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1.
Biochemistry (Mosc) ; 84(11): 1424-1432, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760928

RESUMO

A large body of evidence suggests that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT), as well as expression and function of retinoid receptors, are pivotal features of tumor initiation, progression, and chemoresistance. This is also true for pancreatic ductal adenocarcinoma (PDAC), which represents a clinical challenge due to poor prognosis and increasing incidence. Understanding the above features of cancer cells could open new avenues for PDAC treatment strategies. The aim of this study was to investigate the relation between CSCs, EMT, and retinoid receptors in PDAC after treatment with the chemotherapeutic agents - gemcitabine and 5-fluorouracil. First, we demonstrated the difference in the expression levels of CSC and EMT markers and retinoid receptors in the untreated Mia PaCa-2 and Panc1 cells that also differed in the frequency of spontaneous apoptosis and distribution between the cell cycle phases. Chemotherapy reduced the number of cancer cells in the S phase. Gemcitabine and 5-fluorouracil modulated expression of CSC markers, E-cadherin, and RXRß in Panc1 but not in Mia PaCa-2 cells. We suggest that these effects could be attributed to the difference in the basal levels of expression of the investigated genes. The obtained data could be interesting in the context of future preclinical research.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptor X Retinoide beta/metabolismo , Apoptose/efeitos dos fármacos , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Fluoruracila/farmacologia , Humanos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Receptor X Retinoide beta/genética , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos
3.
Chemotherapy ; 64(3): 146-154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31715603

RESUMO

OBJECTIVE: Colorectal cancer (CRC) remains a major cause of cancer-related death worldwide. Proteasome 26S subunit ATPase 2 (PSMC2) plays vital roles in regulating cell cycle and transcription and has been confirmed to be a gene potentially associated with some human tumors. However, the expression correlation and molecular mechanism of PSMC2 in CRC are still unclear. This study aimed to investigate the role of PSMC2 in malignant behaviors in CRC. METHODS: The high protein levels of PSMC2 in CRC samples were identified by tissue microarray analysis. Lentivirus was used to silence PSMC2 in HCT116 and RKO cells; MTT and colony formation assay were performed to determine cell proliferation. Wound healing and Transwell assay were used to detect cell migration and invasion. Flow cytometry assay was applied to detect cell cycle and apoptosis. RESULT: The results showed that, among the 96 CRC patients, the expression of PSMC2 was a positive correlation with the clinicopathological features of the patients with CRC. Furthermore, the low PSMC2 expression group showed a higher survival rate than the high PSMC2 expression group. The expression levels of PSMC2 in cancer tissue were dramatically upregulated compared with adjacent normal tissues. In vitro, shPSMC2 was designed to inhibit the expression of PSMC2 in CRC cells. Compared with shCtrl, silencing of PSMC2 significantly suppressed cell proliferation, decreased single cell colony formation, enhanced apoptosis, and accelerated G2 phase and/or S phase arrest. CONCLUSION: Survival analysis indicated that high expression of PSMC2 in the CRC samples was associated with poorer survival rate than low expression of PSMC2, while the anti-tumor effect of PSMC2 silencing was also confirmed at the cellular level in vitro. Our results suggested that PSMC2 potentially worked as a regulator for CRC, and the silencing of PSMC2 may be a therapeutic strategy for CRC.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Apoptose , Proliferação de Células , Neoplasias Colorretais/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , Idoso , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Prognóstico , Complexo de Endopeptidases do Proteassoma/genética , RNA Interferente Pequeno/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular
4.
Chem Biol Interact ; 311: 108798, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31433962

