RESUMO
OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined. DESIGN: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA. RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients. CONCLUSION: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.
RESUMO
In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.
Assuntos
Plantas Medicinais , Alcaloides de Pirrolizidina , Alcaloides de Pirrolizidina/toxicidadeRESUMO
Pyrrolizidine alkaloids (PAs) occur as contaminants in plant-based foods and herbal medicines. Following metabolic activation by cytochrome P450 (CYP) enzymes, PAs induce DNA damage, hepatotoxicity and can cause liver cancer in rodents. There is ample evidence that the chemical structure of PAs determines their toxicity. However, more quantitative genotoxicity data are required, particularly in primary human hepatocytes (PHH). Here, the genotoxicity of eleven structurally different PAs was investigated in human HepG2 liver cells with CYP3A4 overexpression and PHH using an in vitro test battery. Furthermore, the data were subject to benchmark dose (BMD) modeling to derive the genotoxic potency of individual PAs. The cytotoxicity was initially determined in HepG2-CYP3A4 cells, revealing a clear structure-toxicity relationship for the PAs. Importantly, experiments in PHH confirmed the structure-dependent toxicity and cytotoxic potency ranking of the tested PAs. The genotoxicity markers γH2AX and p53 as well as the alkaline Comet assay consistently demonstrated a structure-dependent genotoxicity of PAs in HepG2-CYP3A4 cells, correlating well with their cytotoxic potency. BMD modeling yielded BMD values in the range of 0.1-10 µM for most cyclic and open diesters, followed by the monoesters. While retrorsine showed the highest genotoxic potency, monocrotaline and lycopsamine displayed the lowest genotoxicity. Finally, experiments in PHH corroborated the genotoxic potency ranking, and revealed genotoxic effects even in the absence of detectable cytotoxicity. In conclusion, our findings strongly support the concept of grouping PAs into potency classes and help to pave the way for a broader acceptance of relative potency factors in risk assessment.
Assuntos
Neoplasias Hepáticas , Alcaloides de Pirrolizidina , Humanos , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Alcaloides de Pirrolizidina/metabolismo , Hepatócitos , Testes de Mutagenicidade , Neoplasias Hepáticas/metabolismoRESUMO
Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.
Assuntos
Antineoplásicos , Alcaloides de Pirrolizidina , Humanos , Citocromo P-450 CYP3A/metabolismo , Fígado , Hepatócitos , Alcaloides de Pirrolizidina/metabolismo , Dano ao DNA , Antineoplásicos/farmacologiaRESUMO
Nitrosamines occur widespread in food, drinking water, cosmetics, as well as tobacco smoke and can arise endogenously. More recently, nitrosamines have been detected as impurities in various drugs. This is of particular concern as nitrosamines are alkylating agents that are genotoxic and carcinogenic. We first summarize the current knowledge on the different sources and chemical nature of alkylating agents with a focus on relevant nitrosamines. Subsequently, we present the major DNA alkylation adducts induced by nitrosamines upon their metabolic activation by CYP450 monooxygenases. We then describe the DNA repair pathways engaged by the various DNA alkylation adducts, which include base excision repair, direct damage reversal by MGMT and ALKBH, as well as nucleotide excision repair. Their roles in the protection against the genotoxic and carcinogenic effects of nitrosamines are highlighted. Finally, we address DNA translesion synthesis as a DNA damage tolerance mechanism relevant to DNA alkylation adducts.
Assuntos
Nitrosaminas , Dano ao DNA , Alquilação , Reparo do DNA , Alquilantes/farmacologia , Adutos de DNARESUMO
This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.
Assuntos
Alcaloides de Pirrolizidina , Chás de Ervas , Animais , Contaminação de Alimentos/análise , Alcaloides de Pirrolizidina/toxicidade , Medição de Risco , ToxicocinéticaRESUMO
The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-MeG and O6-carboxymethylguanine (O6-CMG) adducts. It is well established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer.
