Amélie Rebillard

Cisplatin-Induced Apoptosis Involves Membrane Fluidification via Inhibition of NHE1 in Human Colon Cancer Cells

We have previously shown that cisplatin triggers an early acid sphingomyelinase (aSMase)-dependent ceramide generation concomitantly with an increase in membrane fluidity and induces apoptosis in HT29 cells. The present study further explores the role and origin of membrane fluidification in cisplatin-induced apoptosis. The rapid increase in membrane fluidity following cisplatin treatment was inhibited by membrane-stabilizing agents such as cholesterol or monosialoganglioside-1. In HT29 cells, these compounds prevented the early aggregation of Fas death receptor and of membrane lipid rafts on cell surface and significantly inhibited cisplatin-induced apoptosis without altering drug intracellular uptake or cisplatin DNA adducts formation. Early after cisplatin treatment, Na+/H+ membrane exchanger-1 (NHE1) was inhibited leading to intracellular acidification, aSMase was activated, and ceramide was detected at the cell membrane. Treatment of HT29 cells with Staphylococcus aureus sphingomyelinase increased membrane fluidity. Moreover, pretreatment with cariporide, a specific inhibitor of NHE1, inhibited cisplatin-induced intracellular acidification, aSMase activation, ceramide membrane generation, membrane fluidification, and apoptosis. Finally, NHE1-expressing PS120 cells were more sensitive to cisplatin than NHE1-deficient PS120 cells. Altogether, these findings suggest that the apoptotic pathway triggered by cisplatin involves a very early NHE1-dependent intracellular acidification leading to aSMase activation and increase in membrane fluidity. These events are independent of cisplatin-induced DNA adducts formation. The membrane exchanger NHE1 may be another potential target of cisplatin, increasing cell sensitivity to this compound.

Cisplatin cytotoxicity: DNA and plasma membrane targets.

Most current anticancer therapies induce tumor cell death through apoptosis where its specific involved pathways are poorly understood. For example, for many DNA-damaging agents, the specific biochemical lesions (DNA adducts) are associated with the induction of apoptosis via the mitochondria death pathway. However, several of these DNA-damaging agents like cisplatin induce apoptosis through plasma membrane disruption, triggering the Fas death receptor pathway. In this review, we focus on the role of early plasma membrane events in cisplatin-induced apoptosis. Special attention is given to changes in plasma membrane fluidity, inhibition of NHE1 exchanger, activation of acid sphingomyelinase and their consequences on the Fas death pathway in response to cisplatin.

Ethanol induces oxidative stress in primary rat hepatocytes through the early involvement of lipid raft clustering

The role of the hepatocyte plasma membrane structure in the development of oxidative stress during alcoholic liver diseases is not yet fully understood. Previously, we have established the pivotal role of membrane fluidity in ethanol‐induced oxidative stress, but no study has so far tested the involvement of lipid rafts. In this study, methyl‐β‐cyclodextrin or cholesterol oxidase, which were found to disrupt lipid rafts in hepatocytes, inhibited both reactive oxygen species production and lipid peroxidation, and this suggested a role for these microstructures in oxidative stress. By immunostaining of lipid raft components, a raft clustering was detected in ethanol‐treated hepatocytes. In addition, we found that rafts were modified by formation of malondialdehyde adducts and disulfide bridges. Interestingly, pretreatment of cells by 4‐methyl‐pyrazole (to inhibit ethanol metabolism) and various antioxidants prevented the ethanol‐induced raft aggregation. In addition, treatment of hepatocytes by a stabilizing agent (ursodeoxycholic acid) or a fluidizing compound [2‐(2‐methoxyethoxy)ethyl 8‐(cis‐2‐n‐octylcyclopropyl)octanoate] led to inhibition or enhancement of raft clustering, respectively, which pointed to a relationship between membrane fluidity and lipid rafts during ethanol‐induced oxidative stress. We finally investigated the involvement of phospholipase C in raft‐induced oxidative stress upon ethanol exposure. Phospholipase C was shown to be translocated into rafts and to participate in oxidative stress by controlling hepatocyte iron content. Conclusion: Membrane structure, depicted as membrane fluidity and lipid rafts, plays a key role in ethanol‐induced oxidative stress of the liver, and its modulation may be of therapeutic relevance. (HEPATOLOGY 2007.)

