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1.
Drug Discov Ther ; 14(1): 58-60, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32147628

RESUMO

The SARS-CoV-2 virus emerged in December 2019 and then spread rapidly worldwide, particularly to China, Japan, and South Korea. Scientists are endeavoring to find antivirals specific to the virus. Several drugs such as chloroquine, arbidol, remdesivir, and favipiravir are currently undergoing clinical studies to test their efficacy and safety in the treatment of coronavirus disease 2019 (COVID-19) in China; some promising results have been achieved thus far. This article summarizes agents with potential efficacy against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Descoberta de Drogas , Alanina/análogos & derivados , Alanina/farmacologia , Amidas/farmacologia , Antivirais/química , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Cloroquina/farmacologia , Estudos Clínicos como Assunto , Humanos , Indóis/farmacologia , Pirazinas/farmacologia , Ribonucleotídeos/farmacologia , Replicação Viral/efeitos dos fármacos
3.
DNA Cell Biol ; 39(3): 368-378, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31971825

RESUMO

Nuclear protein 1 (NUPR1) is a stress-related small molecule and plays important roles in various tumors, including multiple myeloma (MM). Autophagy is essential for maintaining cellular homoeostasis in response to stress and, together with apoptosis, determines cell fate. Previous studies indicate that NUPR1 is involved in cancer progression of MM, but the underlying mechanisms have not been elucidated. In this study, we confirmed that NUPR1 and basal autophagy markers were highly expressed in the bone marrow of MM patients. The overexpression of NUPR1 was correlated with staging (both by Revised International Staging System [RISS] and Durie-Salmon [D-S] Staging System), levels of hemoglobin and calcium, and bone marrow plasma cell ratio in the MM patients. NUPR1 silencing reduced autophagy activities and induced apoptosis in U266 and RPMI 8226. We further observed a decrease in NUPR1 silencing-induced apoptosis in the presence of rapamycin, while an increase in apoptosis after chloroquine and 3-methyladenine treatment. Analysis of the mechanism indicated that PI3K/AKT/mTOR pathway was involved in autophagy-mediated apoptosis upon NUPR1 knockdown. In summary, our results demonstrate that NUPR1 silencing suppresses autophagy activities and induces autophagy-mediated apoptosis in MM cells through the PI3K/AKT/mTOR pathway, which exhibits potential as a treatment strategy for MM.


Assuntos
Apoptose , Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/genética , Transdução de Sinais , Adenina/análogos & derivados , Adenina/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Inativação Gênica , Humanos , Mieloma Múltiplo/genética , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
4.
Nature ; 576(7786): 315-320, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31776516

RESUMO

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.


Assuntos
Microscopia Crioeletrônica , Resistência a Medicamentos/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas de Protozoários/ultraestrutura , Cloroquina/metabolismo , Cloroquina/farmacologia , Resistência a Medicamentos/genética , Concentração de Íons de Hidrogênio , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Quinolinas/metabolismo , Quinolinas/farmacologia
5.
Molecules ; 24(19)2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591298

RESUMO

Malaria and cancer are chronic diseases. The challenge with drugs available for the treatment of these diseases is drug toxicity and resistance. Ferrocene is a potent organometallic which have been hybridized with other compounds resulting in compounds with enhanced biological activity such as antimalarial and anticancer. Drugs such as ferroquine were developed from ferrocene and chloroquine. It was tested in the 1990s as an antimalarial and is still an effective antimalarial. Many researchers have reported ferrocene compounds as potent compounds useful as anticancer and antimalarial agents when hybridized with other pharmaceutical scaffolds. This review will be focused on compounds with ferrocene moieties that exhibit either an anticancer or antimalarial activity.


