RESUMEN
The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804-2818, 2020), we showed that the in vivo metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.
Asunto(s)
Ácidos Aristolóquicos/toxicidad , Carcinogénesis , Carcinógenos/toxicidad , Aductos de ADN/metabolismo , ADN de Neoplasias/metabolismo , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
The environmental pollutant benzo[a]pyrene (BaP) is a human carcinogen that reacts with DNA after metabolic activation catalysed by cytochromes P450 (CYP) 1A1 and 1B1 together with microsomal epoxide hydrolase. The azo dye Sudan I is a potent inducer of CYP1A1/2. Here, Wistar rats were either treated with single doses of BaP (150 mg/kg bw) or Sudan I (50 mg/kg bw) alone or with both compounds in combination to explore BaP-derived DNA adduct formation in vivo. Using 32P-postlabelling, DNA adducts generated by BaP-7,8-dihydrodiol-9,10-epoxide were found in livers of rats treated with BaP alone or co-exposed to Sudan I. During co-exposure to Sudan I prior to BaP treatment, BaP-DNA adduct levels increased 2.1-fold in comparison to BaP treatment alone. Similarly, hepatic microsomes isolated from rats exposed to Sudan I prior to BaP treatment were also the most effective in generating DNA adducts in vitro with the activated metabolites BaP-7,8-dihydrodiol or BaP-9-ol as intermediates. DNA adduct formation correlated with changes in the expression and/or enzyme activities of CYP1A1, 1A2 and 1B1 in hepatic microsomes. Thus, BaP genotoxicity in rats in vivo appears to be related to the enhanced expression and/or activity of hepatic CYP1A1/2 and 1B1 caused by exposure of rats to the studied compounds. Our results indicate that the industrially employed azo dye Sudan I potentiates the genotoxicity of the human carcinogen BaP, and exposure to both substances at the same time seems to be hazardous to humans.
Asunto(s)
Benzo(a)pireno/toxicidad , Citocromo P-450 CYP1A1/metabolismo , Aductos de ADN/toxicidad , Hígado/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Naftoles/toxicidad , Animales , Carcinógenos Ambientales/toxicidad , Colorantes/toxicidad , Masculino , Ratas , Ratas WistarRESUMEN
The plant extract aristolochic acid (AA), containing aristolochic acid I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy and Balkan endemic nephropathy, unique renal diseases associated with upper urothelial cancer. Differences in the metabolic activation and detoxification of AAI and AAII and their effects on the metabolism of AAI/AAII mixture in the plant extract might be of great importance for an individual's susceptibility in the development of AA-mediated nephropathies and malignancies. Here, we investigated in vivo metabolism of AAI and AAII after ip administration to Wistar rats as individual compounds and as AAI/AAII mixture using high performance liquid chromatography/electrospray ionization mass spectrometry. Experimental findings were supported by theoretical calculations using density functional theory. We found that exposure to AAI/AAII mixture affected the generation of their oxidative and reductive metabolites formed during Phase I biotransformation and excreted in rat urine. Several Phase II metabolites of AAI and AAII found in the urine of exposed rats were also analyzed. Our results indicate that AAI is more efficiently metabolized in rats in vivo than AAII. Whereas AAI is predominantly oxidized during in vivo metabolism, its reduction is the minor metabolic pathway. In contrast, AAII is mainly metabolized by reduction. The oxidative reaction only occurs if aristolactam II, the major reductive metabolite of AAII, is enzymatically hydroxylated, forming aristolactam Ia. In AAI/AAII mixture, the metabolism of AAI and AAII is influenced by the presence of both AAs. For instance, the reductive metabolism of AAI is increased in the presence of AAII while the presence of AAI decreased the reductive metabolism of AAII. These results suggest that increased bioactivation of AAI in the presence of AAII also leads to increased AAI genotoxicity, which may critically impact AAI-mediated carcinogenesis. Future studies are needed to explain the underlying mechanism(s) for this phenomenon.
