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1.
Gastroenterol Hepatol ; 44(1): 1-12, 2021 Jan.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-33039171

RESUMO

BACKGROUND: Topotecan is an anti-cancer chemotherapy drug with common side effects, including hepatotoxicity. In this study, we aim to investigate the mechanisms of topotecan-induced hepatocellular injury beyond conventional DNA damage. MATERIALS AND METHODS: Methyl Thiazolyl Tetrazolium (MTT) assay was used to detect the inhibitory effect of topotecan on cell proliferation. Western blot was used to detect protein expression. Flow cytometry assay was performed to determine apoptosis rate under topotecan treatment. ASCT2 overexpression was addressed using adenovirus vector. qRT-PCR and western blot assay were used to detect the expression of ASCT2. Glutamine uptake, intracellular glutathione (GSH) and reactive oxygen species (ROS) level were detected by glutamine detection kit, GSH detection kit and ROS detection kit respectively. RESULTS: MTT results showed that topotecan had an inhibitory effect on cell proliferation and induced apoptosis in both L02 and HepG2 cell lines. Topotecan inhibited the expression of glutamine transporter ASCT2 and the uptake of glutamine in both L02 and HepG2 cell lines. The uptake of glutamine and the GSH level was increased in both L02 and HepG2 cell lines after ASCT2 overexpression. The ROS level was inhibited by ASCT2 overexpression upon topotecan treatment in both L02 and HepG2 cell lines. Topotecan-induced hepatocellular apoptosis and proliferation inhibition were attenuated by ASCT2 overexpression in both L02 and HepG2 cell lines. CONCLUSION: Topotecan-induced hepatocytes death is dependent on ASCT2 down-regulation, which causes oxidative stress via inhibiting GSH production.


Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Glutamina/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Estresse Oxidativo/fisiologia , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologia , Sistema ASC de Transporte de Aminoácidos/efeitos dos fármacos , Sistema ASC de Transporte de Aminoácidos/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/genética , Espécies Reativas de Oxigênio/metabolismo
2.
Int Immunopharmacol ; 88: 106791, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32871480

RESUMO

Delvestidine (DLTD) is a monomeric compound isolated from Aconitum leucostomum Worosch, a widely used medicine for local treatment of rheumatoid arthritis (RA). Studies have shown that Aconitum leucostomum Worosch. can inhibit maturation of bone marrow-derived dendritic cells (BMDCs). Further, microRNAs (miRNAs) have regulatory effects on DC maturity and function. However, the mechanism underlying DLTD effects on DC maturity and RA remains to be elucidated. This study investigated whether DLTD-mediated inhibition of DC maturation is regulated by miRNAs. LPS-induced mature BMDCs were treated with DLTD for 48 h. CD80 and CD86 expression on BMDCs was detected by flow cytometry, and levels of inflammatory factors IL-6, IL-23, IL-1ß, and TNF-α were detected by ELISA and PCR. Further, gene expression and miRNA expression profiles were investigated by bioinformatics analysis and verified by PCR. DLTD was found to inhibit CD80 and CD86 expression on the surface of BMDCs and secretion of inflammatory factors IL-6, IL-23, IL-1ß, and TNF-α. In total, 54 differentially expressed miRNAs were detected, including 29 up-regulated and 25 down-regulated miRNAs after DLTD treatment. Analysis of biological information revealed that the differentially expressed target genes mainly regulated biological processes, including cell differentiation, cell cycle, and protein kinase complexes. Additionally, miR-511-3p downstream targets Calcr, Fzd10, and Eps8, were closely related to BMDCs maturation. DLTD may induce BMDCs maturity through regulation of miRNAs that affect Calcr, Fzd10, and Eps8 gene signals.