Assuntos
Aminoquinolinas/farmacologia , Antimaláricos/química , Antineoplásicos/química , Cloroquina/farmacologia , Compostos Ferrosos/farmacologia , Metalocenos/farmacologia , Aminoquinolinas/síntese química , Aminoquinolinas/química , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Antimaláricos/toxicidade , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Cloroquina/química , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Compostos Ferrosos/síntese química , Compostos Ferrosos/química , Humanos , Malária/tratamento farmacológico , Metalocenos/síntese química , Metalocenos/química , Neoplasias/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos
6.
Malar J ; 18(1): 336, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578142

RESUMO

BACKGROUND: Rapid diagnostic tests (RDTs) have been described as a source of genetic material to analyse malaria parasites in proof-of-concept studies. The increasing use of RDTs (e.g., in focal or mass screening and treatment campaigns) makes this approach particularly attractive for large-scale investigations of parasite populations. In this study, the complexity of Plasmodium falciparum infections, parasite load and chloroquine resistance transporter gene mutations were investigated in DNA samples extracted from positive RDTs, obtained in a routine setting and archived at ambient temperature. METHODS: A total of 669 archived RDTs collected from malaria cases in urban, semi-urban and rural areas of central Gabon were used for P. falciparum DNA extraction. Performance of RDTs as a source of DNA for PCR was determined using: (i) amplification of a single copy merozoite surface protein 1 (msp1) gene followed by highly sensitive and automated capillary electrophoresis; (ii) genotyping of the pfcrt gene locus 72-76 using haplotype-specific-probe-based real-time PCR to characterize chloroquine resistance; and, (iii) real-time PCR targeting 18S genes to detect and quantify Plasmodium parasites. RESULTS: Out of the 669 archived RDTs, amplification of P. falciparum nucleic materials had a success rate of 97% for 18S real-time PCR, and 88% for the msp1 gene. The multiplicity of infections (MOI) of the whole population was 2.6 (95% CI 2.5-2.8). The highest number of alleles detected in one infection was 11. The MOI decreased with increasing age (ß = - 0.0046, p = 0.02) and residence in Lambaréné was associated with smaller MOIs (p < 0.001). The overall prevalence of mutations associated with chloroquine resistance was 78.5% and was not associated with age. In Lambaréné, prevalence of chloroquine resistance was lower compared to rural Moyen-Ogooué (ß = - 0.809, p-value = 0.011). CONCLUSION: RDT is a reliable source of DNA for P. falciparum detection and genotyping assays. Furthermore, the increasing use of RDTs allows them to be an alternative source of DNA for large-scale genetic epidemiological studies. Parasite populations in the study area are highly diverse and prevalence of chloroquine-resistant P. falciparum remains high, especially in rural areas.


Assuntos
Bancos de Espécimes Biológicos , DNA de Protozoário/isolamento & purificação , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Adolescente , Adulto , Temperatura Corporal , Criança , Pré-Escolar , Cloroquina/farmacologia , DNA de Protozoário/genética , Resistência a Medicamentos/genética , Feminino , Gabão , Genótipo , Humanos , Malária Falciparum/sangue , Malária Falciparum/diagnóstico , Masculino , Proteínas de Membrana Transportadoras/genética , Proteína 1 de Superfície de Merozoito/genética , Técnicas de Diagnóstico Molecular , Parasitemia , Plasmodium falciparum/efeitos dos fármacos , Estudos Retrospectivos , Adulto Jovem
7.
Cancer Immunol Immunother ; 68(12): 1909-1920, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31641796

RESUMO

Tumour-associated macrophages (TAMs) are the key components in the tumour microenvironment. TAMs have two major subtypes, M1 and M2. M1 macrophages are tumour inhibitory, while M2 macrophages are tumour promotive. Repolarising TAMs from M2 to M1 is a promising strategy in cancer treatment. M1 and M2 macrophages were generated from murine bone marrow-derived macrophages (BMDMs). We found that chloroquine (CQ), an autophagy inhibitor, was able to repolarise M2 macrophages to the anti-tumour M1 phenotype. The repolarised macrophages demonstrated higher phagocytotic activity towards Hep-2 laryngeal tumour cells and re-sensitised Hep-2 cells to cisplatin (CDDP) treatment in vitro. While CQ did not demonstrate cytotoxicity to Hep-2 cells in vitro, CQ treatment reduced Hep-2 laryngeal tumour growth in vivo and improved CDDP treatment outcomes. It seems that CQ-induced M2-to-M1 macrophage repolarisation played an important role in tumour growth inhibition, and the CQ/CDDP combined therapy might have clinical potential in laryngeal cancer treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cisplatino/uso terapêutico , Neoplasias Laríngeas/imunologia , Macrófagos/fisiologia , Animais , Autofagia , Linhagem Celular Tumoral , Proliferação de Células , Transdiferenciação Celular , Cloroquina/farmacologia , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Laríngeas/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Células Th1/imunologia , Células Th2/imunologia
8.
Chemotherapy ; 64(3): 138-145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31639786

