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1.
PLoS Pathog ; 16(3): e1008442, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196533

RESUMO

Latency reversal agents (LRA) variably induce HIV re-expression in CD4 T cells but reservoirs are not cleared. Whether HIV epitope presentation is similar between latency reversal and initial infection of CD4 T cells is unknown yet crucial to define immune responses able to detect HIV-infected CD4 T cells after latency reversal. HIV peptides displayed by MHC comes from the intracellular degradation of proteins by proteasomes and post-proteasomal peptidases but the impact of LRAs on antigen processing is not known. Here we show that HDAC inhibitors (HDCAi) reduced cytosolic proteolytic activities while PKC agonists (PKCa) increased them to a lesser extent than that induced by TCR activation. During the cytosolic degradation of long HIV peptides in LRA-treated CD4 T cells extracts, HDACi and PKCa modulated degradation patterns of peptides and altered the production of HIV epitopes in often opposite ways. Beyond known HIV epitopes, HDACi narrowed the coverage of HIV antigenic fragments by 8-11aa degradation peptides while PKCa broadened it. LRAs altered HIV infection kinetics and modulated CD8 T cell activation in an epitope- and time-dependent manner. Interestingly the efficiency of endogenous epitope processing and presentation to CD8 T cells was increased by PKCa Ingenol at early time points despite low levels of antigens. LRA-induced modulations of antigen processing should be considered and exploited to enhance and broaden HIV peptide presentation by CD4 T cells and to improve immune recognition after latency reversal. This property of LRAs, if confirmed with other antigens, might be exploited to improve immune detection of diseased cells beyond HIV.


Assuntos
Apresentação do Antígeno , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Proteína Quinase C/antagonistas & inibidores , Latência Viral/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Humanos , Proteína Quinase C/imunologia , Latência Viral/imunologia
2.
Chemistry ; 26(6): 1166-1195, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31479550

RESUMO

Bryostatins are a class of naturally occurring macrocyclic lactones with a unique fast developing portfolio of clinical applications, including treatment of AIDS, Alzheimer's disease, and cancer. This comprehensive account summarizes the recent progress (2014-present) in the development of bryostatins, including their total synthesis and biomedical applications. An emphasis is placed on the discussion of bryostatin 1, the most-studied analogue to date. This review highlights the synthetic and biological challenges of bryostatins and provides an outlook on their future development.


Assuntos
Antineoplásicos/química , Briostatinas/síntese química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Briostatinas/farmacologia , Briostatinas/uso terapêutico , Senescência Celular/efeitos dos fármacos , Doenças do Sistema Nervoso Central/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Humanos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo
3.
Mol Pharmacol ; 97(2): 123-131, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31734646

RESUMO

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) expressed in regions of the brain that control motor function, cognition, and motivation. As a result, D2R is involved in the pathophysiology of disorders such as schizophrenia and drug addiction. Understanding the signaling pathways activated by D2R is crucial to finding new therapeutic targets for these disorders. D2R stimulation by its agonist, dopamine, causes desensitization and internalization of the receptor. A previous study found that inhibitors of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) blocked D2R desensitization in neurons in the ventral tegmental area of the brain. In the present study, using a cell-based system, we investigated whether ALK regulates D2R internalization. The ALK inhibitor alectinib completely inhibited dopamine-induced D2R internalization. Since GPCRs can transactivate receptor tyrosine kinases, we also examined if D2R stimulation activated ALK signaling. ALK phosphorylation increased by almost 2-fold after dopamine treatment and ALK coimmunoprecipitated with D2R. To identify the signaling pathways downstream of ALK that might regulate D2R internalization, we used pharmacological inhibitors of proteins activated by ALK signaling. Protein kinase Cγ was activated by dopamine in an ALK-dependent manner, and a protein kinase C inhibitor completely blocked dopamine-induced D2R internalization. Taken together, these results identify ALK as a receptor tyrosine kinase transactivated by D2R that promotes its internalization, possibly through activation of protein kinase C. ALK inhibitors could be useful in enhancing D2R signaling. SIGNIFICANCE STATEMENT: Receptor internalization is a mechanism by which receptors are desensitized. In this study we found that agonist-induced internalization of the dopamine D2 receptor is regulated by the receptor tyrosine kinase ALK. ALK was also transactivated by and associated with dopamine D2 receptor. Dopamine activated protein kinase C in an ALK-dependent manner and a PKC inhibitor blocked dopamine D2 receptor internalization. These results indicate that ALK regulates dopamine D2 receptor trafficking, which has implications for psychiatric disorders involving dysregulated dopamine signaling.


