RESUMO
Cancer-initiating cells (CICs) that are responsible for tumor initiation, propagation, and resistance to standard therapies have been isolated from human solid tumors, including colorectal cancer (CRC). The aim of this study was to obtain an immunological profile of CRC-derived CICs and to identify CIC-associated target molecules for T cell immunotherapy. We have isolated cells with CIC properties along with their putative non-CIC autologous counterparts from human primary CRC tissues. These CICs have been shown to display "tumor-initiating/stemness" properties, including the expression of CIC-associated markers (e.g., CD44, CD24, ALDH-1, EpCAM, Lgr5), multipotency, and tumorigenicity following injection in immunodeficient mice. The immune profile of these cells was assessed by phenotype analysis and by in vitro stimulation of PBMCs with CICs as a source of Ags. CICs, compared with non-CIC counterparts, showed weak immunogenicity. This feature correlated with the expression of high levels of immunomodulatory molecules, such as IL-4, and with CIC-mediated inhibitory activity for anti-tumor T cell responses. CIC-associated IL-4 was found to be responsible for this negative function, which requires cell-to-cell contact with T lymphocytes and which is impaired by blocking IL-4 signaling. In addition, the CRC-associated Ag COA-1 was found to be expressed by CICs and to represent, in an autologous setting, a target molecule for anti-tumor T cells. Our study provides relevant information that may contribute to designing new immunotherapy protocols to target CICs in CRC patients.
Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Vigilância Imunológica/imunologia , Interleucina-4/metabolismo , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Linfócitos T/imunologia , Evasão Tumoral/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Interleucina-4/antagonistas & inibidores , Ativação Linfocitária/imunologia , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Epigallocatechin-3-gallate (EGCG) and tetracycline are two known inhibitors of amyloid aggregation able to counteract the fibrillation of most of the proteins involved in neurodegenerative diseases. We have recently investigated their effect on ataxin-3 (AT3), the polyglutamine-containing protein responsible for spinocerebellar ataxia typeâ 3. We previously showed that EGCG and tetracycline can contrast the aggregation process and toxicity of expanded AT3, although by different mechanisms. Here, we have performed further experiments by using the sole Josephin domain (JD) to further elucidate the mechanism of action of the two compounds. By protein solubility assays and FTIR spectroscopy we have first observed that EGCG and tetracycline affect the JD aggregation essentially in the same way displayed when acting on the full-length expanded AT3. Then, by saturation transfer difference (STD) NMR experiments, we have shown that EGCG binds both the monomeric and the oligomeric JD form, whereas tetracycline can only interact with the oligomeric one. Surface plasmon resonance (SPR) analysis has confirmed the capability of the sole EGCG to bind monomeric JD, although with a KD value suggestive for a non-specific interaction. Our investigations provide new details on the JD interaction with EGCG and tetracycline, which could explain the different mechanisms by which the two compounds reduce the toxicity of AT3.
Assuntos
Amiloide/antagonistas & inibidores , Amiloide/química , Ataxina-3/química , Catequina/análogos & derivados , Proteínas do Tecido Nervoso/química , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Proteínas Repressoras/química , Tetraciclina/química , Amiloide/metabolismo , Ataxina-3/farmacologia , Catequina/química , Catequina/farmacologia , Humanos , Proteínas do Tecido Nervoso/metabolismo , Peptídeos , Espectroscopia de Infravermelho com Transformada de Fourier , Tetraciclina/farmacologiaRESUMO
The insulin-like growth factor receptor I (IGF-IR) plays an essential role in transformation by promoting cell growth and protecting cancer cells from apoptosis. Aberrant IGF-IR signaling is implicated in several types of tumors, including carcinomas of the lung, breast, prostate, pancreas, liver, and colon. However, the contribution of the IGF-IR to the development of the transformed phenotype in urothelial cells has not been clearly established. In this study we demonstrated that the IGF-IR is overexpressed in invasive bladder cancer tissues compared with nonmalignant controls. We have investigated the role of the IGF-IR in bladder cancer by using urothelial carcinoma-derived 5637 and T24 cells. Although activation of the IGF-IR did not appreciably affect their growth, it did promote migration and stimulate in vitro wound closure and invasion. These effects required the activation of the Akt and Mitogen-activated protein kinase (MAPK) pathways as well as IGF-I-induced Akt- and MAPK-dependent phosphorylation of paxillin, which relocated at dynamic focal adhesions and was necessary for promoting motility in bladder cancer cells. Our results provide the first evidence for a role of the IGF-IR in motility and invasion of bladder cancer cells and support the hypothesis that the IGF-IR may play a critical role in the establishment of the invasive phenotype in urothelial neoplasia. Thus, the IGF-IR may also serve as a novel biomarker for bladder cancer.