RESUMO

BACKGROUND: PD-L1 is a membrane protein with inhibitory effects on immune responses, whose expression has been correlated with high aggressiveness and the propensity of melanoma to metastasize. The nitrobenzoxadiazole (NBD) NBDHEX and its analog MC3181 are endowed with strong antitumor activity towards melanoma and a significant ability to reduce its adhesion and invasiveness. Therefore, we investigated whether PD-L1 status could affect cell sensitivity to the cytotoxic effects of NBDs. We then evaluated the effects of NBDHEX on PD-L1 expression and autophagy in melanoma cells. We used the BRAF-mutated A375 melanoma cell line and an A375 variant population enriched for PD-L1+ cells as a model. The cytotoxic effects of NBDs were evaluated in comparison to those of the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. METHODS: The effect of NBDHEX on autophagy was determined by measuring LC3-II and p62 protein levels by Western blot. The cytotoxic activity of the compounds was evaluated by sulforhodamine B assay. PD-L1 expression and plasma membrane localization were analyzed by FACS and Western blot analysis. RESULTS: NBDHEX behaves as a late-autophagy inhibitor in A375 melanoma cells, as previously found in other tumor cell lines. NBDHEX and MC3181 showed strong and comparable cytotoxic activity in both parental and PD-L1+ A375 cells, with IC50 values in the sub-micromolar range. Conversely, cells sorted for high PD-L1 expression had lower sensitivity to both the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. NBDHEX treatment did not change the total expression and cell surface localization of PD-L1 in both parental and PD-L1+ A375 cells. CONCLUSIONS: Our data suggest that NBDs may represent a promising treatment strategy for melanoma with elevated PD-L1 expression.


Assuntos
Autofagia/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Glutationa Transferase/antagonistas & inibidores , Nitrobenzenos/farmacologia , Oxidiazóis/farmacologia , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Cloroquina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/metabolismo , Humanos , Melanoma , Nitrobenzenos/química , Oxidiazóis/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Vemurafenib/farmacologia
9.
Mol Cell Biochem ; 462(1-2): 115-122, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31473883

RESUMO

Rasal2, a Ras-GTPase-activating protein (RasGAP), is a tumor suppressor in Luminal B breast cancer, frequently metastatic and recurrent. Exosomes (Exos) are small membrane vesicles secreted by various cell types, including tumor cells, recognized as vehicles for cell-to-cell communication. Our study aimed to investigate whether Rasal2 regulates breast cancer cell growth via affecting this process. In this paper, we described that Rasal2 knockout (KO) in MCF-7 cells enhanced exosomal release and increased autophagy-related proteins in exosomal fraction, while attenuated by exosome release inhibitor GW4869. Moreover, MCF-7 cells with chloroquine (CQ) treatment boosted Rasal2 KO-induced secretory autophagy. In addition, we presented that exosomes derived from KO MCF-7 cells (KO-exo) significantly promoted breast cancer cell proliferation compared to those from MCF-7 cells transfected with an empty crispr-cas9 plasmid serving as controls (sgNT-exo); however, exosomes purified from KO MCF-7 cells co-cultured with 3-methyladenine ((3-MA + KO)-exo)/CQ ((CQ + KO)-exo) dramatically inhibited/facilitated MCF-7 cell proliferation in contrast to KO-exo group, separately. In conclusion, our findings revealed a new mechanism of Rasal2 in the regulation of breast cancer cell proliferation via autophagy-exo-mediated pathway.


