40403) were neither efficacious in the cellular nor in the animal model. Such outcome is in accordance with their inability to catalyze Asc oxidation and mimic GPx. INNOVATION: We provided here the first evidence how structure-activity relationship between the catalytic potency and the redox properties of Mn complexes controls their ability to impact cellular redox environment and thus enhance the radiation and ascorbate-mediated tumor suppression. CONCLUSIONS: The interplay between the accumulation of cationic MnPs and their potency as catalysts for oxidation of Asc, protein cysteines, and GSH controls the magnitude of their anticancer therapeutic effects.
Asunto(s)
Antineoplásicos/farmacología , Ácido Ascórbico/metabolismo , Manganeso/farmacología , Estructuras Metalorgánicas/farmacología , Neoplasias/metabolismo , Neoplasias/radioterapia , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Manganeso/química , Estructuras Metalorgánicas/química , Ratones , Ratones Endogámicos BALB C , Neoplasias/patología , Oxidación-Reducción , Relación Estructura-ActividadRESUMEN
PURPOSE: Green tea consumption has been associated with decreased risk of certain types of cancers in humans. Induction of detoxification enzymes has been suggested as one of the biochemical mechanisms responsible for the cancer-preventive effect of green tea. We conducted this clinical study to determine the effect of repeated green tea polyphenol administration on a major group of detoxification enzymes, glutathione S-transferases (GST). METHODS: A total of 42 healthy volunteers underwent a 4-week washout period by refraining from tea or tea-related products. At the end of the washout period, a fasting blood sample was collected, and plasma and lymphocytes were isolated for assessment of GST activity and level. Following the baseline evaluation, study participants underwent 4 weeks of green tea polyphenol intervention in the form of a standardized Polyphenon E preparation at a dose that contains 800 mg epigallocatechin gallate (EGCG) once a day. Polyphenon E was taken on an empty stomach to optimize the oral bioavailability of EGCG. Upon completion of the intervention, samples were collected for postintervention GST assessment. RESULTS: Four weeks of Polyphenon E intervention enhanced the GST activity in blood lymphocytes from 30.7 +/- 12.2 to 35.1 +/- 14.3 nmol/min/mg protein, P = 0.058. Analysis based on baseline activity showed that a statistically significant increase (80%, P = 0.004) in GST activity was observed in individuals with baseline activity in the lowest tertile, whereas a statistically significant decrease (20%, P = 0.02) in GST activity was observed in the highest tertile. In addition, Polyphenon E intervention significantly increased the GST-pi level in blood lymphocytes from 2,252.9 +/- 734.2 to 2,634.4 +/- 1,138.3 ng/mg protein, P = 0.035. Analysis based on baseline level showed that this increase was only significant (P = 0.003) in individuals with baseline level in the lowest tertile, with a mean increase of 80%. Repeated Polyphenon E administration had minimal effects on lymphocyte GST-mu and plasma GST-alpha levels. There was a small but statistically significant decrease (8%, P = 0.003) in plasma GST-alpha levels in the highest tertile. CONCLUSIONS: We conclude that 4 weeks of Polyphenon E administration resulted in differential effects on GST activity and level based on baseline enzyme activity/level, with GST activity and GST-pi level increased significantly in individuals with low baseline enzyme activity/level. This suggests that green tea polyphenol intervention may enhance the detoxification of carcinogens in individuals with low baseline detoxification capacity.
Asunto(s)
Catequina/análogos & derivados , Glutatión Transferasa/sangre , Té , Catequina/administración & dosificación , Catequina/farmacología , Femenino , Gutatión-S-Transferasa pi/sangre , Gutatión-S-Transferasa pi/efectos de los fármacos , Glutatión Transferasa/efectos de los fármacos , Humanos , Isoenzimas/sangre , Isoenzimas/efectos de los fármacos , Linfocitos/enzimología , Masculino , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/farmacologíaRESUMEN
Opioids are currently the primary treatment method used to manage both acute and chronic pain. In the past two to three decades, there has been a surge in the use, abuse and misuse of opioids. The mechanism by which opioids relieve pain and induce euphoria is dependent on the drug crossing the blood-brain barrier and accessing the central nervous system. This suggests the blood brain barrier plays a central role in both the benefits and risks of opioid use. The complex physiological responses to opioids that provide the benefits and drive the abuse also needs to be considered in the resolution of the opioid epidemic.
