RESUMO
Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFß1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.
Assuntos
Anticorpos Monoclonais , Carcinoma de Células Escamosas , Transição Epitelial-Mesenquimal , Netrina-1 , Neoplasias Cutâneas , Animais , Humanos , Camundongos , Células A549 , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores de Netrina/antagonistas & inibidores , Receptores de Netrina/deficiência , Receptores de Netrina/genética , Netrina-1/antagonistas & inibidores , Netrina-1/deficiência , Netrina-1/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Modelos Animais de Doenças , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Metástase Neoplásica/tratamento farmacológico , Análise da Expressão Gênica de Célula Única , RNA-Seq , Molécula de Adesão da Célula Epitelial/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Potassium (K+) efflux across the plasma membrane is thought to be an essential mechanism for ATP-induced NLRP3 inflammasome activation, yet the identity of the efflux channel has remained elusive. Here we identified the two-pore domain K+ channel (K2P) TWIK2 as the K+ efflux channel triggering NLRP3 inflammasome activation. Deletion of Kcnk6 (encoding TWIK2) prevented NLRP3 activation in macrophages and suppressed sepsis-induced lung inflammation. Adoptive transfer of Kcnk6-/- macrophages into mouse airways after macrophage depletion also prevented inflammatory lung injury. The K+ efflux channel TWIK2 in macrophages has a fundamental role in activating the NLRP3 inflammasome and consequently mediates inflammation, pointing to TWIK2 as a potential target for anti-inflammatory therapies.
Assuntos
Inflamassomos/metabolismo , Inflamação/fisiopatologia , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Caspase 1/deficiência , Caspase 1/metabolismo , Linhagem Celular , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/fisiopatologia , Macrófagos/transplante , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/deficiência , Quinina/farmacologia , RNA Interferente Pequeno/farmacologia , Receptores Purinérgicos P2X7/deficiência , Receptores Purinérgicos P2X7/metabolismo , Sepse/metabolismo , Sepse/fisiopatologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Lipoprotein(a) is a presumed risk factor for atherosclerotic cardiovascular disease. Olpasiran is a small interfering RNA that reduces lipoprotein(a) synthesis in the liver. METHODS: We conducted a randomized, double-blind, placebo-controlled, dose-finding trial involving patients with established atherosclerotic cardiovascular disease and a lipoprotein(a) concentration of more than 150 nmol per liter. Patients were randomly assigned to receive one of four doses of olpasiran (10 mg every 12 weeks, 75 mg every 12 weeks, 225 mg every 12 weeks, or 225 mg every 24 weeks) or matching placebo, administered subcutaneously. The primary end point was the percent change in the lipoprotein(a) concentration from baseline to week 36 (reported as the placebo-adjusted mean percent change). Safety was also assessed. RESULTS: Among the 281 enrolled patients, the median concentration of lipoprotein(a) at baseline was 260.3 nmol per liter, and the median concentration of low-density lipoprotein cholesterol was 67.5 mg per deciliter. At baseline, 88% of the patients were taking statin therapy, 52% were taking ezetimibe, and 23% were taking a proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitor. At 36 weeks, the lipoprotein(a) concentration had increased by a mean of 3.6% in the placebo group, whereas olpasiran therapy had significantly and substantially reduced the lipoprotein(a) concentration in a dose-dependent manner, resulting in placebo-adjusted mean percent changes of -70.5% with the 10-mg dose, -97.4% with the 75-mg dose, -101.1% with the 225-mg dose administered every 12 weeks, and -100.5% with the 225-mg dose administered every 24 weeks (P<0.001 for all comparisons with baseline). The overall incidence of adverse events was similar across the trial groups. The most common olpasiran-related adverse events were injection-site reactions, primarily pain. CONCLUSIONS: Olpasiran therapy significantly reduced lipoprotein(a) concentrations in patients with established atherosclerotic cardiovascular disease. Longer and larger trials will be necessary to determine the effect of olpasiran therapy on cardiovascular disease. (Funded by Amgen; OCEAN[a]-DOSE ClinicalTrials.gov number, NCT04270760.).
