REVIEW

Targeting Ceramide Metabolism—a Strategy for Overcoming Drug Resistance

Alex Senchenkov, David A. Litvak, Myles C. Cabot

Affiliation of authors: Breast Cancer Research Program and Chemotherapeutics, John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA.

Correspondence to: Myles C. Cabot, Ph.D., Breast Cancer Research Program and Chemotherapeutics, John Wayne Cancer Institute at Saint John's Health Center, 2200 Santa Monica Blvd., Santa Monica, CA 90404 (e-mail: cabot{at}jwci.org).


    ABSTRACT
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
Inherent or acquired drug resistance, which frequently characterizes cancer cells, is caused by multiple mechanisms, including dysfunctional metabolism of the lipid second messenger ceramide. Ceramide, the basic structural unit of the sphingolipids, plays a role in activating cell death signals initiated by cytokines, chemotherapeutic agents, and ionizing radiation. Recent discoveries about the metabolism of ceramide suggest that this agent may have an important influence on the effectiveness of various cancer therapeutics. In particular, the cytotoxic effect of chemotherapy is decreased when generation of ceramide is impaired but is increased when the degradation of ceramide is blocked. Herein, we review the mechanisms of resistance to chemotherapeutic agents in terms of ceramide metabolism.



    DRUG RESISTANCE
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
Cancer cells develop multiple mechanisms to evade drug toxicity (14). In the classic model of multidrug resistance, a membrane-resident glycoprotein, termed P-glycoprotein, acts as a drug efflux pump, lowering intracellular drug levels to sublethal concentrations (5,6). Other causes of multidrug resistance include overexpression of multidrug resistance-associated protein (a second drug efflux pump that is similar to P-glycoprotein) (79), changes in topoisomerase II activity (10,11), and modifications in glutathione S-transferase activity (12). Chemoresistance also may be related to the expression of important apoptosis-associated proteins, such as the bcl-2 family of proteins (13) and the tumor suppressor protein p53 (14,15), the synthesis of vaults (16), and the overexpression of caveolae (17). Studies (1822) suggest that the dysfunctional metabolism of ceramide, a lipid second messenger, may contribute to multidrug resistance. This review summarizes the current understanding of multidrug resistance and ceramide metabolism.


    CERAMIDE FORMATION
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
Ceramide, the basic unit of the lipid sphingomyelin, can be produced de novo or by the hydrolysis of sphingomyelin. Ceramide is produced de novo by ceramide synthase via N-acylation of sphinganine and the addition of a double bond (Fig. 1Go). Ceramide can also be produced when neutral or acidic sphingomyelinases are activated to cleave the bond between ceramide and the phosphoric acid of sphingomyelin (Figs. 1 and 2GoGo). The two long-chain hydrocarbon moieties in ceramide are responsible for the lipid character of the molecule. The addition of galactose to ceramide initiates production of sulfatides (not shown), whereas the addition of glucose generates glucosylceramide, the ganglioside precursor (Fig. 1Go).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. Glycosphingolipid metabolism and sites of drug interaction. Ceramide can be generated de novo via ceramide synthase (cer syn) by the addition of fatty acid to sphinganine and through degradation of sphingomyelin by spingomyelinase (spm ase). The addition of galactose to ceramide yields sulfatides (not shown), and the addition of glucose to ceramide yields glucosylceramide, precursor of gangliosides. Agents that increase ceramide levels and their proposed sites of interaction are shown in boxes. The drugs shown on the left are believed to increase cellular ceramide levels by the de novo synthesis route, although not solely through ceramide synthase, because serine palmitoyltransferase is also involved. Agents listed at the top right have been shown to enhance ceramide formation by activation of sphingomyelinase. Drugs listed at the bottom promote ceramide elevation by hindering glycosylation via the glucosylceramide synthase (gcs) route (ketoconazole, our unpublished data). 4-HPR = N-(4-hydroxyphenyl)retinamide; Ara-C = 1-{beta}-D-arabinofuranosylcytosine (cytarabine); TNF-{alpha}= tumor necrosis factor-{alpha}; PPMP = 1-phenyl-2-hexadecanoylamine-3-pyrrolidino-1-propanol; NB-DNJ = N-butyldeoxynojirimycin; DT388-GM-CSF = a fusion toxin consisting of a truncated dipththeria toxin linked to human granulocyte–macrophage colony-stimulating factor.