RESUMO

Natural products are a valuable source of anticancer agents, with many naturally derived compounds currently used in clinical and preclinical treatments. This study aims to investigate the antiproliferative activity and potential mechanism of action of the xanthoquinodin JBIR-99, isolated from fungi Parengyodontium album MEXU 30,054 and identified by single-crystal X-ray crystallography. Cytotoxicity of xanthoquinodin was evaluated in a panel of human cancer cells lines and CCD-112-CoN normal colon cells, using the sulforhodamine B assay. PC-3 prostate cancer cells were used in biochemical assays including cell cycle, mitochondrial transmembrane potential (MTP), reactive oxygen species (ROS) and caspase activity. Expression levels of apoptosis-pathway-related proteins were analyzed by Western blot. The in vivo toxicity of xanthoquinodin was determined using a zebrafish model. Xanthoquinodin showed cytotoxicity in all cancer cell lines but demonstrated relative selective potency against PC-3 cells with an IC50 1.7 µM. In CCD-112-CoN cells, xanthoquinodin was non-cytotoxic at 100 µM. In PC-3 cells, the compound induced loss of MTP, production of ROS, and cell cycle arrest in S phase. The expression and activity of caspase-3 was increased, which correlates with the upregulation of Cyt c, Bax, nuclear factor kappa-B (NF-κB) (p65) and IKKß, and downregulation of poly ADP ribose polymerase (PARP-1) and Bcl-2. Lastly, xanthoquinodin did not cause any visible developmental toxicity in zebrafish at 50 µM. These results demonstrate xanthoquinodin induces apoptosis in PC-3 prostate cancer cells by activation of both intrinsic and extrinsic apoptotic pathways. In addition, the non-toxic effect in vivo indicates that xanthoquinodin could be a useful lead in the development of a novel, anti-cancer agent that is selective for prostate cancer.


Assuntos
Apoptose/efeitos dos fármacos , Ascomicetos/química , Cromonas/farmacologia , Ascomicetos/metabolismo , Linhagem Celular Tumoral , Cromonas/química , Cristalografia por Raios X , Citocromos c/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Conformação Molecular , Poli(ADP-Ribose) Polimerase-1/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
5.
PLoS Genet ; 15(8): e1008294, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31369547

RESUMO

The DNA damage checkpoint response is controlled by the phosphatidylinositol 3-kinase-related kinases (PIKK), including ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR). ATR forms a complex with its partner ATRIP. In budding yeast, ATR and ATRIP correspond to Mec1 and Ddc2, respectively. ATRIP/Ddc2 interacts with replication protein A-bound single-stranded DNA (RPA-ssDNA) and recruits ATR/Mec1 to sites of DNA damage. Mec1 is stimulated by the canonical activators including Ddc1, Dpb11 and Dna2. We have characterized the ddc2-S4 mutation and shown that Ddc2 not only recruits Mec1 to sites of DNA damage but also stimulates Mec1 kinase activity. However, the underlying mechanism of Ddc2-dependent Mec1 activation remains to be elucidated. Here we show that Ddc2 promotes Mec1 activation independently of Ddc1/Dpb11/Dna2 function in vivo and through ssDNA recognition in vitro. The ddc2-S4 mutation diminishes damage-induced phosphorylation of the checkpoint mediators, Rad9 and Mrc1. Rad9 controls checkpoint throughout the cell-cycle whereas Mrc1 is specifically required for the S-phase checkpoint. Notably, S-phase checkpoint signaling is more defective in ddc2-S4 mutants than in cells where the Mec1 activators (Ddc1/Dpb11 and Dna2) are dysfunctional. To understand a role of Ddc2 in Mec1 activation, we reconstituted an in vitro assay using purified Mec1-Ddc2 complex, RPA and ssDNA. Whereas ssDNA stimulates kinase activity of the Mec1-Ddc2 complex, RPA does not. However, RPA can promote ssDNA-dependent Mec1 activation. Neither ssDNA nor RPA-ssDNA efficiently stimulates the Mec1-Ddc2 complex containing Ddc2-S4 mutant. Together, our data support a model in which Ddc2 promotes Mec1 activation at RPA-ssDNA tracts.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA , DNA de Cadeia Simples/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
6.
J Agric Food Chem ; 67(36): 10245-10255, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31389238

RESUMO

Ginseng has been widely used as a functional food in the world because of its well-defined health benefits. Previous studies have confirmed that AD-1, a new ginsenoside derived from ginseng, can ameliorate thioacetamide-induced liver injury and fibrosis in mice. Simultaneously, amino acid supplementation is getting more attention as an important adjuvant therapy in the improvement of hepatopathy. The aim of this study was to conjugate AD-1 with several selected amino acids and investigate the cytotoxicity of the obtained conjugates in activated t-HSC/Cl-6 cells and normal human liver cells (LO2). Structure-activity relationships of conjugates and underlying mechanisms of the effect are also explored. The results indicated that conjugate 7c remarkably inhibited cell proliferation in activated t-HSC/Cl-6 cells (IC50 = 3.8 ± 0.4 µM) and appeared to be nontoxic to LO2. Besides, conjugate 7c had a relatively good plasma stability. Further study demonstrated that inducing S-phase arrest and activation of mitochondrial-mediated apoptosis were included in the mechanisms underlying the efficiency of conjugate 7c. These findings provided further insight into designing functional foods (ginsenoside and amino acid) for the application in prevention or improvement of liver fibrosis.


