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1.
ACS Chem Neurosci ; 15(14): 2565-2585, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38795037

RESUMO

Alzheimer's disease is a complex neurological disorder linked with multiple pathological hallmarks. The interrelation of therapeutic targets assists in the enhancement of cognitive decline through interference with overall neuronal transmission. We have synthesized and screened various chromone derivatives as potential multitarget-directed ligands for the effective treatment of Alzheimer's disease. The synthesized compounds exhibited multipotent activity against AChE, BuChE, MAO-B, and amyloid ß aggregation. Three potent compounds, i.e., VN-3, VN-14, and VN-19 were identified that displayed remarkable activities against different targets. These compounds displayed IC50 values of 80 nM, 2.52 µM, and 140 nM against the AChE enzyme, respectively, and IC50 values of 2.07 µM, 70 nM, and 450 nM against the MAO-B isoform, respectively. VN-3 displayed potent activity against self-induced Aß1-42 aggregation with inhibition of 58.3%. In the ROS inhibition studies, the most potent compounds reduced the intracellular ROS levels up to 80% in SH-SY5Y cells at 25 µM concentration. The compounds were found to be neuroprotective and noncytotoxic even at a concentration of 25 µM against SH-SY5Y cells. In silico studies showed that the compounds were nicely accommodated in the active sites of the receptors along with thermodynamically stable orientations. Compound VN-19 exhibited a balanced multitargeting profile against AChE, BuChE, MAO-B, and Aß1-42 enzymes and was further evaluated for in vivo activities on the scopolamine-induced zebrafish model. VN-19 was found to ameliorate the cognitive decline in zebrafish brains by protecting them against scopolamine-induced neurodegeneration. Thus, VN-3, VN-14, and VN-19 were identified as potent multitarget-directed ligands with a balanced activity profile against different targets and can be developed as therapeutics for AD.


Assuntos
Doença de Alzheimer , Inibidores da Colinesterase , Cromonas , Escopolamina , Peixe-Zebra , Animais , Escopolamina/farmacologia , Cromonas/farmacologia , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/síntese química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Humanos , Fármacos Neuroprotetores/farmacologia , Peptídeos beta-Amiloides/metabolismo , Modelos Animais de Doenças , Acetilcolinesterase/metabolismo , Ligantes , Monoaminoxidase/metabolismo , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Inibidores da Monoaminoxidase/química
2.
Microb Pathog ; 188: 106538, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38184177

RESUMO

Because of uncontrolled use of antibiotics, emergence of multidrug-resistant Shigella species poses a huge potential of zoonotic transfer from poultry sector. With increasing resistance to current antibiotics, there is a critical need to explore antibiotic alternatives. Using a Shigella flexneri reference strain, we isolated a novel fPSFA phage after inducing with mitomycin C. The phage was found to be stable for wide ranges of temperature -20 °C-65 °C and pH 3 to 11. fPSFA shows a latent period that ranges from 20 to 30 min and generation times of 50-60 min. The genome analysis of phage reveals two major contigs of 23788 bp and 23285 bp with 50.16 % and 39.33 % G + C content containing a total of 80 CDS and 2 tRNA genes. The phage belongs to Straboviridae family and lacks any virulence or antimicrobial resistance gene, thus making it a suitable candidate for treatment of drug-resistant infections. To confirm lytic ability of novel phage, we isolated 54 multidrug-resistant Shigella species from thirty-five poultry fecal samples that shows multiple antibiotic resistance index ranging from 0.15 to 0.75 (from 3 Indian states). The fPSFA showed lytic activity against multidrug-resistant Shigella isolates (73.08 %) (MARI≥0.50). The wide host ranges of fPSFA phage demonstrate its potential to be used as a biocontrol agent.