Assuntos
Quinase do Linfoma Anaplásico/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores de Dopamina D2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Carbazóis/farmacologia , Dopamina/farmacologia , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Piperidinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Biomed Pharmacother ; 121: 109665, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31810137

RESUMO

Colorectal cancer (CRC) is the third most common malignancy and the fourth most common cause of cancer-related death worldwide. The treatment of metastatic CRC considered palliative for many years aiming for an improved life, with little hope of a cure, highlighting the need for developing novel targeted therapy for CRC. Human protein kinases constitute a complicated system with complex internal and external interactions, which stimulates various cellular processes such as cell growth, metabolism, survival, and apoptosis. This study investigated the effect of a combination of atypical Protein Kinase C (PKC) inhibitor (either ICA-I or ζ-Stat) and 5-FU (a thymidylate synthase inhibitor) on CRC cells viability concerning cellular DNA damage. In this study, we took multiple approaches such as colony formation assay, flow cytometry, DNA ladder assay, TUNEL assay, etc. to examine the CRC cell viability and apoptosis as a function of combination treatment. Our findings showed that the combination of atypical PKC inhibitor and 5-FU synergistically reduced the viability of CRC cells and induced apoptosis. Additionally, the DNA ladder and TUNEL assays indicated that there was a notable DNA damage and fragmentation because of lack of thymidylate synthase and due to the deactivation of atypical PKC dependent CDK7. These data suggest that the simultaneous knockdown of upstream atypical PKC protein and downstream DNA damage repairing mechanism would be a useful approach to combat CRC and to improve overall patients' survival rate.


Assuntos
Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Dano ao DNA , Fluoruracila/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Fluoruracila/química , Humanos , Imidazóis/farmacologia , Modelos Biológicos , Organofosfatos/farmacologia , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/química , Fator de Transcrição TFIIH/metabolismo , Ensaio Tumoral de Célula-Tronco
5.
Int J Nanomedicine ; 14: 8835-8846, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31806974

RESUMO

Background: Inflammatory stress caused by protein kinase D (PKD) plays a critical role in damaging chondrocytes and extracellular matrix (ECM) during osteoarthritis (OA). The PKD inhibitor (PKDi) (CRT0066101) has been used to overcome inflammation in different cell types. However, the efficacy of a therapeutic drug can be limited due to off-target distribution, slow cellular internalization, and limited lysosomal escape. In order to overcome this issue, we developed nanosomes carrying CRT0066101 (PKDi-Nano) and tested their efficacy in vitro in chondrocytes. Methods: Chondrocytes were subjected to IL-1ß-induced inflammatory stress treated with either PKDi or PKDi-Nano. Effects of treatment were measured in terms of cytotoxicity, cellular morphology, viability, apoptosis, phosphorylation of protein kinase B (Akt), and anabolic/catabolic gene expression analyses related to cartilage tissue. Results and Discussion: The effects of PKDi-Nano treatment were more pronounced as compared to PKDi treatment. Cytotoxicity and apoptosis were significantly reduced following PKDi-Nano treatment (P < 0.001). Cellular morphology was also restored to normal size and shape. The viability of chondrocytes was significantly enhanced in PKDi-Nano-treated cells (P < 0.001). The data indicated that PKDi-Nano acted independently of the Akt pathway. Gene expression analyses revealed significant increases in the expression levels of anabolic genes with concomitant decreases in the level of catabolic genes. Our results indicate that PKDi-Nano attenuated the effects of IL-1ß via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) pathway. Conclusion: Taken together, these results suggest that PKDi-Nano can be used as a successful strategy to reduce IL1ß-induced inflammatory stress in chondrocytes.


Assuntos
Condrócitos/efeitos dos fármacos , Nanoestruturas/administração & dosagem , Proteína Quinase C/antagonistas & inibidores , Pirimidinas/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Interleucina-1beta/toxicidade , NF-kappa B/metabolismo , Nanoestruturas/química , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/patologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Suínos
6.
Int J Mol Sci ; 21(1)2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31881809

RESUMO

The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca2+. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets.