Assuntos
Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ginsenosídeos/farmacologia , Células Estreladas do Fígado/citologia , Aminoácidos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ginsenosídeos/química , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos
7.
PLoS Genet ; 15(8): e1008136, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381575

RESUMO

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Quinase 3 da Glicogênio Sintase/genética , Hidroxiureia/toxicidade , Nucleotídeos/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética
8.
Food Chem Toxicol ; 131: 110552, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31163220

RESUMO

[OBJECTIVE]: Di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, may act as an endocrine disruptor and cause developmental toxicity. Differentiated human embryonic stem cells (hESCs) were used to investigate the underlying mechanism of the embryotoxicity induced by DEHP. [Materials and Methods] H9-hESCs were treated with DEHP at different concentrations for 10 days, and the cytotoxicity of DEHP on cell proliferation was determined using a cell-microelectronic sensing technique (Real-Time Cellular Analysis: RTCA). Based on the 50% inhibitory proliferation concentration (IC50), differentiated H9-hESCs were treated with DEHP at 0, 50, 100, and 200 µg/ml for 120 h, followed by measurement of its toxic effects on the transcriptome by mRNA microarray and QuantiGene Plex (QGP). Proteins were detected by the iTRAQ-based proteomics method and the proteins related to the PPARγ/PTEN/Akt pathways were measured by western blotting. The progression of the cell cycle and apoptosis were characterized using flow cytometry (FCM). In other experiments, hESCs were pre-treated with GW9662 (20 µM), a specific PPARγ inhibitor, for 30 min, followed by exposure to GW9662 (20 µM) and DEHP (200 µg/ml) for 120 h to observe the underlying mechanism of DEHP's embryotoxicity. [RESULTS]: DEHP inhibited H9-hESC cell proliferation in a dose-dependent manner, with an IC50 of 165.78 µg/ml. FCM results showed that DEHP could markedly induce cell cycle arrest and increase apoptosis. Gene microarray and QPG array analyses indicated that the peroxisome proliferator-activated receptor γ (PPARγ) was an apparent target for DEHP. We further demonstrated that DEHP could activate the PPARγ and upregulate the expression of PTEN downstream genes, and then play a negative role in the AKT signaling pathway. Cells pretreated with PPARγ inhibitor, GW9662, were shown to restore the effect of DEHP on the PPARγ/PTEN/AKT signaling pathway, and induce the recovery of cell cycle arrest and apoptosis. [CONCLUSION]: DEHP inhibited cell proliferation, promoted cell cycle arrest, and induced apoptosis through the PPARγ/PTEN/AKT signaling pathway in differentiated human embryonic stem cells. It suggested that DEHP exposure possibly cause reproductive or developmental toxicity in humans through the PPARγ signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Dietilexilftalato/toxicidade , Disruptores Endócrinos/toxicidade , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Emulsões/síntese química , Emulsões/química , Humanos , PPAR gama/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Plastificantes/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Soroalbumina Bovina/química , Teratogênios/toxicidade
9.
Eur J Med Chem ; 177: 338-349, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158748

RESUMO

A series of styrylquinolines was designed and synthesized based on the four main quinoline scaffolds including oxine, chloroxine and quinolines substituted with a hydroxyl group or chlorine atom at the C4 position. All of the compounds were tested for their anticancer activity on wild-type colon cancer cells (HCT 116) and those with a p53 deletion. Analysis of SAR revealed the importance of electron-withdrawing substituents in the styryl part and chelating properties in the quinoline ring. The compounds that were more active were also tested on a panel of four cancer cell lines with mutations in TP53 tumor suppressor gene. The results suggest that styrylquinolines induce cell cycle arrest and activate a p53-independent apoptosis. The apparent mechanism of action was studied for the most promising compounds, which produced reactive oxygen species and changed the cellular redox balance.


