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1.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 36(3): 279-285, 2024 Mar.
Artículo en Chino | MEDLINE | ID: mdl-38538357

RESUMEN

OBJECTIVE: To investigate the regulatory role of Wilms tumor 1-associating protein (WTAP) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its molecular mechanism. METHODS: (1) Experiment I: H9C2 cardiomyocytes were divided into blank control group and H/R model group. H/R was used to induce myocardial ischemia/reperfusion (I/R) injury model in H9C2 cells. The blank control group was not treated. N6-methyladenosine (m6A) RNA methylation assay kit was used to detect the level of m6A. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the mRNA and protein expression levels of methyltransferases [WTAP, methyltransferase-like proteins (METTL3, METTL14)], respectively. (2) Experiment II: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, and H/R+sh-WTAP group. sh-WTAP was transfected to knock down the expression of WTAP in H/R+sh-WTAP group, and the model establishment method in the other groups was the same as experiment I. At 48 hours after transfection, the apoptosis rate of cells was detected by flow cytometry. The protein expressions of WTAP, activated caspase-3, activated poly (ADP-ribose) polymerase (PARP), activating transcription factor 4 (ATF4), proline-rich receptor-like protein kinase (PERK), phosphorylated PERK (p-PERK) and CCAAT/enhancer-binding protein homologous protein (CHOP) were detected by Western blotting. The positive expression of ATF4 was observed by immunofluorescence staining. (3) Experiment III: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, H/R+sh-WTAP group and H/R+sh-WTAP+ATF4 group. The overexpression plasmid ATF4 was transfected into H9C2 cardiomyocytes, and the modeling method of the other groups were modeled the same as experiment II. The apoptosis rate was detected by flow cytometry. Western blotting was used to detect the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP. RESULTS: (1) Experiment I: the methylation level of m6A in the H/R group was significantly higher than that in the blank control group. RT-qPCR results showed that the gene expressions of METTL3, METTL14 and WTAP in the H/R model group were significantly higher than those in the blank control group, and WTAP was the most significantly up-regulated. Western blotting results showed the same trend. These results suggested that the expression level of methyltransferase WTAP is significantly up-regulated in H/R-induced cardiomyocytes. (2) Experiment II: the apoptosis level in H/R+sh-WTAP group was significantly lower than that in H/R+sh-NC group [(14.16±1.58)% vs. (24.51±2.38)%, P < 0.05]. Western blotting results showed that the protein expressions of WTAP, activated caspase-3, activated PARP, p-PERK, ATF4 and CHOP in the H/R+sh-WTAP group were significantly lower than those in the H/R+sh-NC group. Fluorescence microscopy results showed that the ATF4 positive signal in the H/R+sh-WTAP group was significantly weaker than that in the H/R+sh-NC group [(19.36±1.81)% vs. (32.83±2.69)%, P < 0.01]. The above results suggested that knockdown of WTAP could inhibit H/R-induced cardiomyocyte apoptosis and endoplasmic reticulum stress. (3) Experiment III: the apoptosis level of H/R+sh-WTAP+ATF4 group was significantly higher than that of H/R+sh-WTAP group [(26.61±2.76)% vs. (17.14±0.87)%, P < 0.05]. Western blotting results showed that the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP in the H/R+sh-WTAP+ATF4 group were significantly higher than those in the H/R+sh-WTAP group. These results suggested that overexpression of ATF4 reversed the inhibitory effect of sh-WTAP on endoplasmic reticulum stress and apoptosis in H/R-induced cardiomyocytes. CONCLUSIONS: Methyltransferase WTAP could regulate ATF4 expression, mediate cell apoptosis and endoplasmic reticulum stress, and promote H/R-induced myocardial cell injury.


Asunto(s)
Lesiones Cardíacas , Miocitos Cardíacos , Humanos , Caspasa 3/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Hipoxia/metabolismo , Metiltransferasas/metabolismo , Metiltransferasas/farmacología , Apoptosis , Estrés del Retículo Endoplásmico , Factores de Empalme de ARN/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/farmacología
2.
FASEB J ; 38(6): e23556, 2024 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-38498348

RESUMEN

PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP-1 and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drive bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.


Asunto(s)
Isquemia Encefálica , Oxígeno , Ratones , Animales , Oxígeno/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Hexoquinasa/genética , Hexoquinasa/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Glucosa/metabolismo , Isquemia Encefálica/metabolismo , Glucólisis , Neuronas/metabolismo , Oxidación-Reducción
3.
Cell Chem Biol ; 31(5): 973-988.e4, 2024 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-38335967

RESUMEN

The (poly)pharmacology of drug metabolites is seldom comprehensively characterized in drug discovery. However, some drug metabolites can reach high plasma concentrations and display in vivo activity. Here, we use computational and experimental methods to comprehensively characterize the kinase polypharmacology of M324, the major metabolite of the PARP1 inhibitor rucaparib. We demonstrate that M324 displays unique PLK2 inhibition at clinical concentrations. This kinase activity could have implications for the efficacy and safety of rucaparib and therefore warrants further clinical investigation. Importantly, we identify synergy between the drug and the metabolite in prostate cancer models and a complete reduction of α-synuclein accumulation in Parkinson's disease models. These activities could be harnessed in the clinic or open new drug discovery opportunities. The study reported here highlights the importance of characterizing the activity of drug metabolites to comprehensively understand drug response in the clinic and exploit our current drug arsenal in precision medicine.