 


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2. Chemical structure of sphingomyelin. Sphingomyelin consists of a long-chain hydrophobic ceramide moiety and a phosphocholine polar head group. The aliphatic chains are generally 16–24 carbons long, saturated or unsaturated, and may contain a hydroxy radical.

 

    CERAMIDE CELL SIGNALING AND APOPTOSIS
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
Most research on ceramide-mediated signaling focuses on pathways initiated by the hydrolysis of sphingomyelin (2325). Additionally, the production of ceramide de novo may initiate signaling pathways (26). The production of ceramide is the result of diverse stimuli that include growth factor deprivation (2729), cytokines (3033), ionizing radiation (34,35), heat shock (36), chemotherapy and other cytotoxic agents (26,3739), and various environmental factors, such as stress (40,41) and even diet (42). These stimuli initiate ceramide-mediated signaling pathways. In addition, exposure of cells to receptor-specific ligands, such as 1,25-dihydroxyvitamin D3 (43), tumor necrosis factor-{alpha} (TNF-{alpha}) (39), CD28, or CD95/APO-1/Fas ligand (44), may initiate ceramide-mediated signaling pathways.

Ceramide-mediated cell signaling has been shown to contribute to cell cycle arrest, terminal cell differentiation, and apoptosis (4348), as well as to cell proliferation (49). Ceramide and other sphingolipids act as second messengers modifying a number of target proteins that induce a cascade of enzymatic and transcriptional activity. One ceramide target protein that has been characterized extensively is ceramide-activated protein kinase (50,51). Other downstream target proteins include ceramide-activated protein phosphatases (52,53), the small guanosine triphosphate-binding protein raf-1, and the atypical protein kinase C{zeta}(54). These proteins appear to contribute to proliferation and, possibly, to inflammation by recruiting the mitogen-activated protein kinase pathway (55), which has been described in detail (56). These proteins also may recruit the stress-activated protein kinase/c-JUN N-terminal kinase (SAPK/JNK) pathway (56). The SAPK/JNK pathway stimulates activity of AP-1 nuclear factors (e.g., c-Jun) that promote transcriptional activation of various genes and that appear necessary to apoptosis (57). Studies have shown that disrupting the SAPK/JNK/AP-1 pathway with antisense oligonucleotides to either JNK-1 or c-jun (57) or with dominant negative c-jun mutants blocks ceramide-mediated apoptosis (58).

Ceramide-induced cell death proceeds by at least two pathways. One pathway is transcriptionally dependent, and the other is transcriptionally independent. The transcriptionally dependent pathway is mediated by the interaction of members of the TNF superfamily of receptors (i.e., TNF-{alpha} and CD95/APO-1/Fas receptors) (59) and their specific ligands. In the CD95 receptor pathway, ceramide is generated by acid sphingomyelinase in a complex series of steps (60). This involves several adapter molecules, such as the TNF receptor 1-associated death domain and the Fas-associated death domain (FADD or MORT1). Death domains of each of these adapter molecules are capable of binding with and potentially activating downstream protease caspases that affect apoptosis [reviewed in (44)]. This type of transcription-dependent ceramide signaling may be important in determining the cytotoxic response to certain chemotherapeutic agents. Activation of CD95/APO-1/Fas signaling by ceramide has been shown to mediate doxorubicin-induced apoptosis (61).

The transcription-independent pathway is characterized by the direct activation of acid sphingomyelinase by environmental stresses, such as ionizing radiation and oxidative damage (62). The subsequent production of ceramide, in turn, activates the SAPK/JNK apoptotic pathway. In addition, the transcriptionally independent formation of ceramide also may affect apoptosis-related proteins of the Bcl-2 family. Ceramide may alter the relationship between proapoptotic (i.e., Bax and Bad) and antiapoptotic (i.e., Bcl-2 and Bcl-XL) members of the Bcl-2 family of proteins that are associated with the mitochondrial membrane. Inhibitory proteins, such as Bcl-2, when overexpressed in cells, have the capacity to block ceramide-mediated cell death without altering ceramide generation (6365). By inducing the heterodimerization of proapoptotic proteins with antiapoptotic proteins, ceramide may reverse a cytoprotective signal (from Bcl-2) and initiate cell death. The loss of this cytoprotective signal leads to a loss of mitochondrial membrane integrity and the efflux of additional proapoptotic molecules, such as cytochrome c and apoptosis-inducing factor (39).

