RESUMEN
Thanks to recent progress in cancer research, most children treated for cancer survive into adulthood. Nevertheless, the long-term consequences of anticancer agents are understudied, especially in the pediatric population. We and others have shown that routinely administered chemotherapeutics drive musculoskeletal alterations, which contribute to increased treatment-related toxicity and long-term morbidity. Yet, the nature and scope of these enduring musculoskeletal defects following anticancer treatments and whether they can potentially impact growth and quality of life in young individuals remain to be elucidated. Here, we aimed at investigating the persistent musculoskeletal consequences of chemotherapy in young (pediatric) mice. Four-week-old male mice were administered a combination of 5-FU, leucovorin, irinotecan (a.k.a., Folfiri) or the vehicle for up to 5 wk. At time of sacrifice, skeletal muscle, bones, and other tissues were collected, processed, and stored for further analyses. In another set of experiments, chemotherapy-treated mice were monitored for up to 4 wk after cessation of treatment. Overall, the growth rate was significantly slower in the chemotherapy-treated animals, resulting in diminished lean and fat mass, as well as significantly smaller skeletal muscles. Interestingly, 4 wk after cessation of the treatment, the animals exposed to chemotherapy showed persistent musculoskeletal defects, including muscle innervation deficits and abnormal mitochondrial homeostasis. Altogether, our data support that anticancer treatments may lead to long-lasting musculoskeletal complications in actively growing pediatric mice and support the need for further studies to determine the mechanisms responsible for these complications, so that new therapies to prevent or diminish chemotherapy-related toxicities can be identified.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Camptotecina/análogos & derivados , Animales , Ratones , Masculino , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Músculo Esquelético/efectos de los fármacos , Irinotecán/efectos adversos , Fluorouracilo/efectos adversos , Fluorouracilo/toxicidad , Leucovorina , Camptotecina/efectos adversos , Camptotecina/toxicidad , Antineoplásicos/efectos adversos , Antineoplásicos/toxicidad , Ratones Endogámicos C57BLRESUMEN
To identify additional genetic markers contributing to variability in CPT-11 disposition and toxicity, we assessed impact of the multiple drug-resistant transporters 1, 2, and 3 (MRP1, MRP2, and MRP3) on the intestinal toxicity, pharmacokinetics, tissue distribution and biliary excretion of CPT-11 using a knockout mouse model. Mrp1/3 knockout had minor impact on intestinal toxicity of CPT-11, tissue distribution, biliary excretion, and PK parameter of its active metabolites SN38. Conversely, Mrp2-/- mice, with low carboxylesterase activity, displayed insensitivity to CPT-11 toxicity due to reduced intestinal exposure to SN38. In PK studies, Mrp1/2 knockout significantly increased the AUC of CPT-11 compared to their AUC in FVB mice. However, the AUC of SN38 in Mrp2 -/- mice was decreased by 3.25-fold. Mrp3 knockout only slightly increased SN38 plasma exposure. Lastly, Mrp2/3 knockout increased biliary excretion amount of CPT-11 by 67.2% and 48.5% compared to wild-type mice, respectively. Consequently, Mrp1/3 deficiency didn't change SN38 tissue distribution. Finally, correlation analysis demonstrated that tissue exposure to SN38 was better correlated with toxicity than plasma AUC of SN38. Mrp1/2/3 deficiency showed a minor impact on PK, biliary excretion, distribution and intestinal exposure of SN38, and as a result, did not affect the intestinal toxicity of CPT-11.