Asunto(s)
Ácidos Aristolóquicos/metabolismo , Animales , Ácidos Aristolóquicos/administración & dosificación , Ácidos Aristolóquicos/orina , Cromatografía Líquida de Alta Presión , Teoría Funcional de la Densidad , Inyecciones Intraperitoneales , Masculino , Ratas , Ratas Wistar , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
We studied the in vitro metabolism of the anti-thyroid-cancer drug vandetanib in a rat animal model and demonstrated that N-desmethylvandetanib and vandetanib N-oxide are formed by NADPH- or NADH-mediated reactions catalyzed by rat hepatic microsomes and pure biotransformation enzymes. In addition to the structural characterization of vandetanib metabolites, individual rat enzymes [cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO)] capable of oxidizing vandetanib were identified. Generation of N-desmethylvandetanib, but not that of vandetanib N-oxide, was attenuated by CYP3A and 2C inhibitors while inhibition of FMO decreased formation of vandetanib N-oxide. These results indicate that liver microsomal CYP2C/3A and FMO1 are major enzymes participating in the formation of N-desmethylvandetanib and vandetanib N-oxide, respectively. Rat recombinant CYP2C11 > >3A1 > 3A2 > 1A1 > 1A2 > 2D1 > 2D2 were effective in catalyzing the formation of N-desmethylvandetanib. Results of the present study explain differences between the CYP- and FMO-catalyzed vandetanib oxidation in rat and human liver reported previously and the enzymatic mechanisms underlying this phenomenon.
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Antineoplásicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas/metabolismo , Piperidinas/metabolismo , Quinazolinas/metabolismo , Animales , Humanos , Microsomas Hepáticos , Oxidación-Reducción , RatasRESUMEN
BACKGROUND: Xenobiotic-metabolizing enzymes (XME) play a critical role in the activation and detoxification of several carcinogens. However, the role of XMEs in colorectal carcinogenesis is unclear. METHODS: We investigated the expression of XMEs in human colorectal tissues among patients with stage I-IV colorectal cancer (n = 71) from the ColoCare Study. Transcriptomic profiling using paired colorectal tumor and adjacent normal mucosa tissues of XMEs (GSTM1, GSTA1, UGT1A8, UGT1A10, CYP3A4, CYP2C9, GSTP1, and CYP2W1) by RNA microarray was compared using Wilcoxon rank-sum tests. We assessed associations between clinicopathologic, dietary, and lifestyle factors and XME expression with linear regression models. RESULTS: GSTM1, GSTA1, UGT1A8, UGT1A10, and CYP3A4 were all statistically significantly downregulated in colorectal tumor relative to normal mucosa tissues (all P ≤ 0.03). Women had significantly higher expression of GSTM1 in normal tissues compared with men (ß = 0.37, P = 0.02). By tumor site, CYP2C9 expression was lower in normal mucosa among patients with rectal cancer versus colon cancer cases (ß = -0.21, P = 0.0005). Smokers demonstrated higher CYP2C9 expression levels in normal mucosa (ß = 0.17, P = 0.02) when compared with nonsmokers. Individuals who used NSAIDs had higher GSTP1 tumor expression compared with non-NSAID users (ß = 0.17, P = 0.03). Higher consumption of cooked vegetables (>1×/week) was associated with higher CYP3A4 expression in colorectal tumor tissues (ß = 0.14, P = 0.007). CONCLUSIONS: XMEs have lower expression in colorectal tumor relative to normal mucosa tissues and may modify colorectal carcinogenesis via associations with clinicopathologic, lifestyle, and dietary factors. IMPACT: Better understanding into the role of drug-metabolizing enzymes in colorectal cancer may reveal biological differences that contribute to cancer development, as well as treatment response, leading to clinical implications in colorectal cancer prevention and management.
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Carcinógenos/metabolismo , Neoplasias Colorrectales/patología , Perfilación de la Expresión Génica/estadística & datos numéricos , Mucosa Intestinal/enzimología , Xenobióticos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Aminas/metabolismo , Antioxidantes/metabolismo , Carcinogénesis/patología , Femenino , Compuestos Heterocíclicos/metabolismo , Humanos , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , No Fumadores/estadística & datos numéricos , Hidrocarburos Policíclicos Aromáticos/metabolismo , Estudios Prospectivos , Fumadores/estadística & datos numéricosRESUMEN
Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(-/-) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by 32P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography-mass spectrometry (GC-MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(-/-) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(-/-) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.
