RESUMO
OBJECTIVE: This study sought to evaluate short-term treatment with COX-2 inhibitors and acute changes in colonic PGE2 levels as predictors of long-term efficacy in a genetic model of colorectal cancer. METHODS: Celecoxib oral suspension (40 mg/kg BID) was dosed to Apc-mutant Pirc (F344/NTac-Apcam1137) rats for 4 days (short-term group), or the equivalent dose of 1500 ppm celecoxib was administered in the diet for 4 months (long-term group). Percent inhibition of colonic PGE2 was calculated, and the reduction in colonic PGE2 was assessed in relation to suppression of adenomatous colon polyps. RESULTS: Colonic mucosa PGE2 was fourfold higher in Pirc than in F344 wild-type rats (21 vs. 5.6 pg/mg epithelial tissue), due at least in part to higher COX-2 expression, and this was confirmed by elevated PGE2-d11 levels in Pirc colonic S9 incubations. In the 4-day study, dose-dependent reductions in PGE2 were observed in colonic epithelium (-33% (P>0.05) and -57% (P=0.0012)), after low- and high-dose celecoxib treatments of 4 mg/kg and 40 mg/kg (bid), respectively. In the 4-month study, 1500 ppm celecoxib suppressed colonic epithelium PGE2 by 43.5%, and tumor multiplicity by 80% (P<0.0015). Suppression of plasma 6-keto PGF1α also was corroborated following long-term treatment with 1500 ppm celecoxib (P<0.05). CONCLUSIONS: Acute changes in colonic mucosa PGE2 provided a rapid means of predicting long-term chemopreventive effects from celecoxib, and might be useful for screening of new COX-2 inhibitor compounds.
Assuntos
Celecoxib/farmacologia , Colo/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/metabolismo , Animais , Biomarcadores/metabolismo , Celecoxib/uso terapêutico , Colo/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Ratos Endogâmicos F344 , Resultado do TratamentoRESUMO
Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of ß-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of ß-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and ß-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.
Assuntos
Neoplasias Colorretais/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus gallolyticus subspecies gallolyticus/fisiologia , Animais , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Humanos , Camundongos , Transdução de Sinais , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/patologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) targets multiple organs for tumorigenesis in the rat, including the colon and the skin. PhIP-induced skin tumors were subjected to mutation screening, which identified genetic changes in Hras (7/40, 17.5%) and Tp53 (2/40, 5%), but not in Ctnnb1, a commonly mutated gene in PhIP-induced colon tumors. Despite the absence of Ctnnb1 mutations, ß-catenin was overexpressed in nuclear and plasma membrane fractions from PhIP-induced skin tumors, coinciding with loss of p120-catenin from the plasma membrane, and the appearance of multiple p120-catenin-associated bands in the nuclear extracts. Real-time RT-PCR revealed that p120-catenin isoforms 1 and 4 were upregulated in PhIP-induced skin tumors, whereas p120-catenin isoform 3 was expressed uniformly, compared with adjacent normal-looking tissue. In human epidermoid carcinoma and colon cancer cells, transient transfection of p120-catenin isoform 1A enhanced the viability and cell invasion index, whereas transient transfection of p120-catenin isoform 4A increased cell viability and cell proliferation. Knockdown of p120-catenin revealed a corresponding reduction in the expression of ß-catenin and a transcriptionally regulated target, Ccnd1/Cyclin D1. Co-immunoprecipitation experiments identified associations of ß-catenin with p120-catenin isoforms in PhIP-induced skin tumors and human cancer cell lines. The results are discussed in the context of therapeutic strategies that might target different p120-catenin isoforms, providing an avenue to circumvent constitutively active ß-catenin arising via distinct mechanisms in skin and colon cancer.
