RESUMO
Checkpoint kinase 1 (Chk1) plays an important role in regulation of the cell cycle, DNA damage response and cell death, and represents an attractive target in anticancer therapy. Small-molecule inhibitors of Chk1 have been intensively investigated either as single agents or in combination with various chemotherapeutic drugs and they can enhance the chemosensitivity of numerous tumor types. Here we newly demonstrate that pharmacological inhibition of Chk1 using potent and selective inhibitor SCH900776, currently profiled in phase II clinical trials, significantly enhances cytotoxic effects of the combination of platinum-based drugs (cisplatin or LA-12) and TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in human prostate cancer cells. The specific role of Chk1 in the drug combination-induced cytotoxicity was confirmed by siRNA-mediated silencing of this kinase. Using RNAi-based methods we also showed the importance of Bak-dependent mitochondrial apoptotic pathway in the combined anticancer action of SCH900776, cisplatin and TRAIL. The triple drug combination-induced cytotoxicity was partially enhanced by siRNA-mediated Mcl-1 silencing. Our findings suggest that targeting Chk1 may be used as an efficient strategy for sensitization of prostate cancer cells to killing action of platinum-based chemotherapeutic drugs and TRAIL.
Assuntos
Antineoplásicos , Quinase 1 do Ponto de Checagem , Cisplatino , Neoplasias da Próstata , Ligante Indutor de Apoptose Relacionado a TNF , Humanos , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Compostos Organoplatínicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacosRESUMO
PURPOSE: Although beneficial effects of the dietary n-3 docosahexaenoic acid (DHA) or butyrate in colon carcinogenesis have been implicated, the mechanisms of their action are not fully clear. Here, we investigated modulations of composition of individual phospholipid (PL) classes, with a particular emphasis on cardiolipins (CLs), in colon cells treated with DHA, sodium butyrate (NaBt), or their combination (DHA/NaBt), and we evaluated possible associations between lipid changes and cell fate after fatty acid treatment. METHODS: In two distinct human colon cell models, foetal colon (FHC) and adenocarcinoma (HCT-116) cells, we compared patterns and composition of individual PL classes following the fatty acid treatment by HPLC-MS/MS. In parallel, we measured the parameters reflecting cell proliferation, differentiation and death. RESULTS: In FHC cells, NaBt induced primarily differentiation, while co-treatment with DHA shifted their response towards cell death. In contrast, NaBt induced apoptosis in HCT-116 cells, which was not further affected by DHA. DHA was incorporated in all main PL types, increasing their unsaturation, while NaBt did not additionally modulate these effects in either cell model. Nevertheless, we identified an unusually wide range of CL species to be highly increased by NaBt and particularly by DHA/NaBt, and these effects were more pronounced in HCT-116 cells. DHA and DHA/NaBt enhanced levels of high molecular weight and more unsaturated CL species, containing DHA, which was specific for either differentiation or apoptotic responses. CONCLUSIONS: We identified a wide range of CL species in the colon cells which composition was significantly modified after DHA and NaBt treatment. These specific CL modulations might contribute to distinct cellular differentiation or apoptotic responses.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Fosfolipídeos/química , Apoptose/efeitos dos fármacos , Ácido Butírico/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colo/citologia , Células HCT116 , Humanos , Espectrometria de Massas em TandemRESUMO
Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid present in fish oil, may exert cytotoxic and/or cytostatic effects on colon cancer cells when applied individually or in combination with some anticancer drugs. Here we demonstrate a selective ability of subtoxic doses of DHA to enhance antiproliferative and apoptotic effects of clinically useful cytokine TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in cancer but not normal human colon cells. DHA-mediated stimulation of TRAIL-induced apoptosis was associated with extensive engagement of mitochondrial pathway (Bax/Bak activation, drop of mitochondrial membrane potential, cytochrome c release), activation of endoplasmic reticulum stress response (CHOP upregulation, changes in PERK level), decrease of cellular inhibitor of apoptosis protein (XIAP, cIAP1) levels and significant changes in sphingolipid metabolism (intracellular levels of ceramides, hexosyl ceramides, sphingomyelines, sphingosines; HPLC/MS/MS). Interestingly, we found significant differences in representation of various classes of ceramides (especially C16:0, C24:1) between the cancer and normal colon cells treated with DHA and TRAIL, and suggested their potential role in the regulation of the cell response to the drug combination. These study outcomes highlight the potential of DHA for a new combination therapy with TRAIL for selective elimination of colon cancer cells via simultaneous targeting of multiple steps in apoptotic pathways.