RESUMO
Multiple myeloma (MM) is the second most common malignant haematological disease with a poor prognosis. The limit therapeutic progress has been made in MM patients with cancer relapse, necessitating deeper research into the molecular mechanisms underlying its occurrence and development. A genome-wide CRISPR-Cas9 loss-of-function screening was utilized to identify potential therapeutic targets in our research. We revealed that COQ2 plays a crucial role in regulating MM cell proliferation and lipid peroxidation (LPO). Knockout of COQ2 inhibited cell proliferation, induced cell cycle arrest and reduced tumour growth in vivo. Mechanistically, COQ2 promoted the activation of the MEK/ERK cascade, which in turn stabilized and activated MYC protein. Moreover, we found that COQ2-deficient MM cells increased sensitivity to the LPO activator, RSL3. Using an inhibitor targeting COQ2 by 4-CBA enhanced the sensitivity to RSL3 in primary CD138+ myeloma cells and in a xenograft mouse model. Nevertheless, co-treatment of 4-CBA and RSL3 induced cell death in bortezomib-resistant MM cells. Together, our findings suggest that COQ2 promotes cell proliferation and tumour growth through the activation of the MEK/ERK/MYC axis and targeting COQ2 could enhance the sensitivity to ferroptosis in MM cells, which may be a promising therapeutic strategy for the treatment of MM patients.
Assuntos
Mieloma Múltiplo , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Peroxidação de Lipídeos , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Mieloma Múltiplo/tratamento farmacológicoRESUMO
Gastrin-releasing peptide (GRP) binds to its receptor (GRP receptor [GRPR]) to regulate multiple biological processes, but the function of GRP/GRPR axis in acute kidney injury (AKI) remains unknown. In the present study, GRPR is highly expressed by tubular epithelial cells (TECs) in patients or mice with AKI, while histone deacetylase 8 may lead to the transcriptional activation of GRPR. Functionally, we uncovered that GRPR was pathogenic in AKI, as genetic deletion of GRPR was able to protect mice from cisplatin- and ischemia-induced AKI. This was further confirmed by specifically deleting the GRPR gene from TECs in GRPRFlox/Flox//KspCre mice. Mechanistically, we uncovered that GRPR was able to interact with Toll-like receptor 4 to activate STAT1 that bound the promoter of MLKL and CCL2 to induce TEC necroptosis, necroinflammation, and macrophages recruitment. This was further confirmed by overexpressing STAT1 to restore renal injury in GRPRFlox/Flox/KspCre mice. Concurrently, STAT1 induced GRP synthesis to enforce the GRP/GRPR/STAT1 positive feedback loop. Importantly, targeting GRPR by lentivirus-packaged small hairpin RNA or by treatment with a novel GRPR antagonist RH-1402 was able to inhibit cisplatin-induced AKI. In conclusion, GRPR is pathogenic in AKI and mediates AKI via the STAT1-dependent mechanism. Thus, targeting GRPR may be a novel therapeutic strategy for AKI.
