RESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a prominent cause of various chronic metabolic hepatic diseases with limited therapeutics. Rubicon, an essential regulator in lysosomal degradation, is reported to exacerbate hepatic steatosis in NAFLD mice and patients, indicating its probability of being a therapeutic target for NAFLD treatment. In this study, the therapeutic potential of Rubicon blockage is investigated. Lipid nanoparticles carrying Rubicon-specific CRISPR-Cas9 components exhibited liver accumulation, cell internalization, and Rubicon knockdown. A single administration of the nanoparticles results in attenuated lipid deposition and hepatic steatosis, with lower circulating lipid levels and decreased adipocyte size in NAFLD mice. Furthermore, the increase of phosphatidylcholine and phosphatidylethanolamine levels can be observed in the NAFLD mice livers after Rubicon silencing, along with regulatory effects on metabolism-related genes such as CD36, Gpcpd1, Chka, and Lpin2. The results indicate that knockdown of Rubicon improves glycerophospholipid metabolism and thereby ameliorates the NAFLD progression, which provides a potential strategy for NAFLD therapy via the restoration of Rubicon.
Asunto(s)
Antígenos CD36 , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Glicerofosfolípidos , Nanopartículas , Enfermedad del Hígado Graso no Alcohólico , Animales , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/terapia , Ratones , Glicerofosfolípidos/metabolismo , Sistemas CRISPR-Cas/genética , Antígenos CD36/genética , Antígenos CD36/metabolismo , Ratones Endogámicos C57BL , Masculino , Lípidos , Metabolismo de los Lípidos/genética , Humanos , LiposomasRESUMEN
Acute liver failure (ALF) is a serious inflammatory disorder with high mortality rates, which poses a significant threat to human health. The IL-33/ST2 signal is a crucial regulator in inflammation responses associated with lipopolysaccharide (LPS)-induced macrophages. The IL-17A signaling pathway promotes the release of chemokines and inflammatory cytokines, recruiting neutrophils and T cells under LPS stimulation, thus facilitating inflammatory responses. Here, the potential therapeutic benefits of neutralizing the IL-17A signal and modulating the IL-33/ST2 signal in ALF were investigated. A novel dual-functional fusion protein, anti-IL-17A-sST2, was constructed, which displayed high purity and biological activities. The administration of anti-IL-17A-sST2 resulted in significant anti-inflammatory benefits in ALF mice, amelioration of hepatocyte necrosis and interstitial congestion, and reduction in TNF-α and IL-6. Furthermore, anti-IL-17A-sST2 injection downregulated the expression of TLR4 and NLRP3 as well as important molecules such as MyD88, caspase-1, and IL-1ß. The results suggest that anti-IL-17A-sST2 reduced the secretion of inflammatory factors, attenuated the inflammatory response, and protected hepatic function by regulating the TLR4/MyD88 pathway and inhibiting the NLRP3 inflammasome, providing a new therapeutic approach for ALF.
RESUMEN
Research and development on Nectin-4 antibody-drug conjugates (ADC) have been greatly accelerated since the approval of enfortumab vedotin to treat uroepithelial cancer. During the course of this study, we identified that autophagy serves as a cytoprotective mechanism during Nectin-4-MMAE treatment and proposed a strategy to enhance the antitumor effects of Nectin-4-MMAE in bladder cancer. Nectin-4-MMAE rapidly internalized into bladder cancer cells in 30 minutes and released MMAE, inducing the onset of caspase-mediated apoptosis and leading to the inhibition of tumor cell growth. Transcriptomics showed significant alterations in autophagy-associated genes in bladder cancer cells treated with Nectin-4-MMAE, which suggested autophagy was activated by Nectin-4-MMAE. Furthermore, autophagy activation was characterized by ultrastructural analysis of autophagosome accumulation, immunofluorescence of autophagic flux, and immunoblotting autophagy marker proteins SQSTM1 and LC3 I/II. Importantly, inhibiting autophagy by LY294002 and chloroquine significantly enhances the cytotoxicity effects of Nectin-4-MMAE in bladder cancer cells. Additionally, we detected the participation of the AKT/mTOR signaling cascade in the induction of autophagy by Nectin-4-MMAE. The combination of Nectin-4-MMAE and an autophagy inhibitor demonstrated enhanced antitumor effects in the HT1376 xenograft tumor model. After receiving a single dose of Nectin-4-MMAE, the group that received the combination treatment showed a significant decrease in tumor size compared to the group that received only one type of treatment. Notably, one mouse in the combination treatment group achieved complete remission of the tumor. The combination group exhibited a notable rise in apoptosis and necrosis, as indicated by H&E staining and immunohistochemistry (cleaved caspase-3, ki67). These findings demonstrated the cytoprotective role of autophagy during Nectin-4-MMAE treatment and highlighted the potential of combining Nectin-4-MMAE with autophagy inhibitors for bladder cancer treatment.