Assuntos
Colo/metabolismo , Guanina/análogos & derivados , Mucosa Intestinal/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Linhagem Celular , Colo/citologia , Dano ao DNA , Reparo do DNA , Guanina/metabolismo , Humanos , Mucosa Intestinal/citologiaRESUMO
The seven signal transducers of transcription (STATs) are cytokine-inducible modular transcription factors. They transmit the stimulation of cells with type I interferons (IFN-α/IFN-ß) and type II interferon (IFN-É£) into altered gene expression patterns. The N-terminal domain (NTD) of STAT1 is a surface for STAT1/STAT1 homodimer and STAT1/STAT2 heterodimer formation and allows the cooperative DNA binding of STAT1. We investigated whether the STAT1 NTD-mediated dimerization affected the IFN-induced tyrosine phosphorylation of STAT1, its nuclear translocation, STAT1-dependent gene expression, and IFN-dependent antiviral defense. We reconstituted human STAT1-negative and STAT2-negative fibrosarcoma cells with STAT1, NTD-mutated STAT1 (STAT1AA), STAT1 with a mutated DNA-binding domain (DBD), or STAT2. We treated these cells with IFN-α and IFN-É£ to assess differences between IFN-α-induced STAT1 homo- and heterodimers and IFN-É£-induced STAT1 homodimers. Our data demonstrate that IFNs induce the phosphorylation of STAT1 and STAT1AA at Y701 and their nuclear accumulation. We further reveal that STAT1AA can be phosphorylated in response to IFN-α in the absence of STAT2 and that IFN-É£-induced STAT1AA can activate gene expression directly. However, STAT1AA largely fails to bind STAT2 and to activate IFN-α-induced expression of endogenous antiviral STAT1/STAT2 target proteins. Congruent herewith, both an intact STAT1 NTD and STAT2 are indispensable to establish an antiviral state with IFN-α. These data provide new insights into the biological importance of the STAT1 NTD.
Assuntos
Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/fisiologia , Antivirais/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Fator de Transcrição STAT2/metabolismoRESUMO
Colorectal cancer (CRC) is one of the most common cancers and preventive strategies based on natural compounds are highly desirable. Curcumin, the principal bioactive compound in Curcuma longa, was described to have multiple beneficial health effects. A drawback, however, is the low bioavailability due to its insolubility in water. Here, we studied whether nanoscaled micellar curcumin with improved bioavailability administered in drinking water reduces inflammation and CRC formation in a mouse model. C57BL6 wild-type (WT) mice and a strain defective in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) were used, in which tumors were induced by azoxymethane (AOM) followed by dextran sodium sulfate (DSS). Inflammation and tumor formation were determined by mini-colonoscopy. Micellar curcumin (mCur) administered in drinking water significantly reduced AOM/DSS-induced colorectal inflammation in both WT and MGMT-deficient mice as compared to animals receiving drinking water with micelles not containing curcumin. In line with this, the tumor yield and tumor score were significantly lower in mCur-treated mice compared to the control group. No adverse effects were observed in animals receiving mCur daily for at least three months. Overall, our data show that chronic oral administered micellar curcumin is well tolerated and reduces chemical-induced gut inflammation and CRC formation in mice.Impact: The study shows that micellar curcumin with high bioavailability chronically administered at low and physiologically relevant concentration suppresses inflammation and carcinogenesis in a mouse colorectal tumor model.
Assuntos
Colite , Neoplasias Colorretais , Curcumina , Animais , Azoximetano/toxicidade , Carcinogênese , Colite/induzido quimicamente , Colite/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/prevenção & controle , Curcumina/farmacologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , MicelasRESUMO
The consumption of red meat is associated with an increased risk for colorectal cancer (CRC). Multiple lines of evidence suggest that heme iron as abundant constituent of red meat is responsible for its carcinogenic potential. However, the underlying mechanisms are not fully understood and particularly the role of intestinal inflammation has not been investigated. To address this important issue, we analyzed the impact of heme iron (0.25 µmol/g diet) on the intestinal microbiota, gut inflammation and colorectal tumor formation in mice. An iron-balanced diet with ferric citrate (0.25 µmol/g diet) was used as reference. 16S rRNA sequencing revealed that dietary heme reduced α-diversity and caused a persistent intestinal dysbiosis, with a continuous increase in gram-negative Proteobacteria. This was linked to chronic gut inflammation and hyperproliferation of the intestinal epithelium as attested by mini-endoscopy, histopathology and immunohistochemistry. Dietary heme triggered the infiltration of myeloid cells into colorectal mucosa with an increased level of COX-2 positive cells. Furthermore, flow cytometry-based phenotyping demonstrated an increased number of T cells and B cells in the lamina propria following heme intake, while γδ-T cells were reduced in the intraepithelial compartment. Dietary heme iron catalyzed formation of fecal N-nitroso compounds and was genotoxic in intestinal epithelial cells, yet suppressed intestinal apoptosis as evidenced by confocal microscopy and western blot analysis. Finally, a chemically induced CRC mouse model showed persistent intestinal dysbiosis, chronic gut inflammation and increased colorectal tumorigenesis following heme iron intake. Altogether, this study unveiled intestinal inflammation as important driver in heme iron-associated colorectal carcinogenesis.