Assuntos
Injúria Renal Aguda , Cisplatino , Animais , Camundongos , Cisplatino/efeitos adversos , Necroptose , Injúria Renal Aguda/metabolismo , Rim/metabolismo , Inflamação/metabolismo , Camundongos Endogâmicos C57BLRESUMO
TJP1, an adaptor protein of the adhesive barrier, has been found to exhibit distinct oncogenic or tumor suppressor functions in a cell-type dependent manner. However, the role of TJP1 in kidney renal clear cell carcinoma (KIRC) remains to be explored. The results showed a marked down-regulation of TJP1 in KIRC tissues compared to normal tissues. Low expression of TJP1 was significantly associated with high grade and poor prognosis in KIRC. Autophagosome aggregation and LC3 II conversion demonstrated that TJP1 may induce autophagy signaling in 786-O and OS-RC-2 cells. Knockdown of TJP1 led to a decrease in the expression of autophagy-related genes, such as BECN1, ATG3, and ATG7. Consistently, TJP1 expression showed a significant positive correlation with these autophagy-related genes in KIRC patients. Furthermore, the overall survival analysis of KIRC patients based on the expression of autophagy-related genes revealed that most of these genes were associated with a good prognosis. TJP1 overexpression significantly suppressed cell proliferation and tumor growth in 786-O cells, whereas the addition of an autophagy inhibitor diminished its inhibitory function. Taken together, these results suggest that TJP1 serves as a favorable prognostic marker and induces autophagy to suppress cell proliferation and tumor growth in KIRC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Proteína da Zônula de Oclusão-1 , Autofagia/genética , Carcinoma de Células Renais/genética , Proliferação de Células/genética , Neoplasias Renais/genética , Rim , PrognósticoRESUMO
Bladder cancer (BLCA) is a common genitourinary cancer in patients, and tumour angiogenesis is indispensable for its occurrence and development. However, the indepth mechanism of tumour angiogenesis in BLCA remains elusive. According to recent studies, the tight junction protein family member occludin (OCLN) is expressed at high levels in BLCA tissues and correlates with a poor prognosis. Downregulation of OCLN inhibits tumour angiogenesis in BLCA cells and murine xenografts, whereas OCLN overexpression exerts the opposite effect. Mechanistically, the RT-qPCR analysis and Western blotting results showed that OCLN increased interleukin-8 (IL8) and p-signal transducer and activator of transcription 3 (STAT3) levels to promote BLCA angiogenesis. RNA sequencing analysis and dual-luciferase reporter assays indicated that OCLN regulated IL8 transcriptional activity via the transcription factor STAT4. In summary, our results provide new perspectives on OCLN, as this protein participates in the development of BLCA angiogenesis by activating the IL8/STAT3 pathway via STAT4 and may serve as a novel and unique therapeutic target.
Assuntos
Interleucina-8 , Ocludina , Fator de Transcrição STAT4 , Neoplasias da Bexiga Urinária , Animais , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Camundongos , Neovascularização Patológica/genética , Ocludina/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
The novel biomarker, insulin-like growth factor binding protein 7 (IGFBP7), is used clinically to predict different types of acute kidney injury (AKI) and has drawn significant attention as a urinary biomarker. However, as a secreted protein in the circulation of patients with AKI, it is unclear whether IGFBP7 acts as a key regulator in AKI progression, and if mechanisms underlying its upregulation still need to be determined. Here we found that IGFBP7 is highly expressed in the blood and urine of patients and mice with AKI, possibly via a c-Jun-dependent mechanism, and is positively correlated with kidney dysfunction. Global knockout of IGFBP7 ameliorated kidney dysfunction, inflammatory responses, and programmed cell death in murine models of cisplatin-, kidney ischemia/reperfusion-, and lipopolysaccharide-induced AKI. IGFBP7 mainly originated from kidney tubular epithelial cells. Conditional knockout of IGFBP7 from the kidney protected against AKI. By contrast, rescue of IGFBP7 expression in IGFBP7-knockout mice restored kidney damage and inflammation. IGFBP7 function was determined in vitro using recombinant IGFBP7 protein, IGFBP7 knockdown, or overexpression. Additionally, IGFBP7 was found to bind to poly [ADP-ribose] polymerase 1 (PARP1) and inhibit its degradation by antagonizing the E3 ubiquitin ligase ring finger protein 4 (RNF4). Thus, IGFBP7 in circulation acts as a biomarker and key mediator of AKI by inhibiting RNF4/PARP1-mediated tubular injury and inflammation. Hence, over-activation of the IGFBP7/PARP1 axis represents a promising target for AKI treatment.