Cisplatin-induced apoptosis involves a Fas-ROCK-ezrin-dependent actin remodelling in human colon cancer cells

In human colon cancer cells, cisplatin-induced apoptosis involves the Fas death receptor pathway independent of Fas ligand. The present study explores the role of ezrin and actin cytoskeleton in relation with Fas receptor in this cell death pathway. In response to cisplatin treatment, a rapid and transient actin reorganisation is observed at the cell membrane by fluorescence microscopy after Phalloidin-FITC staining. This event is dependent on the membrane fluidification studied by electron paramagnetic resonance and necessary for apoptosis induction. Moreover, early after the onset of cisplatin treatment, ezrin co-localised with Fas at the cell membrane was visualised by membrane microscopy and was redistributed with Fas, FADD and procaspase-8 into membrane lipid rafts as shown on Western blots. In fact, cisplatin exposure results in an early small GTPase RhoA activation demonstrated by RhoA-GTP pull down, Rho kinase (ROCK)-dependent ezrin phosphorylation and actin microfilaments remodelling. Pretreatment with latrunculin A, an inhibitor of actin polymerisation, or specific extinction of ezrin or ROCK by RNA interference prevents both cisplatin-induced actin reorganisation and apoptosis. Interestingly, specific extinction of Fas receptor by RNA interference abrogates cisplatin-induced ROCK-dependent ezrin phosphorylation, actin reorganisation and apoptosis suggesting that Fas is a key regulator of cisplatin-induced actin remodelling and is indispensable for apoptosis. Thus, these findings show for the first time that phosphorylation of ezrin by ROCK via Fas receptor is involved in the early steps of cisplatin-induced apoptosis.

Cytotoxicity of TRAIL/Anticancer Drug Combinations in Human Normal Cells

Abstract:  TRAIL (TNF‐α‐Related Apoptosis‐Inducing Ligand) is a promising anticancer agent. In fact, it induces apoptosis in cancer cells and not in most normal cells. Nevertheless, certain cancer cells are resistant to TRAIL‐induced apoptosis and this could limit TRAILs efficiency in cancer therapy. To overcome TRAIL resistance, a combination of TRAIL with chemotherapy could be used in cancer treatment. However, sensitivity of human normal cells to such combinations is not well known. We showed in this study that TRAIL/cisplatin, in contrast to TRAIL/5‐fluorouracil, was toxic toward human primary hepatocytes and resting lymphocytes. Furthermore, both combinations are toxic toward PHA‐IL2‐activated lymphocytes. In contrast, freshly isolated neutrophils are resistant to TRAIL in combination or not with anticancer drugs.

Ceramide in Chemotherapy of Tumors

It is well known that tumor formation arises from the imbalance between cell death and proliferation. For many years, cancer research has engaged an important part of its efforts to find new therapeutic strategies based on cell death induction. One of the predominant ways to kill tumor cells is to trigger apoptosis by chemotherapy. However tumor responsiveness to chemotherapy is dependent on different biological factors including cancer types, genetics and pharmacogenetics. Although molecular mechanisms involved in chemotherapy-induced apoptosis are diverse and depend on cell-type and drugs used, a common pathway leading to tumor cell death has been shown to implicate the generation of a simple cellular sphingolipid, ceramide. Ceramide is released by the activity of neutral or acidic sphingomyelinases or de novo synthesis during treatment with chemotherapy. This review in particular focuses on enzymes involved in chemotherapy-induced cell death such as neutral or acidic sphingomyelinases and ceramide synthases, the role of ceramide in cellular effects of chemotherapy at the plasma membrane or the mitochondria and the induction of cell death by ceramide. It also includes recent advances on novel patented sphingolipid compounds and cancer therapeutic strategies based on ceramide release.

Prostate cancer and physical activity: adaptive response to oxidative stress.

Prostate cancer is the most common form of cancer affecting men in the Western world. Its relative incidence increases exponentially with age and a steady increase is observed with extended life span. A sedentary lifestyle represents an important risk factor and a decrease in prostate cancer prevalence is associated with exercise. However, the molecular mechanisms involved in this process remain unknown. We hypothesize that reactive oxygen species generated by physical exercise are a key regulatory factor in prostate cancer prevention. Aging is correlated with increased oxidative stress (OS), which in turn provides a favorable environment for tumorigenesis. Running training is known to enhance the antioxidant defense system, reducing oxidative stress. In this context, the decrease in OS induced by exercise may delay the development of prostate cancer. This review focuses on oxidative stress-based mechanisms leading to prostate cancer sensitization to exercise, which could have some impact on the development of novel cancer therapeutic strategies.