Assuntos
Neoplasias Colorretais , Heme , Animais , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/patologia , Dieta , Heme/toxicidade , Inflamação/patologia , Mucosa Intestinal/patologia , Ferro , Camundongos , RNA Ribossômico 16SRESUMO
Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1-/-) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1-/- mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1-/- animals were then crossed with O6-methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1-/-/MGMT-/- double knockout (DKO) mice developed more, but much smaller tumors than MGMT-/- animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar O6-methylguanine levels. Studies with PARP-1-/- cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to O6-methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.
Assuntos
Neoplasias Colorretais/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/prevenção & controle , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Heme/toxicidade , Neoplasias Intestinais/induzido quimicamente , Óxido Nítrico/toxicidade , Animais , Células 3T3 BALB , Células CACO-2 , Carcinogênese/induzido quimicamente , Linhagem Celular , Ensaio Cometa , Cricetinae , Heme/química , Humanos , Camundongos , Mutagênese , Mutação , Óxido Nítrico/química , Carne Vermelha/toxicidade , Fatores de Risco , Análise de Célula ÚnicaRESUMO
In mammals, epoxy-polyunsaturated fatty acids (epoxy-PUFA) are enzymatically formed from naturally occurring all-cis PUFA by cytochrome P450 monooxygenases leading to the generation of cis-epoxy-PUFA (mixture of R,S- and S,R-enantiomers). In addition, also non-enzymatic chemical peroxidation gives rise to epoxy-PUFA leading to both, cis- and trans-epoxy-PUFA (mixture of R,R- and S,S-enantiomers). Here, we investigated for the first time trans-epoxy-PUFA and the trans/cis-epoxy-PUFA ratio as potential new biomarker of lipid peroxidation. Their formation was analyzed in correlation with the formation of isoprostanes (IsoP), which are commonly used as biomarkers of oxidative stress. Five oxidative stress models were investigated including incubations of three human cell lines as well as the in vivo model Caenorhabditis elegans with tert-butyl hydroperoxide (t-BOOH) and analysis of murine kidney tissue after renal ischemia reperfusion injury (IRI). A comprehensive set of IsoP and epoxy-PUFA derived from biologically relevant PUFA (ARA, EPA and DHA) was simultaneously quantified by LC-ESI(-)-MS/MS. Following renal IRI only a moderate increase in the kidney levels of IsoP and no relevant change in the trans/cis-epoxy-PUFA ratio was observed. In all investigated cell lines (HCT-116, HepG2 and Caki-2) as well as C. elegans a dose dependent increase of both, IsoP and the trans/cis-epoxy-PUFA ratio in response to the applied t-BOOH was observed. The different cell lines showed a distinct time dependent pattern consistent for both classes of autoxidatively formed oxylipins. Clear and highly significant correlations of the trans/cis-epoxy-PUFA ratios with the IsoP levels were found in all investigated cell lines and C. elegans. Based on this, we suggest the trans/cis-epoxy-PUFA ratio as potential new biomarker of oxidative stress, which warrants further investigation.
Assuntos
Biomarcadores/metabolismo , Isoprostanos/biossíntese , Estresse Oxidativo , Ácidos Graxos trans/biossíntese , Animais , Caenorhabditis elegans , Rim/lesões , Masculino , Camundongos , Traumatismo por Reperfusão/metabolismoRESUMO
N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O6-methylguanine (O6-MeG), which is repaired by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O6-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to monitor O6-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O6-MeG in CRC cells. Using the highly sensitive and specific UPLC-MS/MS, TMZ-induced O6-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O6-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC-MS/MS measurements and dose-response modeling revealed a non-linear formation of hepatic and colonic O6-MeG adducts in WT, whereas linear O6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC-MS/MS analysis is highly sensitive and specific for O6-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.
Assuntos
Cromatografia Líquida/métodos , Adutos de DNA/análise , Guanina/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Animais , Antineoplásicos Alquilantes/administração & dosagem , Azoximetano/administração & dosagem , Adutos de DNA/imunologia , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Relação Dose-Resposta a Droga , Guanina/análise , Guanina/imunologia , Células HCT116 , Humanos , Immunoblotting/métodos , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia de Fluorescência/métodos , Sensibilidade e Especificidade , Temozolomida/administração & dosagem , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.
