RESUMO
Sulfated glycosaminoglycans (GAGs), or synthetic mimetics thereof, are not favorably viewed as orally bioavailable drugs owing to their high number of anionic sulfate groups. Devising an approach for oral delivery of such highly sulfated molecules would be very useful. This work presents the concept that conjugating cholesterol to synthetic sulfated GAG mimetics enables oral delivery. A focused library of sulfated GAG mimetics was synthesized and found to inhibit the growth of a colorectal cancer cell line under spheroid conditions with a wide range of potencies ( 0.8 to 46 µM). Specific analogues containing cholesterol, either alone or in combination with clinical utilized drugs, exhibited pronounced in vivo anticancer potential with intraperitoneal as well as oral administration, as assessed by ex vivo tertiary and quaternary spheroid growth, cancer stem cell (CSC) markers, and/or self-renewal factors. Overall, cholesterol derivatization of highly sulfated GAG mimetics affords an excellent approach for engineering oral activity.
Assuntos
Glicosaminoglicanos , Sulfatos , Glicosaminoglicanos/farmacologia , Glicosaminoglicanos/metabolismo , Células-Tronco Neoplásicas/metabolismo , BiomiméticaRESUMO
The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) that plays critical roles in cancer. Microarray, computational, thermodynamic, and cellular imaging studies reveal that activation of IGF-1R by its cognate ligand IGF1 is inhibited by shorter, soluble heparan sulfate (HS) sequences (e.g., HS06), whereas longer polymeric chains do not inhibit the RTK, a phenomenon directly opposed to the traditional relationship known for GAG-protein systems. The inhibition arises from smaller oligosaccharides binding in a unique pocket in the IGF-1R ectodomain, which competes with the natural cognate ligand IGF1. This work presents a highly interesting observation on preferential and competing inhibition of IGF-1R by smaller sequences, whereas polysaccharides are devoid of this function. These insights will be of major value to glycobiologists and anti-cancer drug discoverers.
Assuntos
Polissacarídeos , Receptores de Somatomedina , Humanos , Ligantes , Neoplasias/metabolismo , Transdução de Sinais , Receptores de Somatomedina/metabolismoRESUMO
High-dose acetaminophen (AAP) with delayed rescue using n-acetylcysteine (NAC), the FDA-approved antidote to AAP overdose, has demonstrated promising antitumor efficacy in early phase clinical trials. However, the mechanism of action (MOA) of AAP's anticancer effects remains elusive. Using clinically relevant AAP concentrations, we evaluated cancer stem cell (CSC) phenotype in vitro and in vivo in lung cancer and melanoma cells with diverse driver mutations. Associated mechanisms were also studied. Our results demonstrated that AAP inhibited 3D spheroid formation, self-renewal, and expression of CSC markers when human cancer cells were grown in serum-free CSC media. Similarly, anti-CSC activity was demonstrated in vivo in xenograft models - tumor formation following in vitro treatment and ex-vivo spheroid formation following in vivo treatment. Intriguingly, NAC, used to mitigate AAP's liver toxicity, did not rescue cells from AAP's anti-CSC effects, and AAP failed to reduce glutathione levels in tumor xenograft in contrast to mice liver tissue suggesting nonglutathione-related MOA. In fact, AAP mediates its anti-CSC effect via inhibition of STAT3. AAP directly binds to STAT3 with an affinity in the low micromolar range and a high degree of specificity for STAT3 relative to STAT1. These findings have high immediate translational significance concerning advancing AAP with NAC rescue to selectively rescue hepatotoxicity while inhibiting CSCs. The novel mechanism of selective STAT3 inhibition has implications for developing rational anticancer combinations and better patient selection (predictive biomarkers) for clinical studies and developing novel selective STAT3 inhibitors using AAP's molecular scaffold.
Assuntos
Acetaminofen/farmacologia , Antineoplásicos/farmacologia , Radicais Livres/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Antígeno AC133/metabolismo , Acetaminofen/administração & dosagem , Antineoplásicos/administração & dosagem , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Humanos , Interleucina-6/antagonistas & inibidores , Neoplasias Pulmonares , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Heparanase, an endo-ß-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains and plays important roles in cellular growth and metastasis. Heparanase assays reported to-date are labor intensive, complex and/or expensive. A simpler assay is critically needed to understand the myriad roles of heparanase. We reasoned that fluorescent heparin could serve as an effective probe of heparanase levels. Following synthesis and screening, a heparin preparation labeled with DABCYL and EDANS was identified, which exhibited a characteristic increase in signal following cleavage by human heparanase. This work describes the synthesis of this heparin substrate, its kinetic and spectrofluorometric properties, optimization of the heparanase assay, use of the assay in inhibitor screening, and elucidation of the state of heparanase in different cell lines. Our FRET-based assay is much simpler and more robust than all assays reported in the literature as well as a commercially available kit.
