RESUMO
The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore, may play a relevant role in the hypoxic breast cancer stem cell niche.
Assuntos
Antígenos de Neoplasias/biossíntese , Neoplasias da Mama/genética , Anidrases Carbônicas/biossíntese , Hipóxia Celular/genética , MicroRNAs/genética , Antígenos de Neoplasias/genética , Neoplasias da Mama/patologia , Proteínas de Ligação ao Cálcio/biossíntese , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteína Jagged-1 , Células MCF-7 , Proteínas de Membrana/biossíntese , MicroRNAs/biossíntese , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptor Notch3 , Receptores Notch/biossíntese , Proteínas Serrate-Jagged , Fatores de Transcrição da Família Snail , Fatores de Transcrição/biossínteseRESUMO
AIM: To investigate mechanisms by which doxorubicin (DOX) and cisplatin (CIS) cause human ovarian stroma injury. PATIENTS & METHODS: Stromal cells from human cryopreserved ovarian tissue were cultured in the presence of 1 µM DOX and 10 µM CIS. Ovarian damage induced by treatments was evaluated by 'Live/Dead' and sulforhodamine-B assays, the expression of different apoptosis markers. RESULTS: Stromal cell growth was inhibited by DOX and CIS, and this effect was accompanied by apoptosis through mitochondrial pathway activation: Bax, cleaved-caspase 9, cleaved-PARP1 induction and Akt1, Bcl2, phospho-44/42-MAPK/ERK1/2 reduction were observed. CONCLUSION: DOX and CIS induced apoptosis in human ovarian stromal cells. Knowledge of mechanisms by which the drugs act is important to identify possible ways to counteract side effects of chemotherapy on ovaries.
Assuntos
Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Cisplatino/efeitos adversos , Doxorrubicina/efeitos adversos , Ovário/efeitos dos fármacos , Adulto , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Criopreservação , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/efeitos dos fármacosRESUMO
Cancer stem cells (CSCs) are affected by the local micro-environment, the niche, in which inflammatory stimuli and hypoxia act as steering factors. Here, two nuclear receptors (NRs) agonists, i.e. pioglitazone (PGZ), a ligand of peroxisome proliferator activated receptor-γ, and 6-OH-11-O-hydroxyphenanthrene (IIF), a ligand of retinoid X receptors, were investigated for their capability to interference with the cross-talk between breast CSCs and the niche compartment. We found that IIF potentiates the ability of PGZ to hamper the mammospheres-forming capability of human breast tumours and MCF7 cancer cells, reducing the expression of CSCs regulatory genes (Notch3, Jagged1, SLUG, Interleukin-6, Apolipoprotein E, Hypoxia inducible factor-1α and Carbonic anhydrase IX). Notably, these effects are not observed in normal-MS obtained from human breast tissue. Importantly, NRs agonists abolish the capability of hypoxic MCF7 derived exosomes to induce a pro-inflammatory phenotype in mammary glands fibroblasts. Moreover, NRs agonist also directly acts on breast tumour associated fibroblasts to downregulate nuclear factor-κB pathway and metalloproteinases (MMP2 and MMP9) expression and activity. In conclusion, NRs agonists disrupt the inflammatory cross-talk of the hypoxic breast CSCs niche.