Assuntos
Antineoplásicos/farmacologia , Quinolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Estirenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Heme Oxigenase-1/metabolismo , Humanos , Estrutura Molecular , Poli(ADP-Ribose) Polimerase-1/metabolismo , Quinolinas/síntese química , Quinolinas/química , Quinolinas/toxicidade , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Relação Estrutura-Atividade , Estirenos/síntese química , Estirenos/química , Estirenos/toxicidade , Proteína Supressora de Tumor p53/metabolismo
10.
Eur J Med Chem ; 177: 350-361, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158749

RESUMO

The relationship between chemical structure and in vitro cytotoxic activities of a series of azastibocine-framework organoantimony(III) halide complexes against cancerous (HepG2, MDA-MB-231, MCF-7 and HeLa) and nonmalignant (HEK-293) cell lines was studied for the first time. A positive correlation between cytotoxic activity and the length of N→Sb coordinate bond on azastibocine framework of same nitrogen substituent was observed. By comparison, the organoantimony(III) complex 6-cyclohexyl-12-fluoro-5,6,7,12-tetrahydrodibenzo[c,f][1,5]azastibocine (C4) exhibited the highest selectivity index, giving a IC50(nonmalignant)/IC50(cancerous) ratio of up to 8.33. The results of cell cycle analysis indicated that the inhibitory effect of C4 on the cellular viability was caused by cell cycle arrest mainly at the S phase. The necrosis induced by C4 was confirmed by the Trypan blue dye exclusion test and the increase of lactic dehydrogenase (LDH) released in the culture medium. Furthermore, evaluation of the levels of intracellular reactive oxygen species (ROS) in MDA-MB-231 cells, by quantifying the relative fluorescence units (RFU) using spectrofluorometer, indicated that cytotoxic activity of C4 is dependent on the production of ROS. This work established the correlation between cytotoxic activity and N→Sb inter-coordination, a finding that provided theoretical and experimental basis for in-depth design of antimony-based organometallic complexes as potential anticancer agents.


Assuntos
Antimônio/química , Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , L-Lactato Desidrogenase/metabolismo , Estrutura Molecular , Necrose/induzido quimicamente , Espécies Reativas de Oxigênio/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Relação Estrutura-Atividade
11.
Food Funct ; 10(6): 3626-3636, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31162493

RESUMO

In this study, the apoptosis induction and antitumor activity of a novel complex, seleno-ß-lactoglobulin (Se-ß-Lg), on H22 cells were explored. In in vitro experiments, the MTT assay showed that Se-ß-Lg was cytotoxic to H22 cells in a concentration- and time-dependent manner and displayed few proliferation inhibition effects on normal liver L02 cells. Annexin V-FITC/PI and PI staining assays showed that Se-ß-Lg induced apoptosis changes of H22 cells from early to late apoptosis and led to S phase cell cycle arrest. Western blot and Z-VAD-FMK inhibitor assays showed that Se-ß-Lg triggered the Fas/FasL-mediated caspase 8-dependent extrinsic death receptor pathway in H22 cells. In in vivo experiments, Se-ß-Lg effectively repressed the growth of transplanted H22 solid tumors in a dose-dependent manner and exhibited few toxic effects on the host animals. H&E and PI staining of tumor tissues showed that Se-ß-Lg caused the occurrence of typical apoptosis morphology features and dose-dependently increased the proportion of apoptosis peaks (Sub-G1 peak) in H22 solid tumors. These results suggest that Se-ß-Lg has the capacity to induce H22 tumor cell apoptosis in vitro and in vivo and support that Se-ß-Lg can be applied as a functional complex in food.


Assuntos
Lactoglobulinas/farmacologia , Leite/química , Selênio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 8/genética , Caspase 8/metabolismo , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Lactoglobulinas/química , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Selênio/química
12.
Eur J Pharmacol ; 857: 172448, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202802

RESUMO

Phosphodiesterases are promising targets for pharmacological intervention against various diseases. There are already inhibitors of PDE3, PDE4 and PDE5 as approved drugs. However there is an unmet need to discover new chemical scaffolds as PDE inhibitors. The main drawback of most of PDE inhibitors is their non specificity; owing to their structural resemblance to cAMP or cGMP. Natural product compounds offer high structural diversity hence may provide new PDE inhibitors. We decided to screen our institutional natural product compound library of nearly 900 molecules for PDE5 inhibition and explore the selectivity against PDE1-11 and cytotoxicity of the hit molecule/s. Rottlerin was identified as a PDE5 inhibitor. It was found to inhibit other PDEs with varying specificities. Structure activity relationship data and molecular dynamics studies showed that Tyr612, Asp764, Gln817 and Phe820 in the binding pocket of PDE5 play an important role in the activity of rottlerin. As a pan PDE inhibitor, rottlerin was also found to activate the AMPK pathway and induce neurodifferentiation in IMR-32 cells, with the effect more efficient in samples co-treated with cAMP activator Forskolin. Rottlerin at higher concentrations was shown to induce autophagy, apoptosis and G2/S cell cycle arrest in IMR-32 cells.