Assuntos
Apoptose , Carcinógenos/toxicidade , Carcinoma de Células Escamosas/patologia , Cateninas/metabolismo , Proliferação de Células , Neoplasias Colorretais/patologia , Neoplasias Cutâneas/patologia , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Cateninas/antagonistas & inibidores , Cateninas/genética , Movimento Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Imidazóis/toxicidade , Invasividade Neoplásica , Isoformas de Proteínas , RNA Interferente Pequeno/genética , Ratos , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Células Tumorais Cultivadas , delta CateninaRESUMO
Heterocyclic amines (HCAs) produced during high-temperature cooking have been studied extensively in terms of their genotoxic/genetic effects, but recent work has implicated epigenetic mechanisms involving non-coding RNAs. Colon tumors induced in the rat by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have altered microRNA (miRNA) signatures linked to dysregulated pluripotency factors, such as c-Myc and Krüppel-like factor 4 (KLF4). We tested the hypothesis that dysregulated miRNAs from PhIP-induced colon tumors would provide a "PhIP signature" for use in other target organs obtained from a 1-year carcinogenicity bioassay in the rat. Downstream targets that were corroborated in the rat were then investigated in human cancer datasets. The results confirmed that multiple let-7 family members were downregulated in PhIP-induced skin, colon, lung, small intestine, and Zymbal's gland tumors, and were associated with c-myc and Hmga2 upregulation. PhIP signature miRNAs with the profile mir-21high/mir-126low/mir-29clow/mir-215low/mir-145low were linked to reduced Klf4 levels in rat tumors, and in human pan-cancer and colorectal cancer. It remains to be determined whether this PhIP signature has predictive value, given that more than 20 different genotoxic HCAs are present in the human diet, plus other agents that likely induce or repress many of the same miRNAs. Future studies should define more precisely the miRNA signatures of other HCAs, and their possible value for human risk assessment.
Assuntos
Aminas/toxicidade , Testes de Carcinogenicidade/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/análise , Neoplasias/genética , Aminas/química , Animais , Humanos , Imidazóis/toxicidade , Fator 4 Semelhante a Kruppel , Masculino , Ratos Endogâmicos F344RESUMO
BACKGROUND AND AIMS: Colonoscopy provides a means for screening and removal of colon adenomas, preventing such lesions from progressing to late-stage carcinoma. No preclinical model currently exists that closely parallels the clinical scenario with respect to polyp resection and recovery after endoscopy. METHODS: When we used the polyposis in rat colon (Pirc) model, a new polypectomy methodology was developed. A novel PLC classification system (polyp number/location/clockwise orientation) also was devised in order to accurately and reproducibly specify the location of each lesion within the colon. RESULTS: One week after surgery, injuries to the polypectomy site were confined to the submucosa, indicating that little or no damage occurred to the inner muscle layer of the colon. Polypectomy sites occasionally continued to show ulcer formation, whereas others exhibited tissue regeneration. A pilot study (n = 6 animals), involving a total of 37 polypectomies, confirmed that the new methodology could be applied by using either air insufflation or water-assisted techniques, with either hot or cold snare. As a general observation, polyps tended to be more fully distended and less flattened against the colon mucosa by using the water-assisted protocol, increasing the technical ease of ensnaring and resecting lesions. The PLC system proved to be straightforward and facilitated longitudinal studies by allowing the investigator to track each polypectomy site on repeated examination. CONCLUSIONS: The Pirc model was ideally suited to colonoscopy with polypectomy. Because the main cause of morbidity in the Pirc model is blockage of the colon, polypectomy can be used as a preventive strategy and will likely facilitate long-term investigations of single agent and combination therapies with potential direct clinical relevance.
Assuntos
Adenoma/cirurgia , Polipose Adenomatosa do Colo/cirurgia , Pólipos do Colo/cirurgia , Colonoscopia/métodos , Neoplasias Colorretais/cirurgia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Projetos Piloto , RatosRESUMO
Cancer etiology involves complex interactions between genetic and non-genetic factors, with epigenetic mechanisms serving as key regulators at multiple stages of pathogenesis. Poor dietary habits contribute to cancer predisposition by impacting DNA methylation patterns, non-coding RNA expression, and histone epigenetic landscapes. Histone post-translational modifications (PTMs), including acyl marks, act as a molecular code and play a crucial role in translating changes in cellular metabolism into enduring patterns of gene expression. As cancer cells undergo metabolic reprogramming to support rapid growth and proliferation, nuanced roles have emerged for dietary- and metabolism-derived histone acylation changes in cancer progression. Specific types and mechanisms of histone acylation, beyond the standard acetylation marks, shed light on how dietary metabolites reshape the gut microbiome, influencing the dynamics of histone acyl repertoires. Given the reversible nature of histone PTMs, the corresponding acyl readers, writers, and erasers are discussed in this review in the context of cancer prevention and treatment. The evolving 'acyl code' provides for improved biomarker assessment and clinical validation in cancer diagnosis and prognosis.