Asunto(s)
Indoles , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Indoles/farmacología , Indoles/química , Indoles/metabolismo , Animales , Masculino , Ratones , Sinergismo Farmacológico , Línea Celular Tumoral , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología
4.
J Biomol Struct Dyn ; 42(7): 3396-3409, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37216358

RESUMEN

Cancer accounts for the majority of deaths worldwide, and the increasing incidence of breast cancer is a matter of grave concern. Poly (ADP-ribose) polymerase-1 (PARP-1) has emerged as an attractive target for the treatment of breast cancer as it has an important role in DNA repair. The focus of the study was to identify novel PARP-1 inhibitors using a blend of tandem structure-based screening (Docking and e-pharmacophore-based screening) and artificial intelligence (deep learning)-based de novo approaches. The scrutiny of compounds having good binding characteristics for PARP-1 was carried out using a tandem mode of screening along with parameters such as binding energy and ADME analysis. The efforts afforded compound Vab1 (PubChem ID 129142036), which was chosen as a seed for obtaining novel compounds through a trained artificial intelligence (AI)-based model. Resultant compounds were assessed for PARP-1 inhibition; binding affinity prediction and interaction pattern analysis were carried out using the extra precision (XP) mode of docking. Two best hits, Vab1-b and Vab1-g, exhibiting good dock scores and suitable interactions, were subjected to 100 nanoseconds (ns) of molecular dynamics simulation in the active site of PARP-1 and compared with the reference Protein-Ligand Complex. The stable nature of PARP-1 upon binding to these compounds was revealed through MD simulation.Communicated by Ramaswamy H. Sarma.


Asunto(s)
Neoplasias de la Mama , Aprendizaje Profundo , Humanos , Femenino , Simulación de Dinámica Molecular , Poli(ADP-Ribosa) Polimerasa-1/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Simulación del Acoplamiento Molecular , Farmacóforo , Inteligencia Artificial , Unión Proteica , Ligandos
5.
Cell Transplant ; 32: 9636897231211067, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38078417

RESUMEN

BACKGROUND: We tested the hypothesis that overexpression of cellular-prion-protein in adipose-derived mesenchymal stem cells (PrPCOE-ADMSCs) effectively protected the kidney against ischemia-reperfusion (IR) injury in rat. METHODS: Part I of cell culture was categorized into A1(ADMSCs)/A2(ADMSCs+p-Cresol)/A3(PrPCOE in ADMSCs)/A4 (PrPCOE in ADMSCs+p-Cresol). Part II of cell culture was divided into B1(ADMSCs)/B2[ADMSCs+lipopolysaccharide (LPS)]/B3(PrPCOE in ADMSCs)/B4(PrPCOE in ADMSCs+LPS). Sprague-Dawley (SD) rats (n = 50) were equally categorized into groups 1 (sham-operated-control)/2 (IR)/3 (IR+ADMSCs/6.0 × 105 equally divided into bilateral-renal arteries and 6.0 × 105 intravenous administration by 1 h after IR)/4 [IR+PrPCOE-ADMSCs (identical dosage administered as group 3)]/5 [IR+silencing PRNP -ADMSCs (identical dosage administered as group 3)], and kidneys were harvested post-day 3 IR injury. RESULTS: Part I results demonstrated that the cell viability at 24/48/72 h, BrdU uptake/number of mitDNA/APT concentration/mitochondrial-cytochrome-C+ cells and the protein expressions of ki67/PrPC at 72 h-cell culturing were significantly higher in PrPCOE-ADMSCs than in ADMSCs (all P < 0.001). The protein expressions of oxidative-stress (NOX-1/NOX2/NOX4/oxidized protein)/mitochondrial-damaged (p22-phox/cytosolic-cytochrome-C)/inflammatory (p-NF-κB/IL-1ß/TNF-α/IL-6)/apoptotic (cleaved caspase-3/cleaved-PARP) biomarkers were lowest in A1/A3 and significantly higher in A2 than in A4 (all P < 0.001). Part II result showed that the protein expressions of inflammatory (p-NF-κB/IL-1ß/TNF-α/IL-6)/apoptotic (cleaved caspase-3/cleaved-PARP) biomarkers exhibited an identical pattern of part I among the groups (all P < 0.001). The protein expressions of inflammatory (p-NF-κB/IL-1ß/TNF-α/MMP-9)/oxidative-stress (NOX-1/NOX-2/oxidized-protein)/mitochondrial-damaged (cytosolic-cytochrome-C/p22-phox)/apoptotic (cleaved caspase-3/cleaved-PARP/mitochondrial-Bx)/autophagic (beclin-1/ratio of LC3B-II/LC3B-I)/fibrotic (Smad3/TGF-ß) biomarkers and kidney-injury-score/creatinine level were lowest in group 1, highest in group 2, significantly higher in group 5 than in groups 3/4 (all P < 0.0001). CONCLUSION: PrPCOE in ADMSCs rejuvenated these cells and played a cardinal role on protecting the kidney against IR injury.


Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Priones , Daño por Reperfusión , Ratas , Animales , Ratas Sprague-Dawley , Proteínas Priónicas/metabolismo , Caspasa 3/metabolismo , Roedores , Priones/metabolismo , Priones/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos , FN-kappa B/metabolismo , Biogénesis de Organelos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Rejuvenecimiento , Trasplante de Células Madre Mesenquimatosas/métodos , Riñón/metabolismo , Daño por Reperfusión/metabolismo , Biomarcadores/metabolismo , Proliferación Celular , Citocromos/metabolismo , Citocromos/uso terapéutico , Adenosina Trifosfato/metabolismo
6.
Endocr Regul ; 57(1): 279-291, 2023 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38127690