Both the transcriptionally dependent and independent pathways activate downstream molecules of the interleukin 1{beta}-converting enzyme family of caspase proteases, which affect apoptosis. A number of caspases may be activated in the various ceramide signaling pathways. Caspase-8 is the particular caspase associated with the TNF receptor-mediated signal and interacts directly with the adapter molecules FADD/MORT1. Caspase-9 and caspase-2 are mitochondrial-associated proteases and may be activated in either pathway. Both caspase-8 and caspase-9 recruit caspase-3. Ultimately, multiple caspases are activated, leading to proteosome-directed DNA fragmentation and cell death (66,67). The ceramide pathway is a complex system of signal reinforcement and magnification that has not been defined completely. There may be cross-talk at various levels of either the transcriptionally dependent or independent pathways, and nuclear transcription factors, phospholipases, reactive oxygen intermediates, and intracellular calcium may play relevant roles (48).


    CERAMIDE AND RESPONSE TO CHEMOTHERAPY
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
A number of clinically important cytotoxic agents (Table 1Go) appear to be effective because of their ability to activate ceramide-mediated pathways in cancer cells. Drugs can impact ceramide metabolism by promoting ceramide synthesis de novo (Fig. 1Go, left), by activating sphingomyelinase (Fig. 1Go, top right), and/or by blocking glucosylceramide formation (Fig. 1Go, bottom). In each case, the result is an enhanced ceramide-governed cytotoxic response. The drugs are of a diverse nature and include the anthracyclines doxorubicin (Adriamycin) and daunorubicin, the vinca alkaloids vincristine and vinblastine, antiestrogens such as tamoxifen, the novel synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR), and the taxane paclitaxel (Taxol).


View this table:
[in this window]
[in a new window]
 
Table 1. Agents that elicit ceramide generation
 
Both of the anthracyclines doxorubicin and daunorubicin effectively elevate ceramide levels in several cell types. Daunorubicin promotes ceramide formation and apoptosis by stimulating ceramide synthase activity (26). Fumonisin B1, an inhibitor of ceramide synthase (68), inhibits daunorubicin-induced apoptosis (26). Daunorubicin also promotes ceramide generation via hydrolysis of sphingomyelin (69). In Jurkat E6.1 lymphoblastic leukemia cells, daunorubicin increases ceramide levels by stimulating ceramide synthase (70). However, ceramide production did not precede either caspase activation or apoptosis in this cell system. It is unclear whether daunorubicin-induced cell death is truly dependent on ceramide-mediated signaling. Doxorubicin also increases cellular ceramide levels (71,72). Exposure to doxorubicin increases ceramide levels in drug-sensitive MCF-7 cells but not in the doxorubicin-resistant MCF-7-AdrR cells. This suggests that drug resistance may be linked to ceramide metabolism.

Some of the cytotoxic properties of vinca alkaloids (vincristine and vinblastine), widely used in the treatment of leukemia patients, may be due to their ability to increase cellular ceramide. Exposure of ALL-697 leukemia cells to vincristine causes apoptosis after a sustained increase in ceramide (65). The related alkaloid vinblastine also works by increasing cellular ceramide levels. Vinblastine concentrations as low as 1.5 nM cause a concomitant increase in ceramide levels and cell death in KB-3–1 human epidermoid carcinoma cells. However, concentrations as high as 1.0 µM have no effect on ceramide levels in the vinblastine-resistant counterpart KB-V-1 cells (73), again suggesting that drug resistance may be related to ceramide metabolism.