Assuntos
Plaquetas/metabolismo , Proteína Quinase C/metabolismo , Quinase Syk/metabolismo , Difosfato de Adenosina/farmacologia , Plaquetas/citologia , Cálcio/metabolismo , Venenos de Crotalídeos/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Lectinas Tipo C , Maleimidas/farmacologia , Fosfolipase C gama/metabolismo , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Quinase Syk/antagonistas & inibidores , Venenos de Víboras/farmacologia
7.
Molecules ; 24(23)2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31771098

RESUMO

The identification of signaling pathways that are involved in gliomagenesis is crucial for targeted therapy design. In this study we assessed the biological and therapeutic effect of ingenol-3-dodecanoate (IngC) on glioma. IngC exhibited dose-time-dependent cytotoxic effects on large panel of glioma cell lines (adult, pediatric cancer cells, and primary cultures), as well as, effectively reduced colonies formation. Nevertheless, it was not been able to attenuate cell migration, invasion, and promote apoptotic effects when administered alone. IngC exposure promoted S-phase arrest associated with p21CIP/WAF1 overexpression and regulated a broad range of signaling effectors related to survival and cell cycle regulation. Moreover, IngC led glioma cells to autophagy by LC3B-II accumulation and exhibited increased cytotoxic sensitivity when combined to a specific autophagic inhibitor, bafilomycin A1. In comparison with temozolomide, IngC showed a mean increase of 106-fold in efficacy, with no synergistic effect when they were both combined. When compared with a known compound of the same class, namely ingenol-3-angelate (I3A, Picato®), IngC showed a mean 9.46-fold higher efficacy. Furthermore, IngC acted as a potent inhibitor of protein kinase C (PKC) activity, an emerging therapeutic target in glioma cells, showing differential actions against various PKC isotypes. These findings identify IngC as a promising lead compound for the development of new cancer therapy and they may guide the search for additional PKC inhibitors.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/enzimologia , Diterpenos/farmacologia , Euphorbia/química , Glioma/enzimologia , Proteína Quinase C/antagonistas & inibidores , Antineoplásicos/química , Autofagia , Neoplasias Encefálicas/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diterpenos/química , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Humanos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos
8.
Neurochem Res ; 44(12): 2755-2764, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31650360

RESUMO

Pathogens such as bacterial lipopolysaccharide (LPS) play an important role in promoting the production of the inflammatory cytokines interleukin-1 beta (IL-1ß) and tumour necrosis factor-α (TNF-α) in response to infection or damage in microglia. However, whether different signalling pathways regulate these two inflammatory factors remains unclear. The protein kinase C (PKC) family is involved in the regulation of inflammation, and our previous research showed that the activation of the PKC pathway played a key role in the LPS-induced transformation of the adenosine A2A receptor (A2AR) from anti-inflammatory activity to pro-inflammatory activity under high glutamate concentrations. Therefore, in the current study, we investigated the role of PKC in the LPS-induced production of these inflammatory cytokines in mouse primary microglia. GF109203X, a specific PKC inhibitor, inhibited the LPS-induced expression of IL-1ß messenger ribonucleic acid and intracellular protein in a dose-dependent manner. Moreover, 5 µM GF109203X prevented LPS-induced IL-1ß expression but did not significantly affect LPS-induced TNF-α expression. PKC promoted IL-1ß expression by regulating the activity of NF-κB but did not significantly impact the activity of ERK1/2. A2AR activation by CGS21680, an A2AR agonist, facilitated LPS-induced IL-1ß expression through the PKC pathway at high glutamate concentrations but did not significantly affect LPS-induced TNF-α expression. Taken together, these results suggest a new direction for specific intervention with LPS-induced inflammatory factors in response to specific signalling pathways and provide a mechanism for A2AR targeting, especially after brain injury, to influence inflammation by interfering with A2AR.


Assuntos
Ácido Glutâmico/metabolismo , Interleucina-1beta/metabolismo , Microglia/metabolismo , Proteína Quinase C/metabolismo , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Animais , Indóis/farmacologia , Inflamação/induzido quimicamente , Lipopolissacarídeos , Maleimidas/farmacologia , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenetilaminas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição RelA/metabolismo
9.
Bull Exp Biol Med ; 167(6): 711-715, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31655990

RESUMO

We studied the involvement of protein kinase Mζ (PKMζ) in the mechanisms of amnesia development within 10 days after disruption of conditioned food aversion memory with ZIP (a PKMζ inhibitor). Repeated training performed in 3 days after amnesia induction with ZIP, led to the formation of conditioned food aversion memory, but the number of combined presentations of food and reinforcer stimuli was lower than during the initial training. Repeated training performed in 10 days after amnesia induction also led to memory formation, but the number of combined presentations of the stimuli was similar to that during the initial training. It was hypothesized that at the early stages of ZIP-induced amnesia, residual memory trace can be restored and amplified during repeated training, which led to memory expression at the behavioral level. At the late stages of amnesia, this memory trace was completely erased and repeated training led to the formation of a new memory. Thus, PKMζ inhibition results in the relatively fast impairment of memory retrieval and induces long-term process of memory erasing.