RESUMEN

Objective. The study was performed to elucidate whether nicotinamide (NAm) can attenuate the diabetes-induced liver damage by correction of ammonia detoxifying function and disbalance of NAD-dependent processes in diabetic rats. Methods. After four weeks of streptozotocin-induced diabetes, Wistar male rats were treated for two weeks with or without NAm. Urea concentration, arginase, and glutamine synthetase activities, NAD+ levels, and NAD+/NADH ratio were measured in cytosolic liver extracts. Expression of parp-1 gene in the liver was estimated by quantitative polymerase chain reaction and PARP-1 cleavage evaluated by Western blotting. Results. Despite the blood plasma lipid peroxidation products in diabetic rats were increased by 60%, the activity of superoxide dismutase (SOD) was reduced. NAm attenuated the oxidative stress, but did not affect the enzyme activity in diabetic rats. In liver of the diabetic rats, urea concentration and arginase activity were significantly higher than in the controls. The glutamine synthetase activity was decreased. Decline in NAD+ level and cytosolic NAD+/NADH ratio in the liver of diabetic rats was observed. Western blot analysis demonstrated a significant up-regulation of PARP-1 expression accompanied by the enzyme cleavage in the diabetic rat liver. However, no correlation was seen between mRNA expression of parp-1 gene and PARP-1 protein in the liver of diabetic rats. NAm markedly attenuated PARP-1 cleavage induced by diabetes, but did not affect the parp-1 gene expression. Conclusions. NAm counteracts diabetes-induced impairments in the rat liver through improvement of its detoxifying function, partial restoration of oxidative stress, NAD+ level, normalization of redox state of free cytosolic NAD+/NADH-couples, and prevention of PARP-1 cleavage.


Asunto(s)
Diabetes Mellitus Experimental , Niacinamida , Ratas , Masculino , Animales , Niacinamida/farmacología , Niacinamida/metabolismo , NAD/metabolismo , NAD/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ratas Wistar , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Arginasa/genética , Arginasa/metabolismo , Arginasa/farmacología , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Glutamato-Amoníaco Ligasa/farmacología , Estrés Oxidativo , Hígado/metabolismo , Urea/metabolismo , Urea/farmacología
7.
J Biomed Sci ; 30(1): 91, 2023 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-37936170

RESUMEN

BACKGROUND: Although stimulating autophagy caused by UV has been widely demonstrated in skin cells to exert cell protection, it remains unknown the cellular events in UVA-treated retinal pigment epithelial (RPE) cells. METHODS: Human ARPE-19 cells were used to measure cell viability, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial mass and lysosomal mass by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics, LC3 level, and AMPK translocation after UVA irradiation. RESULTS: We confirmed mitochondrial ROS production and DNA damage are two major features caused by UVA. We found the cell death is prevented by autophagy inhibitor 3-methyladenine and gene silencing of ATG5, and UVA induces ROS-dependent LC3II expression, LC3 punctate and TFEB expression, suggesting the autophagic death in the UVA-stressed RPE cells. Although PARP-1 inhibitor olaparib increases DNA damage, ROS production, and cell death, it also blocks AMPK activation caused by UVA. Interestingly we found a dramatic nuclear export of AMPK upon UVA irradiation which is blocked by N-acetylcysteine and olaparib. In addition, UVA exposure gradually decreases lysosomal mass and inhibits cathepsin B activity at late phase due to lysosomal dysfunction. Nevertheless, cathepsin B inhibitor, CA-074Me, reverses the death extent, suggesting the contribution of cathepsin B in the death pathway. When examining the role of EGFR in cellular events caused by UVA, we found that UVA can rapidly transactivate EGFR, and treatment with EGFR TKIs (gefitinib and afatinib) enhances the cell death accompanied by the increased LC3II formation, ROS production, loss of MMP and mass of mitochondria and lysosomes. Although AMPK activation by ROS-PARP-1 mediates autophagic cell death, we surprisingly found that pretreatment of cells with AMPK activators (A769662 and metformin) reverses cell death. Concomitantly, both agents block UVA-induced mitochondrial ROS production, autophagic flux, and mitochondrial fission without changing the inhibition of cathepsin B. CONCLUSION: UVA exposure rapidly induces ROS-PARP-1-AMPK-autophagic flux and late lysosomal dysfunction. Pre-inducing AMPK activation can prevent cellular events caused by UVA and provide a new protective strategy in photo-oxidative stress and photo-retinopathy.


Asunto(s)
Muerte Celular Autofágica , Humanos , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Catepsina B/metabolismo , Catepsina B/farmacología , Células Epiteliales/metabolismo , Receptores ErbB , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo
8.
Neurotoxicology ; 99: 274-281, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37939858

RESUMEN

Ethanol administration triggers an inflammatory response that leads to a complex series of immune responses including the release of an excessive amount of inflammatory mediators particularly tumor necrosis factor (TNF-α) and nuclear factor-kB (NF-KB) which produce a large amount of reactive oxygen species. The inflammatory-induced cytotoxicity is increased when the PI3-kinase/Akt pathway is inhibited. Some studies have also shown that ethanol suppresses the PI3-kinase signaling pathway induced by receptor activation. Friedelin and Glutinol belong to pentacyclic triterpenoid class and are known for their anti-inflammatory and antioxidant properties. The present study was aimed to elucidate the effects of these phytoconstituents on one of the key ethanol-induced neuronal damage pathways. The pups having (5-7 g average body weight) were used and randomly divided into groups. The control and ethanol treated pups were administered 0.9% normal saline while treated pups received glutinol and friedelin (30 mg/kg subcutaneously) respectively. After four hours all the experimental animals were sacrificed and their brains were collected carefully for protein expression analysis of p-Akt, TNF-α, NF-KB, caspase-3 and PARP-1 employing immunoblotting technique. Hemolytic, DNA protection, chelating power and ß-carotene assays results revealed that freidelin and glutinol are safe for parenteral administration. Glutinol administration with ethanol significantly abridged the ethanol induced over expression of TNF-α, caspase-3 and PARP-1 in pup's brain. Similarly, freidelin attenuated the neurodegeneration by inhibiting the ethanol induced p-JNK and NF-kB expression in pups' brain. This protection may be attributed to the revival of p-Akt signaling for cell survival. It is concluded that the present study demonstrates the neuro-protective effects of friedelin and glutinol via modulating the capase-3 and PARP-1 expression and modulating the neuronal apoptotic pathways.