Assuntos
Anticolesterolemiantes , Aterosclerose , Hipercolesterolemia , Lipoproteína(a) , RNA Interferente Pequeno , Humanos , Anticolesterolemiantes/administração & dosagem , Anticolesterolemiantes/efeitos adversos , Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêutico , Aterosclerose/tratamento farmacológico , Doenças Cardiovasculares/tratamento farmacológico , Método Duplo-Cego , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Lipoproteína(a)/análise , Lipoproteína(a)/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Inibidores de PCSK9/uso terapêutico , Ezetimiba/uso terapêuticoRESUMO
OBJECTIVE: De novo mutations of the voltage-gated sodium channel gene SCN8A cause developmental and epileptic encephalopathy (DEE). Most pathogenic variants result in gain-of-function changes in activity of the sodium channel Nav1.6, poorly controlled seizures, and significant comorbidities. In previous work, an antisense oligonucleotide (ASO) reduced Scn8a transcripts and increased lifespan after neonatal administration to a mouse model. Here, we tested long-term ASO treatment initiated after seizure onset, as required for clinical application. METHODS: ASO treatment was initiated after observation of a convulsive seizure and repeated at 4 to 6 week intervals for 1 year. We also tested the long-term efficacy of an AAV10-short hairpin RNA (shRNA) virus administered on P1. RESULTS: Repeated treatment with the Scn8a ASO initiated after seizure onset provided long-term survival and reduced seizure frequency during a 12 month observation period. A single treatment with viral shRNA was also protective during 12 months of observation. INTERPRETATION: Downregulation of Scn8a expression that is initiated after the onset of seizures is effective for long-term treatment in a model of SCN8A-DEE. Repeated ASO administration or a single dose of viral shRNA prevented seizures and extended survival through 12 months of observation. ANN NEUROL 2024;95:754-759.
Assuntos
Epilepsia , Animais , Camundongos , Modelos Animais de Doenças , Regulação para Baixo/genética , Epilepsia/terapia , Epilepsia/tratamento farmacológico , Mutação , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Convulsões/genética , Canais de Sódio/genéticaRESUMO
The existing knowledge of the involvement of vinculin (VCL) in the control of ovarian cell functions is insufficient. To understand the role of VCL in the control of basic porcine ovarian granulosa cell functions, we decreased VCL activity by small interfering RNA (VCL siRNA). The expression of VCL, accumulation of VCL protein, cell viability, proliferation (accumulation of PCNA and cyclin B1), proportion of proliferative active cells, apoptosis (accumulation of bax, caspase 3, p53, antiapoptotic marker bcl2, and bax/bcl-2 ratio), DNA fragmentation, and release of steroid hormones and IGF-I were analyzed by RTâqPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT assay, TUNEL assay, and ELISA. The suppression of VCL activity inhibited cell viability, the accumulation of the proliferation-related proteins PCNA and cyclin B1, the antiapoptotic protein bcl2, and the proportion of proliferative active cells. Moreover, VCL siRNA inhibited the release of progesterone, estradiol, and IGF-1. VCL siRNA increased the proportion of the proapoptotic proteins bax, caspase 3, p53, the proportion of DNA fragmented cells, and stimulated testosterone release. Taken together, the present study is the first evidence that inhibition of VCL suppresses porcine granulosa cell functions. Moreover, the results suggest that VCL can be a potent physiological stimulator of ovarian functions.