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Esfingolipídeos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citocromos c/metabolismo , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Transdução de Sinais , Esfingolipídeos/química , Esfingolipídeos/classificação , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Intestinal homeostasis is precisely regulated by a number of endogenous regulatory molecules but significantly influenced by dietary compounds. Malfunction of this system may result in chronic inflammation and cancer. Dietary essential n-3 polyunsaturated fatty acids (PUFAs) and short-chain fatty acid butyrate produced from fibre display anti-inflammatory and anticancer activities. Both compounds were shown to modulate the production and activities of TNF family cytokines. Cytokines from the TNF family (TNF- α, TRAIL, and FasL) have potent inflammatory activities and can also regulate apoptosis, which plays an important role in cancer development. The results of our own research showed enhancement of apoptosis in colon cancer cells by a combination of either docosahexaenoic acid (DHA) or butyrate with TNF family cytokines, especially by promotion of the mitochondrial apoptotic pathway and modulation of NF κ B activity. This review is focused mainly on the interaction of dietary PUFAs and butyrate with these cytokines during colon inflammation and cancer development. We summarised recent knowledge about the cellular and molecular mechanisms involved in such effects and outcomes for intestinal cell behaviour and pathologies. Finally, the possible application for the prevention and therapy of colon inflammation and cancer is also outlined.
Assuntos
Colo/patologia , Ácidos Graxos Insaturados/metabolismo , Inflamação/metabolismo , Neoplasias/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Apoptose , Butiratos/metabolismo , Citocinas/metabolismo , Dieta , Ácidos Docosa-Hexaenoicos/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Mitocôndrias/patologia , NF-kappa B/metabolismoRESUMO
We show that the plant quaternary benzo[c]phenanthridine alkaloid sanguilutine (SL) is a strong inducer of caspase-independent non-apoptotic death in human melanoma cells. Necrostatin-1, a specific inhibitor of necroptosis, completely reversed the cytotoxic effect of SL, suggesting that necroptosis was a predominant type of cell death induced by SL in these cells. In addition, we showed that SL can trigger an autophagic response, as confirmed by GFP-LC3 puncta formation and LC3-II accumulation. Interestingly, we observed a significant decrease in the viability of melanoma cells treated with combination of autophagy inhibitors (3-methyladenine, bafilomycin-A1 and LY294002) and SL. Our results further indicated that autophagy may serve as a pro-survival mechanism, delaying the induction of necroptosis in melanoma cells. The ability of SL to induce caspase-independent non-apoptotic cell death (necroptosis) suggests its possible therapeutic potential in the treatment of apoptosis-resistant melanoma tumours. Furthermore, SL might serve as a useful tool for studying the mechanisms of necroptosis and autophagy induction and the interplay between these two processes.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Benzofenantridinas/farmacologia , Melanoma/patologia , Adenina/análogos & derivados , Adenina/farmacologia , Antineoplásicos/antagonistas & inibidores , Benzofenantridinas/antagonistas & inibidores , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Necrose/induzido quimicamente , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is a major contributor to the worldwide cancer burden. Recent studies on HCC have demonstrated dramatic alterations in expression of several cytochrome P450 (CYP) family members that play a crucial role in biotransformation of many drugs and other xenobiotics; however, the mechanisms responsible for their deregulation remain unclear. METHODS: We investigated a potential involvement of miRNAs in downregulation of expression of CYPs observed in HCC tumors. We compared miRNA expression profiles (TaqMan Array Human MicroRNA v3.0 TLDA qPCR) between HCC human patient tumors with strong (CYP-) and weak/no (CYP+) downregulation of drug-metabolizing CYPs. The role of significantly deregulated miRNAs in modulation of expression of the CYPs and associated xenobiotic receptors was then investigated in human liver HepaRG cells transfected with relevant miRNA mimics or inhibitors. RESULTS: We identified five differentially expressed miRNAs in CYP- versus CYP+ tumors, namely miR-29c, miR-125b1, miR-505, miR-653 and miR-675. The two most-upregulated miRNAs found in CYP- tumor samples, miR-29c and miR-653, were found to act as efficient suppressors of CYP1A2 or AHR expression. CONCLUSIONS: Our results revealed a novel role of miR-653 and miR-29c in regulation of expresion of CYPs involved in crucial biotransformation processes in liver, which are often deregulated during liver cancer progression.