Assuntos
Histonas , Neoplasias , Humanos , Histonas/metabolismo , Neoplasias/genética , Medicina de Precisão , Código das Histonas , Metilação de DNA , Processamento de Proteína Pós-Traducional , Epigênese GenéticaRESUMO
Bromodomain Adjacent to Zinc Finger Domain 1A (BAZ1A) is a critical regulator of chromatin remodeling. We sought to clarify the roles of BAZ1A in the etiology of colorectal cancer, including the mechanisms of its alternatively spliced variants. Public databases were examined and revealed high BAZ1A expression in the majority of colorectal cancer patients, which was corroborated in a panel of human colon cancer cell lines. BAZ1A silencing reduced cell viability and increased markers of DNA damage, apoptosis, and senescence, along with the downregulation of Wnt/ß-catenin signaling. The corresponding molecular changes resulted in tumor growth inhibition when BAZ1A-knockout cells were implanted into nude mice. In rescue experiments, a short isoform of BAZ1A that was associated with alternative splicing by the DBIRD complex failed to restore DNA repair activity in colon cancer cells and maintained chemosensitivity to phleomycin treatment, unlike the full-length BAZ1A. A working model proposes that a buried domain in the N-terminus of the BAZ1A short isoform lacks the ability to access linker DNA, thereby disrupting the activity of the associated chromatin remodeling complexes. Given the current interest in RNA splicing deregulation and cancer etiology, additional mechanistic studies are warranted with new lead compounds targeting BAZ1A, and other members of the BAZ family, with a view to improved therapeutic interventions.
Assuntos
Processamento Alternativo , Neoplasias Colorretais , Dano ao DNA , Camundongos Nus , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/efeitos dos fármacos , Animais , Camundongos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Apoptose/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células HCT116RESUMO
ACE2 overexpression in colorectal cancer patients might increase susceptibility to SARS-CoV-2 infection. We report that knockdown, forced overexpression, and pharmacologic inhibition in human colon cancer cells targeted ACE2-BRD4 crosstalk to mediate marked changes in DNA damage/repair and apoptosis. In colorectal cancer patients for whom high ACE2 plus high BRD4 expression is predictive of poor survival, pan-BET inhibition would need to consider proviral/antiviral actions of different BET proteins during SARS-CoV-2 infection.
RESUMO
Epigenetic mechanisms play an important role in the etiology of colorectal cancer (CRC) and other malignancies due, in part, to deregulated bromodomain (BRD) functions. Inhibitors of the bromodomain and extraterminal (BET) family have entered into clinical trials as anticancer agents, and interest has grown in other acetyl 'reader' proteins as therapeutic targets, including non-BET member bromodomain-containing protein 9 (BRD9). We report here that overexpression of BRD9 is associated with poor prognosis in CRC patients, and that siRNA-mediated knockdown of BRD9 decreased cell viability and activated apoptosis in human colon cancer cells, coincident with increased DNA damage. Seeking natural compounds as BRD9 antagonists, molecular docking in silico identified several polyphenols such as Epigallocatechin-3-gallate (EGCG), Equol, Quercetin, and Aspalathin, with favorable binding energies, supported by BROMOscan® (DiscoverX) and isothermal titration calorimetry experiments. Polyphenols mimicked BRD9 knockdown and iBRD9 treatment in reducing colon cancer cell viability, inhibiting colony formation, and enhancing DNA damage and apoptosis. Normal colonic epithelial cells were unaffected, signifying cancer-specific effects. These findings suggest that natural polyphenols recognize and target BRD9 for inhibition, and might serve as useful lead compounds for bromodomain therapeutics in the clinical setting.
