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Cancer survivors may experience long-term cardiovascular complications due to chemotherapeutic drugs such as doxorubicin (DOX). The exact mechanism of delayed DOX-induced cardiotoxicity has not been fully elucidated. Sex is an important risk factor for DOX-induced cardiotoxicity. In the current study, we identified sex differences in delayed DOX-induced cardiotoxicity and determined the underlying molecular determinants of the observed sexual dimorphism. Five-week-old male and female mice were administered intraperitoneal injections of DOX (4 mg/kg/week) or saline for 6 weeks. Echocardiography was performed 5 weeks after the last dose of DOX to evaluate cardiac function. Thereafter, mice were sacrificed and gene expression of markers of apoptosis, senescence, and inflammation was measured by PCR in hearts and livers. Proteomic profiling of the heart from both sexes was conducted to determine differentially expressed proteins (DEPs). Only DOX-treated male, but not female, mice demonstrated cardiac dysfunction, cardiac atrophy, and upregulated cardiac expression of Nppb and Myh7. No sex-related differences were observed in DOX-induced expression of most apoptotic, senescence, and pro-inflammatory markers. However, the gene expression of Trp53 was significantly reduced in hearts of DOX-treated female mice only. The anti-inflammatory marker Il-10 was significantly reduced in hearts of DOX-treated male mice only, while the pro-inflammatory marker Il-1α was significantly reduced in livers of DOX-treated female mice only. Gene expression of Tnf-α was reduced in hearts of both DOX-treated male and female mice. Proteomic analysis identified several DEPs after DOX treatment in a sex-specific manner, including anti-inflammatory acute phase proteins. This is the first study to assess sex-specific proteomic changes in a mouse model of delayed DOX-induced cardiotoxicity. Our proteomic analysis identified several sexually dimorphic DEPs, many of which are associated with the anti-inflammatory marker Il-10.
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Cardiotoxicidad , Cardiopatías , Femenino , Masculino , Ratones , Animales , Cardiotoxicidad/etiología , Caracteres Sexuales , Interleucina-10/toxicidad , Antibióticos Antineoplásicos/toxicidad , Proteómica , Ratones Endogámicos C57BL , Doxorrubicina , Cardiopatías/inducido químicamente , Cardiopatías/genética , Apoptosis , Antiinflamatorios/farmacología , Miocitos Cardíacos , Estrés OxidativoRESUMEN
Childhood cancer survivors have a high risk for premature cardiovascular diseases, mainly due to cardiotoxic cancer treatments such as doxorubicin (DOX). Psychosocial stress is a significant cardiovascular risk factor and an enormous burden in childhood cancer survivors. Although observational studies suggest that psychosocial stress is associated with cardiovascular complications in cancer survivors, there is no translationally relevant animal model to study this interaction. We established a "two-hit" model in which juvenile mice were administered DOX (4 mg/kg/week for 3 weeks), paired to a validated model of chronic subordination stress (CSS) 5 weeks later upon reaching adulthood. Blood pressure, heart rate, and activity were monitored by radio-telemetry. At the end of CSS experiment, cardiac function was assessed by echocardiography. Cardiac fibrosis and inflammation were assessed by histopathologic analysis. Gene expressions of inflammatory and fibrotic markers were determined by PCR. Juvenile exposure to DOX followed by adult-onset CSS caused cardiac fibrosis and inflammation as evident by histopathologic findings and upregulated gene expression of multiple inflammatory and fibrotic markers. Intriguingly, juvenile exposure to DOX blunted CSS-induced hypertension but not CSS-induced tachycardia. There were no significant differences in cardiac function parameters among all groups, but juvenile exposure to DOX abrogated the hypertrophic response to CSS. In conclusion, we established a translationally relevant mouse model of juvenile DOX-induced cardiotoxicity that predisposes to adult-onset stress-induced adverse cardiac remodeling. Psychosocial stress should be taken into consideration in cardiovascular risk stratification of DOX-treated childhood cancer survivors.
