La inhibición de la proteína tirosina fosfatasa 1B protege frente a la apoptosis temprana inducida por paracetamol en hepatocitos

Premio “Consejo General de Colegios Oficiales de Farmacéuticos” en el Concurso Científico 2013 de la Real Academia Nacional de Farmacia.[EN]: Acetaminophen (paracetamol, APAP) is a widely used analgesic and antipyretic drug safe at therapeutic doses but its overdose causes liver injury. Our goal was to explore whether protein tyrosine phosphatase 1B (PTP1B), a negative modulator of survival signaling pathways, plays a role in APAP-induced cell death in hepatocytes. Hepatotoxicity was evaluated in immortalized hepatocytes generated from wild-type (PTP1B+/+) and PTP1B-deficient (PTP1B-/-) mice. Apoptosis occurred as an early event only in APAP-treated PTP1B+/+ hepatocytes. PTP1B deficiency conferred protection against cell cycle arrest and loss of cellular viability. These data suggest that PTP1B as a target against APAP-induced liver failure.[ES]: El paracetamol es un analgésico/antipirético hepatotóxico a dosis altas. Investigamos el papel de la proteína tirosina fosfatasa 1B (PTP1B), un modulador negativo de señalización de supervivencia celular, en la muerte celular temprana (apoptosis) inducida por paracetamol en hepatocitos. En hepatocitos controles se inducía apoptosis en respuesta al paracetamol. Este efecto se encontraba disminuido en hepatocitos deficientes en PTP1B. La falta de PTP1B protegía a los hepatocitos de la parada del ciclo celular y la pérdida de la viabilidad celular tras el tratamiento con paracetamol. Proponemos a la PTP1B como diana terapéutica frente al fallo hepático inducido por sobredosis de paracetamol.We acknowledge the following grant support: SAF2012-33283 (MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Spain).Peer Reviewe

Protection against paracetamol-induced adverse effects in the liver by the inhibition of the protein tyrosine phosphatase 1B

Tesis Doctoral presentada por Maysa Ahmed Abd El-Hamid Mobasher en el Departamento de Bioquímica de la Universidad Autónoma de Madrid para optar al grado de Doctor, y realizada en el Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-IIBM).[EN]: Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. PTP1B is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes as well as in a mouse model of APAP-induced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was up-regulated by APAP in primary human and mouse hepatocytes together with activation of JNK and p38 MAPK, resulting in cell death. Conversely, Akt phosphorylation and the anti-apoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse hepatocytes protected against APAP-induced cell death preventing GSH depletion, ROS generation and activation of JNK and p38 MAPK. APAP-treated PTP1B-/- hepatocytes showed enhanced anti-oxidant defense through the glycogen synthase kinase GSK3β/SKF axis, delaying tyrosine phosphorylation of Nrf2 and its nuclear exclusion, ubiquitination and degradation. IGFIR-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B-/- cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice resulting in less severe APAP hepatotoxicity in PTP1B-/- mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity suggesting a novel therapeutic target against APAPinduced liver failure.In the initial experiments of APAP-mediated toxicity in hepatocytes we found that APAP also increased PTP1B and activated JNK at subtoxic doses. These data prompted us to evaluate the cross-talk between subtoxic doses of APAP and insulin signaling in hepatocytes. Insulin signaling was decreased in hepatocytes stimulated with subtoxic doses of APAP through decreasing IR and IRS1 tyrosine phosphorylation. However, PTP1B deficiency protected against these effects. In an in vivo chronic treatment with APAP, wild-type mice showed a trend towards a decrease in whole body glucose homeostasis whereas in mice lacking PTP1B no deleterious effects were observed.[ES]: El fallo hepático agudo debido a una sobredosis de paracetamol está asociado con una elevada mortalidad. La PTP1B modula negativamente la señalización mediada por los receptores de factores de crecimiento de la súper familia tirosina quinasa. En el hígado, estas rutas confieren protección frente al daño. En esta Tesis Doctoral hemos investigado la expresión de PTP1B en el daño agudo inducido por sobredosis de paracetamol en humanos, así como su papel en la regulación de los mecanismos moleculares responsables del balance supervivencia/muerte celular en el hígado. Hemos encontrado un aumento en la expresión de PTP1B en el tejido hepático procedente de individuos que necesitaron trasplante tras una sobredosis de paracetamol. Así mismo, los niveles de PTP1B aumentaron en hepatocitos humanos y murinos tratados con paracetamol en paralelo con la activación de las quinasas de estrés JNK y P38 MAPK y la inducción de muerte celular. Por el contrario, la activación de Akt y los niveles de proteínas anti-apoptóticas de la familia Bcl2 se encontraron disminuidos. La deficiencia en PTP1B protegía a los hepatocitos frente a la muerte inducida por paracetamol ya que preveía la depleción de GSH, la generación de ROS y la activación de las quinasas de estrés. Además, los hepatocitos PTP1B-/- tratados con APAP presentaron una mayor respuesta antioxidante modulando las quinasas GSK3β/SKF lo que retrasaba la fosforilación en tirosina del Nrf2 responsable de su salida del núcleo y posterior ubiquitinación y degradación. La inhibición de PTP1B también evitaba la disminución de la fosforilación del IGF-IR y Akt. Estos dos mecanismos se modulaban de igual manera en ratones sometidos a una sobredosis de paracetamol, presentando los ratones PTP1B-/- una menor hepatotoxicidad. Estos resultados demostraban que la PTP1B regula los mecanismos moleculares desencadenados por dosis tóxicas paracetamol, lo que sugiere que la PTP1B podría ser una diana terapéutica frente a este daño hepatocelular. La expresión de PTP1B y la activación de las quinasas de estrés también aumentaban en hepatocitos estimulados con dosis subtóxicas de paracetamol, de manera similar a lo que ocurre en situaciones de resistencia a la insulina. Hemos encontrado que dosis subtóxicas de paracetamol disminuyen la señalización de la insulina en los hepatocitos y este efecto se atenúa en ausencia de PTP1B. Por último, presentamos evidencias de que la administración crónica de paracetamol podría alterar la homeostasis glucídica del organismo y esto se evitaría mediante la inhibición de la PTP1B.This work has been founded by a predoctoral fellowship of the JAE program (CSIC). The experimental research was founded by projects SAF2009-01615 and SAF2012-33283 (to AMV) from MINECO.Peer Reviewe