Assuntos
Neoplasias da Mama/patologia , Inflamação/patologia , Células-Tronco Neoplásicas/patologia , PPAR gama/metabolismo , Receptor Cross-Talk , Receptores X de Retinoides/metabolismo , Nicho de Células-Tronco , Antígenos de Neoplasias/metabolismo , Apolipoproteínas E/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Anidrase Carbônica IX , Anidrases Carbônicas/metabolismo , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Ligantes , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Fenantrenos/farmacologia , Pioglitazona , Receptor Cross-Talk/efeitos dos fármacos , Receptores X de Retinoides/agonistas , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Nicho de Células-Tronco/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Tiazolidinedionas/farmacologiaRESUMO
Cancer stem cell survival relies on the activation of inflammatory pathways, which is speculatively triggered by cell autonomous mechanisms or by microenvironmental stimuli. Here, we observed that hypoxic bone marrow stroma-derived transforming growth factor-ß 1 promotes the growth of human breast cancer stem cells as mammospheres. The ensuing Slug-dependent serine 139 phosphorylation of the DNA damage sensor H2AX in breast cancer stem cells induces tumor necrosis factor-α and IL-8 mRNAs, whose stability is enhanced by cytoplasmic ß-catenin. ß-Catenin also up-regulates and binds miR-221, reducing the stability of the miR-221 targets Rad51 and ERα mRNAs. Our data show that the Slug/ß-catenin-dependent activation of DNA damage signaling triggered by the hypoxic microenvironment sustains the proinflammatory phenotype of breast cancer stem cells.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Inflamação/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Comunicação Autócrina/genética , Neoplasias da Mama/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Citocinas/genética , Citocinas/metabolismo , Dano ao DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Inflamação/genética , Células MCF-7 , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Modelos Biológicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fator de Crescimento Transformador beta1/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Colitis is the term used for chronic inflammatory bowel diseases at substantially increased risk of developing a form of colorectal cancer (CRC) known as colitis-associated cancer. In our study we synthesized core-shell polymeric micelles obtained by self-assembly of block copolymers for high efficiency delivery of anti-inflammatory and anti-cancer compounds to colonocytes and colon mucosa. We achieved an efficient intracellular delivery of these hydrophobic compounds (prednisone, retinoic acid and doxorubicin) to cultured colonocytes without cellular toxicity. The efficacy of retinoic acid and doxorubicin administration was significantly increased using these nanosized carriers. Moreover, these polymeric micelles have been shown to overcome the multidrug resistance efflux mechanism effectively delivering doxorubicin to multidrug-resistant colon cancer cells. These nanocarriers are also suitable for selective in vivo delivery of lipophilic drugs by enema administration to the inflamed colon tissue, specifically targeting the inflamed mucosa. FROM THE CLINICAL EDITOR: This team of investigators studied polymeric micelles as highly efficient drug delivery systems enabling intracellular delivery of hydrophobic compounds (prednisone, retinoic acid, and doxorubicin) to cultured colonocytes without cellular toxicity, also demonstrating beneficial in vivo effects.
Assuntos
Colite/tratamento farmacológico , Colo/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Polímeros/administração & dosagem , Animais , Colite/complicações , Colite/patologia , Colo/citologia , Neoplasias do Colo/complicações , Neoplasias do Colo/patologia , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Humanos , Camundongos , Micelas , Nanopartículas/administração & dosagem , Nanopartículas/química , Polímeros/química , Prednisona/administração & dosagem , Tretinoína/administração & dosagemRESUMO
Glioblastoma represent the most common primary brain tumor in adults and are currently considered incurable. We investigated antiproliferative and anti-invasive mechanisms of 6-OH-11-O-hydroxyfenantrene (IIF), a retinoid X receptor ligand, and pioglitazone (PGZ), a peroxisome proliferator-activated receptor gamma activator, in three different glioblastoma cell lines. A dose-dependent reduction of tumor invasion and strong decrease of matrix metalloproteinases 2 and 9 expression was observed, especially when a combination therapy of IIF and PGZ was administered. Combined treatment also markedly reduced proliferation and induced apoptosis in all glioma cell lines tested. This was in particular accompanied by decrease of antiapoptotic proteins Bcl2 and p53, while simultaneously pro-apoptotic cytochrome c, cleaved caspase 3, Bax and Bad levels increased. These in vitro findings were further substantiated in a murine glioma model in vivo, where oral administration of PGZ and IIF resulted in significantly reduced tumor volume and proliferation. Of note, treatment with nuclear receptor ligands was not only effective when the treatment was initiated shortly after the intraparenchymal seeding of the glioma cells, but even when initiated in the last third of the observation period. Collectively, our results demonstrate the effectiveness of a combined treatment of ligands of proliferator-activated receptor and retinoid X receptor against glioblastoma.