Assuntos
Carcinoma Hepatocelular , Citocromo P-450 CYP1A2/metabolismo , Neoplasias Hepáticas , MicroRNAs/metabolismo , Biotransformação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Xenobióticos/metabolismoRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising drug for the treatment of tumors; however, a number of cancer cells are resistant to this cytokine. Among the mechanisms of resistance of small cell lung carcinomas (SCLCs) to TRAIL is the lack of caspase-8 expression. Although methylation of the caspase-8 promoter has been suggested as the main mechanism of caspase-8 silencing, we showed that reduction of the enzymes involved in DNA methylation, DNA methyltransferases (DNMT) 1, 3a and 3b, was not sufficient to significantly restore caspase-8 expression in SCLC cells, signifying that other mechanisms are involved in caspase-8 silencing. We found that combination of the DNMT inhibitor decitabine with an inhibitor of histone deacetylase (HDAC) significantly increased caspase-8 expression in SCLC cells at the RNA and protein levels. Among all studied HDAC inhibitors, valproic acid (VPA) and CI-994 showed prolonged effects on histone acetylation, while combination with decitabine produced the most prominent effects on caspase-8 re-expression. Moreover, a significant reduction of survivin and cIAP-1 proteins level was observed after treatment with VPA. The combination of two drugs sensitized SCLC cells to TRAIL-induced apoptosis, involving mitochondrial apoptotic pathway and was accompanied by Bid cleavage, activation of Bax, and release of cytochrome c. Both initiator caspase-8 and -9 were required for the sensitization of SCLC cells to TRAIL. Thus, efficient restoration of caspase-8 expression in SCLC cells is achieved when a combination of DNMT and HDAC inhibitors is used, suggesting a combination of decitabine and VPA or CI-994 as a potential treatment for sensitization of SCLC cells lacking caspase-8 to TRAIL.
Assuntos
Caspase 8/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Histona Desacetilases/química , Neoplasias Pulmonares/prevenção & controle , Carcinoma de Pequenas Células do Pulmão/prevenção & controle , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Caspase 8/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Decitabina , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carcinoma de Pequenas Células do Pulmão/enzimologia , Carcinoma de Pequenas Células do Pulmão/patologia , Survivina , Ligante Indutor de Apoptose Relacionado a TNF/genética , Células Tumorais Cultivadas , Ácido Valproico/farmacologia , DNA Metiltransferase 3BRESUMO
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediated apoptosis. Combination therapy with platinum complexes may affect TRAIL-induced signaling via modulation of various steps in apoptotic pathways. Here, we show that cisplatin or a more potent platinum(IV) complex LA-12 used in 20-fold lower concentration enhanced killing effects of TRAIL in human colon and prostate cancer cell lines via stimulation of caspase activity and overall apoptosis. Both platinum complexes increased DR5 surface expression in colon cancer cells. Small interfering RNA-mediated DR5 silencing rescued cells from sensitizing effects of platinum drugs on TRAIL-induced caspase-8 activation and apoptosis, showing the functional importance of DR5 in the effects observed. In addition, both cisplatin and LA-12 triggered the relocalization of DR4 and DR5 receptors to lipid rafts and accelerated internalization of TRAIL, which may also affect TRAIL signaling. Collectively, modulations of the initial steps of the extrinsic apoptotic pathway at the level of DR5 and plasma membrane are important for sensitization of colon and prostate cancer cells to TRAIL-induced apoptosis mediated by LA-12 and cisplatin.