Asunto(s)
Analgésicos Opioides/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Trastornos Relacionados con Opioides/epidemiología , Epidemias , HumanosRESUMEN
We have used a keratinocyte in vivo/in vitro cell model to test the hypothesis that hydrogen peroxide acts as a signaling molecule, contributing to proliferation and tumorigenesis. A cell line, 6M90, that produces squamous cell carcinoma (SCC), has high levels of ROS and low levels of catalase. A new cell line, MTOC2, generated from parental 6M90 cells by introduction of a Tet-responsive catalase transgene, effectively expressed higher peroxisomal catalase. Increased catalase expression diminished constitutive ROS and enhanced viability after treatment with hydrogen peroxide. Protein tyrosine phosphatase activity was higher in the MTOC2 cells with high catalase, consistent with detection of a lower level of phosphorylation at tyrosine 1068 of the epidermal growth factor receptor (EGF-R). Transcription of downstream c-fos, AP-1 transactivation and cell proliferation were higher in the low catalase cells. An EGF-R inhibitor, AG1478, blocks the higher AP-1 transactivation and cell proliferation of the low catalase 6M90 cells. Tumorigenesis in SCID mice was greatly diminished in the high catalase cells. Our data suggest that hydrogen peroxide functions as a signaling molecule that can modulate activity of a protein tyrosine phosphatase/(s) resulting in phosphorylation of tryrosine/(s) on the EGF-R. Therefore, catalase acts as a tumor-suppressor gene in part by decreasing EGF-R signaling.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Catalasa/genética , Receptores ErbB/metabolismo , Genes Supresores de Tumor , Especies Reactivas de Oxígeno/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Catalasa/análisis , Catalasa/metabolismo , Línea Celular Tumoral , Proliferación Celular , Genes fos/genética , Humanos , Peróxido de Hidrógeno/toxicidad , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Queratinocitos/patología , Ratones , Ratones SCID , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Factor de Transcripción AP-1/genética , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glucocorticoids are one component of combined treatment regimens for many types of lymphoma due to their ability to induce apoptosis in lymphoid cells. In WEHI7.2 murine thymic lymphoma cells, altering catalase and glutathione peroxidase activity by transfection or the use of chemical agents modulates the ability of glucocorticoids to induce apoptosis. This suggests that the oxidative stress response is important in determining the glucocorticoid sensitivity of the cells. For glutathione peroxidase and catalase to detoxify reactive oxygen species (ROS), reducing equivalents in the form of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) are ultimately required. The major source of NADPH in the cell is glucose 6-phosphate dehydrogenase (G6PDH). Therefore, we created G6PDH-overexpressing WEHI7.2 variants to test whether G6PDH activity is a key determinant of glucocorticoid sensitivity in WEHI7.2 cells. G6PDH-overexpressing WEHI7.2 cells were more sensitive to oxidative stress and glucocorticoids. The G6PDH-overexpressing WEHI7.2 variants appeared similar to cells undergoing glucose deprivation with decreased adenosine triphosphate (ATP) synthesis by the mitochondria and increased basal levels of ROS. Overexpression of G6PDH also sensitized the cells to other standard lymphoma chemotherapeutics including cyclophosphamide, doxorubicin, and vincristine. The decreased ATP and elevated ROS due to G6PDH overexpression may be key factors in increasing the sensitivity of the WEHI7.2 cells to lymphoma chemotherapeutics.