Acid sphingomyelinase deficiency protects from cisplatin-induced gastrointestinal damage

Cisplatin is one of the most effectively used chemotherapeutic agents for cancer treatment. However, in humans, important cytotoxic side effects are observed including dose-limiting renal damage and profound gastrointestinal symptomatology. The toxic responses to cisplatin in mice are similar to those in human patients. Here, we evaluated whether the acid sphingomyelinase (Asm) mediates at least some of the toxic in vivo effects of cisplatin. To this end, we determined the toxic effects of a single intraperitoneal dose of cisplatin (27 mg/kg) in wild type (Asm+/+) and Asm-deficient mice (Asm−/−). Tissue injury and apoptosis were determined histologically on hematoxylin–eosin and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling) stainings 3, 12, 36 and 72 h after treatment. Our results revealed severe toxicity of cisplatin in Asm+/+ mice with increased numbers of apoptotic cells in the thymus and small intestine. In marked contrast, Asm−/− mice were resistant to cisplatin and no apoptosis was observed in these organs after treatment. Moreover, cisplatin treatment primarily triggered apoptosis of endothelial cells in microvessels of intestine and thymus, an effect that was absent in mice lacking Asm. The data thus suggest that at least some toxic effects of cisplatin are mediated by the Asm in vivo resulting in early death of endothelial cells and consecutive organ damage.

NPC1 repression contributes to lipid accumulation in human macrophages exposed to environmental aryl hydrocarbons

AIMS Aryl hydrocarbons (AHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene (BP), are environmental contaminants promoting the development of atherosclerosis-related cardiovascular diseases. In order to identify molecular mechanisms involved in these effects, we have analysed AH-mediated regulation of the lipid trafficking Niemann-Pick type C1 protein (NPC1) and its contribution to AH-induced macrophage lipid accumulation. METHODS AND RESULTS Exposure of primary human macrophages to TCDD and BP decreased NPC1 mRNA expression in a time-dependent manner. NPC1 protein expression and NPC1-related acid sphingomyelinase activity were reduced in parallel. NPC1 was also similarly down-regulated in mice exposed to BP. Moreover, TCDD and BP were demonstrated to trigger lipid accumulation in human macrophages, as assessed by Oil Red O and Nile Red staining and cholesterol determination. Such lipid loading occurred at least partly in endosomal/lysosomal compartments as demonstrated by immunolabelling of lipid vesicles by the lysosome-associated membrane protein 1. These cellular phenotypic effects were found to be similar to those triggered by knock-down of NPC1 expression using siRNAs and were counteracted by NPC1 overexpression, thus supporting the contribution of NPC1 to AH-mediated lipid accumulation in macrophages. Finally, both NPC1 down-expression and lipid accumulation in response to TCDD were found to be abolished through knock-down of the AH receptor (AHR), a ligand-activated transcription factor mediating many effects of AHs. CONCLUSION Our data have shown that contaminants such as TCDD and BP repress NPC1 expression in macrophages in an AHR-dependent manner, which likely contributes to macrophage lipid accumulation caused by these environmental chemicals. Thus, NPC1 appears to be a new molecular target regulated by environmental AHs and putatively involved in their deleterious cardiovascular effects.

TRAIL (TNF-Related Apoptosis-Inducing Ligand) Induces Necrosis-Like Cell Death in Tumor Cells at Acidic Extracellular pH

How tumor microenvironment, more specifically low extracellular pH (6.5), alters cell response to TNF‐related apoptosis‐inducing ligand (TRAIL)‐based cancer therapy has yet to be determined. The aim of the current work was to test the effect of acidic extracellular pH on TRAIL‐induced cell death in human HT29 colon carcinoma and HepG2 hepatocarcinoma cell lines as well as in human primary hepatocytes. We found an increase in TRAIL sensitivity at low extracellular pH, which is partially inhibited by Bcl‐2 expression in HT29 cells. At low extracellular pH, TRAIL induced a new form of cell death, sharing necrotic and apoptotic features in tumor cells. By contrast, human primary hepatocytes were resistant to TRAIL‐induced cell death even at acidic extracellular pH.