Assuntos
Movimento Celular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Paxilina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Invasividade Neoplásica , Paxilina/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/genética , Transdução de Sinais/fisiologiaRESUMO
Ras oncoproteins play a crucial role in the onset, maintenance, and progression of the most common and deadly human cancers. Despite extensive research efforts, only a few mutant-specific Ras inhibitors have been reported. We show that cmp4-previously identified as a water-soluble Ras inhibitor- targets multiple steps in the activation and downstream signaling of different Ras mutants and isoforms. Binding of this pan-Ras inhibitor to an extended Switch II pocket on HRas and KRas proteins induces a conformational change that down-regulates intrinsic and GEF-mediated nucleotide dissociation and exchange and effector binding. A mathematical model of the Ras activation cycle predicts that the inhibitor severely reduces the proliferation of different Ras-driven cancer cells, effectively cooperating with Cetuximab to reduce proliferation even of Cetuximab-resistant cancer cell lines. Experimental data confirm the model prediction, indicating that the pan-Ras inhibitor is an appropriate candidate for medicinal chemistry efforts tailored at improving its currently unsatisfactory affinity.
RESUMO
CML is a hematopoietic stem-cell disorder emanating from breakpoint cluster region/Abelson murine leukemia 1 (BCR/ABL) translocation. Introduction of different TKIs revolutionized treatment outcome in CML patients, but CML LSCs seem insensitive to TKIs and are detectable in newly diagnosed and resistant CML patients and in patients who discontinued therapy. It has been reported that CML LSCs aberrantly express some CD markers such as CD26 that can be used for the diagnosis and for targeting. In this study, we confirmed the presence of CD26+ CML LSCs in newly diagnosed and resistant CML patients. To selectively target CML LSCs/progenitor cells that express CD26 and to spare normal HSCs/progenitor cells, we designed a venetoclax-loaded immunoliposome (IL-VX). Our results showed that by using this system we could selectively target CD26+ cells while sparing CD26- cells. The efficiency of venetoclax in targeting CML LSCs has been reported and our system demonstrated a higher potency in cell death induction in comparison to free venetoclax. Meanwhile, treatment of patient samples with IL-VX significantly reduced CD26+ cells in both stem cells and progenitor cells population. In conclusion, this approach showed that selective elimination of CD26+ CML LSCs/progenitor cells can be obtained in vitro, which might allow in vivo reduction of side effects and attainment of treatment-free, long-lasting remission in CML patients.
RESUMO
Recent epidemiological studies have demonstrated that the consumption of healthy foods that are particularly rich in polyphenols might reduce the incidence of cancer and neurodegenerative diseases. In particular, chlorogenic acids (CGAs) occur ubiquitously in food and represent the most abundant polyphenols in the human diet. A number of beneficial biological effects of CGAs, such as anti-inflammatory activity, anti-carcinogenic activity, and protection against neurodegenerative diseases, have been reported. However, the molecular mechanisms at the base of these biological activities have not yet been investigated in depth. By combining NMR spectroscopy, molecular docking, surface plasmon resonance and ex vivo assays of the Ras-dependent breast cancer cell line MDA-MB-231, we contribute to the elucidation of the molecular basis of the activity of CGAs and natural extracts from green and roasted coffee beans as chemoprotective dietary supplements.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Café/química , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ácido Clorogênico/análogos & derivados , Ácido Clorogênico/química , Ácido Clorogênico/isolamento & purificação , Ácido Clorogênico/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteína Oncogênica p21(ras)/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Polifenóis/química , Polifenóis/isolamento & purificação , Polifenóis/farmacologia , Ácido Quínico/análogos & derivados , Ácido Quínico/química , Ácido Quínico/isolamento & purificação , Ácido Quínico/farmacologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Cancer cells have an increased demand for amino acids and require transport even of non-essential amino acids to support their increased proliferation rate. Besides their major role as protein synthesis precursors, the two proteinogenic sulfur-containing amino acids, methionine and cysteine, play specific biological functions. In humans, methionine is essential for cell growth and development and may act as a precursor for cysteine synthesis. Cysteine is a precursor for the biosynthesis of glutathione, the major scavenger for reactive oxygen species. METHODOLOGY AND PRINCIPAL FINDINGS: We study the effect of K-ras oncogene activation in NIH3T3 mouse fibroblasts on transport and metabolism of cysteine and methionine. We show that cysteine limitation and deprivation cause apoptotic cell death (cytotoxic effect) in both normal and K-ras-transformed fibroblasts, due to accumulation of reactive oxygen species and a decrease in reduced glutathione. Anti-oxidants glutathione and MitoTEMPO inhibit apoptosis, but only cysteine-containing glutathione partially rescues the cell growth defect induced by limiting cysteine. Methionine limitation and deprivation has a cytostatic effect on mouse fibroblasts, unaffected by glutathione. K-ras-transformed cells-but not their parental NIH3T3-are extremely sensitive to methionine limitation. This fragility correlates with decreased expression of the Slc6a15 gene-encoding the nutrient transporter SBAT1, known to exhibit a strong preference for methionine-and decreased methionine uptake. CONCLUSIONS AND SIGNIFICANCE: Overall, limitation of sulfur-containing amino acids results in a more dramatic perturbation of the oxido-reductive balance in K-ras-transformed cells compared to NIH3T3 cells. Growth defects induced by cysteine limitation in mouse fibroblasts are largely-though not exclusively-due to cysteine utilization in the synthesis of glutathione, mouse fibroblasts requiring an exogenous cysteine source for protein synthesis. Therapeutic regimens of cancer involving modulation of methionine metabolism could be more effective in cells with limited methionine transport capability.