Assuntos
Injúria Renal Aguda , Inibidor Tecidual de Metaloproteinase-2 , Adenosina Difosfato Ribose , Animais , Biomarcadores , Cisplatino/toxicidade , Inflamação , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Lipopolissacarídeos , Camundongos , Camundongos Knockout , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 µM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1ß as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg-1 · d-1, i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/farmacologia , Glomérulos Renais/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Flavanonas/administração & dosagem , Injeções Intraperitoneais , Glomérulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Relação Estrutura-AtividadeRESUMO
Colorectal cancer (CRC) is the third most malignant tumour worldwide, with high mortality and recurrence. Chemoresistance is one of the main factors leading to metastasis and poor prognosis in advanced CRC patients. By analysing the Gene Expression Omnibus data set, we found higher hexokinase 2 (HK2) expression levels in patients with metastatic CRC than in those with primary CRC. Moreover, we observed higher enrichment in oxaliplatin resistance-related gene sets in metastatic CRC than in primary CRC. However, the underlying relationship has not yet been elucidated. In our study, HK2 expression was significantly elevated in CRC patients. Gene set enrichment analysis (GSEA) revealed multi-drug resistance and epithelial-mesenchymal transition (EMT) pathways related to high HK2 expression. Our results showed that knockdown of HK2 significantly inhibited vimentin and Twist1 expression and promoted TJP1 and E-cadherin expression in CRC cells. Additionally, transcriptional and enzymatic inhibition of HK2 by 3-bromopyruvate (3-bp) impaired oxaliplatin resistance in vitro and in vivo. Mechanistically, HK2 interacts with and stabilized Twist1 by preventing its ubiquitin-mediated degradation, which is related to oxaliplatin resistance, in CRC cells. Overexpression of Twist1 reduced the apoptosis rate by HK2 knockdown in CRC cells. Collectively, we discovered that HK2 is a crucial regulator that mediates oxaliplatin resistance through Twist1. These findings identify HK2 and Twist1 as promising drug targets for CRC chemoresistance.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Hexoquinase/metabolismo , Proteínas Nucleares/metabolismo , Oxaliplatina/farmacologia , Proteína 1 Relacionada a Twist/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Diabetic nephropathy (DN) is one of the major complications of diabetes and contributes significantly towards end-stage renal disease. Previous studies have identified the gene encoding carnosinase (CN-1) as a predisposing factor for DN. Despite this fact, the relationship of the level of serum CN-1 and the progression of DN remains uninvestigated. Thus, the proposed study focused on clarifying the relationship among serum CN-1, indicators of renal function and tissue injury, and the progression of DN. A total of 14 patients with minimal changes disease (MCD) and 37 patients with DN were enrolled in the study. Additionally, 20 healthy volunteers were recruited as control. Further, DN patients were classified according to urinary albumin excretion rate into two groups: DN with microalbuminuria (n = 11) and DN with macroalbuminuria (n = 26). Clinical indicators including urinary protein components, serum carnosine concentration, serum CN-1 concentration and activity, and renal biopsy tissue injury indexes were included for analyzation. The serum CN-1 concentration and activity were observed to be the highest, but the serum carnosine concentration was the lowest in DN macroalbuminuria group. Moreover, within DN group, the concentration of serum CN-1 was positively correlated with uric acid (UA, r = 0.376, p = 0.026) and serum creatinine (SCr, r = 0.399, p = 0.018) and negatively correlated with serum albumin (Alb, r = - 0.348, p = 0.041) and estimated glomerular filtration rate (eGRF, r = - 0.432, p = 0.010). Furthermore, the concentration of serum CN-1 was discovered to be positively correlated with indicators including 24-h urinary protein-creatinine ratio (24 h-U-PRO/CRE, r = 0.528, p = 0.001), urinary albumin-to-creatinine ratio (Alb/CRE, r = 0.671, p = 0.000), urinary transferrin (TRF, r = 0.658, p = 0.000), retinol-binding protein (RBP, r = 0.523, p = 0.001), N-acetyl-glycosaminidase (NAG, r = 0.381, p = 0.024), immunoglobulin G (IgG, r = 0.522, p = 0.001), cystatin C (Cys-C, r = 0.539, p = 0.001), beta-2-microglobulin (ß2-MG, r = 0.437, p = 0.009), and alpha-1-macroglobulin (α1-MG, r = 0.480, p = 0.004). Besides, in DN with macroalbuminuria group, serum CN-1 also showed a positive correlation with indicators of fibrosis, oxidative stress, and renal tubular injury. Taken together, our data suggested that the level of CN-1 was increased as clinical DN progressed. Thus, the level of serum CN-1 might be an important character during the occurrence and progression of DN. Our study will contribute significantly to future studies focused on dissecting the underlying mechanism of DN.