Asunto(s)
Autofagia , Moléculas de Adhesión Celular , Morfolinas , Nectinas , Neoplasias de la Vejiga Urinaria , Autofagia/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Humanos , Animales , Línea Celular Tumoral , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/genética , Ratones , Morfolinas/farmacología , Morfolinas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Oligopéptidos/farmacología , Apoptosis/efectos de los fármacos , Ratones Desnudos , Cromonas/farmacología , Cloroquina/farmacología , Cloroquina/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Ratones Endogámicos BALB C , Femenino , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Macrophages are the origin of most foam cells in the early stage of atherosclerotic plaques. However, the mechanism involved in the formation of macrophage-derived foam cell formation remains unclear. Here, we revealed that the hedgehog (Hh) signaling is critical in autophagy-lysosome pathway regulation and macrophage-derived foam cell formation. Inhibition of Hh signaling by vismodegib ameliorated lipid deposition and oxidative stress level in atherosclerotic plaques in high-fat diet-fed apoE-/- mice. For mechanistic study, how the Hh signaling modulate the process of foam cell formation were accessed afterward. Unexpectedly, we found that suppression of Hh signaling in apoE-/- mice had no significant impact on circulating cholesterol levels, indicating that Hh pathway modulate the procession of atherosclerotic plaque not through a traditional lipid-lowing mechanism. Instead, vismodegib was found to accelerate autophagosomes maturation as well as cholesterol efflux in macrophage-derived foam cell and in turn improve foam cell formation, while autophagy inhibitors (LY294002 or CQ) administration significantly attenuated vismodegib-induced cholesterol efflux and reversed the effect on foam cell formation. Therefore, our result demonstrated that inhibition of the Hh signaling pathway increases cholesterol efflux and ameliorates macrophage-derived foam cell formation by promoting autophagy in vitro. Our data thus suggested a novel therapeutic target of atherosclerosis and indicated the potential of vismodegib to treat atherosclerosis.
Asunto(s)
Anilidas , Aterosclerosis , Placa Aterosclerótica , Piridinas , Animales , Ratones , Células Espumosas/metabolismo , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/metabolismo , Proteínas Hedgehog/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Transducción de Señal , Colesterol/metabolismo , Lípidos/farmacología , Autofagia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismoAsunto(s)
Antiinfecciosos , Neoplasias , Surfactantes Pulmonares , Humanos , Antibacterianos , Penicilinas , Neoplasias/terapiaRESUMEN
Immunotherapies including adaptive immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T cells, have developed the treatment of cancer in clinic, and most of them focus on activating T cell immunity. Although these strategies have obtained unprecedented clinical responses, only limited subsets of cancer patients could receive long-term benefits, highlighting the demand for identifying novel targets for the new era of tumor immunotherapy. Innate immunity has been demonstrated to play a determinative role in the tumor microenvironment (TME) and influence the clinical outcomes of tumor patients. A thorough comprehension of the innate immune cells that infiltrate tumors would allow for the development of new therapeutics. In this review, we outline the role and mechanism of innate immunity in TME. Moreover, we discuss innate immunity-based cancer immunotherapy in basic and clinical studies. Finally, we summarize the challenges in sufficiently motivating innate immune responses and the corresponding strategies and measures to improve anti-tumor efficacy. This review could aid the comprehension of innate immunity and inspire the creation of brand-new immunotherapies for the treatment of cancer.