Asunto(s)
Camptotecina , Intestinos , Ratones , Animales , Irinotecán , Camptotecina/toxicidad , Distribución TisularRESUMEN
Spodoptera frugiperda is an economically important agricultural pest and poses a serious threat to food security globally. Its management is gravely challenged by its high polyphagous nature, strong migratory ability, and massive fecundity. Chlorantraniliprole (CHL) is widely utilized in controlling S. frugiperda, its intensive application and over-reliance pose adverse health risks, development of resistance, toxicity to beneficial insects, natural enemies, and environmental contamination. To address S. frugiperda resistance to CHL and its inherent challenges, this study explores the synergistic effects of camptothecin (CPT) with CHL in its management. The binary mixed adversely induced the larvae weight and mortality when compared to single-treated. CHL + CPT (1:20 mg/L) had the highest larvae mortality of (73.80 %) with a high antagonistic factor (0.90), while (1:10 mg/L) with (66.10%) mortality exhibited a high synergistic factor (1.43). Further, CHL + CPT (1:10 mg/L) considerably altered the midgut epithelial cell, peritrophic membrane, microvilli, basement membrane, and regenerative cells. For biochemical analysis, CHL + CPT (1:10 mg/L) significantly decreased glutathione-S-transferase (1-chloro-2,4-dinitrobenzene CDNB) and cytochrome P450 (7-ethoxycoumarin O-deethylation) activities in the midgut in a dose and time dependent manner. Based on RNA-Seq analysis, a total of 4,373 differentially expressed genes (DEGs) were identified from the three treatments. CPT vs CK (Control) had 1694 (968 up-, 726 down-regulated), CHL vs CK with 1771 (978 up-, 793 down-regulated), and CHL + CPT vs CK had 908 (394 up-, 514 down-regulated) DEGs. The enrichment analysis disclosed significant pathways such as metabolism of xenobiotics by cytochrome P450, glutathione metabolism, TOLL and IMD (Immune Deficiency) signaling pathway, longevity regulating pathway. This study provides basis to expatiate on the molecular toxicological mechanism of CHL + CPT in management of fall armyworm.
Asunto(s)
Camptotecina , Insecticidas , Larva , Spodoptera , Sinergismo Farmacológico , Spodoptera/anatomía & histología , Spodoptera/efectos de los fármacos , Spodoptera/crecimiento & desarrollo , Spodoptera/fisiología , Camptotecina/administración & dosificación , Camptotecina/farmacología , Camptotecina/toxicidad , Larva/anatomía & histología , Larva/efectos de los fármacos , Seguridad Alimentaria , Insecticidas/administración & dosificación , Insecticidas/farmacología , Insecticidas/toxicidad , Animales , Metabolismo Secundario , Peso Corporal/efectos de los fármacos , Glutatión Transferasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/administración & dosificación , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Inhibidores Enzimáticos del Citocromo P-450/toxicidad , Perfilación de la Expresión Génica , Ontología de Genes , RNA-Seq , Reproducibilidad de los ResultadosRESUMEN
The adverse effects (diarrhea and neutropenia) of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) are associated with genetic variants of uridine diphosphate glucuronosyltransferase 1A subfamilies (UGT1As). UGT1As are enzymes that metabolize the active form of irinotecan, 7-ethyl-10 hydroxycamptothecin (SN-38), by glucuronidation in the liver. They are widely known as predictive factors of severe adverse effects, such as neutropenia and diarrhea. Some studies have suggested that variants of UGT1As affect SN-38 glucuronidation activities, thus exerting severe adverse effects. We aimed to identify UGT1A isoforms that show SN-38 glucuronidation activity and determine the relationship between UGT1A variants and SN-38 glucuronidation in vitro. We found that UGT1A1 and UGT1A6-UGT1A10 displayed SN-38 glucuronidation activity. Among these, UGT1A1 was the most active. Furthermore, the variants of these isoforms showed decreased SN-38 glucuronidation activity. In our study, we compared the different variants of UGT1As, such as UGT1A1.6, UGT1A1.7, UGT1A1.27, UGT1A1.35, UGT1A7.3, UGT1A8.4, UGT1A10M59I, and UGT1A10T202I, to determine the differences in the reduction of glucuronidation. Our study elucidates the relationship between UGT1A variants and the level of glucuronidation associated with each variant. Therefore, testing can be done before the initiation of irinotecan treatment to predict potential toxicities and adverse effects.