Assuntos
Autofagia , Neoplasias da Mama/patologia , Proteínas Ativadoras de GTPase/metabolismo , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Humanos , Células MCF-7
10.
Am J Chin Med ; 47(6): 1405-1418, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31488033

RESUMO

Baicalein (BAI) is a natural flavonoid. It has been shown that BAI has anticancer effects, but the molecular mechanism is still unclear. The aim of the current study was to confirm whether or not BAI triggers autophagy and induces AMPK activation in glioma U251 cells. The Ad-mcherry-GFP-LC3B adenovirus experiments indicated that BAI induces glioma cell autophagy. Western blotting showed that the level of LC3II expression increased with the time and concentration of BAI. Following treatment with chloroquine, the expression of LC3 was enhanced Immunofluorescence also confirmed this result. At the same time, cleaved caspase-3, DAPI staining, and JC-1 staining revealed that apoptosis was also induced in the induction of autophagy. In addition, we found that BAI activates phosphorylation of AMPK, which is further confirmed using compound C in this process. When the phosphorylation of AMPK was inhibited, autophagy, and apoptosis were also inhibited. In conclusion, BAI induces autophagy and apoptosis through AMPK pathway. Surprisingly, our research provides new insight with the function of anticancer of BAI, and the potential of the promotion in glioma cell apoptosis might be related to autophagy activation. These results demonstrate the anticancer activity of BAI, which can be used as potential therapeutic agents for cancer therapy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos Fitogênicos , Apoptose/efeitos dos fármacos , Flavanonas/farmacologia , Glioma/metabolismo , Glioma/patologia , Transdução de Sinais/efeitos dos fármacos , Caspase 3/metabolismo , Cloroquina/farmacologia , Flavanonas/uso terapêutico , Expressão Gênica , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação/efeitos dos fármacos , Fitoterapia , Células Tumorais Cultivadas
11.
Invest Ophthalmol Vis Sci ; 60(12): 3708-3716, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31479110

RESUMO

Purpose: Sjögren syndrome (SS) is an autoimmune disease characterized by the inflammatory destruction of salivary and lacrimal glands (LG). Chloroquine (CQ) was known as an immunomodulatory drug and in the inhibition of autophagy. The purpose of the study is to investigate the effect of CQ on the development of dry eye in NOD-LtJ mice. Methods: NOD-LtJ mice were observed, during which the occurrence of dry eye was confirmed by tear secretion, corneal staining, and the infiltration of foci into the LG from 13-week-old mice. Intraperitoneal (IP) administration of CQ was performed in 13-week-old mice for 4 weeks and maintained untreated for another 4 weeks. Additionally, CQ was injected IP in 19-week-old mice for 2 weeks from when the disease was fully developed. Results: Interestingly, the expression of autophagy marker ATG5 and LC3B-II was observed in the LG from week 5. When CQ had been administered for 4 weeks from week 13 and then maintained untreated for 4 weeks, tear secretion, corneal staining score, foci formation in the LG, conjunctival goblet cells and proinflammatory cytokine expressions were significantly better than untreated mice. The infiltration of immune cells and the expression of autophagy markers in LG were decreased in the CQ group. These indices improved significantly as well when the 19-week-old mice with severe clinical phenotypes had been treated with CQ for 2 weeks. Conclusions: This study demonstrated that autophagy was induced in the early stages of the SS model and that CQ treatment in the early stages could inhibit disease progression.


Assuntos
Antirreumáticos/farmacologia , Cloroquina/farmacologia , Modelos Animais de Doenças , Síndromes do Olho Seco/prevenção & controle , Síndrome de Sjogren/complicações , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/sangue , Biomarcadores/metabolismo , Córnea/metabolismo , Córnea/patologia , Síndromes do Olho Seco/diagnóstico , Síndromes do Olho Seco/etiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Injeções Intraperitoneais , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Proteínas Associadas aos Microtúbulos/sangue , Reação em Cadeia da Polimerase em Tempo Real , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/metabolismo , Lágrimas/fisiologia
12.
Nat Commun ; 10(1): 4300, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541097