Assuntos
Bacteriófagos , Shigella , Animais , Prófagos/genética , Aves Domésticas , Genoma Viral , Bacteriófagos/genética , Genômica , Antibacterianos/farmacologia
3.
Cell Signal ; 113: 110953, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38084837

RESUMO

BACKGROUND: Cancer Stem Cells (CSCs) have emerged as a critical mediator in recurrence and resistance in cancers. Kindlin-isoform (1 and 2) binds with cytoplasmic ß-tail of integrin and are essential co-activators of integrin function. Given their important function in regulating cancer hallmarks such as cell proliferation, invasion, migration, and metastasis, we hypothesize that it might play a critical role in CSC growth, survival, and self-renewal of colon cancer. MATERIALS AND METHODS: Using knockdown approaches, we inhibited Kindlin-2 expression in HCT116 and HT29 colon cancer cells. Extreme limiting dilution and self-renewal assay were performed to measure the role of Kindlin in colonic CSC. Standard methods such as qRT-PCR and western blotting were carried out to understand the signaling cascade by which Kindlin regulates CSC marker expression and downstream targets. RESULTS: Our data show isoform-specific upregulation of Kindlin-2 in colonic CSCs. The silencing of Kindlin-2 reduces colonosphere formation, decreases CSC size, and self-renewal marker genes such as CD-133, CXCR-4, LGR-5, and C-MYC. Kindlin-2 silencing reduces colonosphere proliferation, invasion, and migration of colonic CSCs. Mechanistically, Kindlin-2 silencing reduces the expression, and nuclear localization of ß-catenin, and decreases ß-catenin target genes such as C-MYC, cyclin D1, DKK-1, and Snail-1. CONCLUSION: Our study delineates the isoform-specific activity of Kindlin-2 in regulating Colonic CSC. Isoform-specific targeting of Kindlin-2 may be a novel strategy to tackle this devastating disease.


Assuntos
Neoplasias do Colo , beta Catenina , Humanos , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/metabolismo , Integrinas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Isoformas de Proteínas/metabolismo , Via de Sinalização Wnt/genética
4.
Curr Cancer Drug Targets ; 23(4): 278-292, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306454

RESUMO

AIMS: Development of anticancer agents targeting tubulin protein. BACKGROUND: Tubulin protein is being explored as an important target for anticancer drug development. Ligands binding to the colchicine binding site of the tubulin protein act as tubulin polymerization inhibitors and arrest the cell cycle in the G2/M phase. OBJECTIVE: Synthesis and screening of benzotriazole-substituted 2-phenyl quinazolines as potential anticancer agents. METHODS: A series of benzotriazole-substituted quinazoline derivatives have been synthesized and evaluated against human MCF-7 (breast), HeLa (cervical) and HT-29 (colon) cancer cell lines using standard MTT assays. RESULTS: ARV-2 with IC50 values of 3.16 µM, 5.31 µM, 10.6 µM against MCF-7, HELA and HT29 cell lines, respectively displayed the most potent antiproliferative activities in the series while all the compounds were found non-toxic against HEK293 (normal cells). In the mechanistic studies involving cell cycle analysis, apoptosis assay and JC-1 studies, ARV-2 and ARV-3 were found to induce mitochondria-mediated apoptosis. CONCLUSION: The benzotriazole-substituted 2-phenyl quinazolines have the potential to be developed as potent anticancer agents.


Assuntos
Antineoplásicos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Relação Estrutura-Atividade , Polimerização , Células HEK293 , Proliferação de Células , Simulação de Acoplamento Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Quinazolinas/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química
5.
Bioorg Med Chem ; 72: 116976, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36067627

RESUMO

Colchicine binding site represent a crucial target for the anticancer drug development especially in view of emerging drug resistance from the currently available chemotherapeutics. A total of 16 novel 4-N-heterocyclic-2-aryl-6,7,8-trimethoxyquinazolines were synthesized and screened for antiproliferative and tubulin polymerization inhibition potential. The synthesized compounds were evaluated against MCF-7, HeLa and HT-29 cancer cell lines and normal cell line HEK-293 T. In the series, 2­aryl group with 4­bromophenyl substitution displayed IC50 values of 6.37 µM, 17.43 µM, 6.76 µM and 4­chlorophenyl substitution displayed IC50 values of 2.16 µM, 8.53 µM, 10.42 µM against MCF-7, HELA and HT29 cancer cell lines, respectively. In the mechanistic studies involving cell cycle analysis, apoptosis assay and JC-1 studies, both the lead compounds were found to induce mitochondria mediated apoptosis and lead molecule with 4­chlorophenyl substitution displayed significant tubulin polymerization inhibition activity. In the computation studies, lead molecule displayed significant binding affinites in the colchicine domain and showed good thermodynamic stability during 100 ns MD simulation studies. 4-N-Heterocyclic-2-aryl-6,7,8-trimethoxyquinazolines showed appreciable drug like characteristics and can be developed as potent anticancer agents.


