Alteration of the Sphingomyelin/Ceramide Pathway Is Associated with Resistance of Human Breast Carcinoma MCF7 Cells to Tumor Necrosis Factor-α-mediated Cytotoxicity

Abstract

The interference of tumor necrosis factor-␣ (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-B (NF-B) activation. However, the transfected cells remained resistant to TNFinduced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C 6 -ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A 2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.

Adenovirus-mediated transfer of wild-type p53 gene sensitizes TNF resistant MCF7 derivatives to the cytotoxic effect of this cytokine: relationship with c-myc and Rb

“…Several mechanisms have been reported to contribute to the cellular resistance to TNF-induced cell killing (Cai et al, 1997a;JaÈ attela et al, 1992;Liston et al, 1996;Opipari et al, 1992;Rothe et al, 1995;Wong and Goeddel, 1988;Zyad et al, 1994). Recently, we have provided evidence for the existence of an association between the resistance of human breast adenocarcinoma cell line MCF7 to the cytotoxic action of TNF and the loss of p53 function (Cai et al, 1997b).…”

“…1001 cells were derived from RA-1 cells transfected by p55 TNF-R cDNA as described (Cai et al, 1997a). All cell lines were routinely cultured in RPMI 1640 medium containing 5% fetal calf serum, 1% penicillin-streptomycin, 1% L-glutamine at 378C in a humidi®ed atmosphere with 5% CO 2 .…”

Wild-type p53 induced sensitization of mutant p53 TNF-resistant cells: Role of caspase-8 and mitochondria

“…32 MCF7 FADD dominant -negative ( DN ) cells were kindly provided by V Dixit (University of Michigan Medical School, Michigan ). All cell lines were routinely cultured in RPMI 1640 medium containing 5% fetal calf serum, 1% penicillin -streptomycin, 1% L -glutamine at 378C in a humidified atmosphere with 5% CO 2 .…”

Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity

“…However, the merely partial inhibition of AICC after TRAIL-R2 blocking ( Fig. 3.5) indicates that another mechanism is involved that ultimately alters the sensitivity It was shown previously that a combination of fenretinide, as a ceramide inducer, and inhibitors of the ceramide glycosylation pathway, such as PPMP, resulted in a synergistic cytotoxicity in cancer cells [78]. However, when we inhibited GCS by using the GCS inhibitor PPPP we found a down regulation of the ch14.18-target antigen GD2 resulting in a nearly complete abrogation of the 4-HPR-mediated increase of GD2-dependent cytotoxicity of effector cells and complement (Fig.…”

“…It has been recently reported, that a MDR phenotype is often associated with aberrant ceramide metabolism [77]. In this context, TNF-α-resistant MCF7 breast cancer cells have been characterized by the inability of their neutral or acidic sphingomyelinases to generate ceramide [78]. Furthermore, in order to prevent ceramide accumulation and ceramide induced apoptosis, MDR tumor cells often exhibit an increased activity and/or expression of enzymes involved in ceramide clearance such as sphingosine kinase (converts sphingosine into sphingosine-1-phosphate, S1P)), sphingomyelin synthase (converts ceramide into sphingomyelin) and glucosylceramide synthase (GCS), which catalyzes the glycosylation of ceramide leading to the formation of glucosylceramide [70,71,72].…”