RESUMO

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Cruzamentos Genéticos , Resistência a Medicamentos/genética , Proteínas de Membrana Transportadoras/genética , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Animais , Anopheles/parasitologia , Culicidae/parasitologia , Descoberta de Drogas , Feminino , Expressão Gênica , Genes de Protozoários , Malária/tratamento farmacológico , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Masculino , Plasmodium falciparum/genética
13.
Malar J ; 18(1): 325, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547827

RESUMO

BACKGROUND: Activation of hypnozoites of vivax malaria causes multiple clinical relapses, which contribute to the Plasmodium vivax burden and continuing transmission. Artemisinin-based combination therapy (ACT) is effective against blood-stage P. vivax but requires co-administration with primaquine to achieve radical cure. The therapeutic efficacy of primaquine depends on the generation of a therapeutically active metabolite via cytochrome P450 2D6 (CYP2D6). Impaired CYP2D6 metabolism has been associated with primaquine treatment failure. This study investigated the association between impaired CYP2D6 genotypes, drug-exposure to the long-acting ACT component (schizonticidal drugs) and tolerance and efficacy. METHODS: Adult patients with acute vivax malaria were enrolled in a recently completed trial and treated with artesunate-mefloquine, chloroquine or artemether-lumefantrine. All received concomitant primaquine (0.5 mg/kg/day for 7-9 days). The association between efficacy and safety and drug exposure was explored using area-under-the-curve (AUC) and half-life (t1/2) estimates obtained by non-compartmental analysis of the long half-life drugs. Parasite recurrences by day 63 were categorized as related relapses or re-infections/unrelated hypnozoite activation by genotyping three microsatellite loci and two polymorphic loci of merozoite surface antigen-1. The CYP2D6 genotype was identified with Taqman assays by real-time PCR to 9 polymorphisms (8 SNPs and one deletion). Impaired CYP2D6 activity was inferred using the Activity Score System. RESULTS: Most recurrences in the ASMQ (67%), CQ (80%) and AL (85%) groups were considered related relapses. Eight of nine (88.9%) of the patients with impaired CYP2D6 activity relapsed with related parasite compared to 18/25 (72%) with normal activity (RR = 1.23, 0.88; 1.72, p = 0.40). There were no associations between the measured PK parameters and recurrence. Patients with longer chloroquine half-lives had more pruritus (RR = 1.09, 1.03; 1.14, p = 0.001). Higher CQ AUCs were associated with reduced falls in haemoglobin by day 14 (Coef - 0.02, - 0.005; - 0.03, p = 0.01). All regimens were well tolerated. CONCLUSION: Genotyping of P. vivax showed that activation of related (homologous) hypnozoites was the most frequent cause of recurrence. The high proportion of the impaired CYP2D6 activity among patients with recurrent infections suggests that slow primaquine metabolism might influence related relapse rates in Brazil among patients receiving primaquine for radical cure, although confirmatory studies are needed. There was no association between drug exposure of the long-acting ACT component (schizonticidal drugs) and risk of related relapse. ACT was well tolerated. These results provide further re-assurance about the safety and efficacy of ACT when combined with short course primaquine to treat uncomplicated malaria vivax in Brazil. Trial registration RBR-79s56s ( http://www.ensaiosclinicos.gov.br/rg/RBR-79s56s/ ).


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Cloroquina/farmacologia , Primaquina/farmacologia , Adulto , Idoso , Antimaláricos/farmacocinética , Artemisininas/farmacocinética , Brasil , Cloroquina/farmacocinética , Combinação de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Primaquina/farmacocinética , Adulto Jovem
14.
Eur Cytokine Netw ; 30(2): 43-58, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31486403