Asunto(s)
Neoplasias , Humanos , Neoplasias/patología , Inmunidad Innata , Inmunoterapia , Linfocitos T , Biología , Microambiente TumoralRESUMEN
Background: Only a subset of B-cell lymphoma (BCL) patients can benefit from immune checkpoint inhibitors targeting PD-1/PD-L1. Materials & methods: In the A20 model, SIRPα-Fc and anti-PD-L1 were employed to target CD47 and PD-L1 simultaneously. Flow cytometry, immunofluorescence and quantitative polymerase chain reaction were used to unravel the potential mechanisms. Results: Simultaneously targeting CD47 and PD-L1 activated CD8+ T cells with an increased release of effector molecules. Furthermore, infiltration of F4/80+iNOS+ M1 macrophages was enhanced by the dual therapy. Conclusion: Anti-CD47 therapy could sensitize BCL tumors to anti-PD-L1 therapy in a CD8+ T-cell- and M1-macrophage-dependent manner by promoting cytotoxic lymphocyte infiltration, which may provide a potential strategy for BCL treatment by simultaneously targeting CD47 and PD-L1.
Immune checkpoint inhibitors targeting PD-1/PD-L1 have become effective agents for cancer treatment. However, only a minority of patients benefit from this treatment in the clinic because of the limited response rate. Targeting CD47/SIRPα restores macrophage function and improves the response of antitumor immunity. Here, combination immunotherapy targeting CD47/SIRPα and PD-1/PD-L1 was investigated to increase the response rate and antitumor effect of PD-L1 monotherapy in B-cell lymphoma (BCL). This study broadens the application of the combination therapy and provided a promising strategy for B-cell lymphoma treatment by simultaneous targeting of PD-1/PD-L1 and CD47/SIRPα axis.
Asunto(s)
Linfoma de Células B , Neoplasias , Humanos , Antígeno CD47 , Linfocitos T CD8-positivos , Inmunoterapia , Linfoma de Células B/tratamiento farmacológico , Macrófagos , Antígeno B7-H1/metabolismoRESUMEN
The pathogenesis of diabetic kidney disease (DKD) is complicated. Current clinical treatments fail to achieve satisfactory efficacy in the prevention of DKD progression, it urgently needs novel and effective treatment for DKD. In this study, we firstly demonstrated that renal lipid metabolism abnormality and inflammation significantly changed in DKD conditions by mining public transcriptomic data of DKD patient samples. KEGG analysis further exhibited the critical role of vascular endothelial growth factor B (VEGF-B) and interleukin 17A (IL-17A) signal pathways in DKD progression, indicating that VEGF-B and IL-17A might be the promising targets for DKD treatment. Then the potential of a novel combination therapy, anti-VEGF-B plus anti-IL-17A antibody, was evaluated for DKD treatment. Our results demonstrated that simultaneous blockade of VEGF-B and IL-17A signaling with their neutralizing antibodies alleviated renal damage and ameliorated renal function. The therapeutic effectiveness was not only related to the reduced lipid deposition especially the neutral lipids in kidney but also associated with the decreased inflammation response. Moreover, the therapy alleviated renal fibrosis by reducing collagen deposition and the expression of fibronectin and α-SMA in kidney tissues. RNA-seq analysis indicated that differential expression genes (DEGs) in db/db mice were significantly clustered into lipid metabolism, inflammation, fibrosis and DKD pathology-related pathways, and 181 of those DEGs were significantly reversed by the combinatory treatment, suggesting the underlying mechanism of administration of anti-VEGF-B and anti-IL-17A antibodies in DKD treatment. Taken together, this study identified that renal lipid metabolism abnormality and inflammation were critically involved in the progression of DKD, and simultaneous blockade of VEGF-B and IL-17A signaling represents a potential DKD therapeutic strategy.