Signalling pathways involved in paracetamol-induced hepatotoxicity: New insights on the role of protein tyrosine phosphatase 1B

Acute hepatic failure secondary to paracetamol poisoning is associated with high mortality. Paracetamol-induced hepatotoxicity causes oxidative stress that triggers signalling pathways and ultimately leads to lethal hepatocyte injury. We will review the signalling pathways activated by paracetamol in the liver emphasizing the role of protein tyrosine phosphatase 1B (PTP1B) in the balance between cell death and survival in hepatocytes. PTP1B has emerged as a key modulator of the antioxidant system mediated by the nuclear factor erythroid-2-related factor 2 (Nrf2) in hepatic cells in response to paracetamol overdose. Also, this phosphatase modulates the classical survival pathways triggered by the activation of the insulin-like growth factor-I (IGF-I) signalling cascade. Therefore, PTP1B is a novel therapeutic target against paracetamol-induced liver failure.We also acknowledge the following grant support: SAF2012-33283 (MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Spain).Peer Reviewe

Protein tyrosine phosphatase 1B modulates GSK3ß/Nrf2 and IGF-1R signalling pathways in acetaminophen-induced hepatotoxicity

Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes, as well as in a mouse model of APAPinduced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was upregulated by APAP in primary human and mouse hepatocytes together with the activation of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), resulting in cell death. Conversely, Akt phosphorylation and the antiapoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse protects hepatocytes against APAP-induced cell death, preventing glutathione depletion, reactive oxygen species (ROS) generation and activation of JNK and p38 MAPK. APAP-treated PTP1B / hepatocytes showed enhanced antioxidant defense through the glycogen synthase kinase 3 (GSK3)b/Src kinase family (SKF) axis, delaying tyrosine phosphorylation of the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) and its nuclear exclusion, ubiquitination and degradation. Insulin-like growth factor-I receptor-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B / cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice, resulting in less severe APAP hepatotoxicity in PTP1B / mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity, suggesting a novel therapeutic target against APAPinduced liver failure. CellPeer Reviewe

The Toll-Like Receptor 3 Agonist Polyriboinosinic Polyribocytidylic Acid Increases the Numbers of NK Cells with Distinct Phenotype in the Liver of B6 Mice