Asunto(s)
Camptotecina , Neutropenia , Humanos , Irinotecán , Camptotecina/toxicidad , Camptotecina/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Diarrea/inducido químicamente , Neutropenia/inducido químicamenteRESUMEN
BACKGROUND AND PURPOSE: Opioids are commonly used for the management of cancer-associated pain and chemotherapy-induced diarrhoea. The chemotherapeutic irinotecan (CPT-11) causes severe gastrointestinal (GI) toxicity due to deconjugation of inactive metabolite SN-38 glucuronide (SN-38G) by bacterial ß-glucuronidases to the active 7-ethyl-10-hydroxycamptothecin (SN-38). Opioids are known to cause gut microbial dysbiosis, this study evaluated whether CPT-11 anti-tumour efficacy and GI toxicity are exacerbated by opioid co-administration. EXPERIMENTAL APPROACH: Eight-week-old C57BL/6 male mice were co-administration with CPT-11 ± opioid. 16S rRNA sequencing was used for gut microbiome analysis. LC-MS analyses of plasma and intestinal extracts were performed to investigate the pharmacokinetic profile of CPT-11. Histological analysis and quantitative real-time polymerase chain reaction were used to determine the severity of intestinal tissue damage. Human liver microsome In vitro assay was performed to confirm the effects of opioids on CPT-11 metabolism. KEY RESULTS: Gut microbiome analysis showed that morphine treatment induced enrichment of ß-glucuronidase-producing bacteria in the intestines of CPT-11-treated mice, resulting in SN-38 accumulation and exacerbation of GI toxicity in the small intestine. Oral administration of both antibiotics and glucuronidase inhibitor protected mice against GI toxicity induced with CPT-11 and morphine co-administration, implicating a microbiome-dependent mechanism. Additionally, morphine and loperamide decreased the plasma concentration of SN-38 and compromised CPT-11 anti-tumour efficacy, this seemed to be microbiome independent. CONCLUSION AND IMPLICATIONS: Gut microbiota play a significant role in opioid and chemotherapeutic agent drug-drug interactions. Inhibition of gut microbial glucuronidase may also prevent adverse GI effects of CPT-11 in patients on opioids.
Asunto(s)
Antineoplásicos Fitogénicos , Neoplasias , Humanos , Ratones , Masculino , Animales , Irinotecán , Analgésicos Opioides/farmacología , Disbiosis , Modelos Animales de Enfermedad , ARN Ribosómico 16S , Antineoplásicos Fitogénicos/toxicidad , Ratones Endogámicos C57BL , Camptotecina/toxicidad , Bacterias , Glucuronidasa/metabolismo , Glucuronidasa/farmacología , Derivados de la Morfina/farmacologíaRESUMEN
Plasticizers/phthalates play a facilitating role in the development of cancer and help the tumor to grow and metastasize. Camptothecin (CPT) and its derivatives are known to have anticancer properties of inhibiting cell growth, promoting cell apoptosis, and increasing autophagy. Therefore, in this study, we investigated whether the presence of di(2-ethylhexyl) phthalate (DEHP) could hinder apoptosis and autophagy caused by CPT in non-small cell lung cancer (NSCLC) cells. We found that DEHP interferes with CPT-induced apoptosis and autophagy and increases the prosurvival pathway by reducing the DNA damage marker γ-H2AX and activating the Akt and NF-κB pathways. Furthermore, we also confirmed that combining DEHP with 3-MA has additive effects in inhibiting autophagy and apoptosis in NSCLC cells. Taken together, our findings show that DEHP could affect CPT-induced anticancer treatment and provide evidence to show that DEHP induces chemoresistance in CPT-based chemotherapy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Dietilhexil Ftalato , Neoplasias Pulmonares , Humanos , FN-kappa B/metabolismo , Dietilhexil Ftalato/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Plastificantes/toxicidad , Camptotecina/toxicidadRESUMEN
Irinotecan is a first-line treatment for colorectal cancer and the prodrug of 7-ethyl-10-hydroxy-camptothecin (SN-38). However, its fatal gastrointestinal (GI) toxicity raises serious concern. In liver, irinotecan generates its inactive metabolite, SN-38G via UDP-glucuronosyltransferase (UGT)1A1. Subsequently, SN-38G is excreted into GI tract where it is reactivated by microbiome to yield the toxic metabolite, SN-38. Activation of toll-like receptor (TLR)/myeloid differentiation primary response 88 (MyD88) by bacterial endotoxin decreases drug-metabolizing enzymes. In this study, we treated C57BL6/J mice with 50 mg/kg irinotecan once daily until observing grade 4 diarrhea. Mice were sacrificed on day0, day2 and day8. Based on the finding in C57BL6/J mice, we repeated the treatment in Tlr2-/-, Tlr4-/- and Myd88-/- mice to determine the impact of inflammation on UGT metabolism. Our toxicity study in C57BL6/J mice showed that mice started bloody diarrhea after 6 days' injection of irinotecan. Ugt1a1 expression in GI tract started decreasing after 24h since first dose, before the onset of diarrhea. In Tlr4-/- and Myd88-/- mice, no Ugt1a1 reduction was observed in distal GI tract after irinotecan injection. In Tlr2-/- mice, intestinal Ugt1a1 expression was down-regulated. Our results indicate that after two doses of irinotecan, mice started losing capability of detoxifying SN-38. TLR4 plays more important role in Ugt1a1 reduction than TLR2, despite that TLR2 and TLR4 share MyD88 as common adaptor protein. We concluded that irinotecan reduced intestinal Ugt1a1 via TLR4/MyD88 pathway, which eventually triggers the onset of diarrhea. Our finding unveils a novel mechanism underlying irinotecan-induced diarrhea and provides a new direction to prevent chemotherapy side effect.