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , PPAR gama/uso terapêutico , Tretinoína/análogos & derivados , Análise de Variância , Animais , Anexina A5/metabolismo , Bromodesoxiuridina/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Citocromos c/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Ligantes , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/fisiopatologia , PPAR gama/metabolismo , Pioglitazona , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Receptor X Retinoide gama/metabolismo , Sais de Tetrazólio , Tiazóis , Tiazolidinedionas/uso terapêutico , Transfecção/métodos , Tretinoína/uso terapêutico , Ensaio Tumoral de Célula-Tronco/métodos , Proteína X Associada a bcl-2/metabolismoRESUMO
A large number of basic researches and observational studies suggested the cancer preventive activity of vitamin E, but large-scale human intervention trials have yielded disappointing results and actually showed a higher incidence of prostate cancer although the mechanisms underlying the increased risk remain largely unknown. Here we show through in vitro and in vivo studies that vitamin E produces a marked inductive effect on carcinogen-bioactivating enzymes and a pro-oxidant status promoting both DNA damage and cell transformation frequency. First, we found that vitamin E in the human prostate epithelial RWPE-1 cell line has the remarkable ability to upregulate the expression of various phase-I activating cytochrome P450 (CYP) enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), giving rise to supraphysiological levels of reactive oxygen species. Furthermore, our rat model confirmed that vitamin E in the prostate has a powerful booster effect on CYP enzymes associated with the generation of oxidative stress, thereby favoring lipid-derived electrophile spread that covalently modifies proteins. We show that vitamin E not only causes DNA damage but also promotes cell transformation frequency induced by the PAH-prototype benzo[a]pyrene. Our findings might explain why dietary supplementation with vitamin E increases the prostate cancer risk among healthy men.
Assuntos
Transformação Celular Neoplásica/induzido quimicamente , Sistema Enzimático do Citocromo P-450/metabolismo , Suplementos Nutricionais/toxicidade , Neoplasias Experimentais/induzido quimicamente , Neoplasias da Próstata/induzido quimicamente , Vitamina E/toxicidade , Células 3T3 , Animais , Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Linhagem Celular , Transformação Celular Neoplásica/genética , Dano ao DNA/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Estresse Oxidativo/efeitos dos fármacos , Próstata/citologia , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos , Vitamina E/administração & dosagemRESUMO
Current literature agrees on the notion that efficient DNA repair favors longevity across evolution. The DNA damage response machinery activates inflammation and type I interferon signaling. Both pathways play an acknowledged role in the pathogenesis of a variety of age-related diseases and are expected to be detrimental for human longevity. Here, we report on the anti-inflammatory molecular make-up of centenarian's fibroblasts (low levels of IL-6, type 1 interferon beta, and pro-inflammatory microRNAs), which is coupled with low level of DNA damage (measured by comet assay and histone-2AX activation) and preserved telomere length. In the same cells, high levels of the RNAseH2C enzyme subunit and low amounts of RNAseH2 substrates, i.e. cytoplasmic RNA:DNA hybrids are present. Moreover, RNAseH2C locus is hypo-methylated and RNAseH2C knock-down up-regulates IL-6 and type 1 interferon beta in centenarian's fibroblasts. Interestingly, RNAseH2C locus is hyper-methylated in vitro senescent cells and in tissues from atherosclerotic plaques and breast tumors. Finally, extracellular vesicles from centenarian's cells up-regulate RNAseH2C expression and dampen the pro-inflammatory phenotype of fibroblasts, myeloid, and cancer cells. These data suggest that centenarians are endowed with restrained DNA damage-induced inflammatory response, that may facilitate their escape from the deleterious effects of age-related chronic inflammation.
Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Inflamação/metabolismo , Longevidade/genética , Ribonuclease H/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/química , Neoplasias da Mama/genética , Dano ao DNA/efeitos dos fármacos , Vesículas Extracelulares/imunologia , Feminino , Fibroblastos/enzimologia , Loci Gênicos , Humanos , Inflamação/genética , Interferon beta/metabolismo , Interleucina-6/metabolismo , Longevidade/fisiologia , Masculino , Metilação , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Placa Aterosclerótica/química , Placa Aterosclerótica/genética , Ribonuclease H/genética , Homeostase do Telômero/genética , Adulto JovemRESUMO
There is high current interest in the chemopreventive potential of Brassica vegetables (cruciferae), particularly due to their content in glucosinolates (GL), which upon myrosinase hydrolysis release the corresponding isythiocyanates (ITC). Some ITCs, such as sulforaphane (SFN) from broccoli ( Brassica oleacea italica), have been found to possess anticancer activity through induction of apoptosis in selected cell lines, as well as indirect antioxidant activity through induction of phase II detoxifying enzymes. Japanese daikon ( Raphanus sativus L.) is possibly the vegetable with the highest per capita consumption within the Brassicaceae family. Thanks to a recently improved gram scale production process, it was possible to prepare sufficient amounts of the GL glucoraphasatin (GRH) as well as the corresponding ITC 4-methylthio-3-butenyl isothiocyanate (GRH-ITC) from its sprouts. This paper reports a study on the cytotoxic and apoptotic activities of GRH-ITC compared with the oxidized counterpart 4-methylsulfinyl-3-butenyl isothiocyanate (GRE-ITC) on three human colon carcinoma cell lines (LoVo, HCT-116, and HT-29) together with a detailed kinetic investigation of the direct antioxidant/radical scavenging ability of GRH and GRH-ITC. Both GRH-ITC and GRE-ITC reduced cell proliferation in a dose-dependent manner and induced apoptosis in the three cancer cell lines. The compounds significantly ( p < 0.05) increased Bax and decreased Bcl2 protein expression, as well as producing caspase-9 and PARP-1 cleavage after 3 days of exposure in the three cancer cell lines. GRH-ITC treatment was shown to have no toxicity with regard to normal human lymphocytes (-15 +/- 5%) in comparison with SFN (complete growth inhibition). GRH and GRH-ITC were able to quench the 2,2-diphenyl-1-picrylhydrazyl radical, with second-order rate constants of 14.0 +/- 2.8 and 43.1 +/- 9.5 M(-1) s(-1), respectively (at 298 K in methanol), whereas the corresponding value measured here for the reference antioxidant alpha-tocopherol was 425 +/- 40 M (-1) s (-1). GRH reacted with H2O2 and tert-butyl hydroperoxide in water (pH 7.4) at 37 degrees C, with rate constants of 1.9 +/- 0.3 x 10(-2) and 9.5 +/- 0.3 x 10(-4) M(-1) s (-1) (paralleling recently developed synthetic antioxidants) being quantitatively (>97%) converted to GRE. It is demonstrated that GRH-ITC has interesting antioxidant/radical scavenging properties, associated with a selective cytotoxic/apoptotic activity toward three human colon carcinoma cell lines, and very limited toxicity on normal human T-lymphocytes.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Isotiocianatos/farmacologia , Raphanus/química , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/química , Cinética , Brotos de Planta/químicaRESUMO
We conducted an in vitro study combining a rexinoid, 6-OH-11-O-hydroxyphenanthrene (IIF), and epigallocatechin-3-gallate (EGCG), which is the main catechin of green tea, on BE(2)-C, a neuroblastoma cell line representative of the high-risk group of patients. Neuroblastoma is the most common malignancy of childhood: high-risk patients, having N-MYC over-expression, undergo aggressive therapy and show high mortality or an increased risk of secondary malignancies. Retinoids are used in neuroblastoma therapy with incomplete success: the association of a second molecule might improve the efficacy. BE(2)-C cells were treated by EGCG and IIF, individually or in combination: cell viability, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, was reduced, EGCG+IIF being the most effective treatment. Apoptosis occurred and the EGCG+IIF treatment decreased N-MYC protein expression and molecular markers of invasion (MMP-2, MMP-9 and COX-2). Zymography demonstrated nearly 50% inhibition of MMP activity. When BE(2)-C cells were grown in non-adherent conditions to enrich the tumor-initiating cell population, BE(2)-C-spheres were obtained. After 48 h and 72 h treatment, EGCG+IIF limited BE(2)-C-sphere formation and elicited cell death with a reduction of N-MYC expression. We concluded that the association of EGCG to IIF might be applied without toxic effects to overcome the incomplete success of retinoid treatments in neuroblastoma patients.