RESUMEN
Introduction: The pathogenic mechanisms of diabetic nephropathy (DN) include podocyte injury, inflammatory responses and metabolic disorders. Although the antagonism of Angiopoietin-like protein 3 (ANGPTL3) can alleviate proteinuria symptoms by inhibiting the activation of integrin αvß3 on the surface of podocytes, it can not impede other pathological processes, such as inflammatory responses and metabolic dysfunction of glucolipid. Interleukin-22 (IL-22) is considered to be a pivotal molecule involved in suppressing inflammatory responses, initiating regenerative repair, and regulating glucolipid metabolism. Methods: Genes encoding the mIL22IgG2aFc and two chains of anti-ANGPTL3 antibody and bifunctional protein were synthesized. Then, the DN mice were treated with intraperitoneal injection of normal saline, anti-ANGPTL3 (20 mg/kg), mIL22Fc (12 mg/kg) or anti-ANGPTL3 /IL22 (25.3 mg/kg) and irrigation of positive drug losartan (20mg/kg/d) twice a week for 8 weeks. Results: In this research, a novel bifunctional fusion protein (anti-ANGPTL3/IL22) formed by the fusion of IL-22 with the C-terminus of anti-ANGPTL3 antibody exhibited favorable stability and maintained the biological activity of anti-ANGPTL3 and IL-22, respectively. The fusion protein showed a more pronounced attenuation of proteinuria and improved dysfunction of glucolipid metabolism compared with mIL22Fc or anti-ANGPTL3. Our results also indicated that anti-ANGPTL3/IL22 intervention significantly alleviated renal fibrosis via inhibiting the expression of the inflammatory response-related protein nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) p65 and NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome. Moreover, transcriptome analysis revealed the downregulation of signaling pathways associated with injury and dysfunction of the renal parenchymal cell indicating the possible protective mechanisms of anti-ANGPTL3/IL22 in DN. Conclusion: Collectively, anti-ANGPTL3/IL22 bifunctional fusion protein can be a promising novel therapeutic strategy for DN by reducing podocyte injury, ameliorating inflammatory response, and enhancing renal tissue recovery.
Asunto(s)
Diabetes Mellitus , Nefropatías Diabéticas , Ratones , Animales , Nefropatías Diabéticas/patología , Proteína 3 Similar a la Angiopoyetina , Interleucinas/uso terapéutico , Proteinuria/tratamiento farmacológico , Interleucina-22RESUMEN
Introduction: Although PD-1/L1 mAb has demonstrated clinical benefits in certain cancer types, low response rate and resistance remain the main challenges for the application of these immune checkpoint inhibitors (ICIs). 4-1BB is a co-stimulator molecule expressed in T cells, which could enhance T cell proliferation and activation. Herein, the synergetic antitumor effect and underlying mechanism of 4-1BB agonist combined with PD-1/PD-L1 blockade were determined in B-cell lymphoma (BCL). Methods: Subcutaneous transplantation BCL tumor models and metastasis models were established to evaluate the therapeutic effect of PD-L1 antibody and/or 4-1BB agonist in vivo. For the mechanistic study, RNA-seq was applied to analyze the tumor microenvironment and immune-related signal pathway after combination treatment. The level of IFN-γ, perforin, and granzyme B were determined by ELISA and Real-time PCR assays, while tumor-infiltrating T cells were measured by flow cytometry and immunohistochemical analysis. CD4/CD8 specific antibodies were employed to deplete the related T cells to investigate the role CD4+ and CD8+ T cells played in combination treatment. Results: Our results showed that combining anti-PD-L1 ICI and 4-1BB agonists elicited regression of BCL and significantly extended the survival of mice compared to either monotherapy. Co-targeting PD-L1 and 4-1BB preferentially promoted intratumoral cytotoxic lymphocyte infiltration and remodeled their function. RNA-sequence analysis uncovered a series of up-regulated genes related to the activation and proliferation of cytotoxic T lymphocytes, further characterized by increased cytokines including IFN-γ, granzyme B, and perforin. Furthermore, depleting CD8+ T cells not CD4+ T cells totally abrogated the antitumor efficacy, indicating the crucial function of the CD8+ T cell subset in the combination therapy. Discussion: In summary, our findings demonstrated that 4-1BB agonistic antibody intensified the antitumor immunity of anti-PD-1/PD-L1 ICI via promoting CD8+ T cell infiltration and activation, providing a novel therapeutic strategy to BCL.