Assuntos
Nefropatias Diabéticas/enzimologia , Dipeptidases/sangue , Adulto , Biomarcadores , Estudos de Casos e Controles , Creatinina/sangue , Cistatina C/sangue , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Progressão da Doença , Feminino , Taxa de Filtração Glomerular , Humanos , Rim/lesões , Rim/fisiopatologia , Falência Renal Crônica/sangue , Falência Renal Crônica/enzimologia , Falência Renal Crônica/patologia , Falência Renal Crônica/fisiopatologia , Masculino , Pessoa de Meia-IdadeRESUMO
Diabetic nephropathy (DN) is a common microvascular complication of diabetes and the main cause of end-stage nephropathy (ESRD). Inflammation and fibrosis play key roles in the development and progression of diabetic nephropathy. By using in vivo and in vitro DN models, our laboratory has identified the protective role of carnosine (CAR) on renal tubules. Our results showed that carnosine restored the onset and clinical symptoms as well as renal tubular injury in DN. Furthermore, carnosine decreased kidney inflammation and fibrosis in DN mice. These results were consistent with high glucose (HG)-treated mice tubular epithelial cells (MTECs). Using web-prediction algorithms, cellular thermal shift assay (CETSA) and molecular docking, we identified glycine N-methyltransferase (GNMT) as a carnosine target. Importantly, we found that GNMT, a multiple functional protein that regulates the cellular pool of methyl groups by controlling the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), was down-regulated significantly in the serum of Type 1 DM patients and renal tissues of DN mice. Moreover, using cultured TECs, we confirmed that the increased GNMT expression by transient transfection mimicked the protective role of carnosine in reducing inflammation and fibrosis. Conversely, the inhibition of GNMT expression abolished the protective effects of carnosine. In conclusion, carnosine might serve as a promising therapeutic agent for DN and GNMT might be a potential therapeutic target for DN.
Assuntos
Carnosina/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/enzimologia , Glicina N-Metiltransferase/metabolismo , Inflamação/enzimologia , Rim/enzimologia , Rim/patologia , Adulto , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Carnosina/química , Carnosina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fibrose , Glucose/toxicidade , Humanos , Inflamação/patologia , Rim/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Terapia de Alvo Molecular , EstreptozocinaRESUMO
Alcohol consumption causes renal injury and compromises kidney function. The underlying mechanism of the alcoholic kidney disease remains largely unknown. In the present study, an alcoholic renal fibrosis animal model was first employed which mice received liquid diet containing alcohol for 4 to 12 weeks. The Masson's Trichrome staining analysis showed that kidney fibrosis increased at week 8 and 12 in the animal model that was further confirmed by albumin assay, Western blot, immunostaining and real-time PCR of fibrotic indexes (collagen I and α-SMA). In vitro analysis also confirmed that alcohol significantly induced fibrotic response (collagen I and α-SMA) in HK2 tubular epithelial cells. Importantly, both in vivo and in vitro studies showed alcohol treatments decreased Smad7 and activated Smad3. We further determined how the alcohol affected the balance of Smad7 (inhibitory Smad) and Smad3 (regulatory Smad). Genome-wide methylation sequencing showed an increased DNA methylation of many genes and bisulfite sequencing analysis showed an increased DNA methylation of Smad7 after alcohol ingestion. We also found DNA methylation of Smad7 was mediated by DNMT1 in ethyl alcohol (EtOH)-treated HK2 cells. Knockdown of Nox2 or Nox4 decreased DNMT1 and rebalanced Smad7/Smad3 axis, and thereby relieved EtOH-induced fibrotic response. The inhibition of reactive oxygen species by the intraperitoneal injection of apocynin attenuated renal fibrosis and restored renal function in the alcoholic mice. Collectively, we established novel in vivo and in vitro alcoholic kidney fibrosis models and found that alcohol induces renal fibrosis by activating oxidative stress-induced DNA methylation of Smad7. Suppression of Nox-mediated oxidative stress may be a potential therapy for long-term alcohol abuse-induced kidney fibrosis.