Assuntos
Catequina/análogos & derivados , Células-Tronco Neoplásicas/efeitos dos fármacos , Neuroblastoma/patologia , Fenantrenos/farmacologia , Apoptose/efeitos dos fármacos , Catequina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Células-Tronco Neoplásicas/metabolismo , Chá/químicaRESUMO
Epidermal growth factor receptor (EGFR) expression is an important marker in breast carcinoma pathology and is considered a pivotal molecule for cancer cell proliferation, invasion and metastasis. We investigated the effects of epigallocatechin-3-gallate (EGCG), the most active green tea catechin, in combination with 6-OH-11-O-hydroxyphenanthrene (IIF), a synthetic retinoid X receptor-γ (RXRγ) agonist, on three breast carcinoma cell lines: MCF-7, MCF-7TAM and MDA-MB-231. EGFR and AKT activation and molecular markers of cell motility and migration (CD44, extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN), MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMPs)) were studied after EGCG and IIF treatments. The EGCG + IIF treatment was the most active in down-regulating EGFR phosphorylation at Tyr1068 in all the investigated cell lines; p473AKT was also down-regulated in MCF-TAM cells. EGCG + IIF was also the most active treatment in reducing the expression of markers of invasion and migration in all the three cell lines: CD44, EMMPRIN, MMP-2 and -9 expression decreased, whereas TIMPs were up-regulated. Zymography and scratch assay also confirmed the reduced invasion tendency. We considered that EGCG and IIF treatments could alter the molecular network based on EGFR, CD44 and EMMPRIN expression interdependence and reduced the migration tendency in MCF-7, MCF-7TAM and MDA-MB-231 cells. These events only occurred in association with AKT inactivation in MCF-7TAM cells. In conclusion, the combination of EGCG and IIF significantly attenuated the invasive behaviour of breast carcinoma cells.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Catequina/análogos & derivados , Movimento Celular/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Invasividade Neoplásica/patologia , Fenantrenos/farmacologia , Basigina/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Catequina/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
The recapitulation of primary tumour heterogenity and the existence of a minor sub-population of cancer cells, capable of initiating tumour growth in xenografts on serial passages, led to the hypothesis that cancer stem cells (CSCs) exist. CSCs are present in many tumours, among which is breast cancer. Breast CSCs (BCSCs) are likely to sustain the growth of the primary tumour mass, as well as to be responsible for disease relapse and metastatic spreading. Consequently, BCSCs represent the most significant target for new drugs in breast cancer therapy. Both the hypoxic condition in BCSCs biology and pro-inflammatory cytokine network has gained increasing importance in the recent past. Breast stromal cells are crucial components of the tumours milieu and are a major source of inflammatory mediators. Recently, the anti-inflammatory role of some nuclear receptors ligands has emerged in several diseases, including breast cancer. Therefore, the use of nuclear receptors ligands may be a valid strategy to inhibit BCSCs viability and consequently breast cancer growth and disease relapse.
RESUMO
Molecules with synergistic effects often enhance the benefits of cancer therapy. We observed that the major catechin of green tea, (-)-Epigallocatechin-3-gallate (EGCG), induced retinoid X receptor-γ (RXRγ) expression in the SK-Ch-A1 cholangiocarcinoma cell line and in two colon carcinoma cell lines (LoVo and the derivative multi-drug resistant LoVoMDR). On this basis, we analyzed the effects of EGCG in combination with an RXRγ ligand, 6-OH-11-O-hydroxyphenantrene (IIF), or with a ligand of retinoic acid receptor, all-trans-retinoic acid (RA). IIF alone and in combination with EGCG activated the retinoic X response elements and induced the germ cell nuclear factor. In parallel, EGCG induced 67 kDa laminin receptor expression alone and in combination with IIF. We observed a synergistic growth inhibition with EGCG and IIF in combination at lower doses. These effects were accompanied by apoptosis activation through the mitochondrial pathway. Moreover, in LoVo cell line we observed an induction of Forkhead box O3 expression, another molecule involved in apoptosis activation. Finally, metalloproteinase activity and extracellular matrix metalloproteinase inducer (EMMPRIN) expression were inhibited and tumor cell invasion was strongly reduced in the SK-Ch-A1 cell line after treatment with EGCG and IIF. In conclusion, the use of specific RXR ligands in combination with catechins could open a new perspective in gastrointestinal tumor chemoprevention.
