Marie-Thérèse Dimanche-Boitrel

Cisplatin-Induced CD95 Redistribution into Membrane Lipid Rafts of HT29 Human Colon Cancer Cells

We have shown previously that the death receptor CD95 could contribute to anticancer drug-induced apoptosis of colon cancer cells. In addition, anticancer drugs cooperate with CD95 cognate ligand or agonistic antibodies to trigger cancer cell apoptosis. In the present study, we show that the anticancer drug cisplatin induces clustering of CD95 at the surface of the human colon cancer cell line HT29, an event inhibited by the inhibitor of acid sphingomyelinase (aSMase) imipramine. The cholesterol sequestering agent nystatin also prevents cisplatin-induced CD95 clustering and decreases HT29 cell sensitivity to cisplatin-induced apoptosis and the synergy between cisplatin and anti-CD95 agonistic antibodies. CD95, together with the adaptor molecule Fas-associated death domain and procaspase-8, is redistributed into cholesterol- and sphingolipid-enriched cell fractions after cisplatin treatment, suggesting plasma membrane raft involvement. Interestingly, nystatin prevents the translocation of the aSMase to the extracellular surface of plasma membrane and the production of ceramide, suggesting that these early events require raft integrity. In addition, nystatin prevents cisplatin-induced transient increase in plasma membrane fluidity that could be required for CD95 translocation. Together, these results demonstrate that cisplatin activates aSMase and induces ceramide production, which triggers the redistribution of CD95 into the plasma membrane rafts. Such redistribution contributes to cell death and sensitizes tumor cells to CD95-mediated apoptosis.

Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of ΔΨm, the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.

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.

p27Kip1 induces drug resistance by preventing apoptosis upstream of cytochrome c release and procaspase-3 activation in leukemic cells.

The cyclin-dependent kinase inhibitor p27Kip1 has been implicated as a drug resistance factor in tumor cells grown as spheroids or confluent monolayers. Here, we show that p27Kip1 overexpression also induces resistance to drug-induced apoptosis and cytotoxicity in human leukemic cells growing in suspension. The anti-apoptotic effect of p27Kip1 is not restricted to DNA-damaging agents but extends to the tubulin poison vinblastin, agonistic anti-Fas antibodies and macromolecule synthesis inhibitors. To further identify at which level this protein interferes with the cell death pathway, we investigated its influence on caspase activation and mitochondrial changes. Exposure of mock-transfected U937 cells to 50 μM etoposide activates procaspase-3 and the long isoform of procaspase-2 and induces mitochondrial potential decrease and cytochrome c release from mitochondria to the cytosol. All these events are prevented by p27Kip1 overexpression. p27Kip1 does not modulate Bcl-2, Bcl-XL, Mcl-1 and Bax protein level in leukemic cells but suppresses Mcl-1 expression decrease observed in mock-transfected U937 cells undergoing etoposide-induced cell death. We conclude that p27Kip1 prevents cell death upstream of the final pathway common to many apoptotic stimuli that involves cytochrome c release from mitochondria and activation of downstream caspases.

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.

Milk fermented by Propionibacterium freudenreichii induces apoptosis of HGT-1 human gastric cancer cells.

Background Gastric cancer is one of the most common cancers in the world. The “economically developed countries” life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate. Methodology/Principal Findings A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy. Conclusions/Significance Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.

Targeting the ceramide system in cancer

Sphingolipids, in particular ceramide, have been described as important components of cellular signalling pathways. Ceramide can be produced via multiple mechanisms including through the hydrolysis of sphingomyelin by acid and neutral sphingomyelinase or by a de novo synthesis pathway. Recent studies have identified sphingomyelinases and ceramide synthases as important targets for γ-irradiation and chemotherapeutic drugs. Likewise, common cancer treatment modalities, such as γ-irradiation and many chemotherapeutic agents, induce cell death via the generation of ceramide. This suggests that the manipulation of ceramide production and metabolism could offer promising means for the enhancement of anti-tumor therapies. The focus of this mini-review will be to discuss contemporary evidence suggesting that ceramide forming pathways and ceramide itself are important targets for the treatment of tumors and the development of novel tumor treatment strategies.

Reversal of tumor resistance to apoptotic stimuli by alteration of membrane fluidity: therapeutic implications.