Assuntos
Metilação de DNA/genética , Etanol/efeitos adversos , Nefropatias/genética , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Proteína Smad7/metabolismo , Acetofenonas/farmacologia , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibrose , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/patologia , Túbulos Renais/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
MLKL is a central mediator for necroptosis. Its knockout significantly relieves acute kidney injury (AKI). However, its upstream regulatory mechanism in AKI has not been fully elucidated. We recently reviewed how microRNAs (miRNAs), a type of well-studied epigenetic regulator, play critical roles in AKI. Here, we evaluated miRNAs that potentially target MLKL and evaluated their function in human tubular epithelial cells in response to toxic and ischemic insults. TargetScan analysis showed that miR-194-5P, miR-338-3P, miR-500a-3P, and miR-577 had MLKL binding sites. Although all 4 miRNAs are reduced in AKI, our data show that only hsa-miR-500a-3P was significantly suppressed in cisplatin-treated human tubular epithelial (HK2) cells. We further found that hsa-miR-500a-3P alleviated cisplatin-induced HK2 cell death, which was confirmed by transmission electron microscopy and flow cytometry. In addition, overexpression of hsa-miR-500a-3P decreased kidney injury molecule-1 mRNA and protein levels. Real-time PCR, ELISA, and immunofluorescence data show that hsa-miR-500a-3P protected against inflammatory response, evidenced by decreased monocyte chemotactic protein-1 and proinflammatory cytokines TNF-α and IL-8. Further, hsa-miR-500a-3P attenuated P65 NF-κB phosphorylation and promoter activity. Mechanistically, luciferase reporter assay showed that hsa-miR-500a-3P bound the 3'UTR of MLKL, thereby suppressing phosphorylation and membrane translocation of MLKL. In agreement with these findings, we identified that overexpression of hsa-miR-500a-3P attenuated cell injury and the inflammatory response in response to sodium azide treatment in an in vitro model. Results show that circulating exosomes from patients with AKI down-regulated miR-500a-3P, which suppressed cell injury and inflammation in HK2 cells. hsa-miR-500a-3P alleviated toxic and ischemic insults that were triggered by cell necroptosis and the inflammatory response in human HK2 cells by targeting MLKL. This may serve as a novel therapeutic target for treatment of AKI.-Jiang, L., Liu, X.-Q., Ma, Q., Yang, Q., Gao, L., Li, H.-D., Wang, J.-N., Wei, B., Wen, J., Li, J., Wu, Y.-G., Meng, X.-M. hsa-miR-500a-3P alleviates kidney injury by targeting MLKL-mediated necroptosis in renal epithelial cells.
Assuntos
Injúria Renal Aguda/genética , Apoptose/genética , Células Epiteliais/patologia , Rim/patologia , MicroRNAs/genética , Necrose/genética , Proteínas Quinases/genética , Regiões 3' não Traduzidas/genética , Linhagem Celular , Regulação para Baixo/genética , Exossomos/genética , Humanos , Inflamação/genética , Interferon-alfa/genética , Interleucina-8/genética , Necrose/patologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genéticaRESUMO
Acute kidney injury (AKI) is a destructive clinical condition induced by multiple insults including ischemic reperfusion, nephrotoxic drugs and sepsis. It is characterized by a sudden decline in renal function, in addition to excessive inflammation, oxidative stress and programmed cell death of renal tubular epithelial cells. RIPK1-mediated necroptosis plays an important role in AKI. In the present study, we evaluated the treatment effects of Compound-71 (Cpd-71), a novel RIPK1 inhibitor, by comparing with Necrostatin-1 (Nec-1), a classic RIPK1 inhibitor, which has several drawbacks like the narrow structure-activity relationship (SAR) profile, moderate potency and non-ideal pharmacokinetic properties, in vivo and in vitro Our results showed that pretreatment of Cpd-71 attenuated cisplatin-induced renal injury, restored renal function and suppressed renal inflammation, oxidative stress and cell necroptosis. In addition, Cpd-71 inhibited renal damage while reducing the up-regulated serum creatinine (Cr) and blood urea nitrogen (BUN) levels in established AKI mice model. Consistently, we confirmed that Cpd-71 exhibited more effectively suppressive effect on cisplatin-induced renal tubular cell necroptosis than Nec-1, by physically binding to the allosteric type III ligand binding site of RIPK1, thereby reduced RIPK1 kinase activity, RIPK1/RIPK3 complex formation and phosphor-MLKL membrane translocation by molecular docking, Western blot, co-immunoprecipitation and cellular thermal shift assay (CETSA). Taken together, we currently showed that targeting RIPK1 with Cpd-71 may serve as a promising clinical candidate for AKI treatment.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Antineoplásicos/administração & dosagem , Cisplatino/efeitos adversos , Necroptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Injúria Renal Aguda/genética , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/fisiopatologia , Animais , Humanos , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologiaRESUMO
E-cadherin is a major component of tubular adherent proteins that maintain intercellular contacts and cell polarity in epithelial tissue. It is involved in pathological processes of renal cell carcinoma and fibrotic diseases via epithelial-mesenchymal transition. Although studies have shown E-cadherin is significantly downregulated in acute kidney injury (AKI), its function in AKI is unknown. Here, we evaluated cell damage and inflammation in cisplatin-stimulated tubular epithelial cell lines after disrupting E-cadherin and restoring it with PPBICA, a small molecule identified by high-throughput screening. We also determined the therapeutic potential of restoring E-cadherin in vivo. Results show cisplatin reduced E-cadherin expression both in mouse kidney and proximal tubular epithelial cell lines (mTECs). PPBICA restored E-cadherin levels, which increased cell viability while attenuating programmed cell death. This may be mediated via deactivation of the RIPK1/RIPK3 axis and decreased caspase3 cleavage. In addition, PPBICA suppressed inflammatory response in cisplatin-treated mTECs, which correlated with suppressed NF-κB phosphorylation and promoter activity. In contrast, disruption of E-cadherin promoted cell damage and inflammation. PPBICA failed to further attenuate kidney damage in E-cadherin knockdown cells, indicating that PPBICA protects against mTECs through E-cadherin restoration. We also found that peritoneal injection of PPBICA in mice prevented loss of renal function and tubular damage by suppressing NF-κB-driven renal inflammation and RIPK-regulated programmed cell death. This was driven by restoration of E-cadherin in cisplatin nephropathy. Additionally, PPBICA attenuated cisplatin-induced kidney damage in an established AKI model, indicating its therapeutic potential in the treatment of AKI. In conclusion, E-cadherin plays functional roles in tubule integrity, programmed cell death, and renal inflammation. Our results underscore the potential of E-cadherin restoration as a novel therapeutic strategy for AKI.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Caderinas/metabolismo , Cisplatino/efeitos adversos , Substâncias Protetoras/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Inflamação/metabolismo , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
A series of structurally new diheteroaryl thioether analogs was designed, prepared and screened toward MGC-803, MKN-45, EC-109 and H1650. Most of the target compounds displayed moderate to potent antiproliferative activities. Among them, compound 5 showed the best antiproliferative activity against the tested cell lines with the half maximal inhibitory concentration (IC50) values below 10µM. In addition, flow cytometry analysis showed that compound 5 increased Bax expression, down-regulated expression of Bcl-2, cleaved caspases-3/9, finally inducing apoptosis of MKN-45 cells as well asarrested the cell cycle at G2/M phase. This study suggests that the diheteroaryl thioethers are a class of emerging chemotypes for developing antitumor agents or biological probes, and compound 5 could serve as a good starting point to design new apoptosis inducers.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Sulfetos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Sulfetos/síntese química , Sulfetos/químicaRESUMO
BACKGROUND/AIMS: Human SIRT1 is reported to be involved in tumorgenesis, mainly due to its modulating effect on p53 by deacetylation on lysine382. A large quantity of SIRT1 inhibitors was applied in chemotherapeutic study, but few of them were applied into clinical trials. METHODS AND RESULTS: In the current study, a novel series of compounds with 1,4-bispiperazinecarbodithioic acid methyl esters scaffold were characterized to have inhibitory potency to SIRT1 by molecular docking and biochemical evaluation. Further cell level study revealed that one of the most potent SIRT1 inhibitors, compound 3a, is cell active. It can upregulate the amount of p53 by accumulating the K382 acetylation of p53, which lead to the stabilization of p53 in human gastric cancer cell line MGC-803 cells. Meanwhile, we also found compound 3a can inactivate SIRT2 in cells, which suggests the compound as a non-selective SIRT inhibitor. CONCLUSION: All these findings indicate that compound 3a is a potent, reversible and cell active SIRT1 inhibitor and deserves further investigation as an anticancer agent or a biological tool.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Tiocarbamatos/farmacologia , Triazóis/farmacologia , Acetilação/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Sirtuína 1/metabolismo , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/metabolismo , Tiocarbamatos/química , Triazóis/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Baicalin is one of the active ingredients in the skullcap, with a variety of pharmacological effects, such as blood pressure reduction, sedation, liver-protection, gallbladder-protection, anti-bacteria, and anti-inflammation. In our study, baicalin was first characterized as a LSD1 inhibitor with an IC50 of 3.01µM and showed strong LSD1 inhibitory effect in cells. Baicalin may serve as a template for designing flavone-based LSD1 inhibitors.