Asunto(s)
Lupanos , FN-kappa B , Neuroprotección , Factor de Necrosis Tumoral alfa , Animales , Encéfalo , Caspasa 3/metabolismo , Etanol/toxicidad , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Lupanos/farmacología
9.
Biol Reprod ; 109(1): 65-72, 2023 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-37104616

RESUMEN

Endocrine disrupting chemicals are present in the environment and/or in consumer products. These agents have the capacity to mimic and/or antagonize endogenous hormones and thus perturb the endocrine axis. The male reproductive tract expresses steroid hormone (androgen and estrogen) receptors at high levels and is a major target for endocrine disrupting chemicals. In this study, Long-Evans male rats were exposed to dichlorodiphenyldichloroethylene, a metabolite of dichlorodiphenyltrichloroethane and a chemical present in the environment, in drinking water at 0.1 and 10 µg/L for 4 weeks. At the end of exposure, we measured steroid hormone secretion and analyzed steroidogenic proteins, including 17ß-hydroxysteroid dehydrogenase, 3ß-hydroxysteroid dehydrogenase, steroidogenic acute regulatory protein, aromatase, and the LH receptor. We also analyzed Leydig cell apoptosis (poly-(ADP-ribose) polymerase) and caspase-3 in the testes. Testicular testosterone (T) and 17ß-estradiol (E2) were both affected by exposure to dichlorodiphenyldichloroethylene by displaying altered steroidogenic enzyme expression. Dichlorodiphenyldichloroethylene exposure also increased the expression of enzymes mediating the pathway for programmed cell death, including caspase 3, pro-caspase 3, PARP, and cleaved PARP. Altogether, the present results demonstrate that dichlorodiphenyldichloroethylene directly and/or indirectly can target specific proteins involved in steroid hormone production in the male gonad and suggest that exposure to environmentally relevant dichlorodiphenyldichloroethylene levels has implications for male reproductive development and function.


Asunto(s)
Disruptores Endocrinos , Testículo , Ratas , Animales , Masculino , Caspasa 3/metabolismo , Disruptores Endocrinos/toxicidad , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas Long-Evans , Testosterona/farmacología , Células Intersticiales del Testículo/metabolismo , Estradiol/farmacología , Esteroides/metabolismo , Receptores de Estrógenos/metabolismo
10.
Altern Ther Health Med ; 29(5): 410-416, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37052975

RESUMEN

Objective: Poly (ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in DNA damage repair, gene transcription, cell growth, death and apoptosis. In our study, we aimed to explore the dynamic role of PARP-1 in chondrocyte (CH) degeneration in vitro. Methods: We used the primary CHs and treated them with interleukin-1 beta for up to 5 days. (IL-1ß) to induce degeneration. Meanwhile, we used AG-14361 (AG) to inhibit endogenous PARP-1 expression. Cell survival and collagen II expression were used to define the cell function of CHs. In addition, other metabolic indicators were measured containing the reactive oxygen species (ROS) level, 8-Hydroxy-2'-deoxyguanosine (8-OH-dG), IL-1ß, tumor necrosis factor alpha (TNF-α) and caspase 3/9 expression. Results: With IL-1ß treatment, the PARP1 expression of CHs was gradually increased from day 1 to day 5, accompanied by a reduction in cell survival and collagen II expression, and an increase in ROS, 8-OH-dG, IL-1ß, TNF-α and caspase 3/9 levels. We suppressed PARP1 expression on the first day of IL-1ß stimulation and found severe destruction of cell survival and collagen II content with a higher expression of caspase 3/9. However, when we cultured the CHs with AG from day 3 of the 5-day IL-1ß stimulation, cell survival and collagen II expression were rescued, and the ROS, 8-OH-dG, IL-1ß, TNF-α, and caspase 3/9 were downregulated. Conclusions: On day 1 of degeneration, increased PARP-1 played a protective role in CHs. However, from days 3 to 5 of degeneration, the accumulated PARP-1 presented a more destructive function in CHs.


Asunto(s)
Inhibidores de Poli(ADP-Ribosa) Polimerasas , Factor de Necrosis Tumoral alfa , Humanos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/farmacología , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Caspasa 3/metabolismo , Caspasa 3/farmacología , Condrocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , 8-Hidroxi-2'-Desoxicoguanosina/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina/farmacología , Apoptosis
11.
Biochimie ; 211: 96-109, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-36934779