Assuntos
Carcinógenos/toxicidade , Instabilidade Cromossômica/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Imidazóis/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quinase 1 do Ponto de Checagem/metabolismo , Aberrações Cromossômicas , Cricetinae , Adutos de DNA , Quebras de DNA de Cadeia Dupla , Receptores com Domínio Discoidina/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
Nitroglycerin (GTN) and other organic nitrates are widely used vasodilators. Their side effects are development of nitrate tolerance and endothelial dysfunction. Given the potential of GTN to induce nitro-oxidative stress, we investigated the interaction between nitro-oxidative DNA damage and vascular dysfunction in experimental nitrate tolerance. Cultured endothelial hybridoma cells (EA.hy 926) and Wistar rats were treated with GTN (ex vivo: 10-1000 µM; in vivo: 10, 20 and 50 mg/kg/day for 3 days, s.c.). The level of DNA strand breaks, 8-oxoguanine and O (6)-methylguanine DNA adducts was determined by Comet assay, dot blot and immunohistochemistry. Vascular function was determined by isometric tension recording. DNA adducts and strand breaks were induced by GTN in cells in vitro in a concentration-dependent manner. GTN in vivo administration leads to endothelial dysfunction, nitrate tolerance, aortic and cardiac oxidative stress, formation of DNA adducts, stabilization of p53 and apoptotic death of vascular cells in a dose-dependent fashion. Mice lacking O (6)-methylguanine-DNA methyltransferase displayed more vascular O (6)-methylguanine adducts and oxidative stress under GTN therapy than wild-type mice. Although we were not able to prove a causal role of DNA damage in the etiology of nitrate tolerance, the finding of GTN-induced DNA damage such as the mutagenic and toxic adduct O (6)-methylguanine, and cell death supports the notion that GTN based therapy may provoke adverse side effects, including endothelial function. Further studies are warranted to clarify whether GTN pro-apoptotic effects are related to an impaired recovery of patients upon myocardial infarction.
Assuntos
Dano ao DNA , Tolerância a Medicamentos/fisiologia , Endotélio Vascular/efeitos dos fármacos , Nitroglicerina/toxicidade , Vasodilatadores/toxicidade , Animais , Western Blotting , Ensaio Cometa , Modelos Animais de Doenças , Immunoblotting , Imuno-Histoquímica , Camundongos , Estresse Oxidativo , Ratos , Ratos WistarRESUMO
Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.
Assuntos
Autofagia/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Ácido Tióctico/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos Alquilantes/farmacologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Cisteína/metabolismo , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/genética , Reparo do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Glutationa/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Células HCT116/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Temozolomida , Ácido Tióctico/análogos & derivados , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epidemiological studies indicate that N-nitroso compounds (NOC) are causally linked to colorectal cancer (CRC). NOC induce DNA alkylations, including O (6)-methylguanine (O (6)-MeG) and N-methylated purines, which are repaired by O (6)-MeG-DNA methyltransferase (MGMT) and N-alkyladenine-DNA glycosylase (AAG)-initiated base excision repair, respectively. In view of recent evidence of nonlinear mutagenicity for NOC-like compounds, the question arises as to the existence of threshold doses in CRC formation. Here, we set out to determine the impact of DNA repair on the dose-response of alkylation-induced CRC. DNA repair proficient (WT) and deficient (Mgmt (-/-), Aag (-/-) and Mgmt (-/-)/Aag (-/-)) mice were treated with azoxymethane (AOM) and dextran sodium sulfate to trigger CRC. Tumors were quantified by non-invasive mini-endoscopy. A non-linear increase in CRC formation was observed in WT and Aag (-/-) mice. In contrast, a linear dose-dependent increase in tumor frequency was found in Mgmt (-/-) and Mgmt (-/-)/Aag (-/-) mice. The data were corroborated by hockey stick modeling, yielding similar carcinogenic thresholds for WT and Aag (-/-) and no threshold for MGMT lacking mice. O (6)-MeG levels and depletion of MGMT correlated well with the observed dose-response in CRC formation. AOM induced dose-dependently DNA double-strand breaks in colon crypts including Lgr5-positive colon stem cells, which coincided with ATR-Chk1-p53 signaling. Intriguingly, Mgmt (-/-) mice displayed significantly enhanced levels of γ-H2AX, suggesting the usefulness of γ-H2AX as an early genotoxicity marker in the colorectum. This study demonstrates for the first time a non-linear dose-response for alkylation-induced colorectal carcinogenesis and reveals DNA repair by MGMT, but not AAG, as a key node in determining a carcinogenic threshold.