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Doxorrubicina , Estrés Psicológico , Animales , Cardiotoxicidad/metabolismo , Cardiotoxicidad/patología , Modelos Animales de Enfermedad , Doxorrubicina/metabolismo , Doxorrubicina/toxicidad , Fibrosis , Inflamación/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Estrés OxidativoRESUMEN
Doxorubicin (DOX) is one of the most widely used chemo-therapeutic agents in pediatric oncology. DOX elicits an inflammatory response in multiple organs, which contributes to DOX-induced adverse effects. Cancer itself causes inflammation leading to multiple pathologic conditions. The current study investigated the inflammatory response to DOX and tumors using an EL4-lymphoma, immunocompetent, juvenile mouse model. Four-week old male C57BL/6N mice were injected subcutaneously with EL4 lymphoma cells (5 × 104 cells/mouse) in the flank region, while tumor-free mice were injected with vehicle. Three days following tumor implantation, both tumor-free and tumor-bearing mice were injected intraperitoneally with either DOX (4 mg/kg/week) or saline for 3 weeks. One week after the last DOX injection, the mice were euthanized and the hearts, livers, kidneys, and serum were harvested. Gene expression and serum concentration of inflammatory markers were quantified using real-time PCR and ELISA, respectively. DOX treatment significantly suppressed tumor growth in tumor-bearing mice and caused significant cardiac atrophy in tumor-free and tumor-bearing mice. EL4 tumors elicited a strong inflammatory response in the heart, liver, and kidney. Strikingly, DOX treatment ameliorated tumor-induced inflammation paradoxical to the effect of DOX in tumor-free mice, demonstrating a widely divergent effect of DOX treatment in tumor-free versus tumor-bearing mice.
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Antibióticos Antineoplásicos/uso terapéutico , Doxorrubicina/uso terapéutico , Inflamación/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/farmacología , Biomarcadores/metabolismo , Línea Celular Tumoral , Citocinas/sangre , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Corazón/efectos de los fármacos , Inflamación/sangre , Inflamación/etiología , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Linfoma/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismoRESUMEN
Cardiovascular complications have been frequently reported in cancer patients and survivors, mainly because of various cardiotoxic cancer treatments. Despite the known cardiovascular toxic effects of these treatments, they are still clinically used because of their effectiveness as anti-cancer agents. In this review, we discuss the growing body of evidence suggesting that inhibition of the cytochrome P450 1B1 enzyme (CYP1B1) can be a promising therapeutic strategy that has the potential to prevent cancer treatment-induced cardiovascular complications without reducing their anti-cancer effects. CYP1B1 is an extrahepatic enzyme that is expressed in cardiovascular tissues and overexpressed in different types of cancers. A growing body of evidence is demonstrating a detrimental role of CYP1B1 in both cardiovascular diseases and cancer, via perturbed metabolism of endogenous compounds, production of carcinogenic metabolites, DNA adduct formation, and generation of reactive oxygen species (ROS). Several chemotherapeutic agents have been shown to induce CYP1B1 in cardiovascular and cancer cells, possibly via activating the Aryl hydrocarbon Receptor (AhR), ROS generation, and inflammatory cytokines. Induction of CYP1B1 is detrimental in many ways. First, it can induce or exacerbate cancer treatment-induced cardiovascular complications. Second, it may lead to significant chemo/radio-resistance, undermining both the safety and effectiveness of cancer treatments. Therefore, numerous preclinical studies demonstrate that inhibition of CYP1B1 protects against chemotherapy-induced cardiotoxicity and prevents chemo- and radio-resistance. Most of these studies have utilized phytochemicals to inhibit CYP1B1. Since phytochemicals have multiple targets, future studies are needed to discern the specific contribution of CYP1B1 to the cardioprotective and chemo/radio-sensitizing effects of these phytochemicals.