RESUMEN

Diabetes and ulcerative colitis are chronic diseases associated with inflammation, dysbiosis, impaired immune function and infection risk. In patients with type 1 diabetes enteropathy, gastrointestinal manifestations are seen relatively frequently. The current investigation was aimed to decipher the role of 3-aminobenzamide (3-AB) in ulcerative colitis associated Diabetes mellitus in male BALB/c mice. Ulcerative colitis associated Diabetes mellitus experimental murine model was developed by 3 cycles (each cycle consists of seven days) of Dextran Sulphate Sodium (DSS; 2.5 %w/v) with recovery time of one week in-between along with Streptozotocin (STZ; 40 mg/kg; i.p. x 5 days; consecutively) was given at the Ist recovery period. As an intervention, 3-aminobenzamide (3-AB; 5 and 10 mg/kg; intraperitoneally) was given beginning with the second DSS cycle and then continue till sacrifice. 3-aminobenzamide treatment significantly reduced the severity of colitis-associated diabetes mellitus by altering the expression of a number of molecular targets, including sirtuin 1 (SIRT 1), proliferating cell nuclear antigen (PCNA), poly[ADP-ribose] polymerase 1 (PARP-1), cysteine protease-1 (Caspase-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkBp65), NLR family pyrin domain containing 3 (NLRP3), insulin growth factor 1 (IGF-1), interleukin-1ß (IL-1ß), interleukin-10 (IL-10) and ß-catenin. Further, 3-AB at high dose (10 mg/kg; intraperitoneally) significantly restored the epithelial tight junction integrity as evaluated by TEM analysis and restored occludin expression analysed by immunofluorescence analysis. Present study revealed that the high dose of 3-AB (10 mg/kg; intraperitoneally) showed significant and consistent protective effects against colitis associated Diabetes mellitus by modulating various molecular targets.


Asunto(s)
Colitis Ulcerosa , Colitis , Diabetes Mellitus , Masculino , Ratones , Animales , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteínas Quinasas Activadas por AMP/metabolismo , Ratones Endogámicos BALB C , Colitis/inducido químicamente , Colitis/complicaciones , Colitis/tratamiento farmacológico , Ratones Endogámicos C57BL , Colon/metabolismo , Modelos Animales de Enfermedad , Diabetes Mellitus/metabolismo
12.
Eur J Nucl Med Mol Imaging ; 50(7): 2081-2099, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36849748

RESUMEN

PURPOSE: Currently, there are multiple active clinical trials involving poly(ADP-ribose) polymerase (PARP) inhibitors in the treatment of glioblastoma. The noninvasive quantification of baseline PARP expression using positron emission tomography (PET) may provide prognostic information and lead to more precise treatment. Due to the lack of brain-penetrant PARP imaging agents, the reliable and accurate in vivo quantification of PARP in the brain remains elusive. Herein, we report the synthesis of a brain-penetrant PARP PET tracer, (R)-2-(2-methyl-1-(methyl-11C)pyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamide ([11C]PyBic), and its preclinical evaluations in a syngeneic RG2 rat glioblastoma model and healthy nonhuman primates. METHODS: We synthesized [11C]PyBic using veliparib as the labeling precursor, performed dynamic PET scans on RG2 tumor-bearing rats and calculated the distribution volume ratio (DVR) using simplified reference region method 2 (SRTM2) with the contralateral nontumor brain region as the reference region. We performed biodistribution studies, western blot, and immunostaining studies to validate the in vivo PET quantification results. We characterized the brain kinetics and binding specificity of [11C]PyBic in nonhuman primates on FOCUS220 scanner and calculated the volume of distribution (VT), nondisplaceable volume of distribution (VND), and nondisplaceable binding potential (BPND) in selected brain regions. RESULTS: [11C]PyBic was synthesized efficiently in one step, with greater than 97% radiochemical and chemical purity and molar activity of 148 ± 85 MBq/nmol (n = 6). [11C]PyBic demonstrated PARP-specific binding in RG2 tumors, with 74% of tracer binding in tumors blocked by preinjected veliparib (i.v., 5 mg/kg). The in vivo PET imaging results were corroborated by ex vivo biodistribution, PARP1 immunohistochemistry and immunoblotting data. Furthermore, brain penetration of [11C]PyBic was confirmed by quantitative monkey brain PET, which showed high specific uptake (BPND > 3) and low nonspecific uptake (VND < 3 mL/cm3) in the monkey brain. CONCLUSION: [11C]PyBic is the first brain-penetrant PARP PET tracer validated in a rat glioblastoma model and healthy nonhuman primates. The brain kinetics of [11C]PyBic are suitable for noninvasive quantification of available PARP binding in the brain, which posits [11C]PyBic to have broad applications in oncology and neuroimaging.


Asunto(s)
Glioblastoma , Ratas , Animales , Glioblastoma/diagnóstico por imagen , Glioblastoma/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Distribución Tisular , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Tomografía de Emisión de Positrones/métodos , Primates
13.
J Am Heart Assoc ; 12(4): e027769, 2023 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-36802924

RESUMEN

Background Hypertension and vascular toxicity are major unwanted side effects of antiangiogenic drugs, such as vascular endothelial growth factor inhibitors (VEGFis), which are effective anticancer drugs but have unwanted side effects, including vascular toxicity and hypertension. Poly (ADP-ribose) polymerase (PARP) inhibitors, used to treat ovarian and other cancers, have also been associated with elevated blood pressure. However, when patients with cancer receive both olaparib, a PARP inhibitor, and VEGFi, the risk of blood pressure elevation is reduced. Underlying molecular mechanisms are unclear, but PARP-regulated transient receptor potential cation channel, subfamily M, member 2 (TRPM2), a redox-sensitive calcium channel, may be important. We investigated whether PARP/TRPM2 plays a role in VEGFi-induced vascular dysfunction and whether PARP inhibition ameliorates the vasculopathy associated with VEGF inhibition. Methods and Results Human vascular smooth muscle cells (VSMCs), human aortic endothelial cells, and wild-type mouse mesenteric arteries were studied. Cells/arteries were exposed to axitinib (VEGFi) alone and in combination with olaparib. Reactive oxygen species production, Ca2+ influx, protein/gene analysis, PARP activity, and TRPM2 signaling were assessed in VSMCs, and nitric oxide levels were determined in endothelial cells. Vascular function was assessed by myography. Axitinib increased PARP activity in VSMCs in a reactive oxygen species-dependent manner. Endothelial dysfunction and hypercontractile responses were ameliorated by olaparib and a TRPM2 blocker (8-Br-cADPR). VSMC reactive oxygen species production, Ca2+ influx, and phosphorylation of myosin light chain 20 and endothelial nitric oxide synthase (Thr495) were augmented by axitinib and attenuated by olaparib and TRPM2 inhibition. Proinflammatory markers were upregulated in axitinib-stimulated VSMCs, which was reduced by reactive oxygen species scavengers and PARP-TRPM2 inhibition. Human aortic endothelial cells exposed to combined olaparib and axitinib showed nitric oxide levels similar to VEGF-stimulated cells. Conclusions Axitinib-mediated vascular dysfunction involves PARP and TRPM2, which, when inhibited, ameliorate the injurious effects of VEGFi. Our findings define a potential mechanism whereby PARP inhibitor may attenuate vascular toxicity in VEGFi-treated patients with cancer.