Asunto(s)
Apoptosis , Glucosa/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Linfoma/metabolismo , Adenosina Trifosfato/análisis , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/análisis , Caspasas/metabolismo , Catalasa/metabolismo , Línea Celular Tumoral , Dexametasona/farmacología , Dexametasona/uso terapéutico , Predicción , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Glucosafosfato Deshidrogenasa/análisis , Glutatión/análisis , Glutatión/metabolismo , Disulfuro de Glutatión/análisis , Disulfuro de Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Linfoma/tratamiento farmacológico , Linfoma/patología , Ratones , NADP/análisis , NADP/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , TransfecciónRESUMEN
P-glycoprotein (PgP), a drug efflux pump in blood-brain barrier endothelial cells, is a major clinical obstacle for effective central nervous system drug delivery. Identifying PgP regulatory pathways that can be exploited clinically is critical for improving central nervous system drug delivery. We previously found that PgP activity increases in rat brain microvessels concomitant with decreased central nervous system drug delivery in response to acute peripheral inflammatory pain. In the current study, we tested the hypothesis that PgP traffics to the luminal plasma membrane of the microvessel endothelial cells from intracellular stores during peripheral inflammatory pain. Using immunofluorescence microscopy, we detected PgP in endothelial cell nuclei and in the luminal plasma membrane in control animals. Following peripheral inflammatory pain, luminal PgP staining increased while staining in the nucleus decreased. Biochemical analysis of nuclear PgP content confirmed our visual observations. Peripheral inflammatory pain also increased endothelial cell luminal staining of polymerase 1 and transcript release factor/cavin1 and serum deprivation response protein/cavin2, two caveolar scaffold proteins, without changing caveolin1 or protein kinase C delta binding protein/cavin3 location. Our data (a) indicate that PgP traffics from stores in the nucleus to the endothelial cell luminal membrane in response to peripheral inflammatory pain; (b) provide an explanation for our previous observation that peripheral inflammatory pain inhibits central nervous system drug uptake; and (c) suggest a novel regulatory mechanism for PgP activity in rat brain.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Endotelio Vascular/metabolismo , Inflamación Neurogénica/metabolismo , Barrera Hematoencefálica/diagnóstico por imagen , Carragenina/farmacología , Caveolina 1/metabolismo , Endotelio Vascular/diagnóstico por imagen , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microscopía Fluorescente , Microvasos/diagnóstico por imagen , Microvasos/metabolismo , Inflamación Neurogénica/diagnóstico por imagen , Transporte de Proteínas , Cloruro de Sodio/farmacologíaRESUMEN
Dexamethasone-treated WEHI7.2 mouse thymoma cells readily undergo apoptosis. WEHI7.2 variants that overexpress catalase (CAT38) or Bcl-2 (Hb12) show a delay or lack of apoptosis, respectively, when treated with dexamethasone. This is accompanied by a delay or lack of cytochrome c release from the mitochondria suggesting that alterations in the signaling phase of apoptosis are responsible for the observed resistance. Because membranes are a rich source of signaling molecules, we have used 31P NMR spectroscopy to compare phospholipids and their metabolites in WEHI7.2, CAT38 and Hb12 cells after dexamethasone treatment. Increased lysophosphatidylcholine (lysoPtdC) content accompanied phosphatidylserine (PtdS) externalization in the WEHI7.2 cells. Both changes were delayed in CAT38 cells suggesting phosphatidylcholine (PtdC) metabolites may play a role in steroid-induced apoptotic signaling. The steroid-resistant Hb12 cells showed a dramatic increase in glycerophosphocholine (GPC) content, suggesting increased phospholipid turnover may contribute to the anti-apoptotic mechanism of Bcl-2.
Asunto(s)
Apoptosis/efectos de los fármacos , Catalasa/metabolismo , Dexametasona/farmacología , Fosfolípidos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Espectroscopía de Resonancia Magnética , Ratones , Mitocondrias/metabolismo , Fosfatidilserinas/metabolismoRESUMEN
Glucocorticoids induce apoptosis in lymphocytes by causing the release of cytochrome c into the cytosol; however, the events in the signaling phase between translocation of the steroid-receptor complex to the nucleus and the release of cytochrome c have not been elucidated. Previously, we found that, in response to steroid treatment, WEHI7.2 mouse thymic lymphoma cells overexpressing catalase (CAT38) show delayed apoptosis (delayed cytochrome c release) compared to the parental cells, while Bcl-2 overexpressing cells (Hb12) are protected from steroid-induced apoptosis. In lymphocytes, glucocorticoid treatment decreases glucose uptake. Both glucose deprivation and the attendant ATP drop are known inducers of apoptosis. Therefore, we used (31)P and (1)H NMR spectroscopy to compare metabolic profiles of WEHI7.2, CAT38 and Hb12 cells in the presence and absence of dexamethasone to determine: (1) whether glucocorticoid effects on glucose metabolism contribute to the mechanism of steroid-induced apoptosis; and (2) whether catalase or Bcl-2 overexpression altered metabolism thereby providing a mechanism of steroid resistance. Loss of mitochondrial hexokinase activity was correlated to the induction of apoptosis in WEHI7.2 and CAT38 cells. CAT38 and Hb12 cells have an altered basal metabolism which includes increases in hexokinase activity, lactate production when subcultured into new medium, use of mitochondria for ATP production and potentially increased glutaminolysis. These data suggest that: (1) glucocorticoid effects on glucose metabolism may contribute to the mechanism of steroid-induced lymphocyte apoptosis; and (2) the altered metabolism seen in catalase and Bcl-2 overexpressing cells may contribute to both the steroid resistance and increased tumorigenicity of these variants.