Assuntos
Progesterona , Proteína Supressora de Tumor p53 , Feminino , Suínos , Animais , Ciclina B1/metabolismo , Ciclina B1/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Vinculina/genética , Vinculina/metabolismo , Progesterona/farmacologia , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proliferação de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Células Cultivadas , Fator de Crescimento Insulin-Like I/metabolismoRESUMO
BACKGROUND: Imatinib has become an exceptionally effective targeted drug for treating gastrointestinal stromal tumors (GISTs). Despite its efficacy, the resistance to imatinib is common in GIST patients, posing a significant challenge to the effective treatment. METHODS: The expression profiling of TRIM21, USP15, and ACSL4 in GIST patients was evaluated using Western blot and immunohistochemistry. To silence gene expression, shRNA was utilized. Biological function of TRIM21, USP15, and ACSL4 was examined through various methods, including resistance index calculation, colony formation, shRNA interference, and xenograft mouse model. The molecular mechanism of TRIM21 and USP15 in GIST was determined by conducting Western blot, co-immunoprecipitation, and quantitative real-time PCR (qPCR) analyses. RESULTS: Here we demonstrated that downregulation of ACSL4 is associated with imatinib (IM) resistance in GIST. Moreover, clinical data showed that higher levels of ACSL4 expression are positively correlated with favorable clinical outcomes. Mechanistic investigations further indicated that the reduced expression of ACSL4 in GIST is attributed to excessive protein degradation mediated by the E3 ligase TRIM21 and the deubiquitinase USP15. CONCLUSION: These findings demonstrate that the TRIM21 and USP15 control ACSL4 stability to maintain the IM sensitive/resistant status of GIST.
Assuntos
Antineoplásicos , Neoplasias Gastrointestinais , Tumores do Estroma Gastrointestinal , Humanos , Animais , Camundongos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Resistencia a Medicamentos Antineoplásicos/genética , RNA Interferente Pequeno/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Linhagem Celular Tumoral , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Proteases Específicas de Ubiquitina/farmacologiaRESUMO
Small interfering RNA (siRNA) highlights the immense therapeutic potential for cancer treatment. The major challenge in siRNA therapy is the effective RNA nanodrug delivery system, which is facilitated by the ligand and the carrier. In this study, we analyzed the binding specificity of linear RGD and circular RGD to αVß3 integrins by mapping the morphology using super-resolution direct stochastic optical reconstruction microscopy. Meanwhile, the binding dynamics was investigated using single-molecule force spectroscopy. Then, the effects of the ligand and carrier on RNA nanodrug cell entry dynamic parameters were evaluated at the single particle level by the force tracing technique. Furthermore, the delivery efficiency of RNA nanodrugs was assessed using AFM-based nanoindentation at the single cell level. This report will provide valuable insights for rational design strategies aiming to achieve improved efficiency for nanodrug delivery systems.
Assuntos
RNA Interferente Pequeno , Ligantes , Humanos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/química , Oligopeptídeos/química , Sistemas de Liberação de Medicamentos , Portadores de Fármacos/química , Microscopia de Força Atômica , Nanopartículas/químicaRESUMO
Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.