Asunto(s)
Ácidos Aristolóquicos/toxicidad , Daño del ADN , Fibroblastos/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Mutágenos/toxicidad , Proteína p53 Supresora de Tumor/genética , Animales , Ácidos Aristolóquicos/metabolismo , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica/efectos de los fármacos , Pruebas de Función Renal , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mutágenos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genéticaRESUMEN
The metabolism of vandetanib, a tyrosine kinase inhibitor used for treatment of symptomatic/progressive medullary thyroid cancer, was studied using human hepatic microsomes, recombinant cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs). The role of CYPs and FMOs in the microsomal metabolism of vandetanib to N-desmethylvandetanib and vandetanib-N-oxide was investigated by examining the effects of CYP/FMO inhibitors and by correlating CYP-/FMO-catalytic activities in each microsomal sample with the amounts of N-desmethylvandetanib/vandetanib-N-oxide formed by these samples. CYP3A4/FMO-activities significantly correlated with the formation of N-desmethylvandetanib/ vandetanib-N-oxide. Based on these studies, most of the vandetanib metabolism was attributed to N-desmethylvandetanib/vandetanib-N-oxide to CYP3A4/FMO3. Recombinant CYP3A4 was most efficient to form N-desmethylvandetanib, while FMO1/FMO3 generated N-oxide. Cytochrome b5 stimulated the CYP3A4-catalyzed formation of N-desmethylvandetanib, which is of great importance because CYP3A4 is not only most efficient in generating N-desmethylvandetanib, but also most significant due to its high expression in human liver. Molecular modeling indicated that binding of more than one molecule of vandetanib into the CYP3A4-active center can be responsible for the high efficiency of CYP3A4 N-demethylating vandetanib. Indeed, the CYP3A4-mediated reaction exhibits kinetics of positive cooperativity and this corresponded to the in silico model, where two vandetanib molecules were found in CYP3A4-active center.
Asunto(s)
Antineoplásicos/farmacología , Citocromo P-450 CYP3A/metabolismo , Enzimas/metabolismo , Oxidación-Reducción , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Animales , Antineoplásicos/química , Citocromo P-450 CYP3A/química , Relación Dosis-Respuesta a Droga , Enzimas/química , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Piperidinas/química , Inhibidores de Proteínas Quinasas/química , Quinazolinas/química , Conejos , Ratas , Proteínas RecombinantesRESUMEN
Balkan endemic nephropathy is a chronic tubulointerstitial disease with insidious onset, slowly progressing to end-stage renal disease and frequently associated with urothelial carcinoma of the upper urinary tract (UTUC). It was described in South-East Europe at the Balkan peninsula in rural areas around tributaries of the Danube River. After decades of intensive investigation, the causative factor was identified as the environmental phytotoxin aristolochic acid (AA) contained in Aristolochia clematitis, a common plant growing in wheat fields that was ingested through home-baked bread. AA initially was involved in the outbreak of cases of rapidly progressive renal fibrosis reported in Belgium after intake of root extracts of Aristolochia fangchi imported from China. A high prevalence of UTUC was found in these patients. The common molecular link between Balkan and Belgian nephropathy cases was the detection of aristolactam-DNA adducts in renal tissue and UTUC. These adducts are not only biomarkers of prior exposure to AA, but they also trigger urothelial malignancy by inducing specific mutations (A:T to T:A transversion) in critical genes of carcinogenesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particularly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies.
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Ácidos Aristolóquicos/toxicidad , Nefropatía de los Balcanes/inducido químicamente , Carcinoma de Células Transicionales/inducido químicamente , Exposición a Riesgos Ambientales/efectos adversos , Neoplasias Renales/inducido químicamente , Neoplasias Ureterales/inducido químicamente , Animales , Aristolochia , Nefropatía de los Balcanes/diagnóstico , Nefropatía de los Balcanes/patología , Nefropatía de los Balcanes/terapia , Carcinógenos/toxicidad , Aductos de ADN , Humanos , Tamizaje MasivoRESUMEN
Although ellipticine (Elli) is an efficient anticancer agent, it exerts several adverse effects. One approach to decrease the adverse effects of drugs is their encapsulation inside a suitable nanocarrier, allowing targeted delivery to tumour tissue whereas avoiding healthy cells. We constructed a nanocarrier from apoferritin (Apo) bearing ellipticine, ApoElli, and subsequently characterized. The nanocarrier exhibits a narrow size distribution suggesting its suitability for entrapping the hydrophobic ellipticine molecule. Ellipticine was released from ApoElli into the water environment under pH 6.5, but only less than 20% was released at pH 7.4. The interaction of ApoElli with microsomal membrane particles containing cytochrome P450 (CYP) biotransformation enzymes accelerated the release of ellipticine from this nanocarrier making it possible to be transferred into this membrane system even at pH 7.4 and facilitating CYP-mediated metabolism. Reactive metabolites were formed not only from free ellipticine, but also from ApoElli, and both generated covalent DNA adducts. ApoElli was toxic in UKF-NB-4 neuroblastoma cells, but showed significantly lower cytotoxicity in non-malignant fibroblast HDFn cells. Ellipticine either free or released from ApoElli was concentrated in the nuclei of neuroblastoma cells, concentrations of which being significantly higher in nuclei of UKF-NB-4 than in HDFn cells. In HDFn the higher amounts of ellipticine were sequestrated in lysosomes. The extent of ApoElli entering the nuclei in UKF-NB-4 cells was lower than that of free ellipticine and correlated with the formation of ellipticine-derived DNA adducts. Our study indicates that the ApoElli form of ellipticine seems to be a promising tool for neuroblastoma treatment.