Assuntos
Antineoplásicos , Neoplasias do Colo , Humanos , Polifenóis/farmacologia , Simulação de Acoplamento Molecular , RNA Interferente Pequeno , Equol , Quercetina , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Apoptose , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Dano ao DNARESUMO
There is growing interest in the crosstalk between the gut microbiome, host metabolomic features, and disease pathogenesis. The current investigation compared long-term (26 week) and acute (3 day) dietary spinach intake in a genetic model of colorectal cancer. Metabolomic analyses in the polyposis in rat colon (Pirc) model and in wild-type animals corroborated key contributions to anticancer outcomes by spinach-derived linoleate bioactives and a butanoate metabolite linked to increased α-diversity of the gut microbiome. Combining linoleate and butanoate metabolites in human colon cancer cells revealed enhanced apoptosis and reduced cell viability, paralleling the apoptosis induction in colon tumors from rats given long-term spinach treatment. Mechanistic studies in cell-based assays and in vivo implicated the linoleate and butanoate metabolites in targeting histone deacetylase (HDAC) activity and the interferon-γ (IFN-γ) signaling axis. Clinical translation of these findings to at-risk patients might provide valuable quality-of-life benefits by delaying surgical interventions and drug therapies with adverse side effects.
Assuntos
Ácido Butírico , Neoplasias do Colo , Dieta , Ácido Linoleico , Spinacia oleracea , Animais , Neoplasias do Colo/patologia , Humanos , Interferon gama/uso terapêutico , Metabolômica , RatosRESUMO
NADPH oxidase/dual-oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa-B (NFκB)-mediated inflammation and inflammation-associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In the PhIP-induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB-p50 and NFκB-p65 were all highly overexpressed compared with their levels in adjacent normal-looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco-2 cells there was a strong apoptotic response, with increased levels of cleaved caspase-3, -6, -7 and poly(ADP-ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide-induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1ß. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP-induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.
Assuntos
Neoplasias do Colo/enzimologia , NADPH Oxidases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Western Blotting , Carcinógenos/toxicidade , Estudos de Casos e Controles , Ciclo Celular , Proliferação de Células , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Feminino , Humanos , Imidazóis/toxicidade , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
Complex interrelationships govern the dynamic interactions between gut microbes, the host, and exogenous drivers of disease outcome. A multi-omics approach to cancer prevention by spinach (SPI) was pursued for the first time in the polyposis in rat colon (Pirc) model. SPI fed for 26 weeks (10% w/w, freeze-dried in the diet) exhibited significant antitumor efficacy and, in the Apc-mutant genetic background, ß-catenin remained highly overexpressed in adenomatous polyps. However, in both wild type and Apc-mutant rats, increased gut microbiome diversity after SPI consumption coincided with reversal of taxonomic composition. Metagenomic prediction implicated linoleate and butanoate metabolism, tricarboxylic acid cycle, and pathways in cancer, which was supported by transcriptomic and metabolomic analyses. Thus, tumor suppression by SPI involved marked reshaping of the gut microbiome along with changes in host RNA-miRNA networks. When colon polyps were compared with matched normal-looking tissues via metabolomics, anticancer outcomes were linked to SPI-derived linoleate bioactives with known anti-inflammatory/ proapoptotic mechanisms, as well as N-aceto-2-hydroxybutanoate, consistent with altered butanoate metabolism stemming from increased α-diversity of the gut microbiome. In colon tumors from SPI-fed rats, L-glutamate and N-acetylneuraminate also were reduced, implicating altered mitochondrial energetics and cell surface glycans involved in oncogenic signaling networks and immune evasion. In conclusion, a multi-omics approach to cancer prevention by SPI provided mechanistic support for linoleate and butanoate metabolism, as well as tumor-associated changes in L-glutamate and N-acetylneuraminate. Additional factors, such as the fiber content, also warrant further investigation with a view to delaying colectomy and drug intervention in at-risk patients.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Pólipos Adenomatosos/metabolismo , Neoplasias do Colo/dietoterapia , Microbioma Gastrointestinal/fisiologia , Spinacia oleracea , Animais , Ácido Butírico/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Neoplasias do Colo/patologia , Dieta , Ácido Glutâmico/metabolismo , Ácido Linoleico/metabolismo , Masculino , Mitocôndrias/metabolismo , Ácidos Neuramínicos/metabolismo , Ratos , Ratos Endogâmicos F344 , VerdurasRESUMO
There is growing evidence that DNA repair factors have clinical value for cancer treatment. Nucleotide excision repair (NER) proteins, including excision repair cross-complementation group 2 (ERCC2), play a critical role in maintaining genome integrity. Here, we examined ERCC2 expression following epigenetic combination drug treatment. Attention was drawn to ERCC2 for three reasons. First, from online databases, colorectal cancer (CRC) patients exhibited significantly reduced survival when ERCC2 was overexpressed in colon tumors. Second, ERCC2 was the most highly downregulated RNA transcript in human colon cancer cells, plus Ercc2 in rat tumors, after treatment with the histone deacetylase 3 (HDAC3) inhibitor sulforaphane (SFN) plus JQ1, which is an inhibitor of the bromodomain and extraterminal domain (BET) family. Third, as reported here, RNA-sequencing of polyposis in rat colon (Pirc) polyps following treatment of rats with JQ1 plus 6-methylsulfinylhexyl isothiocyanate (6-SFN) identified Ercc2 as the most highly downregulated gene. The current work also defined promising second-generation epigenetic drug combinations with enhanced synergy and efficacy, especially in metastasis-lineage colon cancer cells cultured as 3D spheroids and xenografts. This investigation adds to the growing interest in combination approaches that target epigenetic 'readers', 'writers', and 'erasers' that are deregulated in cancer and other pathologies, providing new avenues for precision oncology and cancer interception.