Assuntos
Amantadina/análogos & derivados , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias/metabolismo , Compostos Organoplatínicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Amantadina/farmacologia , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Separação Celular , Citometria de Fluxo , Imunofluorescência , Humanos , Microscopia Confocal , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
The resistance of transformed epithelial cells to a detachment-induced apoptosis (anoikis) can significantly affect their susceptibility to anticancer therapy. We showed that detachment of both fetal (FHC) and adenocarcinoma (HT-29) human colon epithelial cells resulted in the activation of the pro-survival Akt pathway, and significant changes in integrin-linked kinase (ILK) and focal adhesive kinase (FAK) phosphorylation. We demonstrated a detachment-induced and PI3K/Akt-mediated resistance to apoptotic effects of TRAIL, which was not associated with any changes in the cell surface TRAIL death receptor levels. Instead, a modulation of downstream intracellular signaling events was suggested to be involved. Our results may have important implications for optimization of new strategies in treatment of cancers at different stages of development.
Assuntos
Anoikis/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Epiteliais/patologia , Feto/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HT29 , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Preferential uptake of photosensitizer by tumour tissue is an elementary prerequisite of effective and successful photodynamic therapy (PDT). Therefore intracellular concentration of photosensitizer is one of the limiting factors affecting PDT efficiency. Hypericin (HY) has found applications in photodynamic diagnostics solely due to its high specificity for tumour cells and tissues. However, here we suggest that not only HY uptake, but importantly also the cell ability to manage oxidative stress induced by HY-PDT can be important decisive factors finally affecting the cell death response. We showed that despite the higher accumulation of HY in FHC human fetal colon epithelial cells compared to HT-29 colon adenocarcinoma cells, the cytotoxic effects of this photosensitizer were more pronounced in the latter cell line, and this was associated with enhanced accumulation of HY-PDT-induced reactive oxygen species (ROS).
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Perileno/análogos & derivados , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Antracenos , Linhagem Celular , Colo/citologia , Células Epiteliais/efeitos da radiação , Feto , Humanos , Microscopia de Fluorescência , Perileno/uso terapêutico , Perileno/toxicidade , Fármacos Fotossensibilizantes/toxicidade , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cytochrome P450 family 1 (CYP1) enzymes contribute both to metabolism of xenobiotics and to the control of endogenous levels of ligands of the aryl hydrocarbon receptor (AhR). Their activities, similar to other CYPs, can be altered in tumor tissues. Here, we examined a possible role of proliferative/survival pathways signaling, which is often deregulated in tumor cells, and possible links with p300 histone acetyltransferase (a transcriptional co-activator) in the control of CYP1 expression, focusing particularly on CYP1A1. Using cell models derived from human liver, we observed that the induction of CYP1A1 expression, as well as other CYP1 enzymes, was reduced in exponentially growing cells, as compared with their non-dividing counterparts. The siRNA-mediated inhibition of proliferation/pro-survival signaling pathway effectors (such as ß-catenin and/or Hippo pathway effectors YAP/TAZ) increased the AhR ligand-induced CYP1A1 mRNA levels in liver HepaRG cells, and/or in colon carcinoma HCT-116 cells. The activation of proliferative Wnt/ß-catenin signaling in HCT-116 cells reduced both the induction of CYP1 enzymes and the binding of p300 to the promoter of CYP1A1 or CYP1B1 genes. These results seem to indicate that aberrant proliferative signaling in tumor cells could suppress induction of CYP1A1 (or other CYP1 enzymes) via competition for p300 binding. This mechanism could be involved in modulation of the metabolism of both endogenous and exogenous substrates of CYP1A1 (and other CYP1 enzymes), with possible further consequences for alterations of the AhR signaling in tumor cells, or additional functional roles of CYP1 enzymes.