Asunto(s)
Antineoplásicos/farmacología , Apoferritinas/farmacología , Citocromo P-450 CYP3A/metabolismo , Aductos de ADN/metabolismo , Portadores de Fármacos , Elipticinas/farmacología , Nanopartículas , Neuroblastoma/tratamiento farmacológico , Antineoplásicos/química , Apoferritinas/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Aductos de ADN/genética , Composición de Medicamentos , Liberación de Fármacos , Elipticinas/química , Histonas/metabolismo , Humanos , Neuroblastoma/enzimología , Neuroblastoma/genética , Neuroblastoma/patología , FosforilaciónRESUMEN
OBJECTIVES: Vandetanib¸ lenvatinib, and cabozantinib are tyrosine kinase inhibitors (TKIs) targeting VEGFR subtypes 1 and 2, EGFR and the RET-tyrosine kinase, thus considered as multiple TKIs. These TKIs have already been approved for treating patients suffering from thyroid cancer and renal cell carcinoma. Ellipticine, a DNA-damaging drug, is another anticancer agent that is effective against certain tumors of the thyroid gland, ovarian carcinoma, breast cancer and osteolytic breast cancer metastasis. Its anticancer efficiency is dictated by its oxidation with cytochrome P450 (CYP) and peroxidase enzymes. A number of studies testing the effectiveness of individual anticancer drugs, the pharmacological efficiencies of which are affected by their metabolism, alone or in a combination with other cytostatics demonstrated that such combination can have both positive and negative effects on treatment regimen. The aim of this study was to study the effect of vandetanib, lenvatinib and cabozantinib on oxidation of ellipticine which dictates its pharmacological efficiency. METHODS: Ellipticine oxidation catalyzed by hepatic microsomes, recombinant CYP enzymes and peroxidases (horseradish peroxidase, lactoperoxidase and myeloperoxidase) and the effect of TKIs (vandetanib, lenvatinib and cabozantinib) on this oxidation were analyzed by HPLC used for separation of ellipticine metabolites and quantification of their amounts formed during oxidation. RESULTS: The CYP enzymatic system oxidizes ellipticine up to five metabolites (9-hydroxy-, 12-hydroxy-, 13-hydroxy-, 7-hydroxyellipticine, and ellipticine N2- oxide), while peroxidases form predominantly ellipticine dimer. Ellipticine oxidation catalyzed by rat and human hepatic microsomes was inhibited by vandetanib and cabozantinib, but essentially no inhibition was caused by lenvatinib. Of individual CYP enzymes catalyzing oxidation of ellipticine, TKIs inhibited oxidation of ellipticine catalyzed by CYP2D6 > 2D1 > 2C9 > 3A1 > 3A4, the CYP enzymes participating in ellipticine oxidation to metabolites increasing the ellipticine anticancer efficiency. On the contrary, they have essentially no inhibition effect on ellipticine oxidation catalyzed by CYP1A1 and 1A2, which are the enzymes that predominantly detoxify this drug. All tested TKIs had essentially no effect on oxidation of ellipticine by used peroxidases. CONCLUSION: The results found demonstrate that TKIs vandetanib, lenvatinib and cabozantinib cause a decrease in oxidative activation of DNA-damaging drug ellipticine by several CYP enzymes in vitro which might lead to a decrease in its pharmacological efficiency. In contrast, they practically do not influence its detoxification catalyzed by CYP1A1, 1A2 and peroxidases. The present study indicates that tested TKIs seem not to have a potency to increase ellipticine anticancer efficiency.