Asunto(s)
Antineoplásicos Fitogénicos , Glucuronosiltransferasa , Irinotecán , Factor 88 de Diferenciación Mieloide , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Camptotecina/toxicidad , Diarrea/inducido químicamente , Diarrea/metabolismo , Glucuronosiltransferasa/metabolismo , Irinotecán/efectos adversos , Ratones , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Uridina DifosfatoRESUMEN
OBJECTIVES: Colorectal cancer continues to have one of the highest incidents of occurrence with a rising rate of diagnosis among people under the age of 50. Chemotherapy with irinotecan results in severe gastrointestinal dose-limiting toxicity that is caused by the glucuronidated form of the active metabolite (SN-38G). This study evaluates herbal compounds and analogs to biomodulate the metabolism of IR to decrease dose-limiting toxicity while increasing the amount of the active metabolite. METHODS: In vitro metabolism using human liver microsomes was conducted with white willow bark (WWB) extract, select specific components of WWB, and analogues to evaluate biomodulation of the IR metabolism. Samples were analyzed using liquid chromatography-tandem mass spectrometry to measure metabolites between reactions with and without herbals components. RESULTS: WWB showed an optimal decrease (>80%) in SN-38G and a corresponding increase in SN-38 levels (128%) at a concentration of near 200 µg/mL. Tannic acid produced a 75% decrease in SN-38G with a 130% increase in SN-38 at 10 µg/mL, whereas the treatment with beta-pentagalloyl glucose and various analogues decreased SN-38G by 70% and increased SN-38 by 20% at 10 µg/mL. CONCLUSIONS: These results suggest naturally occurring compounds from WWB may have the potential to increase potency by increasing the conversion of IR to SN-38 and decrease dose-limiting toxicity of IR chemotherapy by reducing glucuronidation of SN-38.
Asunto(s)
Antineoplásicos Fitogénicos , Camptotecina , Antineoplásicos Fitogénicos/toxicidad , Camptotecina/análogos & derivados , Camptotecina/toxicidad , Glucuronatos , Glucurónidos/metabolismo , Glucurónidos/farmacología , Humanos , IrinotecánRESUMEN
Lack of coordination between the DNA replication and transcription machineries can increase the frequency of transcription-replication conflicts, leading ultimately to DNA damage and genomic instability. A major source of these conflicts is the formation of R-loops, which consist of a transcriptionally generated RNA-DNA hybrid and the displaced single-stranded DNA. R-loops play important physiological roles and have been implicated in human diseases. Although these structures have been extensively studied, many aspects of R-loop biology and R-loop-mediated genome instability remain unclear. We found that in cancer cells, tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) interacted with PARP1 and localized to R-loops in response to DNA-damaging agent camptothecin (CPT), which is associated with R-loop formation. PARP1-mediated PARylation was required for recruitment of TonEBP to the sites of R-loop-associated DNA damage. Loss of TonEBP increased levels of R-loop accumulation and DNA damage, and promoted cell death in response to CPT. These findings suggest that TonEBP mediates resistance to CPT-induced cell death by preventing R-loop accumulation in cancer cells.