Asunto(s)
Antineoplásicos , Linfoma de Células B , Neoplasias , Animales , Ratones , Antineoplásicos/uso terapéutico , Granzimas , Linfoma de Células B/tratamiento farmacológico , Perforina , Microambiente TumoralRESUMEN
Protein modifications such as post-translational modifications (PTMs) and sequence variants (SVs) occur frequently during protein biosynthesis and have received great attention by biopharma industry and regulatory agencies. In this study, an aberrant peak near light chain (LC) was observed in the non-reduced capillary electrophoresis sodium dodecyl sulfate (nrCE-SDS) electrophoretogram during cell line development of one bispecific antibody (BsAb) product, and the detected mass was about 944 Da higher than LC. The corresponding peak was then enriched by denaturing size-exclusion chromatography (SEC-HPLC) and further characterized by nrCE-SDS and peptide mapping analyses. De novo mass spectra/mass spectra (MS/MS) analysis revealed that the aberrant peak was LC related sequence variant, with the truncated C-terminal sequence "SFNR" ("GEC"deleted) linked with downstream SV40 promotor sequence "EAEAASASELFQ". The unusual sequence was further confirmed by comparing with the direct synthetic peptide "SFNREAEAASASELFQ". It was demonstrated by mRNA sequencing of the cell pool that the sequence variant was caused by aberrant splicing at the transcription step. The prepared product containing this extension variant maintained well-folded structure and good functional properties though the LC/Heavy chain (HC) inter-chain disulfide was not formed. Several control strategies to mitigate the risk of this LC related sequence variant were also proposed.
RESUMEN
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a metabolic disease mainly on account of hypercholesterolemia and may progress to cirrhosis and hepatocellular carcinoma. The discovery of effective therapy for NAFLD is an essential unmet need. Angiopoietin-like protein 3 (ANGPTL3), a critical lipid metabolism regulator, resulted in increased blood lipids and was elevated in NAFLD. Here, we developed a nanobody-heavy chain antibody (VHH-Fc) to inhibit ANGPTL3 for NAFLD treatment. RESULTS: In this study, we retrieved an anti-ANGPTL3 VHH and Fc fusion protein, C44-Fc, which exhibited high affinities to ANGPTL3 proteins and rescued ANGPLT3-mediated inhibition of lipoprotein lipase (LPL) activity. The C44-Fc bound a distinctive epitope within ANGPTL3 when compared with the approved evinacumab, and showed higher expression yield. Meanwhile, C44-Fc had significant reduction of the triglyceride (~ 44.2%), total cholesterol (~ 36.6%) and LDL-cholesterol (~ 54.4%) in hypercholesterolemic mice and ameliorated hepatic lipid accumulation and liver injury in NAFLD mice model. CONCLUSIONS: We discovered a VHH-Fc fusion protein with high affinity to ANGPTL3, strong stability and also alleviated the progression of NAFLD, which might offer a promising therapy for NAFLD.
Asunto(s)
Proteína 3 Similar a la Angiopoyetina , Enfermedad del Hígado Graso no Alcohólico , Proteínas Similares a la Angiopoyetina/metabolismo , Animales , LDL-Colesterol , Lípidos , Ratones , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Triglicéridos/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: Indoleamine-2,3-dioxygenase 1 (IDO1), which catalyzes the degradation of L-tryptophan (L-Trp) to N-formyl kynurenine (NFK) in the first and rate-limiting step of Kynurenine (KYN) pathway has been identified as a promising therapeutic target for cancer immunotherapy. The small molecule Epacadostat developed by Incyte Corp is the most advanced IDO1 inhibitor in clinical trials. METHODS: In this study, various amidine derivatives were individually installed as the polar capping group onto the amino ethylene side chain to replace the sulfamoylamino moiety of Epacadostat to develop novel IDO1 inhibitors. A series of novel 1,2,5-oxadiazol-3-carboximidamide derivatives were designed, prepared, and evaluated for their inhibitory activities against human IDO1 enzyme and cellular IDO1. RESULTS: In vitro human IDO1 enzyme and cellular IDO1 assay results demonstrate that the inhibitory activities of compound 13a and 13b were comparable to Epacadostat, with the enzymatic IC50 values of 49.37nM and 52.12nM and cellular IC50 values of 12.34nM and 14.34nM, respectively. The anti-tumor efficacy of 13b is slightly better than Epacadosta in Lewis Lung Cancer (LLC) tumor-bearing mice model. CONCLUSION: 13b is a potent IDO1 inhibitor with therapeutic potential in tumor immunotherapy.