Assuntos
Amnésia/induzido quimicamente , Lipopeptídeos/farmacologia , Memória de Longo Prazo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Amnésia/psicologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , Condicionamento Psicológico/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Caracois Helix/efeitos dos fármacos , Caracois Helix/fisiologia , Consolidação da Memória/efeitos dos fármacos , Consolidação da Memória/fisiologia , Proteína Quinase C/antagonistas & inibidores , Fatores de Tempo
10.
Cancer Cell ; 36(3): 218-235, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31474570

RESUMO

Atypical protein kinase C (aPKC) isozymes, PKCλ/ι and PKCζ, are now considered fundamental regulators of tumorigenesis. However, the specific separation of functions that determine their different roles in cancer is still being unraveled. Both aPKCs have pleiotropic context-dependent functions that can translate into tumor-promoter or -suppressive functions. Here, we review early and more recent literature to discuss how the different tumor types, and their microenvironments, might account for the selective signaling of each aPKC isotype. This is of clinical relevance because a better understanding of the roles of these kinases is essential for the design of new anti-cancer treatments.


Assuntos
Isoenzimas/metabolismo , Neoplasias/patologia , Proteína Quinase C/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Polaridade Celular/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Camundongos Transgênicos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proto-Oncogenes/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Microambiente Tumoral/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
11.
Mol Cells ; 42(6): 470-479, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31250620

RESUMO

Interstitial cells of Cajal (ICCs) are pacemaker cells that exhibit periodic spontaneous depolarization in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of ghrelin and motilin on the pacemaker potentials of ICCs isolated from the mouse small intestine. Using the whole-cell patch-clamp configuration, we demonstrated that ghrelin depolarized pacemaker potentials of cultured ICCs in a dose-dependent manner. The ghrelin receptor antagonist [D-Lys] GHRP-6 completely inhibited this ghrelin-induced depolarization. Intracellular guanosine 5'-diphosphate-ß-S and pre-treatment with Ca2+free solution or thapsigargin also blocked the ghrelin-induced depolarization. To investigate the involvement of inositol triphosphate (IP3), Rho kinase, and protein kinase C (PKC) in ghrelin-mediated pacemaker potential depolarization of ICCs, we used the IP3 receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C, the Rho kinase inhibitor Y-27632, and the PKC inhibitors staurosporine, Go6976, and rottlerin. All inhibitors except rottlerin blocked the ghrelin-induced pacemaker potential depolarization of ICCs. In addition, motilin depolarized the pacemaker potentials of ICCs in a similar dose-dependent manner as ghrelin, and this was also completely inhibited by [D-Lys] GHRP-6. These results suggest that ghrelin induced the pacemaker potential depolarization through the ghrelin receptor in a G protein-, IP3-, Rho kinase-, and PKC-dependent manner via intracellular and extracellular Ca2+ regulation. In addition, motilin was able to depolarize the pacemaker potentials of ICCs through the ghrelin receptor. Therefore, ghrelin and its receptor may modulate GI motility by acting on ICCs in the murine small intestine.


Assuntos
Grelina/farmacologia , Células Intersticiais de Cajal/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Motilina/farmacologia , Acetofenonas/farmacologia , Amidas/farmacologia , Animais , Benzopiranos/farmacologia , Compostos de Boro/metabolismo , Cálcio/metabolismo , Carbazóis/farmacologia , Motilidade Gastrointestinal/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Células Intersticiais de Cajal/fisiologia , Intestino Delgado/fisiologia , Compostos Macrocíclicos/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Oligopeptídeos/metabolismo , Oxazóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Piridinas/farmacologia , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/metabolismo , Transdução de Sinais , Estaurosporina/farmacologia , Tapsigargina/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
12.
J Pharmacol Exp Ther ; 370(3): 427-435, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31197021