Assuntos
Proliferação de Células/fisiologia , Neoplasias do Colo/patologia , Citocromo P-450 CYP1A1/genética , Fígado/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Neoplasias do Colo/genética , Citocromo P-450 CYP1A1/biossíntese , Proteína p300 Associada a E1A/metabolismo , Indução Enzimática/fisiologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Via de Sinalização Hippo/fisiologia , Humanos , Transdução de Sinais/fisiologia , Via de Sinalização Wnt/fisiologiaRESUMO
BACKGROUND: TRAIL is considered as a promising anti-cancer agent, because of its ability to induce apoptosis in cancer but not in most normal cells. However, growing evidence exist that many cancer cells are resistant to its apoptotic effects. SCLC is a typical example of tumor entity where TRAIL monotherapy is not efficient. RESULTS: We demonstrated that doxorubicin and etoposide markedly sensitized SCLC cells expressing caspase-8 to apoptotic effects of TRAIL. The drug-mediated sensitization of these cells was associated with increase of surface and total DR5 protein level, specific cleavage of cFLIPL, decrease of cFLIPS level, and a strong activation of caspase-8. The involvement of mitochondria-mediated pathway was demonstrated by enhanced Bid cleavage, Bax activation, and cytochrome c release. Activation of caspase-8 induced by combined treatment was shown to occur upstream of mitochondria and effector caspases. CONCLUSIONS: Our results highlight significant applicability of doxorubicin and etoposide in sensitization of SCLC cells expressing caspase-8 to treatment with TRAIL.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Western Blotting , Caspase 8/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteína X Associada a bcl-2RESUMO
In addition to its ability to act as a promising inducer of tumor-specific cell death, TRAIL has also been shown to stimulate signaling pathways leading to cancer cell survival. We examined the changes of anti-apoptotic Mcl-1 protein level following TRAIL treatment of human cell lines representing different stages of colon carcinogenesis-adenocarcinoma (HT-29, HCT116) or secondary metastasis (SW620), together with cell line derived from human fetal colon (FHC). While TRAIL was capable of triggering an anti-apoptotic signaling leading to significant early ERK-mediated transcriptional up-regulation of Mcl-1 in selected colon adenocarcinoma cell lines, none or very limited effects were demonstrated in cell lines derived from colon lymph node metastasis or fetal colon, respectively. We demonstrated an immediate impact of Mcl-1 protein level manipulations on the course of early acute apoptotic response of colon adenocarcinoma cells to TRAIL. It is therefore essential to consider the dynamics of modulation of Mcl-1 level and the balance between TRAIL-induced pro- and anti-apoptotic pathways when predicting the response of cells in different stages of cancer development, and designing the anticancer therapy using TRAIL.
Assuntos
Neoplasias do Colo/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias do Colo/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Metástase Neoplásica/patologiaRESUMO
Hepatocellular carcinoma (HCC) remains a highly prevalent and deadly disease, being among the top causes of cancer-related deaths worldwide. Despite the fact that the liver is the major site of biotransformation, studies on drug metabolizing enzymes in HCC are scarce. It is known that malignant transformation of hepatocytes leads to a significant alteration of their metabolic functions and overall deregulation of gene expression. Advanced stages of the disease are thus frequently associated with liver failure, and severe alteration of drug metabolism. However, the impact of dysregulation of metabolic enzymes on therapeutic efficacy and toxicity in HCC patients is largely unknown. Here we demonstrate a significant down-regulation in European Caucasian patients of cytochromes P450 (CYPs), the major xenobiotic-metabolizing enzymes, in HCC tumour samples as compared to their surrounding non-cancerous (reference) tissue. Moreover, we report for the first time the association of the unique CYP profiles with specific transcriptome changes, and interesting correlations with expression levels of nuclear receptors and with the histological grade of the tumours. Integrated analysis has suggested certain co-expression profiles of CYPs with lncRNAs that need to be further characterized. Patients with large tumours with down-regulated CYPs could be more vulnerable to drug toxicity; on the other hand, such tumours would eliminate drugs more slowly and should be more sensitive to pharmacotherapy (except in the case of pro-drugs where activation is necessary).
