RESUMO
Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Despite recent therapeutic advancements, resistance to 5-fluorouracil (5-FU) remains a major obstacle to the successful treatment of this disease. We have previously identified the ribosomal protein uL3 as a key player in the cell response to 5-FU, and loss of uL3 is associated with 5-FU chemoresistance. Natural products, like carotenoids, have shown the ability to enhance cancer cell response to drugs and may provide a safer choice to defeat chemoresistance in cancer. Transcriptome analysis of a cohort of 594 colorectal patients revealed a correlation between uL3 expression and both progression-free survival and response to treatment. RNA-Seq data from uL3-silenced CRC cells demonstrated that a low uL3 transcriptional state was associated with an increased expression of specific ATP-binding cassette (ABC) genes. Using two-dimensional (2D) and three-dimensional (3D) models of 5-FU-resistant CRC cells stably silenced for uL3, we investigated the effect of a novel therapeutic strategy by combining ß-carotene and 5-FU using nanoparticles (NPs) as a drug delivery system. Our results indicated that the combined treatment might overcome 5-FU chemoresistance, inducing cell cycle arrest in the G2/M phase and apoptosis. Furthermore, the combined treatment significantly reduced the expression levels of analyzed ABC genes. In conclusion, our findings suggest that ß-carotene combined with 5-FU may be a more effective therapeutic approach for treating CRC cells with low levels of uL3.
Assuntos
Neoplasias Colorretais , beta Caroteno , Humanos , beta Caroteno/farmacologia , beta Caroteno/metabolismo , beta Caroteno/uso terapêutico , Proteína Supressora de Tumor p53/genética , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Apoptose , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
Remarkable advances have been made in cancer therapy; however, the high degree of cellular heterogeneity observed during cancer progression is the major driver in the development of resistant phenotypes upon treatment administration [...].
Assuntos
Neoplasias , Humanos , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Carcinogênese/genética , Transformação Celular NeoplásicaRESUMO
The thrombin binding aptamer (TBA) possesses promising antiproliferative properties. However, its development as an anticancer agent is drastically impaired by its concomitant anticoagulant activity. Therefore, suitable chemical modifications in the TBA sequence would be required in order to preserve its antiproliferative over anticoagulant activity. In this paper, we report structural investigations, based on circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR), and biological evaluation of four pairs of enantiomeric heterochiral TBA analogues. The four TBA derivatives of the d-series are composed by d-residues except for one l-thymidine in the small TT loops, while their four enantiomers are composed by l-residues except for one d-thymidine in the same TT loop region. Apart from the left-handedness for the l-series TBA derivatives, CD and NMR measurements have shown that all TBA analogues are able to adopt the antiparallel, monomolecular, 'chair-like' G-quadruplex structure characteristic of the natural D-TBA. However, although all eight TBA derivatives are endowed with remarkable cytotoxic activities against colon and lung cancer cell lines, only TBA derivatives of the l-series show no anticoagulant activity and are considerably resistant in biological environments.
Assuntos
Aptâmeros de Nucleotídeos/genética , Quadruplex G , Ligação Proteica/genética , Trombina/genética , Anticoagulantes/química , Anticoagulantes/uso terapêutico , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Estereoisomerismo , Timidina/genéticaRESUMO
In this paper, we study the T30923 antiproliferative potential and the contribution of its loop residues in six different human cancer cell lines by preparing five T30923 variants using the single residue replacement approach of loop thymidine with an abasic site mimic (S). G-rich oligonucleotides (GRO) show interesting anticancer properties because of their capability to adopt G-quadruplex structures (G4s), such as the G4 HIV-1 integrase inhibitor T30923. Considering the multi-targeted effects of G4-aptamers and the limited number of cancer cell lines tested, particularly for T30923, it should be important to find a suitable tumor line, in addition to considering that the effects also strictly depend on G4s. CD, NMR and non-denaturating polyacrylamide gel electrophoresis data clearly show that all modified ODNs closely resemble the dimeric structure of parallel G4s' parent aptamer, keeping the resistance in biological environments substantially unchanged, as shown by nuclease stability assay. The antiproliferative effects of T30923 and its variants are tried in vitro by MTT assays, showing interesting cytotoxic activity, depending on time and dose, for all G4s, especially in MDA-MB-231 cells with a reduction in cell viability approximately up to 30%. Among all derivatives, QS12 results are the most promising, showing more pronounced cytotoxic effects both in MDA-MB-231 and Hela cells, with a decrease in cell viability from 70% to 60%. In summary, the single loop residue S substitution approach may be useful for designing antiproliferative G4s, considering that most of them, characterized by single residue loops, may be able to bind different targets in several cancer cell pathways. Generally, this approach could be of benefit by revealing some minimal functional structures, stimulating further studies aimed at the development of novel anticancer drugs.