Asunto(s)
Daño del ADN , Replicación del ADN , Inestabilidad Genómica , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Estructuras R-Loop , Factores de Transcripción/metabolismo , Transcripción Genética , Camptotecina/toxicidad , Línea Celular , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Poli ADP RibosilaciónRESUMEN
BACKGROUND: Camptothecin (CPT) and matrine (MAT) have potential as botanical pesticides against several pest species. However, the mechanisms of metabolic and physiological changes in pests induced by CPT and MAT are unknown. In this study, a toxicological test, an NMR-based metabolomic study, an enzymatic test, and an RT quantitative PCR (RT-qPCR) experiment were all conducted to examine the effect of CPT and MAT on Spodoptera litura. RESULTS: CPT (0.5-1%) exerted high toxicity against larvae of S. litura and caused growth stagnation and high mortality of larvae. A variety of metabolites were significantly influenced by 0.5% CPT, including several energy-related metabolites such as trehalose, lactate, succinate, citrate, malate, and fumarate. In contrast, MAT showed low toxicity against larvae and induced almost no changes in hemolymph metabolites of S. litura. Enzymatic tests showed that trehalase activity was significantly decreased in larvae after feeding with 0.5% CPT. RT-qPCR showed that the transcription levels of alanine aminotransferase, malate dehydrogenase, and isocitrate dehydrogenase were decreased while lactate dehydrogenase was increased in the 0.5% CPT-treated group. CONCLUSIONS: These data indicate that one of the important mechanisms of CPT against S. litura larvae is via the inhibition of trehalose hydrolysis and glycolysis. Our findings also suggest that CPT exhibits a stronger toxicological effect than MAT against S. litura, which provides basic information for the application of CPT in the control of S. litura or other lepidoptera pests.
Asunto(s)
Plaguicidas , Alcaloides , Animales , Camptotecina/toxicidad , Larva , Quinolizinas , Spodoptera , MatrinasRESUMEN
DNA damage caused by exogenous or endogenous factors is a common challenge for developing fish embryos. DNA damage repair (DDR) pathways help organisms minimize adverse effects of DNA alterations. In terms of DNA repair mechanisms, sturgeons represent a particularly interesting model due to their exceptional genome plasticity. Sterlet (Acipenser ruthenus) is a relatively small species of sturgeon. The goal of this study was to assess the sensitivity of sterlet embryos to model genotoxicants (camptothecin, etoposide, and benzo[a]pyrene), and to assess DDR responses. We assessed the effects of genotoxicants on embryo survival, hatching rate, DNA fragmentation, gene expression, and phosphorylation of H2AX and ATM kinase. Exposure of sterlet embryos to 1 µM benzo[a]pyrene induced low levels of DNA damage accompanied by ATM phosphorylation and xpc gene expression. Conversely, 20 µM etoposide exposure induced DNA damage without activation of known DDR pathways. Effects of 10 nM camptothecin on embryo development were stage-specific, with early stages, before gastrulation, being most sensitive. Overall, this study provides foundational information for future investigation of sterlet DDR pathways.
Asunto(s)
Daño del ADN , Reparación del ADN , Embrión no Mamífero/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Peces/genética , Animales , Benzo(a)pireno/toxicidad , Camptotecina/toxicidad , Ensayo Cometa , Fragmentación del ADN/efectos de los fármacos , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Etopósido/toxicidad , Femenino , Peces/embriología , Masculino , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidadRESUMEN
It has recently been established that the marked sensitivity of ATM deficient cells to topoisomerase poisons like camptothecin (Cpt) results from unrestrained end-joining of DNA ends at collapsed replication forks that is mediated by the non-homologous end joining [NHEJ] pathway and results in the induction of copious numbers of genomic alterations, termed "toxic NHEJ". Ablation of core components of the NHEJ pathway reverses the Cpt sensitivity of ATM deficient cells, but inhibition of DNA-PKcs does not. Here, we show that complete ablation of DNA-PKcs partially reverses the Cpt sensitivity of ATM deficient cells; thus, ATM deficient cells lacking DNA-PKcs are more resistant to Cpt than cells expressing DNA-PKcs. However, the relative sensitivity of DNA-PKcs proficient ATM deficient cells is inversely proportional to DNA-PKcs expression levels. These data suggest that DNA-PK may phosphorylate an ATM target (that contributes to Cpt resistance), explaining partial rescue of Cpt sensitivity in cells expressing high levels of DNA-PKcs. Although crippling NHEJ function by mutagenic blockade of the critical ABCDE autophosphorylation sites in DNA-PKcs also sensitizes cells to Cpt, this sensitization apparently occurs by a distinct mechanism from ATM ablation because blockade of these sites actually rescues ATM deficient cells from toxic NHEJ. These data are consistent with autophosphorylation of the ABCDE sites (and not ATM mediated phosphorylation) in response to Cpt-induced damage. In contrast, blockade of S3205 (an ATM dependent phosphorylation site in DNA-PKcs) that minimally impacts NHEJ, increases Cpt sensitivity. In sum, these data suggest that ATM and DNA-PK cooperate to facilitate Cpt-induced DNA damage, and that ATM phosphorylation of S3205 facilitates appropriate repair at collapsed replication forks.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Reparación del ADN por Unión de Extremidades , Replicación del ADN , Proteína Quinasa Activada por ADN/metabolismo , Camptotecina/toxicidad , ADN/metabolismo , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Inhibidores de Topoisomerasa I/toxicidadRESUMEN