Assuntos
Acetofenonas/farmacologia , Benzopiranos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Acetofenonas/metabolismo , Autofagia/efeitos dos fármacos , Benzopiranos/metabolismo , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Inibidores de Fosfodiesterase/metabolismo , Diester Fosfórico Hidrolases/química , Conformação Proteica , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
13.
Eur J Pharmacol ; 855: 137-148, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31059711

RESUMO

Selective pharmacologic targeting of cell cycle regulators is a potent anti-cancer therapeutic strategy. Here, we show that caspase-3-mediated p21 cleavage involves p53 independent of triptolide (TPL)-induced S phase arrest in human type 1 nasopharyngeal carcinoma (NPC) cells. Coimmunoprecipitation studies demonstrated that TPL causes S phase cell cycle arrest by suppressing the formation of cyclin A-phosphor (p)-cyclin-dependent kinas 2 (CDK2) (Thr 39) complexes. Ectopic expression of constitutively active protein kinase B1 (Akt1) blocks the induction of S phase arrest and the suppression of cyclin A expression and CDK2 Thr 39 phosphorylation by TPL. Expression of the phosphomimetic mutant CDK2 (T39E) rescues the cells from TPL-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TPL-triggered S phase arrest. Treatment with TPL induces an increase in the formation of complexes between unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and p85α in the plasma membrane. Decreased microRNA (miR)-144 expression and increased PTEN expression after TPL treatment were demonstrated, and TPL-enhanced p85α-PTEN complexes and inhibitory effects on Akt (Ser 473) phosphorylation and S phase arrest were suppressed by ectopic PTEN short hairpin RNA or miR-144 expression. Knockdown of endogenous miR-144 by miR-144 Trap upregulated PTEN expression and accordingly enhanced p85α-PTEN complex formation and S phase arrest. Collectively, the effect of TPL on S phase arrest in human NPC cells is likely to enhance the p85α-PTEN interaction in the plasma membrane by suppressing miR-144 expression, resulting in the attenuation of cyclin A-p-CDK2 (Thr 39) complex formation via Akt inactivation.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Diterpenos/farmacologia , Regulação para Baixo/efeitos dos fármacos , MicroRNAs/genética , Neoplasias Nasofaríngeas/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fenantrenos/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Compostos de Epóxi/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Treonina/metabolismo , Quinases Ativadas por p21/metabolismo
14.
Int J Mol Med ; 44(1): 115-124, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059073

RESUMO

Musashi RNA­binding protein 1 (MSI1) is highly expressed in several types of cancer; however, its role in oral squamous cell carcinoma (OSCC) remains unknown. The purpose of this study was to investigate the probable mechanism underlying the involvement of MSI1 in OSCC. The results demonstrated that MSI1 was upregulated in OSCC tissues, but not in adjacent healthy tissues. MSI1 silencing resulted in decreased cell proliferative, invasive and migrative capacity. In addition, MSI1 silencing led to cell cycle arrest at the S phase, downregulation of c­Myc and cyclin D1, and upregulation of p21 and p27 levels. Additional studies demonstrated that MSI1 suppression inhibited the activation of signal transducer and activator of transcription 3 (STAT3) signaling. Accordingly, the findings of the present study suggested that MSI1 silencing can suppress OSCC cell proliferation and progression, in part by inhibiting the activation of the c­Myc/STAT3 pathway.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Fator de Transcrição STAT3/genética
15.
J Med Food ; 22(5): 444-450, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084542