Assuntos
Afibrinogenemia/metabolismo , Fibrina/biossíntese , Fibrinogênio/biossíntese , Técnicas de Silenciamento de Genes , Lipossomos/farmacologia , RNA Interferente Pequeno , Afibrinogenemia/genética , Animais , Plaquetas/metabolismo , Modelos Animais de Doenças , Feminino , Fibrina/genética , Fibrinogênio/genética , Humanos , Masculino , Camundongos , Nanopartículas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
INTRODUCTION: The ventromedial hypothalamic nucleus (VMN) is an estrogen receptor (ER)-rich structure that regulates glucostasis. The role of nuclear but not membrane G protein-coupled ER-1 (GPER) in that function has been studied. METHODS: Gene silencing and laser-catapult microdissection/immunoblot tools were used to examine whether GPER regulates transmitter and energy sensor function in dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN counter-regulatory nitrergic and γ-Aminobutyric acid (GABA) neurons. RESULTS: Intra-VMN GPER siRNA administration to euglycemic animals did not affect VMNdm or -vl nitrergic neuron nitric oxide synthase (nNOS), but upregulated (VMNdm) or lacked influence on (VMNvl) GABA nerve cell glutamate decarboxylase65/67 (GAD) protein. Insulin-induced hypoglycemia (IIH) caused GPER knockdown-reversible augmentation of nNOS, 5'-AMP-activated protein kinase (AMPK), and phospho-AMPK proteins in nitrergic neurons in both divisions. IIH had dissimilar effects on VMNvl (unchanged) versus VMNdm (increased) GABAergic neuron GAD levels, yet GPER knockdown affected these profiles. GPER siRNA prevented hypoglycemic upregulation of VMNvl and -dm GABA neuron AMPK without altering pAMPK expression. CONCLUSIONS: Outcomes infer that GPER exerts differential control of VMNdm versus -vl GABA transmission during glucostasis and is required for hypoglycemic upregulated nitrergic (VMNdm and -vl) and GABA (VMNdm) signaling. Glycogen metabolism is reported to regulate VMN nNOS and GAD proteins. Data show that GPER limits VMNvl glycogen phosphorylase (GP) protein expression and glycogen buildup during euglycemia but mediates hypoglycemic augmentation of VMNvl GP protein and glycogen content; VMNdm glycogen mass is refractory to GPER control. GPER regulation of VMNvl glycogen metabolism infers that this receptor may govern local counter-regulatory transmission in part by astrocyte metabolic coupling.
Assuntos
Hipoglicemia , Núcleo Hipotalâmico Ventromedial , Ratos , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/farmacologia , Ratos Sprague-Dawley , Norepinefrina/metabolismo , Norepinefrina/farmacologia , Receptores de Estrogênio/metabolismo , Hipoglicemia/metabolismo , Glicogênio/metabolismo , Glicogênio/farmacologia , Hipoglicemiantes/farmacologia , Ácido gama-Aminobutírico/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/farmacologiaRESUMO
After RNAi was first discovered over 20 years ago, siRNA-based therapeutics are finally becoming reality. However, the delivery of siRNA has remained a challenge. In our previous research, we found that spermine-based poly(ß-amino ester)s are very promising for siRNA delivery. However, the role of hydrophobic modification in siRNA delivery of spermine-based poly(ß-amino ester)s is not fully understood yet. In the current work, we synthesized spermine-based poly(ß-amino ester)s with different percentages of oleylamine side chains, named P(SpOABAE). The chemical structures of the polymers were characterized by 1H NMR. The polymers showed efficient siRNA encapsulation determined by SYBR Gold assays. The hydrodynamic diameters of the P(SpOABAE) polyplexes from charge ratio N/P 1 to 20 were 30-100 nm except for aggregation phenomena observed at N/P 3. Morphology of the polyplexes was visualized by atomic force microscopy, and cellular uptake was determined by flow cytometry in H1299 cells, where all the polyplexes showed significantly higher cellular uptake than hyperbranched polyethylenimine (25 kDa). The most hydrophobic P(SpOABAE) polyplexes were able to achieve more than 90% GFP knockdown in H1299/eGFP cells. The fact that gene silencing efficacy increased with hydrophobicity but cellular uptake was affected by both charge and hydrophobic interactions highlights the importance of endosomal escape. For pulmonary administration and improved storage stability, the polyplexes were spray-dried. Results confirmed the maintained siRNA activity after storage for 3 months at room temperature, indicating potential for dry powder inhalation.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , RNA Interferente Pequeno , Espermina , RNA Interferente Pequeno/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Espermina/química , Humanos , Administração por Inalação , Polímeros/química , Pós/química , Linhagem Celular TumoralRESUMO
The simultaneous delivery of CpG oligonucleotide along with short interfering RNA (siRNA) has the potential to significantly boost the anticancer impact of siRNA medications. Our previous research demonstrated that Curdlan nanoparticles functionalized with adenosine are capable of selectively delivering therapeutic siRNA to cancerous cells through endocytosis mediated by adenosine receptors. Herein, we synthesized a dual-ligand-functionalized Curdlan polymer (denoted by CuMAN) to simultaneously target tumor cells and tumor-associated macrophages (TAMs). CuMAN nanoparticles containing CpG and siRNA demonstrated enhanced uptake by B16F10 tumor cells and bone marrow-derived macrophages, which are facilitated by AR on tumor cells and mannose receptor on macrophages. This led to increased release of pro-inflammatory cytokines in both in vitro and in vivo settings. The synergistic effect of CpG on TAMs and RNAi on tumor cells mediated by the CuMAN nanoparticle not only suppressed the tumor growth but also strongly inhibited the lung metastasis. Our findings indicate that the CuMAN nanoparticle has potential as an effective dual-targeting delivery system for nucleic acid therapeutics.