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Citocromo P-450 CYP1B1/antagonistas & inhibidores , Neoplasias Cardíacas/tratamiento farmacológico , Oncología Médica , Terapia Molecular Dirigida , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Cardiotoxicidad/complicaciones , Neoplasias Cardíacas/radioterapia , HumanosRESUMEN
Doxorubicin (DOX) is an effective chemotherapeutic agent used to treat a wide variety of malignancies. In addition to its multi-organ toxicity, DOX treatment has been shown to induce systemic inflammation in patients and experimental animals. Inflammation alters the expression of hepatic cytochrome P450 (CYP) enzymes, which play important roles in drug metabolism and DOX-induced toxicity. Significant sex differences have been reported in DOX-induced toxicity; however, sex differences in DOX-induced systemic inflammation and the potential effects on hepatic CYP expression have not been determined. In the current work, male and female C57Bl/6 mice were administered DOX (20 mg/kg by intraperitoneal injection), and groups of mice were sacrificed 24 and 72 h after DOX administration. DOX elicited a systemic inflammatory response in both male and female mice, but the inflammatory response was stronger in male mice. DOX altered the expression of hepatic CYP isoforms in a sex-dependent manner. Most notably, inhibition of Cyp2c29 and Cyp2e1 was stronger in male than in female mice, which paralleled the sex differences in systemic inflammation. Therefore, sex differences in DOX-induced systemic inflammation may lead to sexually dimorphic drug interactions, in addition to contributing to the previously reported sexual dimorphism in specific DOX-induced organ toxicity.
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Antibióticos Antineoplásicos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Doxorrubicina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/enzimología , Animales , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Femenino , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Inyecciones Intraperitoneales , Interleucina-6/sangre , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Caracteres Sexuales , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Antibodies are commonly used to detect or isolate proteins from biological samples. Much attention has been paid to the potential for poorly-characterized antibodies to lead to misleading results, but antibody-independent artefacts may also occur. Here, we recount two examples of antibody-independent artefacts that have confounded the interpretation of results in our search for molecular entities associated with memory loss in Alzheimer's disease (AD). First, when using biotin-avidin systems for antibody detection, endogenous biotinylated proteins created spurious bands in Western blots of brain lysates from AD patients and transgenic mouse models of AD. These artefactual bands occurred in a transgene- and strain-dependent manner. A second, unexpected artefact occurred when Protein A-conjugated Sepharose beads were used to deplete lysates of endogenous immunoglobulins prior to immunopurification of target proteins. In these assays, Protein A shed from the beads, then bound to (and was eluted from) an immunoaffinity matrix designed to capture AD-related proteins. The Protein A then bound detection antibodies when the immunoaffinity eluates were analyzed by Western blot. Both of these artefacts-the endogenous biotinylated proteins and the Protein A artefact-can be monitored by including an "irrelevant" antibody as an experimental control (e.g., running a parallel protocol in which the antibody directed against the target of interest is replaced by a non-specific antibody).
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Irreversible cardiotoxicity limits the clinical application of doxorubicin (DOX). DOX-induced cardiotoxicity has been associated with induction of senescence and activation of the p38 MAPK pathway. Losmapimod (LOSM), an orally active p38 MAPK inhibitor, is an anti-inflammatory agent with cardioprotective effects. Nevertheless, the effect of LOSM against DOX-induced cardiotoxicity has not been reported. In this study, we determined the effects of LOSM on DOX-induced chronic cardiotoxicity in C57BL/6â¯N mice. Five-week-old C57BL/6â¯N mice were fed diet containing LOSM (estimated daily intake 12â¯mg/kg/day) or a control diet for four days. Thereafter, mice were randomized to receive six weekly intraperitoneal injections of either DOX (4â¯mg/kg) or saline. Three days after the last injection, cardiac function was assessed by trans-thoracic echocardiography. Activation of p38, JNK, and ERK1/2 MAPKs were assessed by immunoblotting in the heart and liver. Gene expressions of senescence, inflammatory, oxidative stress, and mitochondrial function markers were quantified using real-time PCR and serum inflammatory markers were assessed by Luminex. Our results demonstrated that LOSM attenuated p38 MAPK activation, ameliorated DOX-induced cardiac dysfunction, and abrogated DOX-induced expression of the senescence marker p21Cip1. Additionally, LOSM demonstrated anti-inflammatory effects, with reduced cardiac Il-1α and Il-6 gene expression in DOX-treated mice. Systemic inflammation, assessed by serum cytokine levels, showed decreased IL-6 and CXCL1 in both DOX-treated mice and mice on LOSM diet. LOSM significantly increased mitofusin2 gene expression, which may enhance mitochondrial fusion. These findings underscore the potential therapeutic efficacy of p38 MAPK inhibition, exemplified by LOSM, in ameliorating DOX-induced cardiotoxicity, senescence, and inflammation.