Assuntos
Corantes Fluorescentes/química , Glucuronidase/química , Heparina/análogos & derivados , Heparina/química , Naftalenossulfonatos/química , p-Dimetilaminoazobenzeno/análogos & derivados , Animais , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Heparina/síntese química , Humanos , Células MCF-7 , Naftalenossulfonatos/síntese química , Células Sf9 , Spodoptera , p-Dimetilaminoazobenzeno/síntese química , p-Dimetilaminoazobenzeno/químicaRESUMO
Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here, we show for the first time that G2.2, a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide, selectively inhibits colonic CSCs in vivo G2.2-reduced CSCs (CD133+/CXCR4+, Dual hi) induced HT-29 and HCT 116 colon xenografts' growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact, G2.2 robustly inhibited CSCs' abundance (measured by levels of CSC markers, e.g., CD133, DCMLK1, LGR5, and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly, G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly, G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions, blood coagulation profile, and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro, in vivo, and ex vivo mechanistic studies, we showed that G2.2's inhibition of CSC self-renewal was mediated through activation of p38α, uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence, G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs.
Assuntos
Materiais Biomiméticos/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Relação Dose-Resposta a Droga , Células HCT116 , Células HT29 , Heparina/química , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA viruses are characterized by a high mutation rate, a buffer against environmental change. Nevertheless, the means by which random mutation improves viral fitness is not well characterized. Here we report the X-ray crystal structure of the receptor-binding domain (RBD) of the human coronavirus, HCoV-229E, in complex with the ectodomain of its receptor, aminopeptidase N (APN). Three extended loops are solely responsible for receptor binding and the evolution of HCoV-229E and its close relatives is accompanied by changing loop-receptor interactions. Phylogenetic analysis shows that the natural HCoV-229E receptor-binding loop variation observed defines six RBD classes whose viruses have successively replaced each other in the human population over the past 50 years. These RBD classes differ in their affinity for APN and their ability to bind an HCoV-229E neutralizing antibody. Together, our results provide a model for alphacoronavirus adaptation and evolution based on the use of extended loops for receptor binding.
Assuntos
Coronavirus Humano 229E/genética , Coronavirus Humano 229E/fisiologia , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Antígenos CD13/química , Antígenos CD13/metabolismo , Coronavirus Humano 229E/patogenicidade , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Evolução Molecular , Variação Genética , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Filogenia , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/química , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/ß mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.Heparan sulfate (HS) of specific length, i.e., hexasaccharide (HS06), but not longer or shorter sequences, selectively inhibit cancer stem cells (CSCs) through isoform specific activation of p38 mitogen-activated protein kinase. These findings will have major implication for developing chemical probes to decipher complex signaling events that govern cancer stem cells. Additionally, there are direct implications for designing glycosaminoglycan based cancer therapies to selectively target CSCs that escape killing by traditional chemotherapy threatening cancer relapse.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias Colorretais/metabolismo , Heparitina Sulfato/metabolismo , Células-Tronco Neoplásicas/fisiologia , Neoplasias Pancreáticas/metabolismo , Autorrenovação Celular , Descoberta de Drogas , Ativação Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Heparitina Sulfato/química , Humanos , Transdução de Sinais , Fator de Transcrição 4/metabolismo , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We observed a co-upregulation of the insulin-like growth factor receptor (IGF-1R)/AKT/mammalian target of rapamycin (mTOR) [InAT] axis and the mevalonate-isoprenoid biosynthesis (MIB) pathways in colorectal cancer stem cells (CSCs) in an unbiased approach. Hence, we hypothesized that the InAT axis might regulate the MIB pathway to govern colorectal CSCs growth. Stimulation (IGF-1) or inhibition (IGF-1R depletion and pharmacological inhibition of IGF-1R/mTOR) of the InAT axis produced induction or attenuation of CSC growth as well as expression of CSC markers and self-renewal factors respectively. Intriguingly, activation of the InAT axis (IGF-1) caused significant upregulation of the MIB pathway genes (both mRNA and protein); while its inhibition produced the opposite effects in colonospheres. More importantly, supplementation with dimethylallyl- and farnesyl-PP, MIB metabolites downstream of isopentenyl-diphosphate delta isomerase (IDI), but not mevalonate and isopentenyl-pp that are upstream of IDI, resulted in a near-complete reversal of the suppressive effect of the InAT axis inhibitors on CSCs growth. The latter findings suggest a specific regulation of the MIB pathway by the InAT axis distal to the target of statins that inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Effects of IGF-1R inhibition on colonic CSCs proliferation and the MIB pathway were confirmed in an 'in vivo' HCT-116 xenograft model. These observations establish a novel mechanistic link between the InAT axis that is commonly deregulated in colorectal cancer and the MIB pathway in regulation of colonic CSCs growth. Hence, the InAT-MIB corridor is a novel target for developing paradigm shifting optimum anti-CSCs therapies for colorectal cancer.