Assuntos
Antineoplásicos , Aptâmeros de Nucleotídeos , Quadruplex G , Inibidores de Integrase de HIV , Neoplasias , Antineoplásicos/química , Aptâmeros de Nucleotídeos/química , Inibidores de Integrase de HIV/farmacologia , Células HeLa , Humanos , Neoplasias/tratamento farmacológico , TimidinaRESUMO
Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.
Assuntos
Transformação Celular Neoplásica , Neoplasias/metabolismo , Biogênese de Organelas , Ribossomos , Animais , Apoptose , Autofagia , Ciclo Celular , Movimento Celular , Nucléolo Celular/metabolismo , Citosol/metabolismo , Reparo do DNA , Estresse do Retículo Endoplasmático , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/fisiologia , Ribossomos/fisiologiaRESUMO
In this paper, we report our investigations on five T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties.
Assuntos
Antineoplásicos/química , Aptâmeros de Nucleotídeos/química , Neoplasias Colorretais/genética , Neoplasias Pulmonares/genética , Timidina/genética , Substituição de Aminoácidos , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Neoplasias Colorretais/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Quadruplex G , Células HCT116 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Espectroscopia de Ressonância MagnéticaRESUMO
Colorectal cancer (CRC) is the second deadliest cancer worldwide despite significant advances in both diagnosis and therapy. The high incidence of CRC and its poor prognosis, partially attributed to multi-drug resistance and antiapoptotic activity of cancer cells, arouse strong interest in the identification and development of new treatments. S-Adenosylmethionine (AdoMet), a natural compound and a nutritional supplement, is well known for its antiproliferative and proapoptotic effects as well as for its potential in overcoming drug resistance in many kinds of human tumors. Here, we report that AdoMet enhanced the antitumor activity of 5-Fluorouracil (5-FU) in HCT 116p53+/+ and in LoVo CRC cells through the inhibition of autophagy, induced by 5-FU as a cell defense mechanism to escape the drug cytotoxicity. Multiple drug resistance is mainly due to the overexpression of drug efflux pumps, such as P-glycoprotein (P-gp). We demonstrate here that AdoMet was able to revert the 5-FU-induced upregulation of P-gp expression and to decrease levels of acetylated NF-κB, the activated form of NF-κB, the major antiapoptotic factor involved in P-gp-related chemoresistance. Overall, our data show that AdoMet, was able to overcome 5-FU chemoresistance in CRC cells by targeting multiple pathways such as autophagy, P-gp expression, and NF-κB signaling activation and provided important implications for the development of new adjuvant therapies to improve CRC treatment and patient outcomes.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , NF-kappa B/metabolismo , S-Adenosilmetionina/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , NF-kappa B/genética , Células Tumorais CultivadasRESUMO
MicroRNAs (miRNAs) are attractive therapeutic targets and promising candidates as molecular biomarkers for various therapy-resistant tumors. However, the association between miRNAs and drug resistance in melanoma remains to be elucidated. We used an integrative genomic analysis to comprehensively study the miRNA expression profiles of drug-resistant melanoma patients and cell lines. MicroRNA-181a and -181b (miR181a/b) were identified as the most significantly down-regulated miRNAs in resistant melanoma patients and cell lines. Re-establishment of miR-181a/b expression reverses the resistance of melanoma cells to the BRAF inhibitor dabrafenib. Introduction of miR-181 mimics markedly decreases the expression of TFAM in A375 melanoma cells resistant to BRAF inhibitors. Furthermore, melanoma growth was inhibited in A375 and M14 resistant melanoma cells transfected with miR-181a/b mimics, while miR-181a/b depletion enhanced resistance in sensitive cell lines. Collectively, our study demonstrated that miR-181a/b could reverse the resistance to BRAF inhibitors in dabrafenib resistant melanoma cell lines. In addition, miR-181a and -181b are strongly down-regulated in tumor samples from patients before and after the development of resistance to targeted therapies. Finally, melanoma tissues with high miR-181a and -181b expression presented favorable outcomes in terms of Progression Free Survival, suggesting that miR-181 is a clinically relevant candidate for therapeutic development or biomarker-based therapy selection.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , MicroRNAs/biossíntese , Proteínas Mitocondriais/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , Fatores de Transcrição/biossíntese , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Feminino , Genômica , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , MicroRNAs/genética , Proteínas Mitocondriais/genética , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , Fatores de Transcrição/genéticaRESUMO