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Introduction: Doxorubicin (DOX), a chemotherapeutic drug, induces senescence and increases the secretion of senescence-associated secretory phenotype (SASP) in endothelial cells (ECs), which contributes to DOX-induced inflammaging. Metformin, an anti-diabetic drug, demonstrates senomorphic effects on different models of senescence. However, the effects of metformin on DOX-induced endothelial senescence have not been reported before. Senescent ECs exhibit a hyper-inflammatory response to lipopolysachharide (LPS). Therefore, in our current work, we identified the effects of metformin on DOX-induced endothelial senescence and LPS-induced hyper-inflammation in senescent ECs. Methods: ECs were treated with DOX ± metformin for 24 h followed by 72 h incubation without DOX to establish senescence. Effects of metformin on senescence markers expression, SA-ß-gal activity, and SASP secretion were assessed. To delineate the molecular mechanisms, the effects of metformin on major signaling pathways were determined. The effect of LPS ± metformin was determined by stimulating both senescent and non-senescent ECs with LPS for an additional 24 h. Results: Metformin corrected DOX-induced upregulation of senescence markers and decreased the secretion of SASP factors and adhesion molecules. These effects were associated with a significant inhibition of the JNK and NF-κB pathway. A significant hyper-inflammatory response to LPS was observed in DOX-induced senescent ECs compared to non-senescent ECs. Metformin blunted LPS-induced upregulation of pro-inflammatory SASP factors. Conclusion: Our study demonstrates that metformin mitigates DOX-induced endothelial senescence phenotype and ameliorates the hyper-inflammatory response to LPS. These findings suggest that metformin may protect against DOX-induced vascular aging and endothelial dysfunction and ameliorate infection-induced hyper-inflammation in DOX-treated cancer survivors.
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Sex is a salient risk factor in the development of doxorubicin-induced cardiotoxicity. Sex differences in the heart's ability to respond to hypertrophic stimuli in doxorubicin-exposed animals have not been reported. We identified the sexual dimorphic effects of isoproterenol in mice pre-exposed to doxorubicin. Male and female intact or gonadectomized C57BL/6N mice underwent five weekly intraperitoneal injections of 4 mg/kg doxorubicin followed by a five-week recovery period. Fourteen days of subcutaneous isoproterenol injections (10 mg/kg/day) were administered after the recovery period. Echocardiography was used to assess heart function one and five weeks after the last doxorubicin injection and on the fourteenth day of isoproterenol treatment. Thereafter, mice were euthanized, and the hearts were weighed and processed for histopathology and gene expression analysis. Doxorubicin did not produce overt cardiac dysfunction in male or female mice before starting isoproterenol treatment. The chronotropic response to a single isoproterenol injection was blunted by doxorubicin, but the inotropic response was maintained in both males and females. Pre-exposure to doxorubicin caused cardiac atrophy in both control and isoproterenol-treated male mice but not in female mice. Counterintuitively, pre-exposure to doxorubicin abrogated isoproterenol-induced cardiac fibrosis. However, there were no sex differences in the expression of markers of pathological hypertrophy, fibrosis, or inflammation. Gonadectomy did not reverse the sexually dimorphic effects of doxorubicin. Additionally, pre-exposure to doxorubicin abrogated the hypertrophic response to isoproterenol in castrated male mice but not in ovariectomized female mice. Therefore, pre-exposure to doxorubicin caused male-specific cardiac atrophy that persisted after isoproterenol treatment, which could not be prevented by gonadectomy.