Assuntos
Nanopartículas , RNA Interferente Pequeno , beta-Glucanas , Animais , beta-Glucanas/química , beta-Glucanas/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/química , Nanopartículas/química , Camundongos , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/farmacologia , Melanoma Experimental/patologia , Melanoma Experimental/tratamento farmacológico , Linhagem Celular Tumoral , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Ligantes , Sistemas de Liberação de Medicamentos/métodos , Macrófagos Associados a Tumor/efeitos dos fármacosRESUMO
Blood pressure management involves antihypertensive therapies blocking the renin-angiotensin system (RAS). Yet, it might be inadequate due to poor patient adherence or the so-called RAS escape phenomenon, elicited by the compensatory renin elevation upon RAS blockade. Recently, evidence points toward targeting hepatic AGT (angiotensinogen) as a novel approach to block the RAS pathway that could circumvent the RAS escape phenomenon. Removing AGT, from which all angiotensins originate, should prevent further angiotensin generation, even when renin rises. Furthermore, by making use of a trivalent N-acetylgalactosamine ligand-conjugated small interfering RNA that specifically targets the degradation of hepatocyte-produced mRNAs in a highly potent and specific manner, it may be possible in the future to manage hypertension with therapy that is administered 1 to 2× per year, thereby supporting medication adherence. This review summarizes all current findings on AGT small interfering RNA in preclinical models, making a comparison versus classical RAS blockade with either ACE (angiotensin-converting enzyme) inhibitors or AT1 (angiotensin II type 1) receptor antagonists and AGT suppression with antisense oligonucleotides. It ends with discussing the first-in-human study with AGT small interfering RNA.
Assuntos
Angiotensinogênio , Hipertensão , Humanos , Acetilgalactosamina , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Angiotensinogênio/genética , Angiotensinogênio/metabolismo , Pressão Sanguínea , Hipertensão/terapia , Hipertensão/tratamento farmacológico , Renina/metabolismo , Sistema Renina-Angiotensina/fisiologia , RNA Interferente Pequeno/farmacologiaRESUMO
Neuroinflammation and microglia polarization play pivotal roles in brain injury induced by intracerebral hemorrhage (ICH). Despite the well-established involvement of CXC motif chemokine ligand 16 (CXCL16) in regulating inflammatory responses across various diseases, its specific functions in the context of neuroinflammation and microglial polarization following ICH remain elusive. In this study, we investigated the impact of CXCL16 on neuroinflammation and microglia polarization using both mouse and cell models. Our findings revealed elevated CXCL16 expression in mice following ICH and in BV2 cells after lipopolysaccharide (LPS) stimulation. Specific silencing of CXCL16 using siRNA led to a reduction in the expression of neuroinflammatory factors, including IL-1ß and IL-6, as well as decreased expression of the M1 microglia marker iNOS. Simultaneously, it enhanced the expression of anti-inflammatory factors such as IL-10 and the M2 microglia marker Arg-1. These results were consistent across both mouse and cell models. Intriguingly, co-administration of the PI3K-specific agonist 740 Y-P with siRNA in LPS-stimulated cells reversed the effects of siRNA. In conclusion, silencing CXCL16 can positively alleviate neuroinflammation and M1 microglial polarization in BV2 inflammation models and ICH mice. Furthermore, in BV2 cells, this beneficial effect is mediated through the PI3K/Akt pathway. Inhibition of CXCL16 could be a novel approach for treating and diagnosing cerebral hemorrhage.