RESUMO

The Ca2+-sensing receptor (CaSR) detects small changes in extracellular calcium (Ca2+ e) concentration ([Ca2+]e) and transduces the signal into modulation of various signaling pathways. Ca2+-induced relaxation of isolated phenylephrine-contracted mesenteric arteries is mediated by the CaSR of the perivascular nerve. Elucidation of the regulatory mechanisms involved in vascular CaSR signaling may provide insights into the physiologic functions of the receptor and identify targets for the development of new treatments for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is a critical regulator of multiple signaling pathways and can phosphorylate the CaSR leading to receptor desensitization. In this study, we used automated wire myography to investigate the effects of CaSR mutation and small-interfering RNA downregulation of PKCα on CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive (SS) rats on a low-salt diet. The data showed minimal relaxation responses of arteries to Ca2+ e in wild-type (SS) and CaSR mutant (SS-Casrem1Mcwi) rats. Mutation of the CaSR gene had no significant effect on relaxation. PKCα expression was similar in wild-type and mutant rats, and small-interfering RNA downregulation of PKCα and/or inhibition of PKC with the Ca2+-sensitive GÓ§ 6976 resulted in a >80% increase in relaxation. Significant differences in EC50 values were observed between treated and untreated controls (P < 0.05 analysis of variance). The results indicate that PKCα plays an important role in the regulation of CaSR-mediated relaxation of mesenteric arteries, and its downregulation or pharmacological inhibition may lead to an increased Ca2+ sensitivity of the receptor and reversal of age-related changes in vascular tone. SIGNIFICANCE STATEMENT: G protein-coupled CaSR signaling leads to the regulation of vascular tone and may, therefore, play a vital role in blood pressure regulation. The receptor has several PKC phosphorylation sites in the C-terminal intracellular tail that mediate desensitization. We have previously shown that activation of the CaSR in neuronal cells leads to PKC phosphorylation, indicating that protein kinase C is an important regulator of CaSR function. Therefore, PKC in the CaSR signaling pathway in mesenteric arteries is a potential target for the development of new therapeutic approaches to treat hypertension and age-related vascular dysfunction. The present studies show that small-interfering RNA downregulation of PKCα and pharmacological inhibition of PKC enhanced CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive rats.


Assuntos
Cálcio/metabolismo , Regulação para Baixo , Espaço Extracelular/metabolismo , Artérias Mesentéricas/citologia , Artérias Mesentéricas/fisiologia , Proteína Quinase C/metabolismo , Vasodilatação , Animais , Espaço Extracelular/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Endogâmicos Dahl , Receptores de Detecção de Cálcio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
13.
Int J Pharm ; 566: 756-764, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31175987

RESUMO

Nowadays many drugs with improved therapeutic efficacy are discovered but cannot be utilized due to their low solubility and insufficient bioavailability. An example of such a drug molecule is a protein kinase C inhibitor that influences an enzyme which plays an important role in several signal transduction cascades. The aim of this study was to formulate a stable nanoparticle dispersion of the PKC inhibitor encapsulated into PLGA nanoparticles (NPs). Encapsulation of the PKC inhibitor into PLGA NPs of 100-200 nm diameter should provide a targeted delivery to the inflammation sites. The NPs were prepared via nanoprecipitation and different surfactants were investigated: Fully and partially hydrolyzed poly(vinyl alcohol) (PVA, Mowiol X-88 and X-98), poloxamers (Pluronic F68 and F127) and polysorbates (Tween 20 and 80). From all surfactants tested, only NPs prepared with partially hydrolyzed PVA (Mowiol X-88) provided the desired stability throughout the downstream processes. These NPs were subsequently analyzed regarding their particle size, polydispersity, encapsulation efficiency and loading capacity. Dynamic light scattering results revealed that monodisperse NPs of 150-220 nm were formed, a size range that favors targeted delivery. The drug encapsulation efficiency varied from 31 to 75% with a drug loading of 1.3-2%. Moreover, the long-term stability was studied and the residual amount of PVA of the NP solutions was quantified via nuclear magnetic resonance (NMR) measurements. The shell-less hen's egg model was used to test toxic effects (hemorrhage, vascular lysis, thrombosis, hemolysis and lethality) of the NPs in a more complex biological system under dynamic flow conditions.