Asunto(s)
Antineoplásicos , Hipertensión , Neoplasias , Canales Catiónicos TRPM , Humanos , Ratones , Animales , Especies Reactivas de Oxígeno/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Canales Catiónicos TRPM/genética , Axitinib/uso terapéutico , Células Endoteliales/metabolismo , Óxido Nítrico/metabolismo , Antineoplásicos/uso terapéutico , Inhibidores de la Angiogénesis , Neoplasias/tratamiento farmacológico , Hipertensión/tratamiento farmacológico
14.
Laryngoscope ; 133(5): 1146-1155, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-35791892

RESUMEN

OBJECTIVES/HYPOTHESIS: We recently documented that acidic bile, a gastroesophageal reflux content, can cause invasive hypopharyngeal squamous cell carcinoma, by inducing widespread DNA damage and promoting nuclear factor kappa B (NF-κB)-related oncogenic molecular events. Poly or adenosine diphosphate (ADP)-ribose polymerase-1 (PARP-1), a sensitive sensor of DNA damage, may interact with NF-κB. We hypothesized that PARP-1 is activated in hypopharyngeal cells (HCs) with marked DNA damage caused by acidic bile, hence there is an association between PARP-1 and NF-κB activation or its related oncogenic profile, in this process. STUDY DESIGN: In vitro study. METHODS: We targeted PARP-1 and NF-κB(p65), using pharmacologic inhibitors, 1.0 µM Rucaparib (AG014699) and 10 µM BAY 11-7082 {3-[4=methylphenyl)sulfonyl]-(2E)-propenenitrile}, respectively, or silencing their gene expression (siRNAs) and used immunofluorescence, luciferase, cell viability, direct enzyme-linked immunosorbent assays, and qPCR analysis to detect the effect of targeting PARP-1 or NF-κB in acidic bile-induced DNA damage, PARP-1, p-NF-κB, and B-cell lymphoma 2 (Bcl-2) expression, as well as NF-κB transcriptional activity, cell survival, and mRNA oncogenic phenotype in HCs. RESULTS: We showed that (i) PARP-1 is overexpressed by acidic bile, (ii) targeting NF-κB adequately prevents the acidic bile-induced DNA double-strand breaks (DSBs) by gamma H2A histone family member X (γH2AX), oxidative DNA/RNA damage, PARP-1 overexpression, anti-apoptotic mRNA phenotype, and cell survival, whereas (iii) targeting PARP-1 preserves elevated DNA damage, NF-κB activation, and anti-apoptotic phenotype. CONCLUSION: We document for the first time that the activation of PARP-1 is an early event during bile reflux-related head and neck carcinogenesis and that NF-κB can mediate DNA damage and PARP-1 activation. Our data encourage further investigation into how acidic bile-induced activated NF-κB mediates DNA damage in hypopharyngeal carcinogenesis. LEVEL OF EVIDENCE: NA Laryngoscope, 133:1146-1155, 2023.


Asunto(s)
Bilis , FN-kappa B , Humanos , FN-kappa B/metabolismo , Bilis/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Ácidos y Sales Biliares , Carcinogénesis , ARN Mensajero/metabolismo , Daño del ADN , ADN/metabolismo
15.
Cell Mol Neurobiol ; 43(3): 1335-1353, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35840808