Assuntos
Carcinoma Hepatocelular/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Neoplasias Hepáticas/enzimologia , Transcriptoma , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Inativação Metabólica/genética , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Identification of changes of phospholipid (PL) composition occurring during colorectal cancer (CRC) development may help us to better understand their roles in CRC cells. Here, we used LC-MS/MS-based PL profiling of cell lines derived from normal colon mucosa, or isolated at distinct stages of CRC development, in order to study alterations of PL species potentially linked with cell transformation. We found that a detailed evaluation of phosphatidylinositol (PI) and phosphatidylserine (PS) classes allowed us to cluster the studied epithelial cell lines according to their origin: i) cells originally derived from normal colon tissue (NCM460, FHC); ii) cell lines derived from colon adenoma or less advanced differentiating adenocarcinoma cells (AA/C1, HT-29); or, iii) cells obtained by in vitro transformation of adenoma cells and advanced colon adenocarcinoma cells (HCT-116, AA/C1/SB10, SW480, SW620). Although we tentatively identified several PS and PI species contributing to cell line clustering, full PI and PS profiles appeared to be a key to the successful cell line discrimination. In parallel, we compared PL composition of primary epithelial (EpCAM-positive) cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients, with PL profiles of cell lines derived from normal colon mucosa (NCM460) and from colon adenocarcinoma (HCT-116, SW480) cells, respectively. In general, higher total levels of all PL classes were observed in tumor cells. The overall PL profiles of the cell lines, when compared with the respective patient-derived cells, exhibited similarities. Nevertheless, there were also some notable differences in levels of individual PL species. This indicated that epithelial cell lines, derived either from normal colon tissue or from CRC cells, could be employed as models for functional lipidomic analyses of colon cells, albeit with some caution. The biological significance of the observed PL deregulation, or their potential links with specific CRC stages, deserve further investigation.
Assuntos
Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células Epiteliais/metabolismo , Lipidômica , Fosfolipídeos/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/patologia , Humanos , Análise de Componente PrincipalRESUMO
We compared the response of normal (FHC) and cancer (HT-29) human colon epithelial cells to the important apoptotic inducers TNF-alpha, anti-Fas antibody and TNF-related apoptosis inducing ligand (TRAIL). The two cell lines did not respond to TNF-alpha (15 ng/ml), expressed a limited sensitivity to anti-Fas antibody (200 ng/ml) and a different response to TRAIL (100 ng/ml). We studied apoptosis with regard to the changes at the receptor level (DR, DcR and FLIP) and at the level of mitochondria (Bid protein cleavage, Apo2.7 protein expression and caspase-9 activation). Two different approaches were used to sensitize the cells to TRAIL-induced apoptosis: inhibition of protein synthesis (cycloheximide, CHX) and inhibition of the pro-survival MEK/ERK pathway (U0126). While the two cell lines were markedly sensitized to all three TNF family members by CHX, a different degree of response (especially for TRAIL) was obtained when inhibition of the MEK/ERK pathway was achieved. TRAIL-induced apoptosis was significantly enhanced by U0126 co-treatment in the HT-29 cells, but not in the FHC cells. The most significant differences between the HT-29 and FHC cells co-treated with TRAIL and U0126 were demonstrated with regard to the involvement of the mitochondrial apoptotic pathway, suggesting its importance in the regulation of cell sensitivity to the TRAIL-induced apoptosis.
Assuntos
Anticorpos/farmacologia , Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Células Epiteliais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Apoptose , Carcinoma/patologia , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Células Epiteliais/metabolismo , Humanos , Proteínas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismoRESUMO
The short-chain and n-3 polyunsaturated fatty acids exhibit anticancer properties, and they may mutually interact within the colon. However, the molecular mechanisms of their action in colon cancer cells are still not fully understood. Our study focused on the mechanisms responsible for the diverse effects of sodium butyrate (NaBt), in particular when interacting with docosahexaenoic acid (DHA), in distinct colon cancer cell types, in which NaBt either induces cell differentiation or activates programmed cell death involving mitochondrial pathway. NaBt activated autophagy both in HT-29 cells, which are sensitive to induction of differentiation, and in nondifferentiating HCT-116 cells. However, autophagy supported cell survival only in HT-29 cells. Combination of NaBt with DHA-promoted cell death, especially in HCT-116 cells and after longer time intervals. The inhibition of autophagy both attenuated differentiation and enhanced apoptosis in HT-29 cells treated with NaBt and DHA, but it had no effect in HCT-116 cells. NaBt, especially in combination with DHA, activated PPARγ in both cell types. PPARγ silencing