Asunto(s)
Catalasa/metabolismo , Dexametasona/farmacología , Metabolismo Energético , Glucosa/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Adenosina Trifosfato/biosíntesis , Adenosina Trifosfato/metabolismo , Aminoácidos/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Hexoquinasa/metabolismo , Ratones , Resonancia Magnética Nuclear BiomolecularRESUMEN
Cancer chemotherapy is often limited by patient's toxicity and tumor drug resistance indicating that new drug development and modification of existing drugs is critical for improving the therapeutic response. Traditional Chinese medicine is a rich source of potential anticancer agents. In particular, cantharidin (CAN), the active principle ingredient from the blister beetle, Mylabris, has anti-tumor activity, but the cytotoxic mechanism is unknown. In leukemia cells, cantharidin induces apoptosis by a p53-dependent mechanism. Cantharidin causes both DNA single- and double-strand breaks. Colony-forming assays with knockout and transfectant cells lines showed that DNA polymerase beta, but not ERCC1, conferred increased cell survival after cantharidin treatment, indicating that base excision repair (BER), rather than nucleotide excision repair (NER), is important for CAN-induced DNA lesions. Oxidative stress-resistant thymic lymphoma-derived WEHI7.2 variants are also more resistant to cantharidin. These data suggest that cantharidin treatment causes oxidative stress that provokes DNA damage and p53-dependent apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Cantaridina/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Endonucleasas/fisiología , Humanos , Estrés OxidativoRESUMEN
Chronic inflammation is often associated with increased cancer frequency. Continuous exposure to reactive oxygen species, as at the site of chronic inflammation, can result in cells with increased antioxidant defense enzymes. In WEHI7.2 cells, overexpression of catalase or thioredoxin by transfection or selection of a cell population resistant to hydrogen peroxide has resulted in WEHI7.2 variants with altered glucose and energy metabolism. This metabolic change would favor survival in a tumor environment. We conclude that metabolic alterations, due to increased antioxidant enzyme expression, may underlie the increased tumorigenicity seen previously in the variants and contribute to the increased tumor risk associated with chronic inflammation.
Asunto(s)
Antioxidantes/metabolismo , Neoplasias/metabolismo , Adenosina Trifosfato/metabolismo , Aminoácidos/metabolismo , Animales , Ácido Aspártico/metabolismo , Línea Celular Tumoral , Glucólisis , Glicosilación , Hexoquinasa/metabolismo , Ratones , Taurina/metabolismoRESUMEN
Imexon is an aziridine-containing iminopyrrolidone with selective growth-inhibitory potency for multiple myeloma. Our previous research indicates that imexon induces mitochondrial alterations, oxidative stress, and apoptosis. This drug represents an interesting model drug with a nonmyelosuppressive profile to study the basic mechanisms leading to antitumor activity and resistance. The major purpose of this study was to characterize an imexon-resistant RPMI8226/I cell line that was developed from RPMI8226 cells by continuous exposure to imexon. No significant differences were observed in the sensitivity to several cytotoxic drugs, including mitoxantrone, mitomycin C, melphalan, methotrexate, cytarabine, cisplatin, vincristine, and paclitaxel, in the imexon-resistant cells. However, RPMI8226/I cells were cross-resistant to arsenic trioxide, doxorubicin, fluorouracil, etoposide, irinotecan, and especially IFN-alpha. The data from DNA microarray and Western blot analyses indicated that the levels of antiapoptotic proteins Bcl-2 and thioredoxin-2, which reside mainly in the mitochondria, are increased in RPMI8226/I cells. In addition, increased levels of lung resistance protein were detected in imexon-resistant cells. Expression of P-glycoprotein was not detected in RPMI8226/I cells. No loss of mitochondrial membrane potential or increase in the levels of reactive oxygen species was observed in RPMI8226/I cells after exposure to imexon; however, the levels of glutathione are increased in the RPMI8226/I cells. Transmission electron microscopy revealed significant changes in the mitochondrial morphology of RPMI8226/I cells, whereas no ultrastructural changes were observed in other cellular compartments. Imexon-resistant RPMI8226/I myeloma cells appear to have a unique mechanism of resistance that is associated with morphological alterations of mitochondria, increased protection against oxidative stress, elevated levels of glutathione, and enhanced expression of antiapoptotic mitochondrial proteins.