Assuntos
Hemorragia Cerebral , Quimiocina CXCL16 , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Microglia , Doenças Neuroinflamatórias , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Animais , Quimiocina CXCL16/metabolismo , Microglia/metabolismo , Microglia/efeitos dos fármacos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Hemorragia Cerebral/metabolismo , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Doenças Neuroinflamatórias/metabolismo , Masculino , Polaridade Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Inativação Gênica , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/administração & dosagemRESUMO
OBJECTIVE: Despite considerable improvement in therapeutic approaches to chronic myeloid leukemia (CML) treatment, this malignancy is considered incurable due to resistance. However, investigating the molecular mechanism of CML may give rise to the development of extremely efficient targeted therapies that improve the prognosis of patients. Basic leucine zipper transcription factor ATF-like3 (BATF3), as transcription factor, is considered a key regulator of cellular activities and its function has been evaluated in tumor development and growth in several cancer types. This study aimed to evaluate the potential of the cellular impact of siRNA-mediated downregulation of BATF3 on CML cancer cells through cell proliferation, induction of apoptosis, and cell cycle distribution. MATERIALS AND METHODS: The transfection of BATF3 siRNA to K562 CML cells was performed by electroporation device. To measure cellular viability and apoptosis, MTT assay and Annexin V/PI staining were carried out, respectively. Also, cell cycle assay and flow cytometry instrument were applied to assess cell cycle distribution of K562 cells. For more validation, mRNA expression of correlated genes was relatively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The data indicated that siRNA-mediated BATF3 inactivating severely promoted the cell apoptosis. Also, the targeted therapy led to high expression of Caspase-3 gene and Bax/Bcl-2 ratio. Silenced BATF3 also induced cell cycle arrest in phase sub-G1 compared to control. Finally, a noticeable decrement was obtained in c-Myc gene expression through suppression of BATF3 in CML cells. CONCLUSION: The findings of this research illustrated the suppression of BATF3 as an effective targeted therapy strategy for CML.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Apoptose/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
Glioblastoma multiforme (GBM) is among the malignant brain tumors of the central nervous system (CNS). The survival of this disease is about 14 months after diagnosis. To date, temozolomide is known as first-line therapy for glioma. Drug resistance and severe side effects against this drug are important obstacles to the effective treatment of this cancer. Small interfering RNA (siRNA) can adjust the expression of several genes and is used as a new method of gene therapy. Recent studies have shown that siRNAs can increase the sensitivity of cancer cells to chemotherapy drugs. This study aimed to understand the potential role and molecular mechanism of the combination therapy of B7H6-siRNA and temozolomide in glioblastoma cancer. U87 cells were treated with B7H6-siRNA and temozolomide, separately and in combination. Cell viability, stemness, cell migration, and apoptosis were measured. The results of this work presented the synergistic effect of B7H6-siRNA and temozolomide in inhibiting the cancerous features of the U87 cell line. Down-regulating B7H6-siRNA expression inhibited the cell viability of U87 glioblastoma cancer cells and increased their sensitivity to temozolomide. In addition, a noteworthy decrease in cell migration ability and stemness, an increase in apoptosis were observed in the combined groups compared to B7H6-siRNA and temozolomide individually. According to the results, a combination of B7H6-siRNA and temozolomide can be a promising strategy in glioblastoma cancer therapy.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Apoptose , Resistencia a Medicamentos Antineoplásicos , Proliferação de Células , Antineoplásicos Alquilantes/farmacologiaRESUMO
Brain metastasis (BM) is one of the leading causes of cancer-related deaths in patients with advanced non-small cell lung cancer (NSCLC). However, limited treatments are available due to the presence of the blood-brain barrier (BBB). Upregulation of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in NSCLC has been found to promote BM. Conversely, downregulating LPCAT1 significantly suppresses the proliferation and metastasis of lung cancer cells. In this study, we firstly confirmed significant upregulation of LPCAT1 in BM sites compared to primary lung cancer by analyzing scRNA dataset. We then designed a delivery system based on a single-chain variable fragment (scFv) targeting the epidermal growth factor receptor (EGFR) and exosomes derived from HEK293T cells to enhance cell-targeting capabilities and increase permeability. Next, we loaded LPCAT1 siRNA (siLPCAT1) into these engineered exosomes (exoscFv). This novel scFv-mounted exosome successfully crossed the BBB in an animal model and delivered siLPCAT1 to the BM site. Silencing LPCAT1 efficiently arrested tumor growth and inhibited malignant progression of BM in vivo without detectable toxicity. Overall, we provided a potential platform based on exosomes for RNA interference (RNAi) therapy in lung cancer BM.