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Oxadiazoles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Carcinoma Pulmonar de Lewis/patología , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Oxadiazoles/síntesis química , Oxadiazoles/química , Relación Estructura-ActividadRESUMEN
Indoleamine 2,3-dioxygenase 1 (IDO1) is closely associated with immune escape in many tumor tissues, and is considered to be a valuable therapeutic target in cancer immunotherapy. In this study, the modification of amino sidechain was performed with the hydroxyamidine core kept intact to optimize lead compound Epacadostat. 19 new compounds with hydrazide, thietane or sulfonamide moiety as polar capping group in sidechain were prepared and their IDO1 inhibitory activities were evaluated. Sulfonamide 3a showed potent IDO1 inhibition in both enzymatic and cellular assays with the IC50 value of 71 nM and EC50 value of 11 nM, respectively. Furthermore, in vivo Lewis lung cancer (LLC) allograft studies of 3a indicated that it handicapped the tumor growth with similar efficacy to Epacadostat. Molecular docking demonstrated that the change of polar capping group affords influence on the orientation of amino ethylene side chain and forms new hydrogen bonding.
Asunto(s)
Amidinas/farmacología , Antineoplásicos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Amidinas/síntesis química , Amidinas/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Relación Estructura-ActividadRESUMEN
Hepatocellular injury is the pathological hallmark of hepatitis and a crucial driver for the progression of liver diseases, while the treatment options are commonly restricted. Interleukin-22 (IL-22) has attracted special attention as a potent survival factor for hepatocytes that both prevents and repairs the injury of hepatocytes through activation of STAT3 signaling pathway. We hypothesized that the ability to generate potent expression of IL-22 locally for the treatment of severe hepatocellular injury in hepatitis was a promising strategy to enhance efficacy and overcome off-target effects. Accordingly, we developed a polypeptide penetratin-based hybrid nanoparticle system (PDPIA) carrying IL-22 gene by a self-assembly process. This nanocomplex modified with penetratin featured direct translocation across the cellular or endosomal membrane but mild zeta-potential to facilitate the high cellular internalization and endosomal escape of the gene cargos as well as scarcely Kupffer cells uptake. More importantly, PDPIA afforded preferential liver accumulation and predominant hepatocytes internalization following systemic administration, which showed pharmacologically suitable organ and sub-organ-selective properties. Subsequent studies confirmed a considerable protective role of PDPIA in a model of severe hepatitis induced by concanavalin A, evidenced by reduced hepatocellular injury and evaded immune response. The locally expressed IL-22 by PDPIA activated STAT3/Erk signal transduction, and thus promoted hepatocyte regeneration, inhibited reactive oxygen species (ROS) accumulation as well as prevented the dysfunction of mitochondrial. In addition, this system did not manifest side effects or systemic toxicity in mice. Collectively, the high versatility of PDPIA rendered its promising applications might be an effective agent to treat various hepatic disorders.