Assuntos
Aconitum/química , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , MicroRNAs/imunologia , MicroRNAs/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígeno B7-1/efeitos dos fármacos , Antígeno B7-1/metabolismo , Antígeno B7-2/efeitos dos fármacos , Antígeno B7-2/metabolismo , Proteína Semelhante a Receptor de Calcitonina/efeitos dos fármacos , Proteína Semelhante a Receptor de Calcitonina/genética , Diferenciação Celular , Células Cultivadas , Biologia Computacional , Citocinas/genética , Citocinas/metabolismo , Receptores Frizzled/efeitos dos fármacos , Receptores Frizzled/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/genética , Receptores de Citocinas/efeitos dos fármacos , Receptores de Citocinas/genética
3.
Am J Obstet Gynecol ; 223(5): 733.e1-733.e14, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32417359

RESUMO

BACKGROUND: Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress ß1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies. OBJECTIVE: This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. STUDY DESIGN: This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 µM, or control) for 48 hours. Protein and mRNA levels of ß1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction. RESULTS: We found that simvastatin significantly reduced the protein expression of ß1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in ß1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1. CONCLUSION: The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Leiomioma/genética , Mecanotransdução Celular/efeitos dos fármacos , Sinvastatina/farmacologia , Neoplasias Uterinas/genética , Proteínas de Ancoragem à Quinase A/efeitos dos fármacos , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ciclina D1/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina beta1/efeitos dos fármacos , Integrina beta1/genética , Integrina beta1/metabolismo , Leiomioma/metabolismo , Mecanotransdução Celular/genética , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Quinase de Cadeia Leve de Miosina/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Neoplasias Uterinas/metabolismo , Quinases Associadas a rho/efeitos dos fármacos , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
4.
Pharmacol Res ; 158: 104844, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32438035

RESUMO

Glutamine metabolism, described as major energy and building blocks supply to cell growth, has gained great attention. Alanine-Serine-Cysteine Transporter (ASCT2), which belongs to solute carried (SLC) family transporters and is encoded by the SLC1A5 gene serves as a significant role for glutamine transport. Indeed, ASCT2 is often overexpressed in highly proliferative cancer cells to fulfill enhanced glutamine demand. So far, ASCT2 has been proved to be a significant target during the carcinogenesis process, and emerging evidence reveals that ASCT2 inhibitors can provide a benefit strategy for cancer therapy. Herein, we describe the structure of ASCT2, and summarize its related regulatory factors which are associated with antitumor activity. Moreover, this review article highlights the remarkable reform of discovery and development for ASCT2 inhibitors. On the basis of case studies, our perspectives for targeting ASCT2 and development of ASCT2 antagonist are discussed in the final part.


Assuntos
Sistema ASC de Transporte de Aminoácidos/efeitos dos fármacos , Sistema ASC de Transporte de Aminoácidos/genética , Antineoplásicos/farmacologia , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/genética , Neoplasias/genética , Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Sistema ASC de Transporte de Aminoácidos/química , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Regulação da Expressão Gênica , Humanos , Antígenos de Histocompatibilidade Menor/química
5.
Toxicol In Vitro ; 62: 104679, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31676337

RESUMO

Ruthenium complexes are being considered as novel chemotherapeutic alternatives for cancer treatment. In our study, we assessed the antitumoral activities of novel ruthenium complexes coupled to the amino acids proline (RuPro) and threonine (RuThr) in prostate tumor cell lines (DU145) and breast (MCF7), and normal cell lines of the lung fibroblast (GM07492A). Our results revealed that the EC50 of the complexes for DU145 and MCF7 was two times lower than that GM07492A. Moreover, RuPro and RuThr were not able to induce significant genomic instability, cell cycle arrest or cell death in GM07492A, but could induce DNA damage, arrest in G2/M and apoptosis in DU145 and MCF7. Furthermore, BAX, TP53 and ATM were found to be upregulated in DU145 and MCF7 treated with RuPro and RuThr, in which, a higher ASCT2 gene expression was also observed. Using molecular docking, RuPro and RuThr interact with ASCT2, suggesting that this transporter might have a pivotal role in the execution of their activities. Hence, our results with RuPro and RuThr are capable of selectively inducing genetic damage, cell cycle arrest and apoptosis in DU145 and MCF7. We suggest that the selective action of the RuPro and RuThr complexes is related to the higher expression of ASCT2 in the tumor cells.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quelantes/farmacologia , Instabilidade Genômica/efeitos dos fármacos , Prolina/química , Neoplasias da Próstata/tratamento farmacológico , Compostos de Rutênio/farmacologia , Treonina/química , Sistema ASC de Transporte de Aminoácidos/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Feminino , Humanos , Ligantes , Masculino , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Simulação de Acoplamento Molecular , Neoplasias da Próstata/patologia
6.
Mol Cell Biol ; 39(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30348839