RESUMO
Cell growth and proliferation require a complex series of tight-regulated and well-orchestrated events. Accordingly, proteins governing such events are evolutionary conserved, even among distant organisms. By contrast, it is more singular the case of "core functions" exerted by functional analogous proteins that are not homologous and do not share any kind of structural similarity. This is the case of proteins regulating the G1/S transition in higher eukaryotes-i.e., the retinoblastoma (Rb) tumor suppressor Rb-and budding yeast, i.e., Whi5. The interaction landscape of Rb and Whi5 is quite large, with more than one hundred proteins interacting either genetically or physically with each protein. The Whi5 interactome has been used to construct a concept map of Whi5 function and regulation. Comparison of physical and genetic interactors of Rb and Whi5 allows highlighting a significant core of conserved, common functionalities associated with the interactors indicating that structure and function of the network-rather than individual proteins-are conserved during evolution. A combined bioinformatics and biochemical approach has shown that the whole Whi5 protein is highly disordered, except for a small region containing the protein family signature. The comparison with Whi5 homologs from Saccharomycetales has prompted the hypothesis of a modular organization of structural disorder, with most evolutionary conserved regions alternating with highly variable ones. The finding of a consensus sequence points to the conservation of a specific phosphorylation rhythm along with two disordered sequence motifs, probably acting as phosphorylation-dependent seeds in Whi5 folding/unfolding. Thus, the widely disordered Whi5 appears to act as a hierarchical, "date hub" that has evolutionary assayed an original way of modular organization before being supplanted by the globular, multi-domain structured Rb, more suitable to cover the role of a "party hub".
RESUMO
INTRODUCTION: Ras proteins are small GTPases molecular switches that cycle through two alternative conformational states, a GDP-bound inactive state and a GTP-bound active state. In the active state, Ras proteins interact with and modulate the activity of several downstream effectors regulating key cellular processes including proliferation, differentiation, survival, senescence, migration and metabolism. Activating mutations of RAS genes and of genes encoding Ras signaling members have a great incidence in proliferative disorders, such as cancer, immune and inflammatory diseases and developmental syndromes. Therefore, Ras and Ras signaling represent important clinical targets for the design and development of pharmaceutically active agents, including anticancer agents. AREAS COVERED: The authors summarize methods available to down-regulate the Ras pathway and review recent patents covering Ras signaling modulators, as well as methods designed to kill specifically cancer cells bearing activated RAS oncogene. EXPERT OPINION: Targeted therapy approach based on direct targeting of molecules specifically altered in Ras-dependent diseases is pursued with molecules that down-regulate expression or inhibit the biological function of mutant Ras or Ras signaling members. The low success rate in a clinical setting of molecules targeting activated members of the Ras pathway may require development of novel approaches, including combined and synthetic lethal therapies.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/antagonistas & inibidores , Animais , Antineoplásicos/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Genes ras , Humanos , Estrutura Molecular , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Patentes como Assunto , Conformação Proteica , Transdução de Sinais/genética , Relação Estrutura-Atividade , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Mutations of RAS genes are critical events in the pathogenesis of different human tumors and Ras proteins represent a major clinical target for the development of specific inhibitors to use as anticancer agents. Here we present RasGRF1-derived peptides displaying both in vitro and in vivo Ras inhibitory properties. These peptides were designed on the basis of the down-sizing of dominant negative full-length RasGRF1 mutants. The over-expression of these peptides can revert the phenotype of K-RAS transformed mouse fibroblasts to wild type, as monitored by several independent biological readouts, including Ras-GTP intracellular levels, ERK activity, morphology, proliferative potential and anchorage independent growth. Fusion of the RasGRF1-derived peptides with the Tat protein transduction domain allows their uptake into mammalian cells. Chemically synthesized Tat-fused peptides, reduced to as small as 30 residues on the basis of structural constraints, retain Ras inhibitory activity. These small peptides interfere in vitro with the GEF catalyzed nucleotide dissociation and exchange on Ras, reduce cell proliferation of K-RAS transformed mouse fibroblasts, and strongly reduce Ras-dependent IGF-I-induced migration and invasion of human bladder cancer cells. These results support the use of RasGRF1-derived peptides as model compounds for the development of Ras inhibitory anticancer agents.