RESUMO
A clinical trial in patients with familial adenomatous polyposis (FAP) demonstrated that sulindac plus erlotinib (SUL+ERL) had good efficacy in the duodenum and colon, but toxicity issues raised concerns for long-term prevention. We performed a biomarker study in the polyposis in rat colon (Pirc) model, observing phosphorylated Erk inhibition in colon polyps for up to 10 days after discontinuing ERL+SUL administration. In a follow-up study lasting 16 weeks, significant reduction of colon and small intestine (SI) tumor burden was detected, especially in rats given 250 ppm SUL in the diet plus once-a-week intragastric dosing of ERL at 21 or 42 mg/kg body weight (BW). A long-term study further demonstrated antitumor efficacy in the colon and SI at 52 weeks, when 250 ppm SUL was combined with once-a-week intragastric administration of ERL at 10, 21, or 42 mg/kg BW. Tumor-associated matrix metalloproteinase-7 (Mmp7), tumor necrosis factor (Tnf), and early growth response 1 (Egr1) were decreased at 16 weeks by ERL+SUL, and this was sustained in the long-term study for Mmp7 and Tnf. Based on the collective results, the optimal dose combination of ERL 10 mg/kg BW plus 250 ppm SUL lacked toxicity, inhibited molecular biomarkers, and exhibited effective antitumor activity. We conclude that switching from continuous to once-per-week ERL, given at one-quarter of the current therapeutic dose, will exert good efficacy with standard-of-care SUL against adenomatous polyps in the colon and SI, with clinical relevance for patients with FAP before or after colectomy. PREVENTION RELEVANCE: This investigation concludes that switching from continuous to once-per-week erlotinib, given at one-quarter of the current therapeutic dose, will exert good efficacy with standard-of-care sulindac against adenomatous polyps in the colon and small intestine, with clinical relevance for patients with FAP before or after colectomy.