Paclitaxel inhibits microtubule depolymerization and is effective against a number of different solid tumors, including ovarian and breast cancers. Paclitaxel induces programmed cell death in a number of different cell types, including leukemia and breast cancer cells (7476). Studies have shown that the coadministration of paclitaxel with exogenous ceramide substantially inhibits cell proliferation and elicits apoptosis in a synergistic fashion in Jurkat T cells (75) and Tu138 head and neck squamous cell cancer (77). Moreover, studies have shown that the effects of paclitaxel are linked to the de novo synthesis of ceramide in MDA-MB-468 and MCF-7 breast cancer cells (Fig. 3Go, A, MCF-7 cells) (76,78) and that paclitaxel-dependent cytotoxicity was abrogated by blocking ceramide production with L-cycloserine, an inhibitor of ceramide synthesis (78).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Dose–response profiles for ceramide formation induced by various agents in human cancer cells. The representative cell lines include breast cancer (MCF-7), prostate cancer (LNCaP), vincristine-resistant leukemia (HL-60/VCR), and ovarian carcinoma (SKOV-3). Cells were exposed to agents at the concentrations indicated for 24 hours with [3H]palmitic acid in the medium as the ceramide metabolic precursor and ceramide were measured. Data are expressed as the percent of control. 4-HPR = N-(4-hydroxyphenyl)retinamide; DT388-GM-CSF = a fusion toxin consisting of a truncated dipththeria toxin linked to human granulocyte–macrophage colony-stimulating factor.

 
Triphenylethylene antiestrogens, such as tamoxifen, block conversion of ceramide to glucosylceramide (79,80) and, thereby, promote increases in cellular ceramide. This activity is independent of estrogen receptor status. Although increases are moderate (35%), the combination of tamoxifen with agents, such as doxorubicin (72) or the cyclosporin A analogue SDZ PSC 833 (81), has a synergistic effect on ceramide formation. For example, exposure of MCF-7-AdrR cells to tamoxifen, SDZ PSC 833, or tamoxifen plus SDZ PSC 833 increased ceramide levels by 35%, 500%, and 1100%, respectively (81).

The synthetic retinoid 4-HPR elicits apoptosis in human prostate carcinoma cells through genesis of reactive oxygen species, nuclear retinoic acid receptors, or apoptosis-related genes (82). In addition, 4-HPR increases the level of intracellular ceramide in highly drug-resistant human neuroblastoma cell lines (83) and in the prostate cancer cell line LNCaP (Fig. 3Go, B, and our unpublished data). Because the expression of p53 protein is not affected by 4-HPR, the retinoid may form the basis for a novel p53-independent chemotherapy regimen targeting the ceramide pathway. In leukemia cells, 4-HPR but not retinoic acid activates ceramide formation and induces apoptosis (84). Simultaneous treatment of leukemia cells with 4-HPR and the ceramide synthase inhibitor fumonisin B1 decreases ceramide elevation and blocks induction of apoptosis.

DT388-GM-CSF, a fusion toxin consisting of a truncated diphtheria toxin linked to human granulocyte–macrophage colony-stimulating factor (GM-CSF) (85), is toxic to acute myeloid leukemia progenitors bearing the GM-CSF receptor but not to normal marrow cell progenitors (86). The fusion toxin modulates drug resistance in vincristine-resistant HL-60 cells (87). Although one mechanism of action is inactivation of the expression of multidrug resistance proteins, we have shown that DT388-GM-CSF also is a potent agonist of ceramide formation in HL-60/VCR cells (Fig. 3Go, C). In these cells, ceramide is generated via hydrolysis of cellular sphingomyelin, suggesting a sphingomyelinase-governed response. In addition, we have shown that ceramide formation is followed by activation of caspase-9 and caspase-3 in this cell system (88).

Suramin, a polysulfonated naphthylurea introduced in the 1920s for the treatment of African trypanosomiasis, is synergistic with TNF-{alpha} and doxorubicin in human prostate cancer cells (89). Synergy was noted at drug concentrations achieved clinically. A subsequent study (90) showed that bcl-2-transfected prostate cancer cells were resistant to apoptosis induced by doxorubicin; use of a doxorubicin/suramin combination circumvented this resistance. Gill and Windebank (91) reported that suramin disrupts glycolipid metabolism and elicits apoptosis after elevation of ceramide in various cancer cell models. In animal studies, suramin slows the growth of DU145 prostate cancer xenografts in nude mice (92), and it is currently in clinical trials for breast and prostate cancers (9395).