One of the activating factors of the cells of the innate immune system is the agonists of toll-like receptors (TLRs). Our earlier publications detailed how poly(I:C), a TLR3 agonist, elevates the NK cell population and the associated antigen-specific CD8+ T cell responses. This study involved a single treatment of the B6 mice with poly(I:C) intraperitoneally. To perform a detailed phenotypic analysis, mononuclear cells were prepared from each of the liver, peripheral blood, and spleen. These cells were then examined for their NK cell population by flow cytometric analysis following cell staining with indicated antibodies. The findings of the study showed that the NK cell population of the liver with an NK1.1highCD11bhighCD11chigh B220+Ly6G- phenotype was elevated following the treatment with poly(I:C). In the absence of CD11b molecule (CR3-/- mice), poly(I:C) can still increase the remained numbers of NK cells with NK1.1+CD11b- and NK1.1+Ly6G- phenotypes in the liver while their numbers in the blood decrease. After the treatment with anti-AGM1 Ab, which induced depletion of NK1.1+CD11b+ cells and partial depletion of CD3+NK1.1+ and NK1.1+CD11b- cell populations, poly(I:C) normalized the partial decreases in the numbers of NK cells concomitant with increased numbers of NK1.1-CD11b+ cell population in both liver and blood. Regarding mice with a TLR3-/- phenotype, their injection with poly(I:C) resulted in the partial elevation in the NK cell population as compared to wild-type B6 mice. To summarise, the TLR3 agonist poly(I:C) results in the elevation of a subset of liver NK cells expressing the two myeloid markers CD11c and CD11b. The effect of poly(I:C) on NK cells is partially dependent on TLR3 and independent of the presence of CD11b

Protein tyrosine phosphatase 1B deficiency protects against paracetamol-induced hepatotoxicity

Resumen del póster presentado al XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre de 2011.Acetaminophen (APAP) is an analgesic and antipyretic drug safe at therapeutic doses but its overdose causes liver injury. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of growth factor signalling and modulate the balance between survival and cell death. Our purpose was to study if PTP1B plays a role in APAP-induced hepatotoxicity. APAP induced hepatotoxicity was evaluated in immortalized hepatocytes from wild-type (PTP1B+/+) and PTP1B-/- mice. PTP1B deficiency protected against GSH depletion, elevation of ROS, cell cycle arrest and apoptotic/necrotic cell death. At the molecular level, phosphorylation of JNK and p38 was higher in APAPtreated wild-type hepatocytes compared to PTP1B-/- cells. Regarding survival signaling, phosphorylation of IGF-IR, levels of IRS1 and IRS2 and activation of Akt decreased in wild-type hepatocytes but not in PTP1B-/-cells. Therefore, the expression of the anti-apoptotic proteins BclxL and Mcl-1 was reduced in wild-type cells, but it was maintained in PTP1B-/- hepatocytes. We confirmed these results in the liver of wild type and PTP1B-/- mice after ip APAP injection. We found increased JNK phosphorylation at 1-6 h post-injection. Survival signaling including phosphorylation of IGF-IR, levels of IRS1 and activation of Akt was decreased at 6 h after APAP injection in wild-type mice, but not in PTP1B-/- mice. GSH depletion and ROS levels were attenuated in PTP1B-/- livers in parallel with an increase in nuclear Nrf2 accumulation and HO-1 induction. Finally, treatment of human hepatocytes with APAP increased PTP1B expression and activation of JNK and p38, decreasing survival signaling and cellular viability. Our data suggest that PTP1B might be a target against APAPinduced hepatotoxicity.SAF2009-08114 MCINN.Peer Reviewe

The Combined Effect of Licorice Extract and Bone Marrow Mesenchymal Stem Cells on Cisplatin-Induced Hepatocellular Damage in Rats

Drug-induced liver damage is a life-threatening disorder, and one major form of it is the hepatotoxicity induced by the drug cisplatin. In folk medicine, Licorice (Glycyrrhiza glabra (is used for detoxification and is believed to be a potent antioxidant. Currently, the magically self-renewable potential of bone marrow mesenchymal stem cells (BM-MSCs) has prompted us to explore their hepatoregenerative capability. The impact of G. glabra extract (GGE) and BM-MSCs alone and, in combination, on protecting against hepatotoxicity was tested on cisplatin-induced liver injury in rats. Hepatic damage, as revealed by liver histopathology and increased levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and malondialdehyde (MDA), was elevated in rats by received 7 mg/kg of cisplatin intraperitoneally. The combination of GGE and BM-MSCs returned the enzyme levels to near the normal range. It also improved levels of liver superoxide dismutase (SOD) and glutathione (GSH) and reduced MDA levels. Additionally, it was found that when GGE and BM-MSCs were used together, they significantly downregulated caspase9 (Casp9), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and interleukin-1β (IL-1β), which are involved in severe proinflammatory and apoptotic signaling cascades in the liver. Moreover, combining GGE and BM-MSCs led to the normal result of hepatocytes in several examined liver histological sections. Therefore, our findings suggest that GGE may have protective effects against oxidative liver damage and the promising regenerative potential of BM-MSCs