Assuntos
Apoptose/efeitos dos fármacos , Catequina/análogos & derivados , Neoplasias Gastrointestinais/tratamento farmacológico , Fenantrenos/farmacologia , Receptor X Retinoide gama/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Proteína Forkhead Box O3/metabolismo , Neoplasias Gastrointestinais/metabolismo , Humanos , Ligantes , Metaloproteases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Chá/metabolismo , Tretinoína/metabolismoRESUMO
Dynamin GTPase (Dyn) plays a critical role in membrane-remodelling events underlying endocytosis. Studies in Drosophila identified a functional interaction between the Dyn homologue, encoded by the shibire (shi) gene, and Abnormal wing discs (Awd), a nucleoside diphosphate kinase (NDPK) that is the homologue of group I Nme human genes. These Drosophila studies showed that awd mutations enhance mutant shi phenotype and thus indicated the existence of a highly specific interaction between these genes. Furthermore, in human cells, it has been shown that Nme proteins promote Dyn activity in different membrane compartments through spatially controlled supply of GTP. Interestingly, Awd and Nme proteins have been detected in the extracellular environment. While no role has been inferred to extracellular Awd, presence of Nme1 in cancer patient serum is an unfavourable prognostic marker. In the present work, we used Drosophila and human cell line models to investigate the shuttling Awd/Nme1 proteins between intracellular and extracellular spaces. By using classic and reverse genetic approaches, we show that downregulation of Shi/Dyn1 activity enhances extracellular Awd/Nme1 in both Drosophila and human colon cell lines. We extended our analyses to colon cancer cell lines and found that knocking down Dyn1, besides to raise Nme1 extracellular amount, downregulates expression of molecular components that play key roles in tumour invasion. Interestingly, in vivo analyses of Drosophila larval adipocytes show that the conditional block of Shi activity greatly reduces intracellular amount of Awd confirming that Shi plays a key role in controlling the balance between intracellular and extracellular Awd.
Assuntos
Neoplasias do Colo/enzimologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Dinamina I/metabolismo , Dinaminas/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Adipócitos/enzimologia , Animais , Animais Geneticamente Modificados , Neoplasias do Colo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Dinamina I/genética , Dinaminas/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genótipo , Células HT29 , Humanos , Larva/enzimologia , Mutação , Nucleosídeo NM23 Difosfato Quinases/genética , Núcleosídeo-Difosfato Quinase/genética , Fenótipo , Interferência de RNA , TransfecçãoRESUMO
(-)-Epigallocatechin-3-gallate (EGCG) and chemotherapeutic agents cotreatment can improve cytotoxicity against cancer cells. We showed that EGCG and the rexinoid 6-OH-11-O-hydroxyphenanthrene (IIF), given together, were cytotoxic toward MCF-7, MCF-7TAM, and MDA-MB-231, three breast carcinoma cell lines showing different molecular characteristics. Cell growth arrest and apoptosis were greater after EGCG and IIF cotreatment than after individual administration. Cytotoxicity was related to upregulation of 67-kDa laminin receptor (LR67), one of the principal molecular targets of EGCG, and activation of the nuclear retinoic X receptors (RXRs) pathway. Furthermore, the transcription factor Forkhead box O3 (Foxo3a), a protein able to trigger apoptosis through upregulation of genes necessary for cell death, was activated. EGCG and IIF cotreatment produced a significant nuclear import of Foxo3a from the cytoplasm in MCF-7, MCF-7TAM, and MDA-MB-231 cells. In MCF-7TAM cells only, Foxo3a nuclear localization was associated with p473AKT downregulation. For the first time we showed that when EGCG and IIF, two harmless molecules, were given together, they might increase cytotoxicity in three breast carcinoma cell lines, two of them being representative of poorly responsive breast carcinoma types.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Catequina/análogos & derivados , Fenantrenos/administração & dosagem , Receptores X de Retinoides/biossíntese , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Catequina/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Células MCF-7 , Receptores X de Retinoides/genéticaRESUMO