Eukaryotic cells are exposed to many internal and external stimuli that affect their fate. In particular, the exposure to some of these stimuli induces stress triggering a variety of stress responses aimed to re-establish cellular homeostasis. It is now established that the deregulation of stress response pathways plays a central role in cancer initiation and progression, allowing the adaptation of cells to an altered state in the new environment. Autophagy is a tightly regulated pathway which exerts "housekeeping" role in physiological processes. Recently, a growing amount of evidence highlighted the crucial role of autophagy in the regulation of integrated stress responses, including nucleolar and endoplasmic reticulum. In this review, we attempt to afford an overview of the complex role of nucleolar and endoplasmic reticulum stress-response mechanisms in the regulation of autophagy in cancer and cancer treatment.
Assuntos
Autofagia/genética , Núcleo Celular/genética , Retículo Endoplasmático/genética , Neoplasias/genética , Apoptose/genética , Estresse do Retículo Endoplasmático/genética , Humanos , Neoplasias/patologiaRESUMO
The nucleolus is the site of ribosome biogenesis and has been recently described as important sensor for a variety of cellular stressors. In the last two decades, it has been largely demonstrated that many chemotherapeutics act by inhibiting early or late rRNA processing steps with consequent alteration of ribosome biogenesis and activation of nucleolar stress response. The overall result is cell cycle arrest and/or apoptotic cell death of cancer cells. Our previously data demonstrated that ribosomal protein uL3 is a key sensor of nucleolar stress activated by common chemotherapeutic agents in cancer cells lacking p53. We have also demonstrated that uL3 status is associated to chemoresistance; down-regulation of uL3 makes some chemotherapeutic drugs ineffective. Here, we demonstrate that in colon cancer cells, the uL3 status affects rRNA synthesis and processing with consequent activation of uL3-mediated nucleolar stress pathway. Transcriptome analysis of HCT 116p53-/- cells expressing uL3 and of a cell sub line stably depleted of uL3 treated with Actinomycin D suggests a new extra-ribosomal role of uL3 in the regulation of autophagic process. By using confocal microscopy and Western blotting experiments, we demonstrated that uL3 acts as inhibitory factor of autophagic process; the absence of uL3 is associated to increase of autophagic flux and to chemoresistance. Furthermore, experiments conducted in presence of chloroquine, a known inhibitor of autophagy, indicate a role of uL3 in chloroquine-mediated inhibition of autophagy. On the basis of these results and our previous findings, we hypothesize that the absence of uL3 in cancer cells might inhibit cancer cell response to drug treatment through the activation of cytoprotective autophagy. The restoration of uL3 could enhance the activity of many drugs thanks to its pro-apoptotic and anti-autophagic activity.
Assuntos
Nucléolo Celular/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Estresse Fisiológico , Apoptose/genética , Autofagia/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Ribossômico/genética , Proteína Ribossômica L3 , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/genéticaRESUMO
PURPOSE: In order to study novel therapeutic approaches taking advantage of natural compounds showing anticancer and anti-proliferative effects, we focused our interest on S-adenosyl-l-methionine, a naturally occurring sulfur-containing nucleoside synthesized from adenosine triphosphate and methionine by methionine adenosyltransferase, and its potential in overcoming drug resistance in colon cancer cells devoid of p53. RESULTS: In the present study, we demonstrated that S-adenosyl-l-methionine overcomes uL3-mediated drug resistance in p53 deleted colon cancer cells. In particular, we demonstrated that S-adenosyl-l-methionine causes cell cycle arrest at the S phase; inhibits autophagy; augments reactive oxygen species; and induces apoptosis in these cancer cells. CONCLUSIONS: Results reported in this paper led us to propose S-adenosyl-l-methionine as a potential promising agent for cancer therapy by examining p53 and uL3 profiles in tumors to yield a better clinical outcomes.