decreased differentiation and increased apoptosis only in HT-29 cells, therefore we verified the role of caspases in apoptosis, differentiation and also PPARγ activity using a pan-caspase inhibitor. In summary, our data suggest that diverse responses of colon cancer cells to fatty acids may rely on their sensitivity to differentiation, which may in turn depend on distinct engagement of autophagy, caspases and PPARγ. These results contribute to understanding of mechanisms underlying differential effects of NaBt, when interacting with other dietary fatty acids, in colon cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Butiratos/farmacologia , Neoplasias do Colo/patologia , Ácidos Docosa-Hexaenoicos/farmacologia , PPAR gama/metabolismo , Antineoplásicos/farmacologia , Ácido Butírico/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células HCT116 , Células HT29 , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , PPAR gama/genéticaRESUMO
Although Chk1 kinase inhibitors are currently under clinical investigation as effective cancer cell sensitizers to the cytotoxic effects of numerous chemotherapeutics, there is still a considerable uncertainty regarding their role in modulation of anticancer potential of platinum-based drugs. Here we newly demonstrate the ability of one of the most specific Chk1 inhibitors, SCH900776 (MK-8776), to enhance human colon cancer cell sensitivity to the cytotoxic effects of platinum(II) cisplatin and platinum(IV)- LA-12 complexes. The combined treatment with SCH900776 and cisplatin or LA-12 results in apparent increase in G1/S phase-related apoptosis, stimulation of mitotic slippage, and senescence of HCT116 cells. We further show that the cancer cell response to the drug combinations is significantly affected by the p21, p53, and PTEN status. In contrast to their wt counterparts, the p53- or p21-deficient cells treated with SCH900776 and cisplatin or LA-12 enter mitosis and become polyploid, and the senescence phenotype is strongly suppressed. While the cell death induced by SCH900776 and cisplatin or LA-12 is significantly delayed in the absence of p53, the anticancer action of the drug combinations is significantly accelerated in p21-deficient cells, which is associated with stimulation of apoptosis beyond G2/M cell cycle phase. We also show that cooperative killing action of the drug combinations in HCT116 cells is facilitated in the absence of PTEN. Our results indicate that SCH900776 may act as an important modulator of cytotoxic response triggered by platinum-based drugs in colon cancer cells.
Assuntos
Antineoplásicos/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/metabolismo , Neoplasias do Colo/metabolismo , Compostos de Platina/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/genética , Cisplatino/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Searching for new strategies for effective elimination of human prostate cancer cells, we investigated the cooperative cytotoxic action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and two platinum-based complexes, cisplatin or LA-12, and related molecular mechanisms. We demonstrated a notable ability of cisplatin or LA-12 to enhance the sensitivity of several human prostate cancer cell lines to TRAIL-induced cell death via an engagement of mitochondrial apoptotic pathway. This was accompanied by augmented Bid cleavage, Bak activation, loss of mitochondrial membrane potential, activation of caspase-8, -10, -9, and -3, and XIAP cleavage. RNAi-mediated silencing of Bid or Bak in Bax-deficient DU 145 cells suppressed the drug combination-induced cytotoxicity, further underscoring the involvement of mitochondrial signaling. The caspase-10 was dispensable for enhancement of cisplatin/LA-12 and TRAIL combination-induced cell death and stimulation of Bid cleavage. Importantly, we newly demonstrated LA-12-mediated enhancement of TRAIL-induced cell death in cancer cells derived from human patient prostate tumor specimens. Our results provide convincing evidence that employing TRAIL combined with cisplatin/LA-12 could contribute to more effective killing of prostate cancer cells compared to the individual action of the drugs, and offer new mechanistic insights into their cooperative anticancer action.
Assuntos
Amantadina/análogos & derivados , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 10/metabolismo , Cisplatino/farmacologia , Mitocôndrias/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Neoplasias da Próstata/patologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Amantadina/farmacologia , Humanos , Masculino , Mitocôndrias/metabolismo , Neoplasias da Próstata/metabolismoRESUMO
Epithelial cells can be manipulated to undergo apoptosis depending on the balance between pro-survival and apoptotic signals. We showed that TRAIL-induced apoptosis may be differentially regulated by inhibitors of MEK ERK (U0126) or PI3K/Akt (LY294002) pathway in TRAIL-sensitive (HT-29) and TRAIL-resistant (SW620) human epithelial colon cancer cells. U0126 or LY294002 significantly enhanced TRAIL-induced apoptosis in HT-29 cells, but not in SW620 cells. We report a different regulation of the level of an anti-apoptotic Mcl-1 protein under MEK/ERK or PI3K/Akt pathway inhibition and suggest the mechanisms involved. A special attention was paid to the role of the ERK1/2, Akt, and glycogen synthase kinase 3beta.