Assuntos
Polipose Adenomatosa do Colo/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/prevenção & controle , Pólipos do Colo/prevenção & controle , Genes APC , Neoplasias Intestinais/prevenção & controle , Mutação , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/normas , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Pólipos do Colo/genética , Pólipos do Colo/metabolismo , Pólipos do Colo/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Cloridrato de Erlotinib/administração & dosagem , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Masculino , Ratos , Sulindaco/administração & dosagemRESUMO
Methylselenocysteine (MSC) and selenomethionine (SM) are two organoselenium compounds receiving interest for their potential anticancer properties. These compounds can be converted to beta-methylselenopyruvate (MSP) and alpha-keto-gamma-methylselenobutyrate (KMSB), alpha-keto acid metabolites that share structural features with the histone deacetylase (HDAC) inhibitor butyrate. We tested the organoselenium compounds in an in vitro assay with human HDAC1 and HDAC8; whereas SM and MSC had little or no activity up to 2 mM, MSP and KMSB caused dose-dependent inhibition of HDAC activity. Subsequent experiments identified MSP as a competitive inhibitor of HDAC8, and computational modeling supported a mechanism involving reversible interaction with the active site zinc atom. In human colon cancer cells, acetylated histone H3 levels were increased during the period 0.5-48 h after treatment with MSP and KMSB, and there was dose-dependent inhibition of HDAC activity. The proportion of cells occupying G(2)/M of the cell cycle was increased at 10-50 microM MSP and KMSB, and apoptosis was induced, as evidenced by morphological changes, Annexin V staining and increased cleaved caspase-3, -6, -7, -9 and poly(adenosine diphosphate-ribose)polymerase. P21WAF1, a well-established target gene of clinically used HDAC inhibitors, was increased in MSP- and KMSB-treated colon cancer cells at both the messenger RNA and protein level, and there was enhanced P21WAF1 promoter activity. These studies confirm that in addition to targeting redox-sensitive signaling molecules, alpha-keto acid metabolites of organoselenium compounds alter HDAC activity and histone acetylation status in colon cancer cells, as recently observed in human prostate cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Colo/enzimologia , Inibidores de Histona Desacetilases , Cetoácidos/farmacologia , Compostos Organosselênicos/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Acetilação/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Neoplasias do Colo/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Luciferases/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll that exhibits cancer chemopreventive properties, but which also has been studied for its possible cancer therapeutic effects. We report here that human colon cancer cells treated with CHL accumulate in S-phase of the cell cycle, and this is associated with reduced expression levels of p53, p21, and other G(1)/S checkpoint controls. At the same time, E2F1 and E2F4 transcription factors become elevated and exhibit increased DNA binding activity. In CHL-treated colon cancer cells, bromodeoxyuridine pulse-chase experiments provided evidence for the inhibition of DNA synthesis. Ribonucleotide reductase (RR), a pivotal enzyme for DNA synthesis and repair, was reduced at the mRNA and protein level after CHL treatment, and the enzymatic activity was inhibited in a concentration-dependent manner both in vitro and in vivo. Immunoblotting revealed that expression levels of RR subunits R1, R2, and p53R2 were reduced by CHL treatment in HCT116 (p53(+/+)) and HCT116 (p53(-/-)) cells, supporting a p53-independent mechanism. Prior studies have shown that reduced levels of RR small subunits can increase the sensitivity of colon cancer cells to clinically used DNA-damaging agents and RR inhibitors. We conclude that by inhibiting R1, R2, and p53R2, CHL has the potential to be effective in the clinical setting, when used alone or in combination with currently available cancer therapeutic agents.
Assuntos
Anticarcinógenos/farmacologia , Clorofilídeos/farmacologia , Neoplasias do Colo/patologia , Fator de Transcrição E2F4/fisiologia , Ribonucleotídeo Redutases/fisiologia , Fase S/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , DNA/metabolismo , Fator de Transcrição E2F1/análise , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/análise , Humanos , Ribonucleotídeo Redutases/antagonistas & inibidores , Proteína Supressora de Tumor p53/fisiologiaRESUMO
An accurate and reliable UPLC-MS/MS method is reported for the quantification of endogenous Prostaglandin E2 (PGE2) in rat colonic mucosa and polyps. This method adopted the "surrogate analyte plus authentic bio-matrix" approach, using two different stable isotopic labeled analogs - PGE2-d9 as the surrogate analyte and PGE2-d4 as the internal standard. A quantitative standard curve was constructed with the surrogate analyte in colonic mucosa homogenate, and the method was successfully validated with the authentic bio-matrix. Concentrations of endogenous PGE2 in both normal and inflammatory tissue homogenates were back-calculated based on the regression equation. Because of no endogenous interference on the surrogate analyte determination, the specificity was particularly good. By using authentic bio-matrix for validation, the matrix effect and exaction recovery are identically same for the quantitative standard curve and actual samples - this notably increased the assay accuracy. The method is easy, fast, robust and reliable for colon PGE2 determination. This "surrogate analyte" approach was applied to measure the Pirc (an Apc-mutant rat kindred that models human FAP) mucosa and polyps PGE2, one of the strong biomarkers of colorectal cancer. A similar concept could be applied to endogenous biomarkers in other tissues.