Assuntos
Neoplasias do Colo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Deleção de Genes , Proteínas Ribossômicas/metabolismo , S-Adenosilmetionina/farmacologia , Proteína Supressora de Tumor p53/deficiência , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células HCT116 , Humanos , Proteína Ribossômica L3 , Proteínas Ribossômicas/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
In this paper, we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy and electrophoresis methods, on three oligonucleotide sequences, each containing one 3'-3' and two 5'-5' inversion of polarity sites, and four G-runs with a variable number of residues, namely two, three and four (mTG2T, mTG3T and mTG4T with sequence 3'-TGnT-5'-5'-TGnT-3'-3'-TGnT-5'-5'-TGnT-3' in which n = 2, 3 and 4, respectively), in comparison with their canonical counterparts (TGnT)4 (n = 2, 3 and 4). Oligonucleotides mTG3T and mTG4T have been proven to form very stable unprecedented monomolecular parallel G-quadruplex structures, characterized by three side loops containing the inversion of polarity sites. Both G-quadruplexes have shown an all-syn G-tetrad, while the other guanosines adopt anti glycosidic conformations. All oligonucleotides investigated have shown a noteworthy antiproliferative activity against lung cancer cell line Calu 6 and colorectal cancer cell line HCT-116 p53-/-. Interestingly, mTG3T and mTG4T have proven to be mostly resistant to nucleases in a fetal bovine serum assay. The whole of the data suggest the involvement of specific pathways and targets for the biological activity.
Assuntos
Quadruplex G , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos/química , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Células HCT116 , Humanos , Espectroscopia de Ressonância Magnética , Oligonucleotídeos/genética , Oligonucleotídeos/farmacologia , TemperaturaRESUMO
BACKGROUND: The thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative activities. Its chemico-physical and/or biological properties can be tuned by the site-specific replacement of selected residues. METHODS: Four oligodeoxynucleotides (ODNs) based on the TBA sequence (5'-GGTTGGTGTGGTTGG-3') and containing 2'-deoxyuridine (U) or 5-bromo-2'-deoxyuridine (B) residues at positions 4 or 13 have been investigated by NMR and CD techniques. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay) have been tested and compared with two further ODNs containing 5-hydroxymethyl-2'-deoxyuridine (H) residues in the same positions, previously investigated. RESULTS: The CD and NMR data suggest that all the investigated ODNs are able to form G-quadruplexes strictly resembling that of TBA. The introduction of B residues in positions 4 or 13 increases the melting temperature of the modified aptamers by 7⯰C. The replacement of thymidines with U in the same positions results in an enhanced anticoagulant activity compared to TBA, also at low ODN concentration. Although all ODNs show antiproliferative properties, only TBA derivatives containing H in the positions 4 and 13 lose the anticoagulant activity and remarkably preserve the antiproliferative one. CONCLUSIONS: All ODNs have shown antiproliferative activities against two cancer cell lines but only those with U and B are endowed with anticoagulant activities similar or improved compared to TBA. GENERAL SIGNIFICANCE: The appropriate site-specific replacement of the residues in the TT loops of TBA with commercially available thymine analogues is a useful strategy either to improve the anticoagulant activity or to preserve the antiproliferative properties by quenching the anticoagulant ones.
