Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells

ABSTRACT The mild analgesic drug acetaminophen (AAP) induces severe hepatic injury when taken at excessive doses. Recent evidence shows that the initial form of damage is through apoptosis, but this fails to go to completion and degenerates into necrosis. The aim of this study was to elucidate the mechanism through which AAP induces apoptosis using human HuH7 hepatoma cells as an in vitro model system to investigate the initial phase of AAP-induced hepatic injury. AAP-induced apoptosis in HuH7 cells as evidenced by chromatin condensation was preceded by the translocation of Bax to mitochondria and the cytoplasmic release of the proapoptotic factors cytochrome c and Smac/DIABLO. A concomitant loss of mitochondrial membrane potential occurred. Activation of the mitochondrial pathway of apoptosis led to the activation of execution caspases-3 and -7. AAP-induced apoptosis and cell death was blocked by inhibitors of caspases but not by inhibitors of calpains, cathepsins, and serine proteases. Apoptosis was unaffected by inhibitors of the mitochondrial permeability transition pore and by inhibitors of Jun NH 2 -terminal kinases, p38 mitogen-activated protein kinase, or mitogen-activated protein kinase kinase 1/2. However, pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) delayed and decreased the extent of AAP-induced apoptosis. In comparison, endoplasmic reticulum stress-induced but not prooxidant-induced apoptosis of HuH7 cells was sensitive to GSK-3 inhibition. It is concluded that AAP-induced apoptosis involves the mitochondrial pathway of apoptosis that is mediated by GSK-3 and most likely initiated through an endoplasmic reticulum stress response. The mild analgesic acetaminophen (paracetamol, AAP) remains the commonest cause of acute liver failure in the United States and other parts of the world as a result of accidental or deliberate overdose Many of the histochemical and biochemical features of the late stages of AAP toxicity, particularly after high doses, support the conclusion that AAP induces hepatocellular necrosi

Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells

Abstract: 232 Introduction: 61

Bone Marrow Stromal Cells Modulate Mouse ENT1 Activity and Protect Leukemia Cells from Cytarabine Induced Apoptosis

BACKGROUND: Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. METHODS: Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of (3)H-adenosine. RESULTS: Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. CONCLUSION: The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML

Mechanisms of drug-induced apoptosis in liver cells : relevance to in vivo hepatotoxicity

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Mechanisms of Drug-Induced Apoptosis in Liver Cells: Relevance to in vivo Hepatotoxicity.

In response to a cytotoxic insult, a cell can die by necrosis or by apoptosis. Therefore, the overall objective of this investigation was to identify the mechanisms of drug-induced liver cell apoptosis in vitro to help with the discovery of novel markers of apoptosis that could be used for predicting hepatocyte apoptosis in vivo. In this investigation, the capability of drugs to induce apoptosis in liver cells was investigated using two cellular models, namely primary mouse hepatocytes and the hepatoma cell line, HuH7. This study was performed using three model compounds, paracetamol, thapsigargin and duroquinone. The hepatotoxin, paracetamol, is a widely used analgesic drug, can induce fatal liver injury through a combination of apoptosis and necrosis, when taken in large doses. Our work has previously shown that apoptosis plays an essential role in paracetamol-induced hepatic injury since inhibiting apoptosis, prevents the development of acute liver failure. The endoplasmic reticulum (ER) stress inducer, thapsigargin, acts by selectively inhibiting the ER Ca2+ -ATPase, which pumps calcium against a concentration gradient into the ER. Both paracetamol and thapsigargin caused marked cytotoxicity in HuH7 cells as evidenced by chromatin condensation and DNA fragmentation. Similarly, the redox-cycler, duroquinone, caused cell death in the HuH7 cells and primary hepatocytes by both the induction of apoptotic and necrotic cell death. All three compounds caused the activation of executioner caspases-3 and -7 and also the cleavage of caspase-3 cellular substrates including fodrin and cytokeratin 18. Furthermore, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-fmk) protected from paracetamol and thapsigargin-induced cytotoxicity. However, Z-VAD-fmk did not afford protection from duroquinone-induced cell death by oxidative stress in the HuH7 cells. Non-caspase proteases including calpains, cathepsins, serine proteases and the proteasome were not involved in cell death by these compounds. The manifestation of apoptosis was preceded by a loss of mitochondrial membrane potential and the release of cytochrome c and Smac/DIABLO. However, the inhibitors of the membrane permeability transition pore (PTP) cyclosporine A and bongkrekic acid failed to prevent apoptosis. In contrast, the Bcl-2 pro-apoptotic protein Bax, was found to translocate to the mitochondria to allow the release of cytochrome c in the HuH7 cells treated with paracetamol, thapsigargin and duroquinone. It was postulated that as a result of the metabolic activation of paracetamol and the concomitant production of reactive oxygen species (ROS), a cellular stress response was induced in HuH7 cells. To investigate the role of this stress response in the initiation of apoptosis by paracetamol and also thapsigargin and duroquinone, stress-activated protein kinases including c-Jun N-terminal kinase (JNK) and p38 and also MEK1/2 and glycogen synthase kinase-3 (GSK-3) were investigated. Although JNK was shown to be activated, the pharmacological inhibition of JNK, p38 and MEK did not afford protection from cell death by these compounds in the HuH7 cells. However, the inhibition of pro-apoptotic GSK-3 protected significantly from paracetamol and thapsigargin-induced cell death by apoptosis