Assuntos
Indóis/química , Maleimidas/química , Nanopartículas/química , Polímeros/química , Tensoativos/química , Animais , Galinhas , Estabilidade de Medicamentos , Agregação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Indóis/administração & dosagem , Maleimidas/administração & dosagem , Nanopartículas/administração & dosagem , Tamanho da Partícula , Polímeros/administração & dosagem , Proteína Quinase C/antagonistas & inibidores , Ovinos , Tensoativos/administração & dosagem , Zigoto/efeitos dos fármacos
14.
Curr Pharm Des ; 25(10): 1059-1074, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31131745

RESUMO

BACKGROUND: Although protein kinase D1 (PKD1) has been proved to be an efficient target for anticancer drug development, lack of structural details and substrate binding mechanisms are the main obstacles for the development of selective inhibitors with therapeutic benefits. OBJECTIVE: The present study described the in silico dynamics behaviors of PKD1 in binding with selective and non-selective inhibitors and revealed the critical binding site residues for the selective kinase inhibition. METHODS: Here, the three dimensional model of PKD1 was initially constructed by homology modeling along with binding site characterization to explore the non-conserved residues. Subsequently, two known inhibitors were docked to the catalytic site and the detailed ligand binding mechanisms and post binding dyanmics were investigated by molecular dynamics simulation and binding free energy calculations. RESULTS: According to the binding site analysis, PKD1 serves several non-conserved residues in the G-loop, hinge and catalytic subunits. Among them, the residues including Leu662, His663, and Asp665 from hinge region made polar interactions with selective PKD1 inhibitor in docking simulation, which were further validated by the molecular dynamics simulation. Both inhibitors strongly influenced the structural dynamics of PKD1 and their computed binding free energies were in accordance with experimental bioactivity data. CONCLUSION: The identified non-conserved residues likely to play critical role on molecular reorganization and inhibitor selectivity. Taken together, this study explained the molecular basis of PKD1 specific inhibition, which may help to design new selective inhibitors for better therapies to overcome cancer and PKD1 dysregulated disorders.


Assuntos
Simulação de Dinâmica Molecular , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Sítios de Ligação , Simulação de Acoplamento Molecular , Ligação Proteica
15.
J Biol Chem ; 294(23): 9213-9224, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31053639

RESUMO

Chronic or excess glucocorticoid exposure causes lipid disorders such as hypertriglyceridemia and hepatic steatosis. Angptl4 (angiopoietin-like 4), a primary target gene of the glucocorticoid receptor in hepatocytes and adipocytes, is required for hypertriglyceridemia and hepatic steatosis induced by the synthetic glucocorticoid dexamethasone. Angptl4 has also been shown to be required for dexamethasone-induced hepatic ceramide production. Here, we further examined the role of ceramide-mediated signaling in hepatic dyslipidemia caused by chronic glucocorticoid exposure. Using a stable isotope-labeling technique, we found that dexamethasone treatment induced the rate of hepatic de novo lipogenesis and triglyceride synthesis. These dexamethasone responses were compromised in Angptl4-null mice (Angptl4-/-). Treating mice with myriocin, an inhibitor of the rate-controlling enzyme of de novo ceramide synthesis, serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/SPTLC2, decreased dexamethasone-induced plasma and liver triglyceride levels in WT but not Angptl4-/- mice. We noted similar results in mice infected with adeno-associated virus-expressing small hairpin RNAs targeting Sptlc2. Protein phosphatase 2 phosphatase activator (PP2A) and protein kinase Cζ (PKCζ) are two known downstream effectors of ceramides. We found here that mice treated with an inhibitor of PKCζ, 2-acetyl-1,3-cyclopentanedione (ACPD), had lower levels of dexamethasone-induced triglyceride accumulation in plasma and liver. However, small hairpin RNA-mediated targeting of the catalytic PP2A subunit (Ppp2ca) had no effect on dexamethasone responses on plasma and liver triglyceride levels. Overall, our results indicate that chronic dexamethasone treatment induces an ANGPTL4-ceramide-PKCζ axis that activates hepatic de novo lipogenesis and triglyceride synthesis, resulting in lipid disorders.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Ceramidas/metabolismo , Dexametasona/toxicidade , Fígado/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteína 4 Semelhante a Angiopoietina/deficiência , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Hipertrigliceridemia/etiologia , Hipertrigliceridemia/metabolismo , Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase C/antagonistas & inibidores , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismo
16.
J Biol Chem ; 294(27): 10649-10662, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31142616