Assuntos
Anticoagulantes/farmacologia , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Anticoagulantes/síntese química , Anticoagulantes/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular Tumoral , Dicroísmo Circular , Estabilidade de Medicamentos , Quadruplex G , Humanos , Temperatura de TransiçãoRESUMO
BACKGROUND: The thrombin binding aptamer (TBA) is endowed with antiproliferative properties but its potential development is counteracted by the concomitant anticoagulant activity. METHODS: Five oligonucleotides (ODNs) based on TBA sequence (GGTTGGTGTGGTTGG) and containing l-residues or both l-residues and inversion of polarity sites have been investigated by NMR and CD techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay), and their resistance in fetal bovine serum have been tested. RESULTS: CD and NMR data suggest that the investigated ODNs are able to form right- and left-handed G-quadruplex structures. All ODNs do not retain the anticoagulant activity characteristic of TBA but are endowed with a significant antiproliferative activity against two cancerous cell lines. Their resistance in biological environment after six days is variable, depending on the ODN. CONCLUSIONS: A comparison between results and literature data suggests that the antiproliferative activity of the TBA analogues investigated could depends on two factors: a) biological pathways and targets different from those already identified or proposed for other antiproliferative G-quadruplex aptamers, and b) the contribution of the guanine-based degradation products. GENERAL SIGNIFICANCE: Modified TBA analogues containing l-residues and inversion of polarity sites lose the anticoagulant activity but gain antiproliferative properties against two cancer cell lines. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
Assuntos
Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Trombina/farmacologia , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Coagulação Sanguínea/efeitos dos fármacos , Dicroísmo Circular , Estabilidade de Medicamentos , Esterases/química , Quadruplex G , Células HCT116 , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Neoplasias/patologia , Ligação Proteica , Relação Estrutura-Atividade , Trombina/análogos & derivados , Trombina/química , Trombina/metabolismo , Fatores de TempoRESUMO
The nucleolus is the site of ribosome biogenesis, a complex process that requires the coordinate activity of all three RNA polymerases and hundreds of non-ribosomal factors that participate in the maturation of ribosomal RNA (rRNA) and assembly of small and large subunits. Nevertheless, emerging studies have highlighted the fundamental role of the nucleolus in sensing a variety of cellular stress stimuli that target ribosome biogenesis. This condition is known as nucleolar stress and triggers several response pathways to maintain cell homeostasis, either p53-dependent or p53-independent. The mouse double minute (MDM2)-p53 stress signaling pathways are activated by multiple signals and are among the most important regulators of cellular homeostasis. In this review, we will focus on the role of ribosomal proteins in p53-dependent and p53-independent response to nucleolar stress considering novel identified regulators of these pathways. We describe, in particular, the role of ribosomal protein uL3 (rpL3) in p53-independent nucleolar stress signaling pathways.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Homeostase/fisiologia , Humanos , Modelos Biológicos , Proteína Ribossômica L3 , Estresse Fisiológico/fisiologiaRESUMO
Cancer is one of the most common causes of death among adults. Chemotherapy is crucial in determining patient survival and quality of life. However, the development of multidrug resistance (MDR) continues to pose a significant challenge in the management of cancer. In this study, we analyzed the role of human ribosomal protein uL3 (formerly rpL3) in multidrug resistance. Our studies revealed that uL3 is a key determinant of multidrug resistance in p53-mutated lung cancer cells by controlling the cell redox status. We established and characterized a multidrug resistant Calu-6 cell line. We found that uL3 down-regulation correlates positively with multidrug resistance. Restoration of the uL3 protein level re-sensitized the resistant cells to the drug by regulating the reactive oxygen species (ROS) levels, glutathione content, glutamate release, and cystine uptake. Chromatin immunoprecipitation experiments and luciferase assays demonstrated that uL3 coordinated the expression of stress-response genes acting as transcriptional repressors of solute carrier family 7 member 11 (xCT) and glutathione S-transferase α1 (GST-α1), independently of Nuclear factor erythroid 2-related factor 2 (Nrf2). Altogether our results describe a new function of uL3 as a regulator of oxidative stress response genes and advance our understanding of the molecular mechanisms underlying multidrug resistance in cancers.
Assuntos
Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/genética , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Proteína Ribossômica L3RESUMO
L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.