Assuntos
Antineoplásicos , Quinazolinas , Moduladores de Tubulina , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Colchicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Polimerização , Quinazolinas/química , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química
6.
Methods Mol Biol ; 2303: 765-777, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34626421

RESUMO

Cancer stem-like cells (CSC) have been implicated in resistance to conventional chemotherapy as well as invasion and metastasis resulting in tumor relapse in majority of epithelial cancers including colorectal cancer. Hence, targeting CSC by small molecules is likely to improve therapeutic outcomes. Glycosaminoglycans (GAGs) are long linear polysaccharide molecules with varying degrees of sulfation that allows specific GAG-protein interaction which plays a key role in regulating cancer hallmarks such as cellular growth, angiogenesis, and immune modulation. However, identifying selective CSC-targeting GAG mimetic has been marred by difficulties associated with isolating and enriching CSC in vitro. Herein, we discuss two distinct methods, spheroid growth and EMT-transformed cells, to enrich CSC and set up medium- and high-throughput screen to identify selective CSC-targeting agents.


Assuntos
Neoplasias , Células-Tronco Neoplásicas , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal , Humanos
7.
Microb Pathog ; 162: 105267, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34718127

RESUMO

OmpU is a multimeric, cation selective outer membrane protein of Vibrio and related species that non-covalently interact with peptidoglycan layer. Interaction of OmpU with human host cells triggers signaling pathways to promote cytokine secretion, reactive oxygen species production, and caspase independent death in immune and epithelial cells. Non-choleric OmpU imparts resistance to antimicrobial peptides and induces actin cytoskeletal reorganization in the host cells. Further, OmpU isolated from Vibrio species elicits an immune response in several aquaculture hosts. Importantly, in-vivo studies using recombinant OmpU or OmpU derived mimotopes reveal a short-lasting immunity, and protection against Vibrio in the aquaculture sector. In conclusion, OmpU is a key adhesion protein and an important virulence factor for successful colonization of Vibrio species into hosts. This review article provides a broad overview of structural, regulatory, and functional mechanisms of OmpU in normal and disease states.


Assuntos
Vibrioses , Vibrio , Adesinas Bacterianas , Peptídeos Antimicrobianos , Proteínas da Membrana Bacteriana Externa , Humanos , Vibrioses/veterinária
8.
Bioorg Med Chem Lett ; 30(20): 127468, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32768647

RESUMO

A series of triphenyl substituted pyrimidines as analogous of colchicine and combretastatin A-4 was synthesized and evaluated for the antiproliferative potential. The compounds were screened against MDA-MB-231, HCT-116 and HT-29 cell lines using MTT assay. Most of the compounds displayed antiproliferative activity in low to sub micro molar concentration. Amongst the synthesized derivatives, compounds HK-2, HK-10 and HK-13 were found to be effective against all the three cancer cell lines. HK-2 exhibited IC50 values of 3.39 µM, 4.78 µM and 4.23 µM, HK-10 showed IC50 values of 0.81 µM, 5.89 µM, 4.96 µM and HK-13 showed IC50 values 3.24 µM, 4.93 µM and 4.73 µM against MDA-MB-231, HCT-116 and HT-29 cancer cell lines, respectively. HK-10 was found to be the most potent compound in the series with IC50 values of 0.81 µM against MDA-MB-231. In the cell cycle analysis, HK-2 and HK-10 showed cell arrest at G2/M phase of the cell cycle while HK-13 inhibited cell growth at the G1/G0 phase. All the three compounds showed cell death induced through apoptosis. In the docking studies, HK-2, HK-10 and HK-13 were found to fit well in the colchicine binding site of the tubulin. Some of the compounds in the current series were found to be promising against all the three cancer cell lines and may act as potent leads for further development.