RESUMEN

Alzheimer's disease (AD) is characterized by the increase of hippocampal Ca2+ influx-induced apoptosis and mitochondrial oxidative stress (OS). The OS is a stimulator of TRPM2, although N-(p-amylcinnamoyl)anthranilic acid (ACA), 2-aminoethyl diphenylborinate (2/APB), and glutathione (GSH) are non-specific antagonists of TRPM2. In the present study, we investigated the protective roles of GSH and TRPM2 antagonist treatments on the amyloid ß42 peptide (Aß)-caused oxidative neurotoxicity and apoptosis in the hippocampus of mice with AD model. After the isolation of hippocampal neurons from the newborn mice, they were divided into five incubation groups as follows: control, ACA, Aß, Aß+ACA, and Aß+GSH. The levels of apoptosis, hippocampus death, cytosolic ROS, cytosolic Zn2+, mitochondrial ROS, caspase-3, caspase-9, lipid peroxidation, and cytosolic Ca2+ were increased in the primary hippocampus cultures by treatments of Aß, although their levels were decreased in the neurons by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2/APB). The Aß-induced decreases of cell viability, cytosolic GSH, reduced GSH, and GSH peroxidase levels were also increased in the groups of Aß+ACA and Aß+GSH by the treatments of ACA and GSH. However, the Aß-caused changes were not observed in the hippocampus of TRPM2-knockout mice. In conclusion, the present data demonstrate that maintaining the activation of TRPM2 is not only important for the quenching OS and neurotoxicity in the hippocampal neurons of mice with experimental AD but also equally critical to the modulation of Aß-induced apoptosis. The possible positive effects of GSH and TRPM2 antagonist treatments on the amyloid-beta (Aß)-induced oxidative toxicity in the hippocampus of mice. The ADP-ribose (ADPR) is produced via the stimulation of PARP-1 in the nucleus of neurons. The NUT9 in the C terminus of TRPM2 channel acts as a key role for the activation of TRPM2. The antagonists of TRPM2 are glutathione (GSH), ACA, and 2/APB in the hippocampus. The Aß incubation-mediated TRPM2 stimulation increases the concentration of cytosolic-free Ca2+ and Zn2+ in the hippocampus. In turn, the increased concentration causes the increase of mitochondrial membrane potential (ΔΨm), which causes the excessive generations of mitochondria ROS and the decrease of cytosolic GSH and GSH peroxidase (GSH-Px). The ROS production and GSH depletion are two main causes in the neurobiology of Alzheimer's disease. However, the effect of Aß was not shown in the hippocampus of TRPM2-knockout mice. The Aß and TRPM2 stimulation-caused overload Ca2+ entry cause apoptosis and cell death via the activations of caspase-3 (Casp/3) and caspase-9 (Casp/9) in the hippocampus. The actions of Aß-induced oxidative toxicity were modulated in the primary hippocampus by the incubations of ACA, GSH, 2/APB, and PARP-1 inhibitors (PJ34 and DPQ). (↑) Increase. (↓) Decrease.


Asunto(s)
Enfermedad de Alzheimer , Canales Catiónicos TRPM , Ratas , Ratones , Animales , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Caspasa 9/farmacología , Especies Reactivas de Oxígeno/metabolismo , Canales Catiónicos TRPM/metabolismo , Enfermedad de Alzheimer/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas Wistar , Estrés Oxidativo , Apoptosis , Glutatión/metabolismo , Glutatión/farmacología , Hipocampo/metabolismo , Peroxidasas/metabolismo , Peroxidasas/farmacología , Ratones Noqueados , Calcio/metabolismo
16.
J Cardiovasc Transl Res ; 16(3): 624-635, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36414924

RESUMEN

Dexmedetomidine (DEX) is clinically used for sedation of patients in intensive care, which also has been shown to have a strong anti-inflammatory effect on a variety of diseases. Parthanatos is a newly discovered form of programmed cell death. Here, we aimed to explore whether DEX protects cardiomyocytes from parthanatos in chronic heart failure (CHF). The levels of malondialdehyde (MAD), total superoxide dismutase (SOD), and adenosine triphosphate (ATP) were measured by corresponding detection kits. CHF mice model was established by transverse aortic constriction (TAC). PARP-1 expression in cardiac tissues of wild-type CHF mice was evaluated by immunohistochemistry. Flow cytometry was used to detect the effect of N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) on cell death. Masson trichrome staining and hematoxylin and eosin staining were conducted in cardiac tissues to evaluate the histological changes. TUNEL and caspase-1 double-staining and caspase-1 and NLRP3 double-staining were conducted in cardiac tissues to evaluate the effect of DEX on cell death in vivo. The relative expression of parthanatos and NLRP3 inflammasome-related proteins was evaluated by western blotting. MNNG induced parthanatos in mouse HL-1 cardiomyocytes. MNNG-induced parthanatos was promoted by ROS production and NLRP3 inflammasome activation. DEX treatment suppressed MNNG-induced parthanatos via NLRP3 inflammasome activation mediated by ROS. Importantly, DEX inhibited pathological changes and parthanatos in CHF mice. DEX suppressed the ROS/NLRP3 signaling pathway in CHF mice. DEX inhibited parthanatos in cardiomyocytes and in CHF mice by regulating the ROS-mediated NLRP3 inflammasome activation. The PARP-1 activation and NLRP3 inflammasome activation induced by MNNG was inhibited by DEX treatment, thus the generation of ROS was further inhibited, suggesting the inhibitory effect of DEX treatment on parthanatos in cardiomyocytes.


Asunto(s)
Dexmedetomidina , Parthanatos , Ratones , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Dexmedetomidina/farmacología , Miocitos Cardíacos/metabolismo , Metilnitronitrosoguanidina/metabolismo , Metilnitronitrosoguanidina/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Caspasa 1/metabolismo
17.
FEBS J ; 290(6): 1596-1624, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36239430

RESUMEN

Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down nicotinamide adenine dinucleotide+ (NAD+ ), it is also allosterically inhibited by NAD+ . However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here, we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a 'biological trigger' of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP inhibitor, PJ34 restored mitochondrial complex I activity and also prevented subsequent Sarm1 activation in rotenone-treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone-mediated loss of locomotor abilities, and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncover a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders.


Asunto(s)
Proteínas del Dominio Armadillo , Drosophila melanogaster , Mitocondrias , Enfermedades Mitocondriales , NAD , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/inducido químicamente , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , NAD/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Rotenona/farmacología
18.
Phytomedicine ; 108: 154528, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36343549