Several other cytotoxic agents have been shown to elevate cellular ceramide levels. Tepper et al. (96) reported a threefold elevation of ceramide levels in Jurkat T cells whose DNA has been damaged by exposure to the topoisomerase II inhibitor etoposide. Etoposide increases cellular ceramide levels de novo via activation of serine palmitoyltransferase (97). GW1843, a thymidylate synthase inhibitor in clinical development, causes Molt-4 leukemic T cells to undergo apoptosis with activation of both acidic and neutral sphingomyelinases and ceramide production (98). In this study, the kinetics of ceramide formation were consistent with its role in signaling apoptosis. 1,25-Dihydroxyvitamin D3 stimulates hydrolysis of sphingomyelin in leukemia cells (43) and in keratinocytes (99). It also inhibits the growth of prostate adenocarcinoma cells (100), but it is not known whether ceramide is involved in growth inhibition. Two nucleoside analogues, fludarabine and 1-{beta}-D-arabinofuranosylcytosine, promote ceramide generation in leukemia cells. Exposure of HL-60 cells to 1-{beta}-D-arabinofuranosylcytosine causes a time-dependent and dose-dependent increase in ceramide (37) that follows activation of neutral sphingomyelinase. Treatment of B cells (from patients with chronic B-cell lymphocytic leukemia) with fludarabine results in ceramide elevation and in killing by both apoptotic and nonapoptotic mechanisms (101). The topoisomerase I inhibitor irinotecan (CPT-11) increases ceramide levels in 4B1 mouse fibroblasts (102) and in HT-29 human colon cancer cells (our unpublished data). CPT-11 cytotoxicity is blocked by treatment with the ceramide synthesis inhibitor fumonisin B1 [(102); our unpublished data]. Treatment of HL-60 cells with sodium nitroprusside, a nitric oxide donor, is associated with activation of magnesium-dependent neutral sphingomyelinase, generation of ceramide, and apoptosis (103). To our knowledge, this work is the first to show a relationship between sphingolipid metabolites and nitric oxide-mediated cell death signaling; however, the response may be cell type specific.


    CERAMIDE AND RESPONSE TO RADIOTHERAPY
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
In addition to playing an important role in cell death signaling in response to chemotherapeutic agents, ceramide also may be important to radiation-induced cell death. Exposure of bovine aortic endothelial cells to ionizing radiation induces apoptotic signaling as a result of hydrolysis of sphingomyelin to ceramide by a neutral sphingomyelinase (104). Subsequent studies in HL-60 and U-937 cells showed that DNA fragmentation induced by ionizing radiation is accompanied by a decrease in Bcl-2 messenger RNA (mRNA) levels. Exposure of cells to ceramide had the same impact on Bcl-2, suggesting that modulation of Bcl-2 gene expression may be a target of ceramide-mediated apoptosis after exposure to ionizing radiation.

Cellular resistance to ionizing radiation also may be linked to impaired ceramide production or defective ceramide signaling (105,106). Michael et al. (105) demonstrated a direct relationship between resistance to radiation-induced apoptosis and defective ceramide signaling in Burkitt's lymphoma. Acquired defects in ceramide cell death signaling may contribute to the development of radioresistant thymoma cell lines (107). Ceramide also has been shown to participate in molecular events that govern UV radiation-induced apoptosis (108). Moreover, defective radiation-induced ceramide generation and cellular resistance to radiation treatment in sphingomyelinase knockout mice and in patients with Niemann–Pick disease (characterized by a congenital deficiency in sphingomyelinase) can be corrected by transfection of the human acidic sphingomyelinase gene (34).


    CERAMIDE METABOLISM AND MULTIDRUG RESISTANCE
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
Although there is controversy surrounding the role of ceramide in programmed cell death (65,109,110), the resistance of cancer cells to chemotherapy can be reversed by targeting the metabolism of ceramide. Our research strongly indicates that dysfunctional ceramide metabolism contributes to multidrug resistance and that enhancement of the ceramide response enhances cellular response to chemotherapy. Specifically, ceramide glycosylation by the enzyme glucosylceramide synthase (GCS), which forms the noncytotoxic metabolite glucosylceramide and has been noted in some multidrug-resistant cell lines (18,19), may be an important pathway for bypassing apoptosis.