RESUMO

Studies have identified the potential of chemopreventive effects of sulforaphane (SFN); however, the underlying mechanisms of its effect on breast cancer require further elucidation. This study investigated the anticancer effects of SFN that specifically induces G1/S arrest in breast ductal carcinoma (ZR-75-1) cells. The proliferation of the cancer cells after treatment with SFN was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. DNA content and cell cycle status were analyzed through flow cytometry. Our results demonstrated the inhibition of growth in ZR-75-1 cells upon SFN exposure. In addition, SERTAD1 (SEI-1) caused the accumulation of SFN-treated G1/S-phase cells. The downregulation of SEI-1, cyclin D2, and histone deacetylase 3 suggested that in addition to the identified effects of SFN against breast cancer prevention, it may also exert antitumor activities in established breast cancer cells. In conclusion, SFN can inhibit growth of and induce cell cycle arrest in cancer cells, suggesting its potential role as an anticancer agent.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Isotiocianatos/farmacologia , Proteínas Nucleares/genética , Transativadores/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D2/genética , Ciclina D2/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Extratos Vegetais/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transativadores/metabolismo , Fatores de Transcrição , Verduras/química
16.
Environ Mol Mutagen ; 60(8): 740-751, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31095781

RESUMO

The antitumor activity of resveratrol, a polyphenolic compound found mainly in grapes, has been studied in several types of cancer. In bladder cancer, its antiproliferative effects have already been demonstrated; however, its mechanism of action is not completely understood. The aim of this study was to evaluate resveratrol antitumor activity (12.5, 25, 50, 100, 150, 200, and 250 µM) and its possible mechanisms of action in bladder tumor cells with different TP53 gene status (RT4, grade 1, TP53 wild type; 5637-grade 2 and T24-grade 3, TP53 mutated). Cell proliferation, clonogenic survival, morphological changes, cell cycle progression, apoptosis rates, genotoxicity, global methylation, immunocytochemistry for p53 and PCNA and relative expression profiles of the AKT, mTOR, RASSF1A, HOXB3, SRC, PLK1, and DNMT1 were evaluated. Resveratrol decreased cell proliferation and induced DNA damage in all cell lines. Regarding the long-term effects, resveratrol reduced the number of colonies in all cell lines; however, TP53 wild type cells were more resistant. Increased rates of apoptosis were found in the TP53 wild type cells and this was accompanied by AKT, mTOR, and SRC downregulation. In addition, the resveratrol antiproliferative effects in wild type TP53 cells were accompanied by modulation of the DNMT1 gene. In the TP53 mutated cells, cell cycle arrest at S phase with PLK1 downregulation was observed. Additionally, there was modulation of the HOXB3/RASSF1A pathway and nuclear PCNA reduction in the highest-grade cells. In conclusion, resveratrol has antiproliferative activity in bladder tumor cells; however, the mechanisms of action are dependent on TP53 status. Environ. Mol. Mutagen., 60:740-751, 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Resveratrol/farmacologia , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/genética , Dano ao DNA/genética , Humanos , Antígeno Nuclear de Célula em Proliferação/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteína Supressora de Tumor p53/biossíntese
17.
Mol Carcinog ; 58(7): 1248-1259, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31100197

RESUMO

Purpurogallin is a natural compound that is extracted from nutgalls and oak bark and it possesses antioxidant, anticancer, and anti-inflammatory properties. However, the anticancer capacity of purpurogallin and its molecular target have not been investigated in esophageal squamous cell carcinoma (ESCC). Herein, we report that purpurogallin suppresses ESCC cell growth by directly targeting the mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling pathway. We found that purpurogallin inhibits anchorage-dependent and -independent ESCC growth. The results of in vitro kinase assays and cell-based assays indicated that purpurogallin also strongly attenuates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and also directly binds to and inhibits MEK1 and MEK2 activity. Furthermore, purpurogallin contributed to S and G2 phase cell cycle arrest by reducing cyclin A2 and cyclin B1 expression and also induced apoptosis by activating poly (ADP ribose) polymerase (PARP). Notably, purpurogallin suppressed patient-derived ESCC tumor growth in an in vivo mouse model. These findings indicated that purpurogallin is a novel MEK1/2 inhibitor that could be useful for treating ESCC.