RESUMO

The present study demonstrates that monocyte-derived dendritic cells (moDCs) produced in vitro using a GM-CSF and IFN-α differentiation protocol encompass a rare (∼5%) subpopulation of cells showing classical dendritic cell morphology and capable of natural internalization of extracellular self-DNA. We established that DEFB, HMGB1, LL-37 and RAGE antigens, which mediate the process of DNA internalization, are expressed on the surface of moDCs similar to plasmacytoid dendritic cells. However, in constrast to the latter subpopulation, these cells do not produce interleukin (IL)-37. Nonetheless, the process of DNA internalization was not in direct relation to the presence of the above antigens on the surface of these cells. Dendritic cells were sorted into total and non-DNA-internalizing populations and cytokine production was analyzed at 24-48 hours post-DNA treatment. We show that massive secretion of cytokines by dendritic cells is associated with the dsDNA-internalizing subpopulation. A total pool of IFN-moDCs secrete pro-inflammatory "first-wave" cytokines (IL-2, IL-6, IL-8, TNF-α) at both 24 and 48 hours time points. The anti-inflammatory cytokines IL-4 and IL-10 were found to be modestly induced, whereas GM-CSF, G-CSF, and IFN-γ production was strongly induced. Treatment of moDCs with dsDNA results in the up-regulated transcription of IFN-α, IFN-ß, IFN-γ, IL-8, IL-10, and VEGF by 6 hours. Combined dsDNA + chloroquine treatment has a synergistic effect on transcription of only one of the genes tested, with the pro-inflammatory cytokine IFN-ß displaying the strongest fold induction by 24 hours.


Assuntos
DNA/metabolismo , Células Dendríticas/citologia , Endocitose , Espaço Extracelular/metabolismo , Monócitos/citologia , Antígenos de Neoplasias/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Cloroquina/farmacologia , Citocinas/metabolismo , Sondas de DNA/metabolismo , Células Dendríticas/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Feminino , Proteína HMGB1/metabolismo , Humanos , Interferons/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rodaminas/metabolismo , beta-Defensinas/metabolismo
15.
J Toxicol Sci ; 44(8): 565-574, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31378768

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive deposition of droplets in hepatocytes. Patients with NAFLD can be at risk for nonalcoholic steatohepatitis, which can lead to hepatocellular carcinoma. Autophagy is a cellular pathway that is crucial for survival and homeostasis, and which protects against pathophysiological changes like obesity and cancer. We determined the expression of autophagy markers in preneoplastic hepatic lesions and the effects of an autophagy repressor chloroquine (CQ) or inducer amiodarone (AM) in a steatosis-related hepatocarcinogenesis model. Male F344 rats were fed a control diet or high fat diet (HFD), and subjected to initiation and promotion steps with N-nitrosodiethylamine injection at week 0 and a partial hepatectomy at week 3. Several HFD-fed rats were administered 0.1% CQ and 0.5% AM in their drinking water during week 2 and 8. CQ and AM did not improve HFD-induced obesity. AM, but not CQ, significantly decreased the number of glutathione S-transferase placental form-positive preneoplastic liver foci in the liver. Autophagosome markers LC3 and the LC3-binding protein p62 were heterogeneously expressed in the preneoplastic foci. CQ might inhibit autophagy by significantly increased p62/LC3 ratio, while AM might have a potential of inducing autophagy by showing an increased gene expression of the autophagy regulator, Atg5. These results suggest that preneoplastic lesions express autophagosome markers and that AM might decrease steatosis-related early hepatocarcinogenesis by potentially inducing autophagy in HFD-fed rats, while inhibition of autophagy by CQ did not alter the hepatocarcinogenesis. However, an immunohistochemical trial revealed a technical limitation in detecting autophagosome markers because there were variations in each preneoplastic lesion.


Assuntos
Autofagossomos , Autofagia/genética , Dieta Hiperlipídica/efeitos adversos , Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Amiodarona/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Cloroquina/farmacologia , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Masculino , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ratos Endogâmicos F344
16.
Molecules ; 24(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374989