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Carfilzomib (CFZ) is a proteasome inhibitor approved for relapsed/refractory multiple myeloma (MM) but its clinical use is limited by cardiovascular toxicity. The mechanisms of CFZ-induced cardiovascular toxicity are not fully understood but endothelial dysfunction may be a common denominator. Here, we first characterized the direct toxic effects of CFZ on endothelial cells (HUVECs and EA.hy926 cells) and tested whether SGLT2 inhibitors, known to have cardioprotective effects, can protect against CFZ-induced toxicity. To determine the chemotherapeutic effect of CFZ in the presence of SGLT2 inhibitors, MM and lymphoma cells were treated with CFZ with or without canagliflozin. CFZ decreased cell viability and induced apoptotic cell death in endothelial cells in a concentration-dependent manner. CFZ also upregulated ICAM-1 and VCAM-1 and downregulated VEGFR-2. These effects were associated with the activation of Akt and MAPK pathways, inhibition of p70s6k, and downregulation of AMPK. Canagliflozin, but not empagliflozin or dapagliflozin, protected endothelial cells from CFZ-induced apoptosis. Mechanistically, canagliflozin abrogated CFZ-induced JNK activation and AMPK inhibition. AICAR (an AMPK activator) protected from CFZ-induced apoptosis, and compound C (an AMPK inhibitor) abrogated the protective effect of canagliflozin, strongly suggesting that AMPK mediates these effects. Canagliflozin did not interfere with the anticancer effect of CFZ in cancer cells. In conclusion, our findings demonstrate for the first time the direct toxic effects of CFZ in endothelial cells and the associated signaling changes. Canagliflozin abrogated the apoptotic effects of CFZ in endothelial cells in an AMPK-dependent mechanism, without interfering with its cytotoxicity in cancer cells.
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Canagliflozina , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Canagliflozina/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Células Endoteliales/metabolismo , ApoptosisRESUMEN
Doxorubicin (DOX) induces endothelial cell (EC) senescence, which contributes to endothelial dysfunction and cardiovascular complications. Senolytic drugs selectively eliminate senescent cells to ameliorate senescence-mediated pathologies. Previous studies have demonstrated differences between immortalized and primary EC models in some characteristics. However, the response of DOX-induced senescent ECs to senolytics has not been determined across these two models. In the present work, we first established a comparative characterization of DOX-induced senescence phenotypes in immortalized EA.hy926 endothelial-derived cells and primary human umbilical vein EC (HUVECs). Thereafter, we evaluated the senolytic activity of four senolytics across both ECs. Following the DOX treatment, both EA.hy926 and HUVECs shared similar senescence phenotypes characterized by upregulated senescence markers, increased SA-ß-gal activity, cell cycle arrest, and elevated expression of the senescence-associated secretory phenotype (SASP). The potentially senolytic drugs dasatinib, quercetin, and fisetin demonstrated a lack of selectivity against DOX-induced senescent EA.hy926 cells and HUVECs. However, ABT-263 (Navitoclax) selectively induced the apoptosis of DOX-induced senescent HUVECs but not EA.hy926 cells. Mechanistically, DOX-treated EA.hy926 cells and HUVECs demonstrated differential expression levels of the BCL-2 family proteins. In conclusion, both EA.hy926 cells and HUVECs demonstrate similar DOX-induced senescence phenotypes but they respond differently to ABT-263, presumably due to the different expression levels of BCL-2 family proteins.
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Proteínas Proto-Oncogénicas c-bcl-2 , Senoterapéuticos , Compuestos de Anilina , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fenotipo , SulfonamidasRESUMEN
Hypertension is the most significant risk factor for heart failure in doxorubicin (DOX)-treated childhood cancer survivors. We previously developed a two-hit mouse model of juvenile DOX-induced latent cardiotoxicity that is exacerbated by adult-onset angiotensin II (ANGII)-induced hypertension. It is still not known how juvenile DOX-induced latent cardiotoxicity would predispose the heart to pathologic stimuli that do not cause hypertension. Our main objective is to determine the cardiac effects of ANGII (a hypertensive pathologic stimulus) and isoproterenol (ISO, a non-hypertensive pathologic stimulus) in adult mice pre-exposed to DOX as juveniles. Five-week-old male C57BL/6N mice were administered DOX (4 mg/kg/week) or saline for 3 weeks and then allowed to recover for 5 weeks. Thereafter, mice were administered either ANGII (1.4 mg/kg/day) or ISO (10 mg/kg/day) for 14 days. Juvenile exposure to DOX abrogated the hypertrophic response to both ANGII and ISO, while it failed to correct ANGII- and ISO-induced upregulation in the hypertrophic markers, ANP and BNP. ANGII, but not ISO, worsened cardiac function and exacerbated cardiac fibrosis in DOX-exposed mice as measured by echocardiography and histopathology, respectively. The adverse cardiac remodeling in the DOX/ANGII group was associated with a marked upregulation in several inflammatory and fibrotic markers and altered expression of Ace, a critical enzyme in the RAAS. In conclusion, juvenile exposure to DOX causes latent cardiotoxicity that predisposes the heart to a hypertensive pathologic stimulus (ANGII) more than a non-hypertensive stimulus (ISO), mirroring the clinical scenario of worse cardiovascular outcome in hypertensive childhood cancer survivors.