Assuntos
Neoplasias Colorretais/genética , DNA Glicosilases/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Reparo do DNA/genética , Proteínas Supressoras de Tumor/genética , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/patologia , Reparo do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Compostos Nitrosos/toxicidadeRESUMO
Alpha-lipoic acid (LA), which plays a pivotal role in mitochondrial energy metabolism, is an endogenous dithiol compound with an array of antioxidative functions. It has been shown that LA triggers cell death in tumor cell lines, whereas non-transformed cells are hardly affected. In the present study, we analyzed the cytotoxicity of LA on colorectal cancer (CRC) cells differing in their p53 status and investigated a putative synergistic effect with the anticancer drug 5-fluorouracil (5-FU). We show that LA induces a dose-dependent decrease in cell viability, which was independent of the p53 status as attested in isogenic p53-proficient and p53-deficient cell lines. This effect was largely attributable to cell death induction as revealed by Annexin-V/PI staining. LA-treated HCT116 cells underwent caspase-dependent and caspase-independent cell death, which was blocked by the pan-caspase inhibitor zVAD and the RIP-kinase inhibitor Necrostatin-1, respectively. In CaCO-2 and HT29 cells, LA induced caspase-dependent cell demise via activation of caspase-9, caspase-3 and caspase-7 with subsequent PARP-1 cleavage as demonstrated by immunoblot analysis, activity assays and pan-caspase inhibition. Interestingly, LA treatment did neither activate p53 nor induced genotoxic effects as shown by lack of DNA strand breaks and phosphorylation of histone 2AX. Finally, we provide evidence that LA increases the cytotoxic effect induced by the anticancer drug 5-FU as revealed by significantly enhanced cell death rates in HCT116 and CaCO-2 cells. Collectively, these findings demonstrate that LA induces CRC cell death independent of their p53 status and potentiates the cytotoxicity of 5-FU without causing DNA damage on its own, which makes it a candidate for tumor therapy.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/farmacologia , Ácido Tióctico/farmacologia , Antimetabólitos Antineoplásicos/administração & dosagem , Células CACO-2 , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Fluoruracila/administração & dosagem , Células HCT116 , Humanos , Proteína Supressora de Tumor p53/metabolismoRESUMO
The natural compound 1,8-cineol, also known as eucalyptol, is a major constituent of eucalyptus oil. This epoxy-monoterpene is used as flavor and fragrance in consumer goods as well as medical therapies. Due to its anti-inflammatory properties, 1,8-cineol is also applied to treat upper and lower airway diseases. Despite its widespread use, only little is known about the genotoxicity of 1,8-cineol in mammalian cells. This study investigates the genotoxicity and cytotoxicity of 1,8-cineol in human and hamster cells. First, we observed a significant and concentration-dependent increase in oxidative DNA damage in human colon cancer cells, as detected by the Formamidopyrimidine-DNA glycosylase (Fpg)-modified alkaline comet assay. Pre-treatment of cells with the antioxidant N-acetylcysteine prevented the formation of Fpg-sensitive sites after 1,8-cineol treatment, supporting the notion that 1,8-cineol induces oxidative DNA damage. In the dose range of DNA damage induction, 1,8-cineol did neither reduce the viability of colon cancer cells nor affected their cell cycle distribution, suggesting that cells tolerate 1,8-cineol-induced oxidative DNA damage by engaging DNA repair. To test this hypothesis, hamster cell lines with defects in BRCA2 and Rad51, which are essentials players of homologous recombination (HR)-mediated repair, were treated with 1,8-cineol. The monoterpene induced oxidative DNA damage and subsequent DNA double-strand breaks in the hamster cell lines tested. Intriguingly, we detected a significant concentration-dependent decrease in viability of the HR-defective cells, whereas the corresponding wild-type cell lines with functional HR were not affected. Based on these findings, we conclude that 1,8-cineol is weakly genotoxic, inducing primarily oxidative DNA damage, which is most likely tolerated in DNA repair proficient cells without resulting in cell cycle arrest and cell death. However, cells with deficiency in HR were compromised after 1,8-cineol treatment, suggesting a protective role of HR in response to high doses of 1,8-cineol.