OBJECTIVES: Resistance to gemcitabine is one of the main causes of treatment failure in pancreatic cancer. Compelling evidences have shown the involvement of nuclear factor κB (NF-κB) activation in such phenomenon. The protease inhibitor gabexate mesilate has been shown to inhibit NF-κB. We here investigated if combined treatment with this drug could improve gemcitabine antitumoral efficacy in pancreatic cancer cells. METHODS: The effect of gabexate mesilate and gemcitabine, both used at concentrations achievable in human plasma, was assessed on in vitro pancreatic cancer cell growth, invasion, and tumor angiogenesis. The molecular mechanism at the basis of these effects was also investigated. RESULTS: Gabexate mesilate significantly increased gemcitabine anti-invasive and antiangiogenic efficacy. This effect was related to inhibition of gemcitabine-induced NF-κB activation by gabexate mesilate, which prevented RelA/p65 nuclear translocation and resulted in metalloproteinase 2, metalloproteinase 9, vascular endothelial growth factor, and interleukin 8 down-regulation. Combined treatment with gabexate mesilate also inhibited gemcitabine-induced extracellular-regulated kinase 1/2 and AKT activation by increased expression of Raf kinase inhibitor protein and phosphatase and tensin homolog. CONCLUSIONS: Combined treatment with gabexate mesilate sensitizes pancreatic cancer cells to gemcitabine by inhibition of the NF-κB pathway. The efficacy of this therapeutic strategy in pancreatic cancer patients remains to be established and deserves future clinical investigation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Gabexato/farmacologia , NF-kappa B/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacologia , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gabexato/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores de Serina Proteinase/administração & dosagem , Inibidores de Serina Proteinase/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , GencitabinaRESUMO
Cytotoxic effects of the combination of the food components vitexin-2-O-xyloside (X), raphasatin (4-methylsulphanyl-3-butenyl isothiocyanates; G) and (-)-epigallocatechin-3-gallate (E) were investigated in colon (LoVo and CaCo-2) and breast (MDA-MB-231 and MCF-7) cancer cells. Breast cancer cells were more resistant than colon cells to X, G and E inhibition. On the contrary, marked synergistic effects among X, G and E on cell growth were found in both colon cancer cells. Further analysis revealed a G0/G1 arrest of the phase cell progression and apoptosis, linked to modulation of Bax, Bcl2, caspase-9 and poly(ADP-ribose) polymerase as well as Reactive Oxygen Species (ROS) generation in both colon cancer cells, whereas apoptosis and ROS were not significantly detected in normal human lymphocytes. We conclude that the X, G and E mixture might act by mitochondrial pathway activation of apoptosis, possibly elicited by ROS and the mixture may be effective in the chemoprevention of colon cancer.
Assuntos
Apigenina/farmacologia , Apoptose/efeitos dos fármacos , Catequina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/fisiopatologia , Isotiocianatos/farmacologia , Caspase 3/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Sinergismo Farmacológico , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Many whole cell-based assays in use today rely on flat, two-dimensional (2D) glass or plastic substrates that may not produce results characteristic of in vivo conditions. In this study, a three-dimensional (3D) cell-based assay scaffold was fabricated using a gas-in-foam templating technique. The scaffold was made of poly(vinyl alcohol), a water-soluble synthetic polymer with excellent film-forming, emulsifying, and biocompatible properties widely used in the biomedical field. The preliminary rheological studies on the solution of PVA and surfactant permitted us to disclose the significant physical parameters that influence the morphology of the ensuing materials. The scaffolds obtained were subjected to detailed analysis by light microscopy, Scanning Electron Microscopy (SEM), computed X-ray microtomography (µCT), infrared spectroscopy, and mechanical testing. Morphological investigations showed that the produced scaffolds are characterised by average void and interconnect diameters lying in the range of 200-300 and 30-150 µm, respectively, suitable for cell infiltration. Two different cross-linking procedures were adopted in order to modulate the mechanical properties of the PVA scaffolds. One made use of a bi-epoxide (PEGDGE), the other was based on glutaraldehyde (GA). The efficiency in terms of cross-linking density of the two procedures resulted in very different mechanical properties. Furthermore, in this article it is demonstrated how PVA foams can be processed into uniform, porous films suitable to be integrated with multi-well 2D culture plates in order to create a 3D analogue. The PEGDGE cross-linked scaffold was tested on C3A cells, a human hepatocyte cell line, representing an appropriate model for liver toxicity studies. Proliferation and cytotoxicity assays indicated good cell viability throughout the culture time, which was also confirmed by SEM analysis. Typical hepatic functions such as albumin and urea production and induction of Cyp3A4 enzyme activity following drug administration were satisfactory, thus proving the efficiency of this construct in maintaining specific liver functions.