BACKGROUND AND PURPOSE: Irinotecan, used in colorectal cancer therapy, is metabolized by glucuronidation involving different UDP-glucuronosyltransferase (UGT)1A isoforms leading to facilitated elimination from the body. Individuals homozygous for the genetic variants UGT1A1*28 (Gilbert syndrome) and UGT1A7*3 are more susceptible to irinotecan side effects, severe diarrhoea and leukopenia. The aim of this study was to investigate the protective effects and active constituents of coffee during irinotecan therapy using humanized transgenic (htg)UGT1A-WT and htgUGT1A-SNP (carry UGT1A1*28 and UGT1A7*3 polymorphisms) mice. EXPERIMENTAL APPROACH: HtgUGT1A mice were pretreated with coffee or caffeic acid (CA) + caffeic acid phenylethyl ester (CAPE) and injected with irinotecan. The effects of coffee and CA + CAPE were investigated using reporter gene assays, immunoblot, TaqMan-PCR, siRNA analyses and blood counts. KEY RESULTS: Only the combination of the two coffee ingredients, CA and CAPE, mediates protective effects of coffee in a model of irinotecan toxicity by activation of UGT1A genes. Coffee and CA + CAPE significantly increased UGT1A expression and activity along with SN-38 glucuronide excretion in irinotecan-injected htgUGT1A mice, resulting in significant improvement of leukopenia, intestinal oxidative stress and inflammation. CONCLUSION AND IMPLICATIONS: In this study, we identify the compounds responsible for mediating the previously reported coffee-induced activation of UGT1A gene expression. CA and CAPE represent key factors for the protective properties of coffee which are capable of reducing irinotecan toxicity, exerting antioxidant and protective effects. Provided that CA + CAPE do not affect irinotecan efficacy, they might represent a novel strategy for the treatment of irinotecan toxicity.
Asunto(s)
Ácidos Cafeicos , Café , Irinotecán , Leucopenia , Estrés Oxidativo , Animales , Ácidos Cafeicos/farmacología , Camptotecina/toxicidad , Ésteres , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Irinotecán/toxicidad , Leucopenia/inducido químicamente , Leucopenia/prevención & control , RatonesRESUMEN
DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11-RAD50-NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM-DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350-600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN-CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550-600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.
Asunto(s)
Reparación del ADN , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/fisiología , Proteína BRCA1/metabolismo , Camptotecina/toxicidad , Línea Celular , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Endodesoxirribonucleasas/genética , Humanos , Dominios Proteicos , Eliminación de Secuencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Intestinal mucositis refers to mucosal damage caused by cancer treatment, and irinotecan is one of the agents most associated with this condition. Focusing on the development of alternatives to prevent this important adverse effect, we evaluated the activity of the flavonoid luteolin, which has never been tested for this purpose despite its biological potential. EXPERIMENTAL APPROACH: The effects of luteolin were examined on irinotecan-induced intestinal mucositis in mice. Clinical signs were evaluated. Moreover, histological, oxidative, and inflammatory parameters were analysed, as well as the possible interference of luteolin in the anti-tumour activity of irinotecan. KEY RESULTS: Luteolin (30 mg·kg-1 ; p.o. or i.p.) prevented irinotecan-induced intestinal damage by reducing weight loss and diarrhoea score and attenuating the shortening of the duodenum and colon. Histological analysis confirmed that luteolin (p.o.) prevented villous shortening, vacuolization, and apoptosis of cells and preserved mucin production in the duodenum and colon. Moreover, luteolin treatment mitigated irinotecan-induced oxidative stress, by reducing the levels of ROS and LOOH and augmenting endogenous antioxidants, and inflammation by decreasing MPO enzymic activity, TNF, IL-1ß, and IL-6 levels and increasing IL-4 and IL-10. Disruption of the tight junctions ZO-1 and occludin was also prevented by luteolin treatment. Importantly, luteolin did not interfere with the anti-tumour activity of irinotecan. CONCLUSION AND IMPLICATIONS: Luteolin prevents intestinal mucositis induced by irinotecan and therefore could be a potential adjunct in anti-tumour therapy to control this adverse effect, increasing treatment adherence and consequently the chances of cancer remission.