RESUMO

This paper describes a continuation of our efforts in the pursuit of novel antiplasmodial agents with optimized properties. Following our previous discovery of biologically potent asymmetric primaquine (PQ) and halogenaniline fumardiamides (1-6), we now report their significant in vitro activity against the hepatic stages of Plasmodium parasites. Furthermore, we successfully prepared chloroquine (CQ) analogue derivatives (11-16) and evaluated their activity against both the hepatic and erythrocytic stages of Plasmodium. Our results have shown that PQ fumardiamides (1-6) exert both higher activity against P. berghei hepatic stages and lower toxicity against human hepatoma cells than the parent drug and CQ derivatives (11-16). The favourable cytotoxicity profile of the most active compounds, 5 and 6, was corroborated by assays performed on human cells (human breast adenocarcinoma (MCF-7) and non-tumour embryonic kidney cells (HEK293T)), even when glucose-6-phosphate dehydrogenase (G6PD) was inhibited. The activity of CQ fumardiamides on P. falciparum erythrocytic stages was higher than that of PQ derivatives, comparable to CQ against CQ-resistant strain PfDd2, but lower than CQ when tested on the CQ-sensitive strain Pf3D7. In addition, both sets of compounds showed favourable drug-like properties. Hence, quinoline fumardiamides could serve as a starting point towards the development of safer and more effective antiplasmodial agents.


Assuntos
Antimaláricos/química , Cloroquina/química , Malária Falciparum/tratamento farmacológico , Primaquina/química , Antimaláricos/farmacologia , Cloroquina/farmacologia , Eritrócitos/efeitos dos fármacos , Células HEK293 , Humanos , Malária Falciparum/parasitologia , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , Primaquina/farmacologia , Relação Estrutura-Atividade
17.
Respir Res ; 20(1): 184, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31416461

RESUMO

BACKGROUND: Influenza attacks the epithelium of the lung, causing cell death and disruption of the epithelial barrier leading to fluid buildup in the lung and impairment of gas exchange. Limited treatment options for severe influenza pneumonia prioritize the need for the discovery of effective therapies. IL-22 is a cytokine that promotes tissue integrity and has strong promise as a treatment option. While research has been focused on the cytokine itself, there is limited understanding of the regulation of the IL-22 receptor (IL-22Ra1) at the epithelial surface during infection. METHODS: IL-22Ra1 levels were measured by qRT-PCR, western blot and immunofluorescence following H1N1 influenza infection (A/PR/8/34 H1N1) or synthetic TLR3 mimetic, Poly (I:C). Regulation of the receptor was determined using STAT inhibitors (STAT1, STAT3 and PanSTAT inhibitors), TLR3 inhibition, and neutralization of interferon alpha receptor 2 (IFNAR2). Significance was determined by a p-value of greater than 0.05. Significance between two groups was measured using unpaired t-test and significance between more than two groups was measured using one-way ANOVA with Tukey Multiple Comparison Test. RESULTS: Here we show both in vivo and in vitro that IL-22Ra1 was induced as early as 24 h after influenza (H1N1 PR8) infection. This induction was triggered by toll-like receptor 3 (TLR3) as a TLR3 mimetic [Poly (I:C)] also induced IL-22Ra1 and inhibition of endosomal formation required for TLR3 function inhibited this process. This upregulation was dependent upon IFNß signaling through STAT1. Importantly, induction of IL-22Ra1 significantly increased IL-22 signaling as evidenced by pSTAT3 levels following IL-22 treatment. CONCLUSION: Collectively, these data suggest epithelial cells may optimize the beneficial effects of IL-22 through the induction of the IL-22 receptor during viral infection in the lung.


Assuntos
Influenza Humana/metabolismo , Receptores de Interleucina/biossíntese , Fator de Transcrição STAT1/biossíntese , Receptor 3 Toll-Like/biossíntese , Células A549 , Animais , Cloroquina/farmacologia , Humanos , Influenza Humana/patologia , Interferons/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poli I-C/farmacologia
18.
Mem Inst Oswaldo Cruz ; 114: e190054, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31411308