Asunto(s)
Péptidos de Penetración Celular/química , Portadores de Fármacos/química , Hepatitis/terapia , Interleucinas/metabolismo , Nanopartículas/química , Animales , Línea Celular , Supervivencia Celular , Concanavalina A , Dendrímeros/química , Terapia Genética , Hepatitis/etiología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Interleucinas/genética , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Potencial de la Membrana Mitocondrial , Ratones Endogámicos BALB C , Polietilenglicoles/química , Especies Reactivas de Oxígeno/metabolismo , Interleucina-22RESUMEN
Background: Antibody drug conjugate (ADC) showed potent therapeutic efficacy in several types of cancers. The role of autophagy in antitumor effects of ADC remains unclear. Methods: In this study, the ADC, Rituximab-monomethyl auristatin E (MMAE) with a Valine-Citrulline cleavable linker, was designed to investigate its therapeutic efficacy against non-Hodgkin lymphoma (NHL) as well as the underlying mechanisms. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to detect growth inhibition in B-cell lymphoma cell lines, Ramos and Daudi cells, which were treated by Rituximab-MMAE alone or combined with autophagy conditioner. Apoptosis was detected by flow cytometry and immunohistochemistry, and apoptosis inhibitor was employed to discover the relationship between autophagy and apoptosis during the Rituximab-MMAE treatment. Autophagy was determined by three standard techniques which included confocal microscope, transmission electron microscope, and western blots. Ramos xenograft tumors in BALB/c nude mice were established to investigate the antitumor effect of combination use of Rituximab-MMAE and autophagy conditioner in B-NHL therapy. Results: Our results showed that Rituximab-MMAE elicited caspase-3-dependent apoptosis in NHL cells and exhibited potent therapeutic efficacy in vivo. Autophagy, which was characterized by upregulated light chain 3-II expression, and accumulation of autophagosomes, was triggered during the Rituximab-MMAE treatment. Meanwhile, inactivation of Akt/mTOR pathway was shown to be involved in the autophagy triggered by Rituximab-MMAE, indicating a probable mechanism of the ADC-initiated autophagy. Importantly, inhibition of autophagy by chloroquine suppressed the Rituximab-MMAE-induced apoptosis, while activating autophagy by rapamycin significantly enhanced the therapeutic effect of Rituximab-MMAE both in vitro and in vivo. Conclusion: Our data elucidated the critical relationship between autophagy and apoptosis in Rituximab-MMAE-based therapy and highlighted the potential approach for NHL therapy by combined administration of the ADC and autophagy activator.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Autofagia/efectos de los fármacos , Inmunoconjugados/farmacología , Oligopéptidos , Rituximab/farmacología , Animales , Antineoplásicos Inmunológicos/química , Apoptosis/efectos de los fármacos , Autofagosomas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Inmunoconjugados/química , Ratones , Terapia Molecular Dirigida , Oligopéptidos/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Rituximab/química , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma, characterized by extensive microvascular proliferation and invasive tumor growth, is one of the most common and lethal malignancies in adults. Benefits of the conventional anti-angiogenic therapy were only observed in a subset of patients and limited by diverse relapse mechanism. Fortunately, recent advances in cancer immunotherapy have offered new hope for patients with glioblastoma. Herein, we reported a novel dual-targeting therapy for glioblastoma through simultaneous blockade of VEGF and CD47 signaling. Our results showed that VEGFR1D2-SIRPαD1, a VEGF and CD47 bispecific fusion protein, exerted potent anti-tumor effects via suppressing VEGF-induced angiogenesis and activating macrophage-mediated phagocytosis. Meanwhile, autophagy was activated by VEGFR1D2-SIRPαD1 through inactivating Akt/mTOR and Erk pathways in glioblastoma cells. Importantly, autophagy inhibitor or knockdown of autophagy-related protein 5 potentiated VEGFR1D2-SIRPαD1-induced macrophage phagocytosis and cytotoxicity against glioblastoma cells. Moreover, suppression of autophagy led to increased macrophage infiltration, angiogenesis inhibition, and tumor cell apoptosis triggered by VEGF and CD47 dual-targeting therapy, thus eliciting enhanced anti-tumor effects in glioblastoma. Our data revealed that VEGFR1D2-SIRPαD1 alone or in combination with autophagy inhibitor could effectively elicit potent anti-tumor effects, highlighting potential therapeutic strategies for glioblastoma through disrupting angiogenetic axis and CD47-SIRPα anti-phagocytic axis alone or in combination with autophagy inhibition.