Assuntos
Produtos Biológicos/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Histona Desmetilases/antagonistas & inibidores , Produtos Biológicos/síntese química , Produtos Biológicos/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Flavonoides/síntese química , Flavonoides/química , Histona Desmetilases/metabolismo , Humanos , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
A series of 5H-thiazolo[3,2-a]pyrimidin-5-ones were synthesized by the cyclization reactions of S-alkylated derivatives in concentrated H2SO4. Upon treatment of S-alkylated derivatives at different temperatures, intramolecular cyclization to 7-(substituted phenylamino)-5H-thiazolo[3,2-a]pyrimidin-5-ones or sulfonation of cyclized products to sulfonic acid derivatives occurred. The structures of the target compounds were confirmed by IR, ¹H-NMR, (13)C-NMR and HRMS studies. The compounds were evaluated for their preliminary in vitro antibacterial activity against some Gram-positive and Gram-negative bacteria and screened for antitubercular activity against Mycobacterium tuberculosis by the broth dilution assay method. Some compounds showed good antibacterial and antitubercular activities.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Antituberculosos/síntese química , Antituberculosos/farmacologia , Antibacterianos/química , Antituberculosos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Espectroscopia de Prótons por Ressonância Magnética , Espectrofotometria Infravermelho , Ácidos Sulfônicos/químicaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall has been used in Chinese Medicine for thousands of years, especially having anti-inflammatory, sedative, analgesic and other ethnic pharmacological effects. Moreover, Paeoniflorin is the main active ingredient of the Paeonia lactiflora Pall, and most are used in the treatment of inflammation-related autoimmune diseases. In recent years, studies have found that Paeoniflorin has a therapeutic effect on a variety of kidney diseases. AIM OF THE STUDY: Cisplatin (CIS) is limited in clinical use due to its serious side effects, such as renal toxicity, and there is no effective method for prevention. Paeoniflorin (Pae) is a natural polyphenol which has a protective effect against many kidney diseases. Therefore, our study is to explore the effect of Pae on CIS-induced AKI and the specific mechanism. MATERIALS AND METHODS: Firstly, CIS induced acute renal injury model was constructed in vivo and in vitro, and Pae was continuously injected intraperitoneally three days in advance, and then Cr, BUN and renal tissue PAS staining were detected to comprehensively evaluate the protective effect of Pae on CIS-induced AKI. We then combined Network Pharmacology with RNA-seq to investigate potential targets and signaling pathways. Finally, affinity between Pae and core targets was detected by molecular docking, CESTA and SPR, and related indicators were detected in vitro and in vivo. RESULTS: In this study, we first found that Pae significantly alleviated CIS-AKI in vivo and in vitro. Through network pharmacological analysis, molecular docking, CESTA and SPR experiments, we found that the target of Pae was Heat Shock Protein 90 Alpha Family Class A Member 1 (Hsp90AA1) which performs a crucial function in the stability of many client proteins including Akt. RNA-seq found that the KEGG enriched pathway was PI3K-Akt pathway with the most associated with the protective effect of Pae which is consistent with Network Pharmacology. GO analysis showed that the main biological processes of Pae against CIS-AKI include cellular regulation of inflammation and apoptosis. Immunoprecipitation further showed that pretreatment with Pae promoted the Hsp90AA1-Akt protein-protein Interactions (PPIs). Thereby, Pae accelerates the Hsp90AA1-Akt complex formation and leads to a significant activate in Akt, which in turn reduces apoptosis and inï¬ammation. In addition, when Hsp90AA1 was knocked down, the protective effect of Pae did not continue. CONCLUSION: In summary, our study suggests that Pae attenuates cell apoptosis and inflammation in CIS-AKI by promoting Hsp90AA1-Akt PPIs. These data provide a scientific basis for the clinical search for drugs to prevent CIS-AKI.