Asunto(s)
Anilidas/farmacología , Elipticinas/farmacocinética , Oxidación-Reducción/efectos de los fármacos , Peroxidasas/antagonistas & inhibidores , Compuestos de Fenilurea/farmacología , Piperidinas/farmacología , Piridinas/farmacología , Quinazolinas/farmacología , Quinolinas/farmacología , Animales , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Humanos , Microsomas Hepáticos/efectos de los fármacos , RatasRESUMEN
So far multiple differences in prostate cancer-specific amino acids metabolism have been discovered. Moreover, attempts to utilize these alterations for prostate cancer diagnosis and treatment have been made. The prostate cancer metabolism and biosynthesis of amino acids are particularly focused on anaplerosis more than on energy production. Other crucial requirements on amino acids pool come from the serine, onecarbon cycle, glycine synthesis pathway and folate metabolism forming major sources of interproducts for synthesis of nucleobases necessary for rapidly proliferating cells. Considering the lack of some amino acids biosynthetic pathways and/or their extraordinary importance for prostate cancer cells, there is a widespread potential for targeted therapeutic applications with no effect on non-malignant cells. This review summarizes the up-to-date knowledge of the importance of amino acids for prostate cancer pathogenesis with a special emphasis on potential applications of metabolic variabilities in the new oncologic paradigm of precision medicine.
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Aminoácidos/metabolismo , Medicina de Precisión , Neoplasias de la Próstata/metabolismo , Animales , Humanos , MasculinoRESUMEN
Metal-based coordination compounds, including the well-known cytostatic drug cisplatin, are widely used in the anticancer therapy. Generally, they exhibit high cytotoxicity not only towards malignant cells, but also towards non-malignant cells, which represents main problem of their clinical use. Herein, we describe the synthesis, characterization and biological testing of three trinuclear nickel(II) coordination compounds. Central nickel atoms are bridged by trithiocyanurate anion and coordinated by triamine and bis-benzimidazoles, respectively. To delineate a potential usage in anticancer therapy, we encapsulated the most cytotoxic complex into biomacromolecular protein cage apoferritin (FRT), forming FRTNi. FRT encapsulation markedly decreased the hemotoxicity of free Ni compounds. Despite FRTNi can be internalized through passive targeting by enhanced permeability and retention effect, we further introduced active targeting utilizing folate receptor (FR) via folic acid (FA)-modified FRT (FRTNiFA). Using breast cancer cell lines T-47D (FR+), MCF-7 (FR-) and non-malignant mammary gland derived cell line HBL-100 (FR-), we show pronounced FR-dependent internalization of FRTNiFA. Overall, we demonstrate that the FRT macromolecular nanocarrier provides a very low off-target toxicity, which could enable the use of highly toxic Ni compounds in cancer nanomedicine.
Asunto(s)
Complejos de Coordinación/farmacología , Ácido Fólico/farmacología , Proteínas de Unión a Hierro/metabolismo , Níquel/farmacología , Receptores de Superficie Celular/metabolismo , Apoptosis/efectos de los fármacos , Materiales Biocompatibles/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Clonales , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Endocitosis/efectos de los fármacos , Ferritinas/metabolismo , Humanos , LigandosRESUMEN
The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10â¯mg/kg body weight ellipticine (nâ¯=â¯4/group) for 24â¯h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.