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Sex-related differences in cardiovascular diseases are highly complex in humans and model-dependent in experimental laboratory animals. The objective of this work was to comprehensively investigate key sex differences in the response to acute and prolonged adrenergic stimulation in C57Bl/6NCrl mice. Cardiac function was assessed by trans-thoracic echocardiography before and after acute adrenergic stimulation (a single sub-cutaneous dose of isoproterenol 10 mg/kg) in 15 weeks old male and female C57Bl/6NCrl mice. Thereafter, prolonged adrenergic stimulation was achieved by sub-cutaneous injections of isoproterenol 10 mg/kg/day for 14 days in male and female mice. Cardiac function and morphometry were assessed by trans-thoracic echocardiography on the 15th day. Thereafter, the mice were euthanized, and the hearts were collected. Histopathological analysis of myocardial tissue was performed after staining with hematoxylin & eosin, Masson's trichrome and MAC-2 antibody. Gene expression of remodeling and fibrotic markers was assessed by real-time PCR. Cardiac function and morphometry were also measured before and after isoproterenol 10 mg/kg/day for 14 days in groups of gonadectomized male and female mice and sham-operated controls. In the current work, there were no statistically significant differences in the positive inotropic and chronotropic effects of isoproterenol between male and female C57Bl/6NCrl. After prolonged adrenergic stimulation, there was similar degree of cardiac dysfunction, cardiac hypertrophy, and myocardial fibrosis in male and female mice. Similarly, prolonged isoproterenol administration induced hypertrophic and fibrotic genes in hearts of male and female mice to the same extent. Intriguingly, gonadectomy of male and female mice did not have a significant impact on isoproterenol-induced cardiac dysfunction as compared to sham-operated animals. The current work demonstrated lack of significant sex-related differences in isoproterenol-induced cardiac hypertrophy, dysfunction, and fibrosis in C57Bl/6NCrl mice. This study suggests that female sex may not be sufficient to protect the heart in this model of isoproterenol-induced cardiac dysfunction and underscores the notion that sexual dimorphism in cardiovascular diseases is highly model-dependent.
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Cardiopatías/fisiopatología , Caracteres Sexuales , Animales , Biomarcadores/metabolismo , Cardiomegalia/inducido químicamente , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Cardiopatías/inducido químicamente , Cardiopatías/metabolismo , Cardiopatías/patología , Isoproterenol/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , OvariectomíaRESUMEN
Xanomeline is thought to be a M1/M4 functionally selective agonist at muscarinic receptors. We have previously demonstrated that it binds in a unique manner at the M1 receptor. In the current study, we examined the ability of xanomeline to bind to the M3 receptor and determined the long-term consequences of this mode of binding in Chinese hamster ovary cells expressing M3 receptors. Xanomeline binds in a reversible and wash-resistant manner at the M3 receptor and elicits a functional response under both conditions. Long-term exposure to xanomeline resulted in changes in the binding profile of [(3)H]NMS and a decrease in cell-surface receptor density. Additionally, pretreatment with xanomeline was associated with antagonism of the functional response to subsequent stimulation by conventional agonists. Our results indicate that xanomeline binds to and activates the M3 muscarinic receptor in a wash-resistant manner, and that this type of binding results in time-dependent receptor regulation.
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Agonistas Muscarínicos/farmacología , Piridinas/farmacología , Receptor Muscarínico M3/metabolismo , Tiadiazoles/farmacología , Animales , Unión Competitiva , Células CHO , Cricetinae , Cricetulus , Hidrólisis , Fosfatos de Inositol/biosíntesis , Antagonistas Muscarínicos/farmacología , N-Metilescopolamina/farmacología , Fosfatidilinositoles/metabolismo , Ensayo de Unión Radioligante , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/antagonistas & inhibidores , Factores de TiempoRESUMEN
BACKGROUND/AIMS: Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect. METHODS: Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments. RESULTS: The above treatment protocol with xanomeline resulted in similar effects of the binding of [(3)H]NMS and [(3)H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent. CONCLUSION: Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.