RESUMEN

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the fatal cancers and has not effective treatments. Alantolactone (ATL), a terpenoid extracted from traditional Chinese medicinal herb Inula helenium L., confers significant anti-inflammatory, antibacterial and antitumor activity. However, the activity and mechanisms of ATL in ATC remain unclear. PURPOSE: To investigate the potential anti-ATC effects in vitro and in vivo and the mechanisms involved. METHODS: The anti-proliferative activity of Alantolactone (ATL) against ATC cells was analyzed through CCK-8 and colony formation assays. Flow cytometry assay was performed to assess the cell cycle, cell apoptosis, ROS, and mitochondrial membrane potential (ΔΨm), whereas the cellular localization of cytochrome c and calreticulin were determined using cellular immunofluorescence assays. The lactate dehydrogenase (LDH) enzyme activity in the cell culture medium was measured using a commercial LDH kit, whereas ELISA was conducted to assess the secretory function of IL-1ß. Western blot assays were conducted to determine the expression or regulation of proteins associated with apoptosis and pyroptosis. Subcutaneous tumor model of nude mice was established to evaluate the anticancer activity of ATL in vivo. The expression of Ki67, cyclin B1, cleaved-PARP, cleaved-caspase 3, and IL-1ß in the animal tumor tissues was profiled using immunohistochemistry analyses. RESULTS: Our data showed that ATL significantly inhibited the proliferation and colony formation activity of ATC cells. ATL induced ATC cell cycle arrest at G2/M phase, and downregulated the expression of cyclin B1 and CDC2. Furthermore, ATL induced concurrent apoptosis and pyroptosis in the ATC cells, and the cleavage of PARP and GSDME. It also significantly increased the release of LDH and IL-1ß. Mechanically, ATL-mediated increase in ROS suppressed the Bcl-2/Bax ratio, downregulated the mitochondrial membrane potential and increased the release of cytochrome c, leading to caspase 9 and caspase 3 cleavage. We also found that ATL induced the translocation of an immunogenic cell death marker (calreticulin) to the cell membrane. In addition, it inhibited the growth of the ATC subcutaneous xenograft model, and activated proteins associated with apoptosis and pyroptosis, with a high safety profile. CONCLUSION: Taken together, these results firstly demonstrated that ATL exerted an anti-ATC activity by inducing concurrent apoptosis and GSDME-dependent pyroptosis through ROS-mediated mitochondria-dependent caspase activation. Meanwhile, these cell deaths exhibited obvious characteristics of immunogenic cell death, which may synergistically increase the potential of cancer immunotherapy in ATC. Further studies are needed to explore deeper mechanisms for the anti- ATC activity of ATL.


Asunto(s)
Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Ratones , Animales , Humanos , Caspasa 3/metabolismo , Piroptosis , Caspasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ciclina B1/metabolismo , Calreticulina/metabolismo , Calreticulina/farmacología , Citocromos c/metabolismo , Ratones Desnudos , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Apoptosis , Mitocondrias , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral
19.
Folia Histochem Cytobiol ; 60(4): 323-334, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36504133

RESUMEN

INTRODUCTION: As one of the basic components of Astragalus, Astragaloside IV (AS-IV) has a protective effect on endothelial injury caused by diabetes. AS-IV stimulated endothelial progenitor cells (EPCs) to secrete exosomes loaded with miR-21. This study aimed to investigate the effects of AS-IV-mediated EPCs exosomal miR-21 (EPC-exos-miR-21) on high glucose (HG) damaged endothelial cells. MATERIALS AND METHODS: After the isolation of EPCs derived from fetal umbilical cord blood, exosomes of EPCs were obtained by differential centrifugation. The morphology of exosomes was observed by electron microscopy. The particle size distribution of exosomes was detected by Nanoparticle Tracking Analysis. Human umbilical vein endothelial cells (HUVECs) were treated with 33 mM glucose to establish an HG injury model. Flow cytometry and TUNEL assay were used to characterize the surface markers of primary EPCs and the apoptosis of HUVECs. The gene and protein expression were detected by qPCR, immunofluorescence, and Western blotting. A dual luciferase assay was used to verify the targeting relationship of miR-21 with PTEN. RESULTS: HG environment led to time- and dose-dependent inhibition and enhancement of autophagy and apoptosis in HUVECs. AS-IV stimulated EPCs to secrete exosomes loaded with miR-21. Exosomes secreted by EPCs pretreated with AS-IV [EPC-exo(ASIV)] promoted autophagy and inhibited apoptosis in HG-impaired HUVECs. PTEN is a target of miR-21. MiR-21 carried by EPC-exo(ASIV) repressed PTEN expression in HG-impaired HUVECs. In contrast, p-AKT, p-mTOR, p-PI3K, cleaved PARP and PARP levels were upregulated. Compared to the HG group, the expression of autophagy regulatory genes (ATG5, beclin1 and LC3) was enhanced in the EPC-exo(ASIV) group and EPC-exo(ASIV)-miR-21 mimic group. In contrast, apoptosis-positive regulatory genes (Bax, caspase-3 and caspase-9) were attenuated. Further overexpression of PTEN reversed the expression of these genes. CONCLUSIONS: AS-IV-mediated EPC-exos-miR-21 could enhance autophagy and depress apoptosis in HG-damaged endothelial cells via the miR-21/PTEN axis.


Asunto(s)
Células Progenitoras Endoteliales , Exosomas , MicroARNs , Humanos , Células Progenitoras Endoteliales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Exosomas/genética , Exosomas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Apoptosis , Autofagia , Glucosa/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo
20.
Nat Cancer ; 3(10): 1211-1227, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36253486

RESUMEN

Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK-p-Tyr19-CDK9 expression.


Asunto(s)
Quinasa de Linfoma Anaplásico , Antineoplásicos , Neoplasias de la Mama , Quinasa 9 Dependiente de la Ciclina , Animales , Femenino , Humanos , Ratones , Quinasa de Linfoma Anaplásico/metabolismo , Antineoplásicos/farmacología , Biomarcadores , Neoplasias de la Mama/tratamiento farmacológico , Quinasa 9 Dependiente de la Ciclina/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factor B de Elongación Transcripcional Positiva , Tirosina/química , Tirosina/metabolismo , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Estados Unidos
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