RESUMO

Phorbol 12-myristate 13-acetate (PMA) promotes skin cancer in rodents. The mutations found in murine tumors are similar to those found in human skin cancers, and PMA promotes proliferation of human skin cells. PMA treatment of human keratinocytes increases the synthesis of APOBEC3A, an enzyme that converts cytosines in single-stranded DNA to uracil, and mutations in a variety of human cancers are attributed to APOBEC3A or APOBEC3B expression. We tested here the possibility that induction of APOBEC3A by PMA causes genomic accumulation of uracils that may lead to such mutations. When a human keratinocyte cell line was treated with PMA, both APOBEC3A and APOBEC3B gene expression increased, anti-APOBEC3A/APOBEC3B antibody bound a protein(s) in the nucleus, and nuclear extracts displayed cytosine deamination activity. Surprisingly, there was little increase in genomic uracils in PMA-treated wild-type or uracil repair-defective cells. In contrast, cells transfected with a plasmid expressing APOBEC3A acquired more genomic uracils. Unexpectedly, PMA treatment, but not APOBEC3A plasmid transfection, caused a cessation in cell growth. Hence, a reduction in single-stranded DNA at replication forks may explain the inability of PMA-induced APOBEC3A/APOBEC3B to increase genomic uracils. These results suggest that the proinflammatory PMA is unlikely to promote extensive APOBEC3A/APOBEC3B-mediated cytosine deaminations in human keratinocytes.


Assuntos
Citidina Desaminase/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Proteínas/efeitos dos fármacos , Uracila/metabolismo , Carcinógenos/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Citosina/metabolismo , DNA de Cadeia Simples/efeitos dos fármacos , Genômica , Humanos , Queratinócitos/metabolismo , Mutagênese/efeitos dos fármacos , Neoplasias/genética
7.
Zhonghua Wei Chang Wai Ke Za Zhi ; 20(4): 450-454, 2017 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-28440528

RESUMO

OBJECTIVE: To investigate the effect of ASCT2 gene (glutamine transporter) knock-down by shRNA on biological behaviors of colorectal cancer cells. METHODS: shRNA was transfected into colorectal cancer cells Lovo and SW480 to knockdown ASCT2 mediated by Lipofectamine 2000. Reverse transcription-PCR and Western blot were used to examine the mRNA and protein expression of ASCT2. MTT and transwell assay were used to determine the proliferation and invasiveness of Lovo and SW480 cells. Radioactive-tracer was used to detect the uptake of glutamine. RESULTS: ASCT2 mRNA and protein levels were significantly down-regulated by shRNA in Lovo and SW480 cells(P<0.01). MTT and transwell assays showed that ASCT2 knock-down could significantly inhibit the proliferation of Lovo and SW480 cells (A490) and decrease the number of invasive Lovo and SW480 cells from the membrane (both P<0.01). The number of membrane Lovo cells in shASCT group and control group was 46.3±5.9 and 197.7±9.1, respectively while the number of membrane SW480 cells in shASCT group and control group was 29.7±3.8 and 139.0±9.5, respectively. Radioactive-tracer showed that shASCT2 transfection could significantly reduce the uptake of glutamine, with an inhibition rate of 79.15% in Lovo and 67.22% in SW480 cells (both P<0.01). CONCLUSIONS: ASCT2 plays an oncogenic role in colonic cancer, and its promotion mechanism may be associated with glutamine metabolism. ASCT2 may be a novel therapeutic target of colonic cancer.


Assuntos
Sistema ASC de Transporte de Aminoácidos/efeitos dos fármacos , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/fisiologia , Proliferação de Células/genética , Neoplasias Colorretais/genética , Glutamina/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/fisiologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Linhagem Celular Tumoral/fisiologia , Neoplasias Colorretais/fisiopatologia , Regulação para Baixo/efeitos dos fármacos , Técnicas de Silenciamento de Genes/métodos , Glutamina/genética , Glutamina/fisiologia , Humanos , Oncogenes/efeitos dos fármacos , Oncogenes/genética , RNA Mensageiro/fisiologia , RNA Interferente Pequeno/farmacologia , Transfecção
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