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Antineoplásicos/metabolismo , Citocromo-B(5) Reductasa/deficiencia , Citocromos b5/deficiencia , Elipticinas/metabolismo , Hepatocitos/enzimología , Hígado/enzimología , Activación Metabólica , Animales , Antineoplásicos/farmacología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromo-B(5) Reductasa/genética , Citocromos b5/genética , Aductos de ADN/metabolismo , Elipticinas/farmacología , Genotipo , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , FenotipoRESUMEN
DNA hypermethylation is one of the most common epigenetic modifications in prostate cancer (PCa). Several studies have delineated sarcosine as a PCa oncometabolite that increases the migration of malignant prostate cells while decreasing their doubling time. Here, we show that incubation of prostate cells with sarcosine elicited the upregulation of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and pipecolic acid oxidase. This process was accompanied by a considerable increase in the production of the major methyl-donor S-adenosylmethionine (SAMe), together with an elevation of cellular methylation potential. Global DNA methylation analyses revealed increases in methylated CpG islands in distinct prostate cell lines incubated with sarcosine, but not in cells of nonprostate origin. This phenomenon was further associated with marked upregulation of DNA methyltransferases (Dnmts). Epigenetic changes were recapitulated through blunting of Dnmts using the hypomethylating agent 5-azacytidine, which was able to inhibit sarcosine-induced migration of prostate cells. Moreover, spatial mapping revealed concomitant increases in sarcosine, SAMe and Dnmt1 in histologically confirmed malignant prostate tissue, but not in adjacent or nonmalignant tissue, which is in line with the obtained in vitro data. In summary, we show here for the first time that sarcosine acts as an epigenetic modifier of prostate cells and that this may contribute to its oncometabolic role.
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Islas de CpG , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Sarcosina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Línea Celular , Humanos , Masculino , Próstata/patología , Neoplasias de la Próstata/patologíaRESUMEN
Herein, we describe the in vivo effects of doxorubicin (DOX) encapsulated in ubiquitous protein apoferritin (APO) and its efficiency and safety in anti-tumor treatment. APODOX is both passively (through Enhanced Permeability and Retention effect) and actively targeted to tumors through prostate-specific membrane antigen (PSMA) via mouse antibodies conjugated to the surface of horse spleen APO. To achieve site-directed conjugation of the antibodies, a HWRGWVC heptapeptide linker was used. The prostate cancer-targeted and non-targeted nanocarriers were tested using subcutaneously implanted LNCaP cells in athymic mice models, and compared to free DOX. Prostate cancer-targeted APODOX retained the high potency of DOX in attenuation of tumors (with 55% decrease in tumor volume after 3 weeks of treatment). DOX and non-targeted APODOX treatment caused damage to liver, kidney and heart tissues. In contrast, no elevation in liver or kidney enzymes and negligible changes were revealed by histological assessment in prostate cancer-targeted APODOX-treated mice. Overall, we show that the APO nanocarrier provides an easy encapsulation protocol, reliable targeting, high therapeutic efficiency and very low off-target toxicity, and is thus a promising delivery system for translation into clinical use.
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Apoferritinas/uso terapéutico , Doxorrubicina/análogos & derivados , Inmunoconjugados/uso terapéutico , Nanoconjugados/uso terapéutico , Neoplasias de la Próstata/terapia , Animales , Antígenos de Superficie/inmunología , Apoferritinas/efectos adversos , Línea Celular Tumoral , Doxorrubicina/efectos adversos , Doxorrubicina/uso terapéutico , Glutamato Carboxipeptidasa II/inmunología , Corazón/efectos de los fármacos , Xenoinjertos , Humanos , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Próstata/tratamiento farmacológico , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.
Asunto(s)
Carcinógenos/metabolismo , Aductos de ADN/metabolismo , Animales , Marcaje Isotópico , Ratas , Ratas WistarRESUMEN
Endocrine disruptors (EDs) are compounds that interfere with the balance of the endocrine system by mimicking or antagonising the effects of endogenous hormones, by altering the synthesis and metabolism of natural hormones, or by modifying hormone receptor levels. The synthetic estrogen 17α-ethinylestradiol (EE2) and the environmental carcinogen benzo[a]pyrene (BaP) are exogenous EDs whereas the estrogenic hormone 17ß-estradiol is a natural endogenous ED. Although the biological effects of these individual EDs have partially been studied previously, their toxicity when acting in combination has not yet been investigated. Here we treated Wistar rats with BaP, EE2 and estradiol alone or in combination and studied the influence of EE2 and estradiol on: (i) the expression of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver on the transcriptional and translational levels; (ii) the inducibility of these CYP enzymes by BaP in this rat organ; (iii) the formation of BaP-DNA adducts in rat liver in vivo; and (iv) the generation of BaP-induced DNA adducts after activation of BaP with hepatic microsomes of rats exposed to BaP, EE2 and estradiol and with recombinant rat CYP1A1 in vitro. BaP acted as a strong and moderate inducer of CYP1A1 and 1B1 in rat liver, respectively, whereas EE2 or estradiol alone had no effect on the expression of these enzymes. However, when EE2 was administered to rats together with BaP, it significantly decreased the potency of BaP to induce CYP1A1 and 1B1 gene expression. For EE2, but not estradiol, this also correlated with a reduction of BaP-induced CYP1A1 enzyme activity in rat hepatic microsomes. Further, while EE2 and estradiol did not form covalent adducts with DNA, they affected BaP-derived DNA adduct formations in vivo and in vitro. The observed decrease in BaP-DNA adduct levels in rat liver in vivo resulted from the inhibition of CYP1A1-mediated BaP bioactivation by EE2 and estradiol. Our results indicate that BaP genotoxicity mediated through its activation by CYP1A1 in rats in vivo is modulated by estradiol and its synthetic derivative EE2.