RESUMO

Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR2) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR2 coupling to Gαq, Gαs, and ß-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR2 at distinct sites and activate it by biased mechanisms that induce coupling to Gαs, but not to Gαq or ß-arrestins. Because proteases activate PAR2 by irreversible cleavage, and activated PAR2 is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR2 from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR2-dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gßγ to the Golgi, coinciding with PAR2 mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR2-Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR2 responsiveness initially declined, consistent with PAR2 cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gßγ, and protein translation inhibited recovery of PAR2 responsiveness. PKD and Gßγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR2 mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteína Quinase C/metabolismo , Receptor PAR-2/metabolismo , Animais , Catepsinas/metabolismo , Membrana Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Hiperalgesia/prevenção & controle , Elastase de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/antagonistas & inibidores , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Receptor PAR-2/agonistas , Transdução de Sinais/efeitos dos fármacos , Xantenos/administração & dosagem , Xantenos/farmacologia
17.
Cancer ; 125(16): 2732-2746, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31017664

RESUMO

Colorectal cancer (CRC) represents a major public health problem as the second leading cause of cancer-related mortality in the United States. Of an estimated 140,000 newly diagnosed CRC cases in 2018, roughly one-third of these patients will have a primary tumor located in the distal large bowel or rectum. The current standard-of-care approach includes curative-intent surgery, often after preoperative (neoadjuvant) radiotherapy (RT), to increase rates of tumor down-staging, clinical and pathologic response, as well as improving surgical resection quality. However, despite advancements in surgical techniques, as well as sharpened precision of dosimetry offered by contemporary RT delivery platforms, the oncology community continues to face challenges related to disease relapse. Ongoing investigations are aimed at testing novel radiosensitizing agents and treatments that might exploit the systemic antitumor effects of RT using immunotherapies. If successful, these treatments may usher in a new curative paradigm for rectal cancers, such that surgical interventions may be avoided. Importantly, this disease offers an opportunity to correlate matched paired biopsies, radiographic response, and molecular mechanisms of treatment sensitivity and resistance with clinical outcomes. Herein, the authors highlight the available evidence from preclinical models and early-phase studies, with an emphasis on promising developmental therapeutics undergoing prospective validation in larger scale clinical trials. This review by the National Cancer Institute's Radiation Research Program Colorectal Cancer Working Group provides an updated, comprehensive examination of the continuously evolving state of the science regarding radiosensitizer drug development in the curative treatment of CRC.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/radioterapia , Radiossensibilizantes/uso terapêutico , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Produtos Biológicos , Proteínas de Choque Térmico HSP90/metabolismo , Herpesvirus Humano 1 , Humanos , Imunoterapia/métodos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Terapia de Alvo Molecular , National Cancer Institute (U.S.) , Proteína Quinase C/antagonistas & inibidores , Nucleosídeos de Pirimidina/farmacologia , Radiossensibilizantes/farmacologia , Estados Unidos
18.
Arthritis Res Ther ; 21(1): 92, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971291

RESUMO

BACKGROUND: This study aimed to explore the molecular mechanism and clinical relevance of iguratimod in the regulation of human B cell terminal differentiation. METHODS: An in vitro human antibody-secreting cell (ASC) differentiation system was established to test the effect of iguratimod. B cell phenotype and key transcription factors (TFs) relevant to ASC differentiation were analyzed through flow cytometry and qPCR. The COX-2 activity was measured by enzyme immunoassay (EIA). RNA sequencing was used to identify potential targets of iguratimod. We enrolled six treatment-naive rheumatoid arthritis (RA) patients whose blood samples were collected for phenotypic and molecular studies along with 12-week iguratimod monotherapy. RESULTS: Iguratimod inhibited human ASC generation without affecting B cell activation and proliferation. Iguratimod showed only weak COX-2 activity. Gene set enrichment analysis (GSEA) identified that protein kinase C (PKC) pathway was targeted by iguratimod which was confirmed by PKC activity detection. Furthermore, early growth response 1 (EGR1), a target of PKC and a non-redundant TF for ASC differentiation, was found to be the most downregulated gene in iguratimod-treated B cells. Lastly, iguratimod monotherapy decreased peripheral ASCs and was associated with improved disease activity. The expression of major ASC-related TFs, including EGR1, was similarly downregulated in patient blood samples. CONCLUSIONS: Iguratimod inhibits ASC differentiation both in vitro and in RA patients. Our study suggests that PKC/EGR1 axis, rather than COX-2, is critically involved in the inhibitory effect by iguratimod on human ASC differentiation. Iguratimod could have a broader application to treat B cell-related autoimmune diseases in clinics.