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Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/farmacocinética , Piridinas/farmacología , Piridinas/farmacocinética , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/fisiología , Tiadiazoles/farmacología , Tiadiazoles/farmacocinética , Animales , Atropina/farmacología , Sitios de Unión/efectos de los fármacos , Carbacol/farmacología , Línea Celular Transformada , Cricetinae , Interacciones Farmacológicas , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , N-Metilescopolamina/farmacocinética , Fosfatidilinositoles/metabolismo , Pilocarpina/farmacología , Quinuclidinil Bencilato/farmacocinética , Factores de Tiempo , TransfecciónRESUMEN
Cardio-oncology is a clinical/scientific discipline which aims to prevent and/or treat cardiovascular diseases in cancer patients. Although a large number of cancer treatments are known to cause cardiovascular toxicity, they are still widely used because they are highly effective. Unfortunately, therapeutic interventions to prevent and/or treat cancer treatment-induced cardiovascular toxicity have not been established yet. A major challenge for such interventions is to protect the cardiovascular system without compromising the therapeutic benefit of anticancer medications. Intriguingly, the polyphenolic natural compound resveratrol and its analogs have been shown in preclinical studies to protect against cancer treatment-induced cardiovascular toxicity. They have also been shown to possess significant anticancer properties on their own, and to enhance the anticancer effect of other cancer treatments. Thus, they hold significant promise to protect the cardiovascular system and fight the cancer at the same time. In this review, we will discuss the current knowledge regarding the cardio-protective and the anticancer properties of resveratrol and its analogs. Thereafter, we will discuss the challenges that face the clinical application of these agents. To conclude, we will highlight important gaps of knowledge and future research directions to accelerate the translation of these exciting preclinical findings to cancer patient care.
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Antineoplásicos , Cardiotónicos , Enfermedades Cardiovasculares , Neoplasias , Resveratrol , Animales , Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Oncología Médica , Ratones , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológicoRESUMEN
Doxorubicin (DOX) is a chemotherapeutic agent that has been reported to cause nephrotoxicity in rodent models and to a lesser degree in cancer patients. Female rodents have been shown to be protected against several features of DOX-induced nephrotoxicity. Nevertheless, the underlying mechanisms of this sexual dimorphism are not fully elucidated. Therefore, in the current study, we investigated the sex and time-dependent changes in pathological lesions as well as apoptotic and fibrotic markers in response to acute DOX-induced nephrotoxicity. We also determined the effect of acute DOX treatment on the renal expression of the sexually dimorphic enzyme, soluble epoxide hydrolase (sEH), since inhibition of sEH has been shown to protect against DOX-induced nephrotoxicity. Acute DOX-induced nephrotoxicity was induced by a single intra-peritoneal injection of 20 mg/kg DOX to male and female adult C57Bl/6 mice. The kidneys were isolated 1, 3 and 6 days after DOX administration. Histopathology assessment, gene expression of the apoptotic marker, BAX, protein expression of the fibrotic marker, transforming growth factor-ß (TGF-ß), and gene and protein expression of sEH were assessed. DOX administration caused more severe pathological lesions as well as higher induction of the apoptotic and fibrotic markers in kidneys of male than in female mice. Intriguingly, DOX inhibited sEH protein expression in kidneys of male mice sacrificed at 3 and 6 days following administration, suggesting that induction of sEH is not necessary for acute DOX-induced nephrotoxicity. However, DOX-induced inhibition of renal sEH in male mice may protect the kidney from further DOX-induced injury in a negative feedback mechanism. We also observed lower constitutive expressions of TGF-ß and sEH in the kidney of female mice which may contribute, at least in part, to sexual dimorphism of DOX-induced nephrotoxicity.