Asunto(s)
Autofagia/efectos de los fármacos , Antígeno CD47/antagonistas & inhibidores , Glioblastoma/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Xenoinjertos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fagocitosis/efectos de los fármacosRESUMEN
Increased biomedical applications of mesoporous silica nanoparticles (MSNs) raise considerable attention concerning their toxicological effects; the toxicities of MSNs are still undefined and the underlying mechanisms are unknown. We conducted this study to determine the hepatotoxicity of continuous administration of MSNs and the potential mechanisms. MSNs caused cytotoxicity in hepatic L02 cells in a dose- and time-dependent manner. Then, MSNs were shown to elicit NOD-like receptor protein 3 (NLRP3) inflammasome activation in hepatocytes, leading to caspase-1-dependent pyroptosis, a novel manner of cell death. In vivo MSN administration triggered hepatotoxicity as indicated by increased histological injury, serum alanine aminotransferase and serum aspartate aminotransferase. Notably, NLRP3 inflammasome and pyroptosis were also activated during the treatment. Meanwhile, in NLRP3 knockout mice and caspase-1 knockout mice, MSN-induced liver inflammation and hepatotoxicity could be abolished. Furthermore, experiments indicated that MSNs induced mitochondrial reactive oxygen species (ROS) generation, and the ROS scavenger could attenuate the MSN-activated NLRP3 inflammasomes and pyroptosis in the liver. Collectively, these data suggested that MSNs triggered liver inflammation and hepatocyte pyroptosis through NLRP3 inflammasome activation, which was caused by MSN-induced ROS generation. Our study provided novel insights into the hepatotoxicity of MSNs and the underlying mechanisms, and facilitated the potential approach to increase the biosafety of MSNs.
Asunto(s)
Caspasa 1/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Nanopartículas/toxicidad , Piroptosis , Dióxido de Silicio/toxicidad , Animales , Línea Celular , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
CD47-targeting immune checkpoint inhibitors have been investigated for immunotherapy of several cancers, glioblastoma, one of the most common tumors in brain, was still a challenge for CD47-targeting therapy. Herein, we reported novel strategies for glioblastoma therapy via blocking CD47-signal regulatory protein-α (SIRPα) by SIRPα-Fc alone or in combination with autophagy inhibition. Our results showed that SIRPα-Fc increased macrophages-triggered cytotoxicity and phagocytosis of glioblastoma cells then elicited potent anti-tumor efficacy. During the treatment, SIRPα-Fc induced autophagy and autophagic flux in glioblastoma cells and Akt/mammalian target of rapamycin (mTOR) inactivation was participated in the autophagy activation. Inhibition of autophagy by pharmacological agents or small-interfering RNA increased SIRPα-Fc-triggered macrophage phagocytosis and cytotoxicity. Importantly, when compared with SIRPα-Fc treatment, blocking both CD47/SIRPα and autophagy significantly increased infiltration of macrophages and apoptosis of tumor cells, triggering potentiated anti-glioblastoma effect and extended median survival. Further experiments showed that adaptive immune response, including CD8+ T-cell subsets, was also played a crucial role in SIRPα-Fc-induced glioblastoma rejection. Our results indicated that SIRPα-Fc alone or combined with autophagy inhibitors elicited potent anti-glioblastoma effect, highlighting potential therapeutic strategies of glioblastoma via blocking CD47/SIRPα alone or in combination with autophagy inhibitor.
Asunto(s)
Autofagia/inmunología , Antígeno CD47/metabolismo , Glioblastoma/metabolismo , Glioblastoma/terapia , Receptores Inmunológicos/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Glioblastoma/inmunología , Humanos , Inmunoterapia/métodos , Macrófagos/inmunología , Ratones , Fagocitosis/inmunología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
DG-7 (11,14-dihydroxy-7,20-epoxy-20-O-derivative of ent-kaurene diterpenoid) is a novel anti-tumor candidate drug. A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of DG-7 in rat plasma. An aliquot of 50 µL plasma sample was prepared by liquid-liquid extraction with ethyl acetate. Chromatographic separation was accomplished on a Waters XTerra C18 column (2.1 mm × 150 mm, 5 µm) with an isocratic elution system consisting of methanol and water. Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. The optimized fragmentation transitions for MRM were m/z 590.1âm/z 260.0 for DG-7 and m/z 180.3âm/z 110.1 for phenacetin (internal standard). The method was linear over the concentration range of 5-2,500 ng/mL. The intra- and inter-day precisions were less than 7.9% and the accuracy was within ± 9.0%. The mean recovery of DG-7 ranged from 76.8% to 79.2%. The validated method has been successfully applied to a pharmacokinetic study in rats after intravenous administration of DG-7.