Glycine Betaine Relieves Lead-Induced Hepatic and Renal Toxicity in Albino Rats

Lead (Pb) is a widespread and nondegradable environmental pollutant and affects several organs through oxidative mechanisms. This study was conducted to investigate the antioxidant protective effect of glycine betaine (GB) against Pb-induced renal and hepatic injury. Male albino rats (n = 45) were divided into three groups: G1 untreated control, G2 Pb-acetate (50 mg/kg/day), and G3 Pb-acetate (50 mg/kg/day) plus GB (250 mg/kg/day) administered for 6 weeks. For G3, Pb-acetate was administered first and followed by GB at least 4 h after. Pb-acetate treatment (G2) resulted in a significant decrease in renal function, including elevated creatinine and urea levels by 17.4% and 23.7%, respectively, and nonsignificant changes in serum uric acid levels. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphates (ALP) activities were significantly increased with Pb treatment by 37.6%, 59.3%, and 55.1%, respectively. Lipid peroxidation level was significantly increased by 7.8 times after 6 weeks of Pb-acetate treatment. The level of reduced glutathione (GSH-R) significantly declined after Pb-acetate treatment. Pb-acetate treatment also reduced the activities of superoxide dismutase (SOD), glutathione-S-transferase (GST), and glutathione peroxidase (GSH-PX) by 74.1%, 85.0%, and 40.8%, respectively. Treatment of Pb-intoxicated rats with GB resulted in a significant reduction in creatinine, urea, ALT, AST, and lipid peroxidation, as well as a significant increase in the level of GSH-R and in the activities of ALP, SOD, GST, and GSH-PX. The molecular interaction between GB and GSH-PX indicated that the activation of GSH-PX in Pb-intoxicated rats was not the result of GB binding to the catalytic site of GSH-PX. The affinity of GB to bind to the catalytic site of GSH-PX is lower than that of H2O2. Thus, GB significantly mitigates Pb-induced renal and liver injury through the activation of antioxidant enzymes and the prevention of Pb-induced oxidative damage in the kidney and liver

Antiproliferative Effect of <i>Clitoria ternatea</i> Ethanolic Extract against Colorectal, Breast, and Medullary Thyroid Cancer Cell Lines

Clitoria ternatea is a native plant with medicinal and nutritive significance in Asia. The goal of this work was to examine the antiproliferative role of Clitoria ternatea against colorectal (HCT116), breast (MCF-7), and thyroid (TT) cancer cell lines at cellular and molecular levels. A phytochemical analysis, the cytotoxic effect, an apoptotic induction cell cycle analysis, and the expression level of GAX, DIABLO, and NAIP1 genes were assessed. The plant extract exhibited a clear cytotoxic action against the utilized cancer cell lines via a low IC50, foremost by means of cell cycle arrest at the pre-G0, G1, and S phases associated with an apoptotic induction. An apparent raise in the mRNA levels of GAX and DIABLO and a concomitant decrease in the NAIP1 mRNA level were observed in the used cancer cells treated with the IC50 of the plant extract. This study concluded that an ethanolic extract of Clitoria ternatea induced apoptotic cell death, suggesting that it could possibly be utilized as a new source of an apoptosis-inducing anticancer agent for colon, breast, and medullary thyroid cancer cell line treatments with further detailed studies

Antiproliferative Effect of Clitoria ternatea Ethanolic Extract against Colorectal, Breast, and Medullary Thyroid Cancer Cell Lines

Clitoria ternatea is a native plant with medicinal and nutritive significance in Asia. The goal of this work was to examine the antiproliferative role of Clitoria ternatea against colorectal (HCT116), breast (MCF-7), and thyroid (TT) cancer cell lines at cellular and molecular levels. A phytochemical analysis, the cytotoxic effect, an apoptotic induction cell cycle analysis, and the expression level of GAX, DIABLO, and NAIP1 genes were assessed. The plant extract exhibited a clear cytotoxic action against the utilized cancer cell lines via a low IC50, foremost by means of cell cycle arrest at the pre-G0, G1, and S phases associated with an apoptotic induction. An apparent raise in the mRNA levels of GAX and DIABLO and a concomitant decrease in the NAIP1 mRNA level were observed in the used cancer cells treated with the IC50 of the plant extract. This study concluded that an ethanolic extract of Clitoria ternatea induced apoptotic cell death, suggesting that it could possibly be utilized as a new source of an apoptosis-inducing anticancer agent for colon, breast, and medullary thyroid cancer cell line treatments with further detailed studies