RESUMO
AIMS: Cancer stem cell biology is tightly connected to the regulation of the pro-inflammatory cytokine network. The concept of cancer stem cells "inflammatory addiction" leads to envisage the potential role of anti-inflammatory molecules as new anti-cancer targets. Here we report on the relationship between nuclear receptors activity and the modulation of the pro-inflammatory phenotype in breast cancer stem cells. METHODS: Breast cancer stem cells were expanded as mammospheres from normal and tumor human breast tissues and from tumorigenic (MCF7) and non tumorigenic (MCF10) human breast cell lines. Mammospheres were exposed to the supernatant of breast tumor and normal mammary gland tissue fibroblasts. RESULTS: In mammospheres exposed to the breast tumor fibroblasts supernatant, autocrine tumor necrosis factor-α signalling engenders the functional interplay between peroxisome proliferator activated receptor-α and hypoxia inducible factor-1α (PPARα/HIF1α). The two proteins promote mammospheres formation and enhance each other expression via miRNA130b/miRNA17-5p-dependent mechanism which is antagonized by PPARγ. Further, the PPARα/HIF1α interplay regulates the expression of the pro-inflammatory cytokine interleukin-6, the hypoxia survival factor carbonic anhydrase IX and the plasma lipid carrier apolipoprotein E. CONCLUSION: Our data demonstrate the importance of exploring the role of nuclear receptors (PPARα/PPARγ) in the regulation of pro-inflammatory pathways, with the aim to thwart breast cancer stem cells functioning.
Assuntos
Apolipoproteínas E/metabolismo , Neoplasias da Mama/metabolismo , Anidrases Carbônicas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Neoplásicas/metabolismo , PPAR alfa/metabolismo , Apolipoproteínas E/genética , Comunicação Autócrina , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Feminino , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , PPAR alfa/genética , PPAR gama/genética , PPAR gama/metabolismo , Ligação Proteica , Esferoides Celulares , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Natural derivatives of vitamin A, including all-trans-retinoic acid (ATRA), commonly known as retinoids, currently produce favorable results in the treatment of many types of tumors. The rexinoid 6-OH-11-O-hydroxyphenantrene (IIF) is a synthetic derivative of ATRA. Previous in vitro and in vivo studies demonstrated that IIF is able to induce growth inhibition of various cancer cells and is a potent apoptosis-inducing agent with clinical potential. Osteosarcoma (OS) is the most common type of bone cancer, characterized by a rising aggressiveness. Recent evidences suggest that mesenchymal stem cells (MSC) may favour tumor growth and progression. Thus, it is important to investigate whether a compound with potential anti-tumoral properties such as IIF affects not only tumor cells but also MSC. The current study is an attempt to understand the mode of the potential cytotoxicity of IIF on OS cells and MSC. The response to IIF treatment of osteosarcoma SaOS-2, MG63, and U2OS cells and of bone marrow-derived MSC was the subject of investigation. The results showed that IIF significantly inhibited cell growth in OS cell lines and MSC in both a time- and dose-dependent manner, as evaluated by methylene blue assay. This was also associated with altered cell morphology and an increase in cell death with the involvement of apoptosis as demonstrated by NucleoCounter, Hoechst 33342 staining and FACS analysis. No cell death and apoptosis was found in U2OS cells. Analysis of cells treated with 20 and 40µM IIF for 24h by western blot suggests the activation of initiator caspase 9, indicating the involvement of caspases in inducing apoptosis. Furthermore, IIF upregulated the expression of the pro-apoptotic protein Bax and downregulated the anti-apoptotic protein Bcl2. For the first time, our results collectively provide an evidence for cell growth inhibition and activation of apoptosis in human OS cells and MSC by IIF. These results confirm that IIF may be an effective compound for anticancer treatment, including that of OS.