Asunto(s)
Benzo(a)pireno/toxicidad , Citocromo P-450 CYP1A1/biosíntesis , Disruptores Endocrinos/toxicidad , Estradiol/toxicidad , Etinilestradiol/toxicidad , Regulación Enzimológica de la Expresión Génica , Animales , Citocromo P-450 CYP1A1/genética , Sinergismo Farmacológico , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Ratas , Ratas WistarRESUMEN
Human metallothionein-3 (hMT-3), also known as growth inhibitory factor, is predominantly expressed in the central nervous system. hMT-3 is presumed to participate in the processes of heavy metal detoxification, regulation of metabolism and protection against oxidative damage of free radicals in the central nervous system; thus, it could play important neuromodulatory and neuroprotective roles. However, the primary functions of hMT-3 and the mechanism underlying its multiple functions in neuroblastoma have not been elucidated so far. First, we confirmed relatively high expression of hMT-3 encoding mRNA in biopsies (n = 23) from high-risk neuroblastoma subjects. Therefore, we focused on investigation of the impact of hMT-3 up-regulation in N-Myc amplifying neuroblastoma cells. The differentially up-regulated genes involved in biological pathways related to cellular senescence and cell cycle were identified using electrochemical microarray with consequent bioinformatic processing. Further, as experimental verification of microarray data, the cytotoxicity of the cisplatin (CDDP) was examined in hMT-3 and mock cells by MTT and clonogenic assays. Overall, our data strongly suggest that up-regulation of hMT-3 positively correlates with the genes involved in oncogene-induced senescence (CDKN2B and ANAPC5) or apoptosis (CASP4). Moreover, we identified a significant increase in chemoresistance to cisplatin (CDDP) due to hMT-3 up-regulation (24IC50: 7.5 vs. 19.8 µg/ml), indicating its multipurpose biological significance.
RESUMEN
Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) can induce cytochrome P450 1A1 (CYP1A1) via a p53-dependent mechanism. The effect of different p53-activating chemotherapeutic drugs on CYP1A1 expression, and the resultant effect on BaP metabolism, was investigated in a panel of isogenic human colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/-) or TP53(-/-) were treated for up to 48â¯h with 60⯵M cisplatin, 50⯵M etoposide or 5⯵M ellipticine, each of which caused high p53 induction at moderate cytotoxicity (60-80% cell viability). We found that etoposide and ellipticine induced CYP1A1 in TP53(+/+) cells but not in TP53(-/-) cells, demonstrating that the mechanism of CYP1A1 induction is p53-dependent; cisplatin had no such effect. Co-incubation experiments with the drugs and 2.5⯵M BaP showed that: (i) etoposide increased CYP1A1 expression in TP53(+/+) cells, and to a lesser extent in TP53(-/-) cells, compared to cells treated with BaP alone; (ii) ellipticine decreased CYP1A1 expression in TP53(+/+) cells in BaP co-incubations; and (iii) cisplatin did not affect BaP-mediated CYP1A1 expression. Further, whereas cisplatin and etoposide had virtually no influence on CYP1A1-catalysed BaP metabolism, ellipticine treatment strongly inhibited BaP bioactivation. Our results indicate that the underlying mechanisms whereby etoposide and ellipticine regulate CYP1A1 expression must be different and may not be linked to p53 activation alone. These results could be relevant for smokers, who are exposed to increased levels of BaP, when prescribing chemotherapeutic drugs. Beside gene-environment interactions, more considerations should be given to potential drug-environment interactions during chemotherapy.