Assuntos
Antineoplásicos/farmacologia , Pirimidinas/farmacologia , Moduladores de Tubulina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química
9.
Turk J Chem ; 44(6): 1623-1641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33488258

RESUMO

Quinoline moiety is an important scaffold in the field of drug discovery and drug development, with a wide range of pharmacological activities. Quinoline derivatives are potent inhibitors for reverse transcriptase, which is responsible for the conversion of single-stranded viral RNA into double-stranded viral DNA.In the present study, we have designed and synthesized 2 series, namely pyrazoline and pyrimidine containing quinoline derivatives as non nucleoside reverse transcriptase inhibitors (NNRTIs). Eleven compounds were synthesized and characterized by 1H and 13C NMR and mass spectrophotometry. The synthesized compounds were also docked on an HIV reverse transcriptase binding site (PDB: 4I2P); most of these compounds showed good binding interactions with the active domain of the receptor. Most of the compounds displayed a docking score higher than those of standard drugs. Among the synthesized quinoline derivatives, compound 4 exhibited the highest docking score (-10.675).

11.
Cancer Res ; 79(9): 2152-2166, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30635277

RESUMO

Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation of focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same cocultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, this study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology. SIGNIFICANCE: Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration.See related commentary by McAndrews and Kalluri, p. 2099.


Assuntos
Neoplasias da Mama , Exossomos , Animais , Linhagem Celular Tumoral , Movimento Celular , Receptores de Imidazolinas , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos
13.
Mol Cancer ; 17(1): 21, 2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29415725

RESUMO

BACKGROUND: During metastasis, tumor cells move through the tracks of extracellular matrix (ECM). Focal adhesions (FAs) are the protein complexes that link the cell cytoskeleton to the ECM and their presence is necessary for cell attachment. The tumor suppressor Nischarin interacts with a number of signaling proteins such as Integrin α5, PAK1, LIMK1, LKB1, and Rac1 to prevent cancer cell migration. Although previous findings have shown that Nischarin exerts this migratory inhibition by interacting with other proteins, the effects of these interactions on the entire FA machinery are unknown. METHODS: RT-PCR, Western Blotting, invadopodia assays, and immunofluorescence were used to examine FA gene expression and determine whether Nischarin affects cell attachment, as well as the proteins that regulate it. RESULTS: Our data show that Nischarin prevents cell migration and invasion by altering the expression of key focal adhesion proteins. Furthermore, we have found that Nischarin-expressing cells have reduced ability to attach the ECM, which in turn leads to a decrease in invadopodia-mediated matrix degradation. CONCLUSIONS: These experiments demonstrate an important role of Nischarin in regulating cell attachment, which adds to our understanding of the early events of the metastatic process in breast cancer.


Assuntos
Neoplasias da Mama/genética , Adesão Celular/genética , Receptores de Imidazolinas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Podossomos/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Receptores de Imidazolinas/metabolismo , Integrinas/genética , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Biológicos , Podossomos/metabolismo
15.
J Biol Chem ; 292(41): 16833-16846, 2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-28842496

RESUMO

Nischarin (Nisch) is a key protein functioning as a molecular scaffold and thereby hosting interactions with several protein partners. To explore the physiological importance of Nisch, here we generated Nisch loss-of-function mutant mice and analyzed their metabolic phenotype. Nisch-mutant embryos exhibited delayed development, characterized by small size and attenuated weight gain. We uncovered the reason for this phenotype by showing that Nisch binds to and inhibits the activity of AMP-activated protein kinase (AMPK), which regulates energy homeostasis by suppressing anabolic and activating catabolic processes. The Nisch mutations enhanced AMPK activation and inhibited mechanistic target of rapamycin signaling in mouse embryonic fibroblasts as well as in muscle and liver tissues of mutant mice. Nisch-mutant mice also exhibited increased rates of glucose oxidation with increased energy expenditure, despite reduced overall food intake. Moreover, the Nisch-mutant mice had reduced expression of liver markers of gluconeogenesis associated with increased glucose tolerance. As a result, these mice displayed decreased growth and body weight. Taken together, our results indicate that Nisch is an important AMPK inhibitor and a critical regulator of energy homeostasis, including lipid and glucose metabolism.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Linhagem Celular , Glucose/genética , Glucose/metabolismo , Humanos , Receptores de Imidazolinas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado/patologia , Camundongos , Camundongos Mutantes , Mutação , Oxirredução , Ligação Proteica
16.
Oncotarget ; 7(51): 84608-84622, 2016 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-27705927