Assuntos
Cisteína/farmacologia , Ferro/metabolismo , Queratinócitos/metabolismo , Queratinas/biossíntese , Linhagem Celular , Homeostase , Humanos , Queratinócitos/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Regulação para CimaRESUMO
BACKGROUND: Novel polymeric nanoparticles (NPs) specifically designed for delivering chemotherapeutics in the body and aimed at improving treatment activity and selectivity, cover a very relevant area in the field of nanomedicine. Here, we describe how to build a polymer shell of Hyaluronan (HA) and Polyethyleneimine (PEI) on biodegradable NPs of poly(lactic-co-glycolic) acid (PLGA) through electrostatic interactions and to achieve NPs with unique features of sustained delivery of a docetaxel (DTX) drug cargo as well as improved intracellular uptake. RESULTS: A stable PEI or HA/PEI shell could be obtained by careful selection of layering conditions. NPs with exquisite stability in salt and protein-rich media, with size and surface charge matching biological requirements for intravenous injection and endowed with sustained DTX release could be obtained. Cytotoxicity, uptake and activity of both PLGA/PEI/HA and PLGA/PEI NPs were evaluated in CD44(+) (A549) and CD44(-) (Calu-3) lung cancer cells. In fact, PEI-coated NPs can be formed after degradation/dissociation of the surface HA because of the excess hyaluronidases overexpressed in tumour interstitium. There was no statistically significant cytotoxic effect of PLGA/PEI/HA and PLGA/PEI NPs in both cell lines, thus suggesting that introduction of PEI in NP shell was not hampered by its intrinsic toxicity. Intracellular trafficking of NPs fluorescently labeled with Rhodamine (RHO) (RHO-PLGA/PEI/HA and RHO-PLGA/PEI NPs) demonstrated an increased time-dependent uptake only for RHO-PLGA/PEI/HA NPs in A549 cells as compared to Calu-3 cells. As expected, RHO-PLGA/PEI NP uptake in A549 cells was comparable to that observed in Calu-3 cells. RHO-PLGA/PEI/HA NPs internalized into A549 cells showed a preferential perinuclear localization. Cytotoxicity data in A549 cells suggested that DTX delivered through PLGA/PEI/HA NPs exerted a more potent antiproliferative activity than free DTX. Furthermore, DTX-PLGA/PEI NPs, as hypothetical result of hyaluronidase-mediated degradation in tumor interstitium, were still able to improve the cytotoxic activity of free DTX. CONCLUSIONS: Taken together, results lead us to hypothesize that biodegradable NPs coated with a PEI/HA shell represent a very promising system to treat CD44 overexpressing lung cancer. In principle, this novel nanocarrier can be extended to different single drugs and drug combinations taking advantage of the shell and core properties.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/química , Taxoides/administração & dosagem , Antineoplásicos/química , Materiais Biocompatíveis/química , Linhagem Celular/efeitos dos fármacos , Docetaxel , Estabilidade de Medicamentos , Corantes Fluorescentes/farmacocinética , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Ácido Láctico/química , Neoplasias Pulmonares/patologia , Nanopartículas/administração & dosagem , Nanopartículas/toxicidade , Nanoconchas/química , Polietilenoimina/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Taxoides/químicaRESUMO
Despite advancements in therapeutic strategies, the development of drug resistance and metastasis remains a serious concern for the efficacy of chemotherapy against colorectal cancer (CRC). We have previously demonstrated that low expression of ribosomal protein uL3 positively correlates with chemoresistance in CRC patients. Here, we demonstrated that the loss of uL3 increased the metastatic capacity of CRC cells in chick embryos. Metabolomic analysis revealed large perturbations in amino acid and glutathione metabolism in resistant uL3-silenced CRC cells, indicating that uL3 silencing dramatically triggered redox metabolic reprogramming. RNA-Seq data revealed a notable dysregulation of 108 genes related to ferroptosis in CRC patients. Solute Carrier Family 7 Member 11 (SLC7A11) is one of the most dysregulated genes; its mRNA stability is negatively regulated by uL3, and its expression is inversely correlated with uL3 levels. Inhibition of SLC7A11 with erastin impaired resistant uL3-silenced CRC cell survival by inducing ferroptosis. Of interest, the combined treatment erastin plus uL3 enhanced the chemotherapeutic sensitivity of uL3-silenced CRC cells to erastin. The antimetastatic potential of the combined strategy was evaluated in chick embryos. Overall, our study sheds light on uL3-mediated chemoresistance and provides evidence of a novel therapeutic approach, erastin plus uL3, to induce ferroptosis, establishing individualized therapy by examining p53, uL3 and SLC7A11 profiles in tumors.