Assuntos
Colo/metabolismo , Dinoprostona/metabolismo , Animais , Bioensaio/métodos , Biomarcadores/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Neoplasias Colorretais/metabolismo , Inflamação/metabolismo , Marcação por Isótopo/métodos , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodosRESUMO
SCOPE: DNA repair inhibitors have broad clinical applications in tumor types with DNA repair defects, including colorectal cancer (CRC). Structural analogs of the anticancer agent sulforaphane (SFN) were investigated as modifiers of histone deacetylase (HDAC) and histone acetyltransferase (HAT) activity, and for effects on DNA damage/repair pertinent to human CRC. METHODS AND RESULTS: In the polyposis in rat colon (Pirc) model, single oral administration of SFN and structurally related long-chain isothiocyanates (ITCs) decreased histone deacetylase 3 (HDAC3) expression and increased pH2AX levels markedly in adenomatous colon polyps, extending prior observations on HDAC3 inhibition/turnover in cell-based assays. Colon cancer cells at a high initial plating density had diminished cytotoxicity from SFN, whereas novel tetrazole-containing heterocyclic analogs of SFN retained their efficacy. The potent SFN analogs triggered DNA damage, cell cycle arrest, apoptosis, and loss of a key DNA repair regulator, C-terminal binding protein (CtBP) interacting protein (CtIP). These SFN analogs also altered HAT/HDAC activities and histone acetylation status, lowered the expression of HDAC3, P300/CBP-associated factor (PCAF) and lysine acetyltransferase 2A (KAT2A/GCN5), and attenuated homologous recombination (HR)/non-homologous end joining (NHEJ) repair activities in colon cancer cells. CONCLUSION: Novel tetrazole-containing heterocyclic analogs of SFN provide a new avenue for chemosensitization in colon cancer cells via modulation of HAT/HDAC activities and associated DNA damage/repair signaling pathways.
Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Isotiocianatos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Brassica/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Feminino , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Masculino , Mostardeira/química , Ratos , Ratos Endogâmicos F344 , Sulfóxidos , Tetrazóis/farmacologia , Verduras/química , Wasabia/química , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. We recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). In the present investigation, we sought to test whether SFN also might inhibit HDAC activity in vivo. When consumed in the diet at an average daily dose of 7.5 mumol per animal for 21 days, SFN suppressed the growth of human PC-3 prostate cancer cells by 40% in male nude mice. There was a significant decrease in HDAC activity in the xenografts, as well as in the prostates and mononuclear blood cells (MBC), of mice treated with SFN, compared to controls. There also was a trend towards increased global histone acetylation in the xenografts, prostates, and MBC. In human subjects, a single dose of 68 g BroccoSprouts inhibited HDAC activity significantly in peripheral blood mononuclear cells (PBMC) 3 and 6 hrs following consumption. These findings provide evidence that one mechanism through which SFN acts as a cancer chemopreventive agent in vivo is through the inhibition of HDAC activity. Moreover, the data suggest that HDAC activity in PBMC may be used as a biomarker for assessing exposure to novel dietary HDAC inhibitors in human subjects.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Tiocianatos/farmacologia , Animais , Western Blotting , Humanos , Isotiocianatos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Sulfóxidos , Transplante HeterólogoRESUMO
Eomesodermin (Eomes) is a T-box transcription factor that has been implicated in the etiology of colorectal cancer and other human malignancies. We screened a panel of human primary colon cancers and patient-matched controls (n = 30) and detected Eomes overexpression at the mRNA and protein level. Similar results were obtained in a panel of rat colon tumors and adjacent normal-looking colonic mucosa (n = 24). In human colon cancer cells, forced overexpression of Eomes enhanced cell viability and protected against staurosporine-induced apoptosis. On the other hand, knocking down Eomes resulted in reduced cell viability, G2/M cell cycle arrest, and apoptosis induction. The apoptotic mechanism centered on the reciprocal downregulation of anti-apoptotic BIRC5 (Survivin) and upregulation of proapoptotic Bcl-2 modifying factor (BMF). In patients with colorectal cancer, high EOMES expression (n = 95) was associated with poor overall survival compared with individuals exhibiting low EOMES levels (n = 80). We conclude from the current investigation, and prior literature, that Eomes has a divergent role in cancer development, with evidence for tumor suppressor and oncogenic functions, depending on stage and tissue context. Further studies are warranted on the apoptotic mechanisms linked to the reciprocal regulation of BMF and BIRC5 in human colorectal cancers characterized by Eomes overexpression.