Assuntos
Neoplasias Encefálicas , Carcinoma Pulmonar de Células não Pequenas , Exossomos , Neoplasias Pulmonares , Animais , Humanos , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismo , Carcinoma Pulmonar de Células não Pequenas/terapia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , RNA Interferente Pequeno/farmacologia , Exossomos/metabolismo , Células HEK293 , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismoRESUMO
Pancreatic cancer, predominantly pancreatic ductal adenocarcinoma (PDAC), remains a highly lethal malignancy with limited therapeutic options and a dismal prognosis. By targeting the underlying molecular abnormalities responsible for PDAC development and progression, gene therapy offers a promising strategy to overcome the challenges posed by conventional radiotherapy and chemotherapy. This study sought to explore the therapeutic potential of small activating RNAs (saRNAs) specifically targeting the CCAAT/enhancer-binding protein alpha (CEBPA) gene in PDAC. To overcome the challenges associated with saRNA delivery, tetrahedral framework nucleic acids (tFNAs) were rationally engineered as nanocarriers. These tFNAs were further functionalized with a truncated transferrin receptor aptamer (tTR14) to enhance targeting specificity for PDAC cells. The constructed tFNA-based saRNA formulation demonstrated exceptional stability, efficient saRNA release ability, substantial cellular uptake, biocompatibility, and nontoxicity. In vitro experiments revealed successful intracellular delivery of CEBPA-saRNA utilizing tTR14-decorated tFNA nanocarriers, resulting in significant activation of tumor suppressor genes, namely, CEBPA and its downstream effector P21, leading to notable inhibition of PDAC cell proliferation. Moreover, in a mouse model of PDAC, the tTR14-decorated tFNA-mediated delivery of CEBPA-saRNA effectively upregulated the expression of the CEBPA and P21 genes, consequently suppressing tumor growth. These compelling findings highlight the potential utility of saRNA delivered via a designed tFNA nanocarrier to induce the activation of tumor suppressor genes as an innovative therapeutic approach for PDAC.
Assuntos
Aptâmeros de Nucleotídeos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Receptores da Transferrina , Animais , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Receptores da Transferrina/metabolismo , Camundongos , Linhagem Celular Tumoral , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proliferação de Células/efeitos dos fármacos , Terapia Genética/métodos , RNA Interferente Pequeno/farmacologia , Camundongos NusRESUMO
At the time of writing, although siRNA therapeutics are approved for human use, no official regulatory guidance specific to this modality is available. In the absence of guidance, preclinical development for siRNA followed a hybrid of the small molecule and biologics guidance documents. However, siRNA differs significantly from small molecules and protein-based biologics in its physicochemical, absorption, distribution, metabolism and excretion properties, and its mechanism of action. Consequently, certain reports typically included in filing packages for small molecule or biologics may benefit from adaption, or even omission, from an siRNA filing. In this white paper, members of the 'siRNA working group' in the IQ Consortium compile a list of reports included in approved siRNA filing packages and discuss the relevance of two in vitro reports-the plasma protein binding evaluation and the drug-drug interaction risk assessment-to support siRNA regulatory filings. Publicly available siRNA approval packages and the literature were systematically reviewed to examine the role of siRNA plasma protein binding and drug-drug interactions in understanding pharmacokinetic/pharmacodynamic relationships, safety and translation. The findings are summarized into two decision trees to help guide industry decide when in vitro siRNA plasma protein binding and drug-drug interaction studies are warranted.