The CCL2/CCR2 Axis Affects Transmigration and Proliferation but Not Resistance to Chemotherapy of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) has a high mortality rate despite chemotherapy and transplantation. Both CXCR4/SDF-1 and VLA-4/VCAM1 axes are involved in leukemia protection but little is known about the role of CCL2/CCR2 in AML biology and protection against chemotherapy. We measured CCR2 expression in AML cell lines and primary AML cells by flow cytometry (FCM), real time PCR (RT-PCR) and western blot (WB). CCL2 production was quantified by solid phase ELISA in peripheral blood (PB) and bone marrow (BM) serum. We measured chemotaxis in a transwell system with different concentrations of CCL2/CCR2 blockers; cell cycle with BrDU and propidium iodide and proliferation with yellow tetrazolium MTT. We determined synergy in in vitro cell apoptosis combining chemotherapy and CCL2/CCR2 blockade. Finally, we performed chemoprotection studies in an in vivo mouse model. Of 35 patients, 23 (65%) expressed CCR2 by FCM in PB. Two cell lines expressed high levels of CCR2 (THP-1 and murine AML). RT-PCR and WB confirmed CCR2 production. CCL2 solid phase ELISA showed significantly lower levels of CCL2 in PB and BM compared to normal controls. Chemotaxis experiments confirmed a dose-dependent migration in AML primary cells expressing CCR2 and THP-1 cells. A significant inhibition of transmigration was seen after CCL2/CCR2 blockade. Proliferation of CCR2+ AML cell lines was slightly increased (1.4-fold) after axis stimulation. We observed a non-significant increase in phase S THP-1 cells exposed to CCL2 and a concomitant decrease of cells in G1. The chemotherapy studies did not show a protective effect of CCL2 on cytarabine-induced apoptosis or synergy with chemotherapy after CCL2/CCR2 blockade both in vitro and in vivo. In conclusion, CCL2/CCR2 axis is expressed in the majority of monocytoid AML blasts. The axis is involved in cell trafficking and proliferation but no in vitro and in vivo chemotherapy protective effect was seen

Cell proliferation and cell cycle analysis of THP-1 cells exposed to CCL2.

<p>(<b>A</b>) THP-1 cells were seeded on a 96-well plate and incubated with various concentrations of recombinant human CCL2 (10, 50 and 100 ng/ml) for 24 hours and cell proliferation measured by FCM. (<b>B</b>) THP-1 cells were cultured with or without CCL2 (50 ng/ml) for 24 hours. Leukemia cells were harvested, fixed and stained with PI and analyzed by FCM. Each bar represents the mean ± SD of 3 independent experiments (*<i>p</i> < 0.05; **<i>p</i> < 0.01).</p

CCR2 expression in human and murine AML cell lines.

<p>Human AML cells lines THP1, U-937 and Kasumi and murine AML cell line (APL) were cultured to detect CCR2 expression by FCM (<b>A</b>), WB (<b>B</b>) and qPCR (<b>C and D</b>). All cell lines were grown in RPMI medium and were processed for the various studies according to the material and methods section. Positive control samples correspond to PB or BM samples from healthy individuals with 5–8% normal monocytes. Healthy monocytes show above 90% CCR2 expression.</p