Asunto(s)
Mucositis , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Camptotecina/uso terapéutico , Camptotecina/toxicidad , Mucosa Intestinal , Irinotecán/uso terapéutico , Luteolina/farmacología , Luteolina/uso terapéutico , Ratones , Mucositis/inducido químicamente , Mucositis/tratamiento farmacológico , Mucositis/prevención & controlRESUMEN
Systemic chemotherapy-mediated cell toxicity is a major risk factor for cardiovascular disease and atherosclerosis. Life-threatening acute events of the FOLFIRI (irinotecan, folinic acid and 5-fluorouracil) regimen are mainly due to DNA damage induced by antimetabolite and topoisomerase inhibition effects. However, the role of human aortic smooth muscle cells (HaVSMCs) in this process and the mechanisms of oxidative stress, DNA and protein damage and apoptosis have not been investigated. Therefore, the effects of curcumin and quercetin on HaVSMC survival in the generation of molecular and cellular toxicity by FOLFIRI treatment and the involvement of vital cellular signalling pathways were investigated. We analysed both FOLFIRI toxicity and the therapeutic potential of quercetin and curcumin in terms of HaVSMC damage using molecular probe and florescence staining, Random Amplified Polymorphic DNA (RAPD), qRT-PCR and Western blot assays. Our study presents two preliminary findings: (a) in HaVSMCs, FOLFIRI treatment significantly induces oxidative damage to both DNA and protein, leading to a dramatic increase in caspase-dependent apoptotic death through P53-mediated Caspase3-dependent mitochondrial apoptosis, and results in TNF-α/Caspase8-mediated necrotic death, and (b) flavonoids not only regulate the expression of genes encoding antioxidant enzymes and increase DNA damage but also limit programmed and necrotic cell death processes in HaVSMCs. Our results clearly indicate the potential for curcumin and, particularly, quercetin as preventative chemotherapeutic interventions for cardiovascular toxicity induced by the FOLFIRI regime in HaVSMCs.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Camptotecina/análogos & derivados , Curcumina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Quercetina/farmacología , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Camptotecina/toxicidad , Células Cultivadas , Daño del ADN , Fluorouracilo/toxicidad , Humanos , Leucovorina/toxicidad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Necrosis , Estrés Oxidativo/efectos de los fármacos , Transducción de SeñalRESUMEN
Mammalian cells possess multiple pathways for repairing various types of DNA damage. Although the molecular mechanisms of each DNA repair pathway have been analyzed by biochemical analysis and cell biological analysis, interplay between different pathways has not been fully elucidated. In this study, using human Nalm-6-mutant cell lines, we analyzed the relationship between the base excision repair factor DNA polymerase ß (POLß) and DNA ligase IV (LIG4), which is essential for DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). We found that cells lacking both POLß and LIG4 grew significantly more slowly than either single mutant, indicating cooperative functions of the two proteins in normal cell growth. To further investigate the genetic interaction between POLß and LIG4, we examined DNA damage sensitivity of the mutant cell lines. Our results suggested that NHEJ acts as a backup pathway for repairing alkylation damage (when converted into DSBs) in the absence of POLß. Surprisingly, despite the critical role of POLß in alkylation damage repair, cells lacking POLß exhibited increased resistance to camptothecin (a topoisomerase I inhibitor that induces DNA single-strand breaks), irrespective of the presence or absence of LIG4. A LIG4-independent increased resistance associated with POLß loss was also observed with ionizing radiation; however, cells lacking both POLß and LIG4 were more radiosensitive than either single mutant. Taken together, our findings provide novel insight into the complex interplay between different DNA repair pathways.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP)/genética , ADN Polimerasa beta/genética , Camptotecina/toxicidad , Línea Celular , Daño del ADN , ADN Ligasa (ATP)/metabolismo , ADN Polimerasa beta/metabolismo , Resistencia a Medicamentos , Humanos , Mutación , Tolerancia a Radiación , Inhibidores de Topoisomerasa/toxicidadRESUMEN
A novel enzyme-responsive supramolecular polysaccharide assembly composed of disulfide linked adamantane-naphthalimide fluorescent camptothecin prodrug (AdaCPT) and ß-CD modified hyaluronic acid (HACD) was constructed, possessing low cellular cytotoxicity and exhibiting targeted cellular imaging and controlled drug release at specific sites while providing a concurrent means for the real-time tracking of drug delivery.