Assuntos
Proteínas Sanguíneas , Interações Medicamentosas , Produtos Biológicos , Proteínas Sanguíneas/química , Árvores de Decisões , Humanos , Ligação Proteica , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
Choroidal Neovascularization (CNV) is capable of inciting recurrent hemorrhage in the macular region, severely impairing patients' visual acuity. During the onset of CNV, infiltrating M2 macrophages play a crucial role in promoting angiogenesis. To control this disease, our study utilizes the RNA interference (RNAi)-based gene therapy to reprogram M2 macrophages to the M1 phenotype in CNV lesions. We synthesize the mannose-modified siRNA-loaded liposome specifically targeting M2 macrophages to inhibit the inhibitory kappa B kinase ß (IKKß) gene involved in the polarization of macrophages, consequently modulating macrophage polarization state. In vitro and in vivo, the mannose-modified IKKß siRNA-loaded liposome (siIKKß-ML) has been proven to effectively target M2 macrophages to repolarize them to M1 phenotype, and inhibit the progression of CNV. Collectively, our findings elucidate that siIKKß-ML holds the potential to control CNV by reprogramming the macrophage phenotype, indicating a promising therapeutic avenue for CNV management.
Assuntos
Neovascularização de Coroide , Quinase I-kappa B , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Quinase I-kappa B/genética , Quinase I-kappa B/farmacologia , Lipossomos/farmacologia , Manose , Neovascularização de Coroide/genética , Macrófagos , Terapia GenéticaRESUMO
This study investigated the expression of sortilin 1 (SORT1) in cultured human dental pulp-derived stem cells (hDPSCs) and its role in their odontoblastic differentiation. Permanent teeth were extracted from five patients, and the dental pulp was harvested for explant culture. Fluorescence-activated cell sorting was used to analyze the outgrowth of adherent cells and cells that had migrated from the tissue margin. SORT1 expression was detected in hDPSCs simultaneously expressing the mesenchymal stem cell markers CD44 and CD90. The odontoblastic differentiation potential of SORT1-positive hDPSCs was examined via staining for alkaline phosphatase (ALP), an early odontoblastic differentiation marker. ALP staining was more intense in SORT1-positive than in SORT1-negative hDPSCs. Consistently, the expression of mRNA encoding SORT1 and p75NTR, a binding partner of SORT1, increased in SORT1-positive hDPSCs during odontoblastic differentiation. In addition, pro-nerve growth factor (NGF), a ligand for SORT1-p75NTR co-receptor, promoted ALP expression in SORT1-positive hDPSCs, and the interaction between SORT1 and p75NTR was detected using a coimmunoprecipitation assay. The function of SORT1 in odontoblastic differentiation was examined via RNA interference using shRNA targeting SORT1. ALP staining intensity in SORT1/shRNA-transfected cells was markedly lower than in control/shRNA-transfected cells. SORT1 knockdown decreased JUN phosphorylation and recruitment of phosphorylated JUN to the ALP promoter. Collectively, these results indicate that SORT1 is involved in the odontoblastic differentiation of hDPSCs through the JUN N-terminal kinases (JNK)/JUN signaling pathway and that the binding of SORT1 and p75NTR plays an important role in this process.