RESUMO

BACKGROUND: The central repetitive region (CRR) of the Plasmodium vivax circumsporozoite surface protein (CSP) is composed of a repetitive sequence that is characterised by three variants: VK210, VK247 and P. vivax-like. The most important challenge in the treatment of P. vivax infection is the possibility of differential response based on the parasite genotype. OBJECTIVES: To characterise the CSP variants in P. vivax isolates from individuals residing in a malaria-endemic region in Brazil and to profile these variants based on sensitivity to chloroquine and mefloquine. METHODS: The CSP variants were determined by sequencing and the sensitivity of the P. vivax isolates to chloroquine and mefloquine was determined by Deli-test. FINDINGS: Although five different allele sizes were amplified, the sequencing results showed that all of the isolates belonged to the VK210 variant. However, we observed substantial genetic diversity in the CRR, resulting in the identification of 10 different VK210 subtypes. The frequency of isolates that were resistant to chloroquine and mefloquine was 11.8 and 23.8%, respectively. However, we did not observe any difference in the frequency of the resistant isolates belonging to the VK210 subtypes. MAIN CONCLUSION: The VK210 variant is the most frequently observed in the studied region and there is significant genetic variability in the CRR of the P. vivax CSP. Moreover, the antimalarial drug sensitivity profiles of the isolates does not seem to be related to the VK210 subtypes.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Malária Vivax/parasitologia , Mefloquina/farmacologia , Plasmodium vivax/efeitos dos fármacos , Proteínas de Protozoários/genética , Genótipo , Humanos , Testes de Sensibilidade Parasitária , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Reação em Cadeia da Polimerase
19.
Hum Exp Toxicol ; 38(11): 1266-1274, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31446784

RESUMO

Cyclophosphamide (CYP) and methotrexate (MTX) have been evaluated for their ability to induce toxicity in human peripheral blood lymphocytes (PBLs) and the protective role of mitochondrial and lysosomal stabilizing agents. The potential toxicity effects of CYP and MTX were measured in vitro by cellular parameters assays such as cellular viability, reactive oxygen species (ROS) formation, mitochondrial membrane permeability transition (mitochondrial membrane potential (MMP)) collapse, lysosomal membrane damage, intracellular reduced glutathione (GSH), extracellular oxidized glutathione (GSSG), and lipid peroxidation. Separately, human lymphocytes were treated with concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 ng/mL for CYP and 1, 2, 5, and 10 µg/mL for MTX for 6 h. Statistical evaluations showed that CYP and MTX significantly decreased the cell viability at the three highest concentrations when compared with both the negative and solvent controls. In addition, CYP and MTX were significantly induced ROS formation, MMP collapse, lysosomal membrane damage, lipid peroxidation, and GSH depletion compared with the controls. Mitochondrial and lysosomal protective agents like cyclosporine A and chloroquine, respectively, decreased cytotoxicity and oxidative stress induced by CYP and MTX. The present results indicate that CYP and MTX are toxic to human PBLs and their toxicity could be ameliorated by mitochondrial and lysosomal protective agents.


Assuntos
Antineoplásicos/toxicidade , Ciclofosfamida/toxicidade , Imunossupressores/toxicidade , Linfócitos/efeitos dos fármacos , Metotrexato/toxicidade , Substâncias Protetoras/farmacologia , Adolescente , Adulto , Hidroxitolueno Butilado/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cloroquina/farmacologia , Ciclosporina/farmacologia , Glutationa/metabolismo , Humanos , Linfócitos/metabolismo , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Adulto Jovem
20.
Nucleic Acids Res ; 47(16): 8563-8580, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31291457

RESUMO

Creating access to DNA double-strand break (DSB) sites in the chromatin context is an essential step during the repair process, but much remains to be determined about its regulatory mechanisms. Here, using a novel reporter cassette for simultaneous detection of homologous recombination (HR) and nonhomologous end joining (NHEJ) at the same chromosomal site, we report that the efficiency of HR but not NHEJ negatively correlates with nucleosome density. We demonstrate that PARP1 is required for HR by modulating nucleosome density at damage sites. Mechanistic studies indicate that the ATPase domain of BRG1 and the ZnF domain of SIRT1 interact with poly-ADP ribose (PAR) in response to DNA damage, and are responsible for bringing the two factors to broken DNA ends. At DNA damage sites, BRG1 and SIRT1 physically interact, whereupon SIRT1 deacetylates BRG1 at lysine residues 1029 and 1033, stimulating its ATPase activity to remodel chromatin and promote HR.


Assuntos
DNA Helicases/genética , DNA/genética , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Reparo de DNA por Recombinação , Sirtuína 1/genética , Fatores de Transcrição/genética , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Cloroquina/farmacologia , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Fenantrenos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismo
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