Asunto(s)
Antineoplásicos/farmacología , Camptotecina/farmacología , Portadores de Fármacos/química , Colorantes Fluorescentes/farmacología , Profármacos/farmacología , Adamantano/análogos & derivados , Adamantano/síntesis química , Adamantano/farmacología , Adamantano/toxicidad , Animales , Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Camptotecina/síntesis química , Camptotecina/toxicidad , Liberación de Fármacos , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Células HCT116 , Humanos , Ácido Hialurónico/química , Ratones , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Células 3T3 NIH , Naftalimidas/síntesis química , Naftalimidas/farmacología , Naftalimidas/toxicidad , Profármacos/síntesis química , Profármacos/toxicidad , beta-Ciclodextrinas/químicaRESUMEN
DNA damage response (DDR) processes, often caused by oxidative stress, are important in aging and -related disorders. We recently showed that G protein-coupled receptor (GPCR) kinase interacting protein 2 (GIT2) plays a key role in both DNA damage and oxidative stress. Multiple tissue analyses in GIT2KO mice demonstrated that GIT2 expression affects the GPCR relaxin family peptide 3 receptor (RXFP3), and is thus a therapeutically-targetable system. RXFP3 and GIT2 play similar roles in metabolic aging processes. Gaining a detailed understanding of the RXFP3-GIT2 functional relationship could aid the development of novel anti-aging therapies. We determined the connection between RXFP3 and GIT2 by investigating the role of RXFP3 in oxidative stress and DDR. Analyzing the effects of oxidizing (H2O2) and DNA-damaging (camptothecin) stressors on the interacting partners of RXFP3 using Affinity Purification-Mass Spectrometry, we found multiple proteins linked to DDR and cell cycle control. RXFP3 expression increased in response to DNA damage, overexpression, and Relaxin 3-mediated stimulation of RXFP3 reduced phosphorylation of DNA damage marker H2AX, and repair protein BRCA1, moderating DNA damage. Our data suggests an RXFP3-GIT2 system that could regulate cellular degradation after DNA damage, and could be a novel mechanism for mitigating the rate of age-related damage accumulation.
Asunto(s)
Daño del ADN , Proteínas Activadoras de GTPasa/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Estrés Oxidativo , Receptores Acoplados a Proteínas G/metabolismo , Camptotecina/toxicidad , Biología Computacional , Felodipino , Proteínas Activadoras de GTPasa/genética , Regulación de la Expresión Génica/fisiología , Redes Reguladoras de Genes , Células HEK293 , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Inhibidores de Topoisomerasa I/toxicidadRESUMEN
As a tightly controlled cell death process, apoptosis eliminates unwanted cells and plays a vital role in multicellular organisms. Previous study have demonstrated that apoptosis occurred in Spodoptera frugiperda cultured Sf9 cells, which triggered by diverse apoptotic stimuli, including azadirachtin, camptothecin and ultraviolet. Due to its simplicity, high sensitivity and reliable specificity, RT-qPCR has been used widespread for analyzing expression levels of target genes. However, the selection of reference genes influences the accuracy of results profoundly. In this study, eight genes were selected for analyses of their suitability as references for normalizing RT-PCR data in Sf9 cells treated with apoptotic agents. Five algorithms, including NormFinder, BestKeeper, Delta Ct method, geNorm, and RefFinder, were used for stability ranking. Based on comprehensively analysis, the expression stability of selected genes varied in cells with different apoptotic stimuli. The best choices for cells under different apoptosis conditions were listed: EF2 and EF1α for cells treated with azadirachtin; RPL13 and RPL3 for cells treated with camptothecin; EF1α and ß-1-TUB for cells irradiated under ultraviolet; and EF1α and EF2 for combinational analyses of samples. Our results not only facilitate a more accurate normalization for RT-qPCR data, but also provide the reliable assurance for further studies of apoptotic mechanisms under different stimulus in Sf9 cells.