Assuntos
Injúria Renal Aguda , Cisplatino , Humanos , Cisplatino/efeitos adversos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Simulação de Acoplamento Molecular , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/prevenção & controle , Inflamação/induzido quimicamente , Proteínas de Choque Térmico HSP90/uso terapêuticoRESUMO
AIMS: The aim of this study was to explore the fibrogenic effects of ATP-P1Rs axis and ATP-P2Rs axis on alcohol-related liver fibrosis (ALF). MATERIALS AND METHODS: C57BL/6J CD73 knock out (KO) mice were used in our study. 8-12 weeks male mice were used as an ALF model in vivo. In conclusion, after one week of adaptive feeding, 5 % alcohol liquid diet was given for 8 weeks. High-concentration alcohol (31.5 %, 5 g/kg) was administered by gavage twice weekly, and 10 % CCl4 intraperitoneal injections (1 ml/kg) were administered twice weekly for the last two weeks. The mice in the control group were injected intraperitoneally with an equivalent volume of normal saline. Fasting for 9 h after the last injection, blood samples were collected, and related indicators were tested. In vitro, rat hepatic stellate cells (HSCs) were treated with 200 µM acetaldehyde to establish an alcoholic liver fibrosis for 48 h, then tested related indicators. KEY FINDINGS: We found that both adenosine receptors including adenosine A1, A2A, A2B, A3 receptors (A1R, A2AR, A2BR, A3R) and ATP receptors including P2X7, P2Y2 receptors (P2X7R, P2Y2R) were expressed increased in ALF. After CD73 was knocked out, we found that adenosine receptors expression decreased, ATP expression increased, and fibrosis degree decreased. SIGNIFICANCE: Based on the research, we discovered that adenosine plays a more important role in ALF. Therefore, blocking the ATP-P1Rs axis represented a potential treatment for ALF, and CD73 will become a potential therapeutic target.
Assuntos
Etanol , Cirrose Hepática , Ratos , Camundongos , Masculino , Animais , Camundongos Endogâmicos C57BL , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/prevenção & controle , Cirrose Hepática/metabolismo , Etanol/toxicidade , Etanol/metabolismo , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Receptores Purinérgicos P1/metabolismo , Camundongos Knockout , Fígado/metabolismoRESUMO
Tight junction protein 1 (TJP1) is a recently identified prominent regulator of bladder cancer (BLCA) angiogenesis and tumorigenesis. Vascular mimicry (VM) is a newly described tumor feature and is correlated with an increased risk of tumor metastasis. However, the relationship between TJP1 expression and VM in bladder cancer remains elusive. In the present study, we report a novel function for TJP1 in accommodating VM to promote tumor progression. We found that the elevated TJP1 expression was positively related to VM in patients and xenograft tumor models in bladder cancer. Enforced expression of TJP1 increased VM of BLCA cells in vitro and in vivo by elevating Vascular endothelial growth factor A (VEGFA) levels. Furthermore, VM induced by TJP1 overexpression was significantly blocked by the VEGFA and VEGFR inhibitors (Bevacizumab and Sunitinib). Mechanistically, TJP1 promoted VEGFA transcriptional and protein level in a TWIST1-dependent manner. Taken together, our study reveals that TJP1-regulated VEGFA overexpression may indicate a potential therapeutic target for clinical intervention in the early tumor neovascularization of bladder cancer.