In recent years, significant development and improvement have been observed in the treatment of cancer; however, relapses and recurrences occur frequently and there have not been any current therapies to treat such cancers. Cancers resistant to conventional therapies develop several mechanisms to escape death-inducing stimuli. A poorly understood mechanism is the involvement of the cancer cell plasma membrane composition and architecture and their involvement in regulating drug-inducing stimuli leading to cell death. Although the basic structure of the biological membrane was established 80 years ago, study of the physical properties of lipid bilayers still provides significant information regarding membrane organization and dynamics. Membrane fluidity is probably the most important physicochemical property of cell membranes. Alterations of membrane fluidity can seriously affect functional properties of the cell and induction of apoptotic pathways resulting in cell death. The role of membrane fluidity in the apoptotic process is clearly exemplified as it is seriously disrupted as a result of cell injury. The molecular signaling pathways leading to apoptosis are currently promising areas of research investigation and lead to unravel the underlying molecular mechanisms of tumor cells resistance to apoptotic stimuli and hence the development of new effective therapeutic agents. Recent findings indicate that most anticancer agents induce apoptosis, directly or indirectly, through alterations of tumor cell membrane fluidity. The present chapter summarizes the relationship between alterations of tumor cell membrane fluidity and tumor cell response to apoptotic-inducing stimuli. Several potential therapeutic applications directed at tumor cell membrane fluidity are proposed.

Nongenomic effects of cisplatin: acute inhibition of mechanosensitive transporters and channels without actin remodeling

Cisplatin is an antineoplastic drug, mostly documented to cause cell death through the formation of DNA adducts. In patients, it exhibits a range of short-term side effects that are unlikely to be related to its genomic action. As cisplatin has been shown to modify membrane properties in different cell systems, we investigated its effects on mechanosensitive ion transporters and channels. We show here that cisplatin is a noncompetitive inhibitor of the mechanosensitive Na(+)/H(+) exchanger NHE-1, with a half-inhibition concentration of 30 μg/mL associated with a decrease in V(max) and Hill coefficient. We also showed that it blocks the Cl(-) and K(+) mechanosensitive channels VSORC and TREK-1 at similar concentrations. In contrast, the nonmechanosensitive Cl(-) and K(+) channels CFTR and TASK-1 and the Na(+)-coupled glucose transport, which share functional features with VSORC, TREK-1, and NHE-1, respectively, were insensitive to cisplatin. We next investigated whether cisplatin action was due to a direct effect on membrane or to cortical actin remodeling that would affect mechanosensors. Using scanning electron microscopy, in vivo actin labeling, and atomic force microscopy, we did not observe any modification of the Youngs modulus and actin cytoskeleton for up to 60 and 120 μg/mL cisplatin, whereas these concentrations modified membrane morphology. Our results reveal a novel mechanism for cisplatin, which affects mechanosensitive channels and transporters involved in cell fate programs and/or expressed in mechanosensitive organs in which cisplatin elicits strong secondary effects, such as the inner ear or the peripheral nervous system. These results might constitute a common denominator to previously unrelated effects of this drug.

TRAIL Induces Receptor-Interacting Protein 1-Dependent and Caspase-Dependent Necrosis-Like Cell Death under Acidic Extracellular Conditions

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that induces apoptosis in cancer cells but not in most normal cells. How tumor physiology, particularly acidic extracellular pH (pH(e)), would modify sensitivity of cancer cells to TRAIL-induced cell death is not known. We have previously shown that cancer cells, resistant to TRAIL-induced apoptosis at physiologic pH(e) (7.4), could be sensitized to TRAIL at acidic pH(e) (6.5). However, at this acidic pH(e), cell death was necrotic. We show here that, in spite of a necrosis-like cell death morphology, caspases are activated and are necessary for TRAIL-induced cell death at acidic pH(e) in HT29 human colon cancer cells. Furthermore, we observed that, whereas receptor-interacting protein (RIP) was cleaved following TRAIL treatment at physiologic pH(e) (7.4), it was not cleaved following TRAIL treatment at acidic pH(e) (6.5). Moreover, RIP degradation by geldanamycin or decrease expression of RIP by small RNA interference transfection inhibited TRAIL-induced necrosis at acidic pH(e), showing that RIP was necessary for this necrotic cell death pathway. We also show that RIP kinase activity was essential for this cell death pathway. Altogether, we show that, under acidic pH(e) conditions, TRAIL induces a necrosis-like cell death pathway that depends both on caspases and RIP kinase activity. Thus, our data suggest for the first time that RIP-dependent necrosis might be a major death pathway in TRAIL-based therapy in solid tumors with acidic pH(e).