RESUMO

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/metabolismo
17.
Am J Pathol ; 186(4): 844-58, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26878213

RESUMO

The actin cytoskeleton is a crucial regulator of the intestinal mucosal barrier, controlling the assembly and function of epithelial adherens and tight junctions (AJs and TJs). Junction-associated actin filaments are dynamic structures that undergo constant turnover. Members of the actin-depolymerizing factor (ADF) and cofilin protein family play key roles in actin dynamics by mediating filament severing and polymerization. We examined the roles of ADF and cofilin-1 in regulating the structure and functions of AJs and TJs in the intestinal epithelium. Knockdown of either ADF or cofilin-1 by RNA interference increased the paracellular permeability of human colonic epithelial cell monolayers to small ions. Additionally, cofilin-1, but not ADF, depletion increased epithelial permeability to large molecules. Loss of either ADF or cofilin-1 did not affect the steady-state morphology of AJs and TJs but attenuated de novo junctional assembly. The observed defects in AJ and TJ formation were accompanied by delayed assembly of the perijunctional filamentous actin belt. A total loss of ADF expression in mice did not result in a defective mucosal barrier or in spontaneous gut inflammation. However, ADF-null mice demonstrated increased intestinal permeability and exaggerated inflammation during dextran sodium sulfate-induced colitis. Our findings demonstrate novel roles for ADF and cofilin-1 in regulating the remodeling and permeability of epithelial junctions, as well as the role of ADF in limiting the severity of intestinal inflammation.


Assuntos
Cofilina 1/metabolismo , Destrina/metabolismo , Células Epiteliais/metabolismo , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Junções Íntimas/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Citoesqueleto/metabolismo , Destrina/genética , Humanos , Camundongos , Proteínas dos Microfilamentos/metabolismo , Permeabilidade
18.
Oncotarget ; 6(17): 15332-47, 2015 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-25895029

RESUMO

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 Cultivadas
19.
Am J Physiol Gastrointest Liver Physiol ; 308(9): G745-56, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25792565

RESUMO

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis.


Assuntos
Junções Aderentes/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Junções Íntimas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Actomiosina/metabolismo , Junções Aderentes/patologia , Animais , Células CACO-2 , Polaridade Celular , Cistos/metabolismo , Cistos/patologia , Células Epiteliais/patologia , Regulação da Expressão Gênica , Humanos , Mucosa Intestinal/patologia , Camundongos , Proteínas dos Microfilamentos/genética , Morfogênese , Permeabilidade , Interferência de RNA , Transdução de Sinais , Junções Íntimas/patologia , Transfecção
20.
Methods Mol Biol ; 1229: 529-41, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25325978

RESUMO

Cancer stem-like cells (CSC) have been implicated in resistance to conventional chemotherapy as well as invasion and metastasis resulting in tumor relapse in majority of epithelial cancers including colorectal cancer. Hence, targeting CSC by small molecules is likely to improve therapeutic outcomes. Glycosaminoglycans (GAGs) are long linear polysaccharide molecules with varying degrees of sulfation that allows specific GAG-protein interaction which plays a key role in regulating cancer hallmarks such as cellular growth, angiogenesis, and immune modulation. However, identifying selective CSC-targeting GAG mimetic has been marred by difficulties associated with isolating and enriching CSC in vitro. Herein, we discuss two distinct methods, spheroid growth and EMT-transformed cells, to enrich CSC and set up medium- and high-throughput screen to identify selective CSC-targeting agents.


Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Neoplásicas/patologia , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Lentivirus/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Transdução Genética
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