Assuntos
Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptor IGF Tipo 1/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose , Isomerases de Ligação Dupla Carbono-Carbono/genética , Proliferação de Células/fisiologia , Células HCT116 , Hemiterpenos/metabolismo , Hemiterpenos/farmacologia , Humanos , Ácido Mevalônico/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Compostos Organofosforados/metabolismo , Compostos Organofosforados/farmacologia , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos de Poli-Isoprenil/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , Receptor IGF Tipo 1/biossíntese , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Esferoides Celulares , Serina-Treonina Quinases TOR/metabolismo , Terpenos/metabolismo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.
Assuntos
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Peptidil Dipeptidase A , Estrutura Terciária de Proteína , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genéticaRESUMO
We studied the immunogenicity of an anti-SARS subunit vaccine comprised of the fragment of the SARS coronavirus (SARS-CoV) spike protein amino acids 318-510 (S318-510) containing the receptor-binding domain. The S protein fragment was purified from the culture supernatant of stably transformed HEK293T cells secreting a tagged version of the protein. The vaccine was given subcutaneously to 129S6/SvEv mice in saline, with alum adjuvant or with alum plus CpG oligodeoxynucleotides (ODN). Mice immunized with the adjuvanted antigen elicited strong antibody and cellular immune responses; furthermore, adding the CpG ODN to the alum resulted in increased IgG2a antibody titers and a higher number of INF-gamma-secreting murine splenocytes. Mice vaccinated with S318-510 deglycosylated by PNGase F (dgS318-510) showed a lower neutralizing antibody response but had similar numbers of INF-gamma-producing cells in the spleen. This finding suggests that carbohydrate is important for the immunogenicity of the S318-510 protein fragment and provide useful information for designing an effective and safe SARS subunit vaccine.
Assuntos
Glicoproteínas de Membrana , Receptores Virais/metabolismo , Síndrome Respiratória Aguda Grave/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral , Vacinas Virais/imunologia , Compostos de Alúmen , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Feminino , Humanos , Interferon gama/biossíntese , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Oligodesoxirribonucleotídeos , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/administração & dosagemRESUMO
Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by beta-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.
Assuntos
Anticorpos Antivirais/sangue , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Vacinação , Vacinas Virais/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Injeções Intramusculares , Injeções Subcutâneas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Testes de Neutralização , Proteínas do Nucleocapsídeo/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Glicoproteína da Espícula de Coronavírus , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
Voltage-gated potassium channels formed with the cardiac subunit HERG and a polymorphic variant of MinK-related peptide 1 (MiRP1) exhibit increased susceptibility to the antibiotic sulfamethoxazole (SMX) compared with channels formed with wild-type (WT) subunits. Here the molecular bases for SMX high-affinity block are investigated. The polymorphism causes a benign T to A amino acid mutation at position 8 (T8A) that destroys an N-glycosylation site of MiRP1. In vitro disruption of glycosylation by mutagenesis or in vivo by treatment with neuraminidase is associated with increased susceptibility to SMX and to other elementary agents such as divalent cations. Defective glycosylation does not affect the ability of T8A to form stable complexes with HERG, but rather it increases drug susceptibility through structural modifications in the channel complex. We conclude that N-glycosylation may play a key role in the etiology of life-threatening arrhythmia.