Asunto(s)
Doxorrubicina/toxicidad , Riñón/efectos de los fármacos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/genética , Doxorrubicina/administración & dosificación , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Femenino , Fibrosis , Expresión Génica/efectos de los fármacos , Riñón/enzimología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Caracteres Sexuales , Factor de Crecimiento Transformador beta/metabolismo , Proteína X Asociada a bcl-2/genéticaRESUMEN
In a previous study, we reported that levels of two types of protein species-a type of ~55-kDa species and a type of ~15-kDa species-are elevated in the lumbar cerebrospinal fluid (CSF) of cognitively intact elderly individuals who are at risk for Alzheimer's disease (AD). These species are immunoreactive to the monoclonal antibody 6E10, which is directed against amino acids 6-10 of amyloid-ß (Aß), and their levels correlate with levels of total tau and tau phosphorylated at Thr181. In this study, we investigated the molecular composition of these AD-related proteins using immunoprecipitation (IP)/Western blotting coupled with IP/mass spectrometry. We show that canonical Aß1-40/42 peptides, together with amyloid-ß precursor protein (APP) fragments located N-terminally of Aß, are present in the ~55-kDa, 6E10-immunoreactive species. We demonstrate that APP fragments located N-terminally of Aß, plus the N-terminal region of Aß, are present in the ~15-kDa, 6E10-immunoreactive species. These findings add to the catalog of AD-related Aß/APP species found in CSF and should motivate further study to determine whether these species may serve as biomarkers of disease progression.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/líquido cefalorraquídeo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/inmunología , Biomarcadores/líquido cefalorraquídeo , Western Blotting , Progresión de la Enfermedad , Femenino , Humanos , Inmunoprecipitación , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunologíaRESUMEN
A series of xanomeline analogs were synthesized and evaluated for binding at the M(1) muscarinic acetylcholine receptor (M(1) receptor). Specifically, compounds that substitute the O-hexyl chain of xanomeline with polar, ionizable, or conformationally restricted moieties were assessed for their ability to bind to the M(1) receptor in a wash-resistant manner (persistent binding). From our screen, several novel ligands that persistently bind to the M(1) receptor with greater affinity than xanomeline were discovered. Results indicate that persistent binding may arise not only from hydrophobic interactions but also from ionic interactions with a secondary M(1) receptor binding site. Herein, a qualitative model that accounts for both binding scenarios is proposed and applied to understand the structural basis to wash-resistant binding and long-acting effects of xanomeline-based compounds.
Asunto(s)
Piridinas/síntesis química , Piridinas/farmacología , Receptor Muscarínico M1/antagonistas & inhibidores , Tiadiazoles/síntesis química , Tiadiazoles/farmacología , Animales , Células CHO , Ácidos Carboxílicos/química , Cricetinae , Cricetulus , Concentración de Iones de Hidrógeno , Estructura Molecular , Piridinas/química , Receptor Muscarínico M1/metabolismo , Relación Estructura-Actividad , Tiadiazoles/químicaRESUMEN
Unlike other M1 muscarinic acetylcholine receptor agonists, xanomeline demonstrates a unique mode of binding to the receptor. It not only binds reversibly to the receptor's conventional orthosteric site but also binds persistently at a secondary binding domain(s) on the M1 receptor. This results in persistent activation of the receptor even after extensive washout, and allosteric modulation of the orthosteric site. In the current study, we investigated how the effects of very brief exposure (1 min) of intact Chinese hamster ovary cells expressing M1 receptors to xanomeline followed by washout change with time. Pretreatment with xanomeline for 1 min resulted in a concentration-dependent wash-resistant inhibition of [3H]N-methylscopolamine (NMS) binding, with a lower potency than that observed in the continuous presence of xanomeline in the binding assay medium. This effect was associated with wash-resistant receptor activation. Incubation of pretreated and washed cells in control medium for 24 h transformed the monophasic xanomeline wash-resistant binding curve to one that exhibits two distinct potencies. This was the result of the appearance of a new very high-potency binding component without a change in the low-potency state. The delayed effects of persistently bound xanomeline are mainly due to reduction of the maximal binding of [3H]NMS without a change in its affinity. These treatment conditions also reversed persistent receptor activation by xanomeline. Our results imply that brief exposure to xanomeline followed by washing and prolonged waiting may result in delayed receptor desensitization accompanied by internalization or down-regulation.