Assuntos
Antineoplásicos/farmacologia , Benzocicloeptenos/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina A2/biossíntese , Ciclina B1/biossíntese , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Camundongos , Preparações de Plantas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Eur J Pharmacol ; 856: 172400, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31103630

RESUMO

Eosinophils and their granular proteins are crucial for combating allergic airway diseases. Eosinophils derived from HL-60 clone 15 (HC15) cells have been established as a feasible alternative cell model for human primary eosinophils. Simvastatin, a cholesterol-lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Among the granular eosinophil proteins, eosinophil cationic protein (ECP) is the one best recognised in allergic airway diseases. The aim of our study is to investigate the effect and regulatory mechanisms of simvastatin on ECP levels derived from eosinophils. Both HC15 cell counts and ECP levels decreased after simvastatin treatment in the animal and cell models; however, after a cell count adjustment, simvastatin was not observed to exert a significantly inhibitory effect on ECP expression. Real-time polymerase chain reaction and Western blotting analyses demonstrated that simvastatin did not inhibit the intracellular formation or release of ECP. Cell cycle analysis showed that the percentage of HC15 cells in the G1 and S phases significantly increased and decreased, respectively, after simvastatin treatment. Simvastatin inhibited the proliferation of HC15-derived eosinophils by inducing G1/S cell cycle arrest in a dose-dependent manner. Its effect on the cell cycle involved the downregulation of cyclin A but without the presence of mevalonate; therefore, total ECP expression from eosinophils decreased, not by suppressing the actual formation or release of ECP but by arresting the G1/S cell cycle phase and inhibiting subsequent cell proliferation through the mevalonate pathway.


Assuntos
Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Clonais/citologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Masculino , Ácido Mevalônico/farmacologia , Ratos , Ratos Sprague-Dawley , Sinvastatina/antagonistas & inibidores
19.
Mol Cell ; 75(1): 76-89.e3, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31101497

RESUMO

Cyclin-dependent kinases (CDKs) coordinate hundreds of molecular events during the cell cycle. Multiple cyclins are involved, but the global role of cyclin-specific phosphorylation has remained unsolved. We uncovered a cyclin docking motif, LxF, that mediates binding of replication factor Cdc6 to mitotic cyclin. This interaction leads to phospho-adaptor Cks1-mediated inhibition of M-CDK to facilitate Cdc6 accumulation and sequestration in mitosis. The LxF motif and Cks1 also mediate the mutual inhibition between M-CDK and the tyrosine kinase Swe1. Additionally, the LxF motif is critical for targeting M-CDK to phosphorylate several mitotic regulators; for example, Spo12 is targeted via LxF to release the phosphatase Cdc14. The results complete the full set of G1, S, and M-CDK docking mechanisms and outline the unified role of cyclin specificity and CDK activity thresholds. Cooperation of cyclin and Cks1 docking creates a variety of CDK thresholds and switching orders, including combinations of last in, first out (LIFO) and first in, first out (FIFO) ordering.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclinas/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Pontos de Checagem da Fase M do Ciclo Celular/genética , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
20.
Eur J Pharmacol ; 851: 161-173, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30817902

RESUMO

Polyphyllin VI (PP-VI) is one of the major saponins present in Paris polyphylla Sm., a medicinal plant primarily used for cancer treatment in China and India. However, its anti-metastatic activity remains largely unknown. The current study thus investigated the anti-metastatic activity of PP-VI in mouse mammary carcinoma 4T1 and human breast cancer MDA-MB-231 cells. The anti-metastatic effect of PP-VI was investigated at a sub-cytotoxic dose in migration and invasion assays in vitro. Experimental metastasis mouse model was used to examine the anti-metastatic effect of PP-VI in vivo. Additionally, target prediction, real-time PCR, Western blotting and luciferase assay were performed to identify the target gene of a pro-metastatic microRNA, miR-18a in 4T1 cells. The effect of PP-VI on the identified target of miR-18a was further investigated. The results showed that PP-VI impaired the viability of 4T1 and MDA-MB-231 cells. Moreover, when applied at a sub-cytotoxic dose, PP-VI suppressed the metastatic potential of 4T1 and MDA-MB-231 cells. Receptor expressed in lymphoid tissue (RELT)-like 2 (Rell2) was identified as a direct target of miR-18a with anti-metastatic functions in 4T1 and MDA-MB-231 cells. PP-VI treatment resulted in increased expression of Rell2 and decreased level of miR-18a in 4T1 and MDA-MB-231 cells. PP-VI treatment also attenuated miR-18a mimic or Rell2 siRNA-augmented migration of MDA-MB-231 cells. The current work thus demonstrates for the first time that targeted regulation of Rell2 by miR-18a is in part implicated in the anti-metastatic effect of PP-VI in breast cancer cells, providing novel pharmacological insights into the anti-cancer effect of PP-VI.


Assuntos
Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Saponinas/farmacologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/genética
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