Assuntos
Antirreumáticos/farmacologia , Linfócitos B/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Cromonas/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Sulfonamidas/farmacologia , Antirreumáticos/uso terapêutico , Artrite Reumatoide/sangue , Artrite Reumatoide/tratamento farmacológico , Linfócitos B/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Cromonas/uso terapêutico , Proteína 1 de Resposta de Crescimento Precoce/sangue , Humanos , Proteína Quinase C/sangue , Sulfonamidas/uso terapêutico
19.
Oncol Rep ; 41(5): 2703-2716, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896865

RESUMO

Glioblastoma (GBM) is the most common primary brain tumor. Tumor hypoxia is a pivotal factor responsible for the progression of this malignant glioma, and its resistance to radiation and chemotherapy. Thus, improved tumor tissue oxygenation may promote greater sensitivity to anticancer treatment. Protein kinase D1 (PKD1) protects cells from oxidative stress, and its abnormal activity serves an important role in multiple malignancies. The present study examined the effects of various oxygen conditions on the cytotoxic potential of the novel isothiourea derivate N,N'­dimethyl­S­(2,3,4,5,6­pentabromobenzyl)­â€‹isothiouronium bromide (ZKK­3) against the T98G GBM cell line. ZKK­3 was applied at concentrations of 10, 25 and 50 µM, and cells were maintained under conditions of normoxia, anoxia, hypoxia, hyperbaric oxygen (HBO), hypoxia/hypoxia and hypoxia/HBO. The proliferation and viability of neoplastic cells, and protein expression levels of hypoxia­inducible factor 1α (HIF­1α), PKD1, phosphorylated (p)PKD1 (Ser 916) and pPKD1 (Ser 744/748) kinases were evaluated. Oxygen deficiency, particularly regarding hypoxia, could diminish the cytotoxic effect of ZKK­3 at 25 and 50 µM and improve T98G cell survival compared with normoxia. HBO significantly reduced cell proliferation and increased T98G cell sensitivity to ZKK­3 when compared with normoxia. HIF­1α expression levels were increased under hypoxia compared with normoxia and decreased under HBO compared with hypoxia/hypoxia at 0, 10 and 50 µM ZKK­3, suggesting that HBO improved oxygenation of the cells. ZKK­3 exhibited inhibitory activity against pPKD1 (Ser 916) kinase; however, the examined oxygen conditions did not appear to significantly influence the expression of this phosphorylated form in cells treated with the tested compound. Regarding pPKD1 (Ser 744/748), a significant difference in expression was observed only for cells treated with 10 µM ZKK­3 and hypoxia/hypoxia compared with normoxia. However, there were significant differences in the expression levels of both phosphorylated forms of PKD1 under different oxygen conditions in the controls. In conclusion, the combination of isothiourea derivatives and hyperbaric oxygenation appears to be a promising therapeutic approach for malignant glioma treatment.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Oxigenação Hiperbárica , Isotiurônio/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada/métodos , Glioblastoma/patologia , Humanos , Isotiurônio/análogos & derivados , Isotiurônio/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo
20.
PLoS One ; 14(3): e0211309, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30921339

RESUMO

Serine/threonine protein kinase C (PKC) is activated by diacylglycerol that is released from membrane lipids by phospholipase C in response to activation of G protein-coupled receptors or receptor tyrosine kinases. PKC isoforms are particularly relevant for proliferation and differentiation of cells including osteoblasts. Osteoblasts/osteocytes produce fibroblast growth factor 23 (FGF23), a hormone regulating renal phosphate and vitamin D handling. PKC activates NFκB, a transcription factor complex controlling FGF23 expression. Here, we analyzed the impact of PKC on FGF23 synthesis. Fgf23 expression was analyzed by qRT-PCR in UMR106 osteoblast-like cells and in IDG-SW3 osteocytes, and FGF23 protein was measured by ELISA. Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA), a PKC activator, up-regulated FGF23 production. In contrast, PKC inhibitors calphostin C, Gö6976, sotrastaurin and ruboxistaurin suppressed FGF23 formation. NFκB inhibitor withaferin A abolished the stimulatory effect of PMA on Fgf23. PKC is a powerful regulator of FGF23 synthesis, an effect which is at least partly mediated by NFκB.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Proteína Quinase C/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fatores de Crescimento de Fibroblastos/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Osteoblastos/metabolismo , Osteócitos/metabolismo , Fosfatos/metabolismo , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Regulação para Cima/efeitos dos fármacos
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