The cytotoxic potential of many cancer drugs is related to activation of signal transduction pathways that lead to apoptosis (111113). Disruption of this process renders tumor cells drug resistant. TNF-{alpha}-resistant MCF-7 breast cancer cells have been characterized by the inability of their neutral or acidic sphingomyelinases to generate ceramide (20). Using rhabdomyosarcoma cells, Bourteele et al. (114) showed that TNF-{alpha}-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide and inhibition of two ceramide-metabolizing enzymes, GCS and sphingomyelin synthase. In addition, acid ceramidase, which catalyzes ceramide breakdown, is overexpressed in prostate tumor tissue and in prostate tumor cell lines (115). This enzyme may play a role in later stage hormone-insensitive, chemotherapy-refractory prostate cancer. Overexpression of acid ceramidase also protects cells from TNF-{alpha}-induced death, whereas pharmacologic suppression of acid ceramidase by N-oleoylethanolamine restores ceramide accumulation and sensitivity to cytokines (116). These results suggest that the ability of cells to limit ceramide metabolism contributes to chemosensitivity.

Evidence is mounting that the accumulation of a glycosylated form of ceramide, glucosylceramide, may play an important role in the development of drug resistance. Glucosylceramide is an intermediate metabolite in the synthesis and degradation of the more complex gangliosides (Fig. 1Go, right). A number of drug-resistant cancer cell lines accumulate this noncytotoxic metabolite (18,19). Drug-resistant MCF-7-AdrR breast cancer and KB-V-1 cutaneous cancer cells have higher levels of glucosylceramide than their drug-sensitive counterparts, MCF-7 and KB-3–1 (19). The human ovarian adenocarcinoma cell line NIH:OVCAR-3, established from a patient resistant to doxorubicin, melphalan, and cisplatin, also expresses high levels of glucosylceramide (19). Moreover, analysis of tumors from selected cancer patients who failed to respond to chemotherapy treatment demonstrated elevated glucosylceramide levels (19). These findings suggest that elevated glucosylceramide levels in cancer cells or in tumors represent a marker for a drug-resistant phenotype.

The level of activity of GCS, which converts ceramide to glucosylceramide, may determine the multidrug resistance phenotype in cancer cells. To better characterize the influence of GCS on multidrug resistance, we used a retroviral "tetracycline-on" expression system to introduce the GCS gene into drug-sensitive MCF-7 cells. The resulting cell line MCF-7/GCS expresses an 11-fold higher level of GCS activity, is resistant to doxorubicin and exogenous ceramide (21), and is also resistant to TNF-{alpha}-induced cell death (22). Lipid metabolism studies confirmed that TNF-{alpha} causes an increase in ceramide in MCF-7 cells but causes an increase in glucosylceramide in resistant MCF-7/GCS cells. In addition, TNF-{alpha} exposure increases caspase activity in MCF-7 cells but not in MCF-7/GCS. Furthermore, resistance to doxorubicin and TNF-{alpha} in MCF-7/GCS cells is related to hyperglycosylation of ceramide and not to changes in the levels of P-glycoprotein, Bcl-2, or TNF receptor-1 expression (21,22). These results support the theory that GCS activity and the accumulation of cellular glucosylceramide are important to the development of chemotherapy resistance in cancer cells.


    CHEMOSENSITIZATION AND REVERSAL OF DRUG RESISTANCE BY REGULATING CERAMIDE METABOLISM
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
P-glycoprotein is the most widely studied contributor of multidrug resistance. Binding of the calcium channel blocker verapamil and other multidrug resistance modulators to P-glycoprotein inhibits the pump activity of P-glycoprotein and thereby reverses drug resistance (117,118). Among the P-glycoprotein-inactivating compounds are calcium channel blockers, cyclosporin A and its analogue SDZ PSC 833, and tamoxifen (119,120). However, additional mechanisms of action have been postulated for these agents because clinical efficacy does not always correlate with multidrug resistance phenotype (121). Radin and colleagues (122124) described application of glycosphingolipid metabolism in cancer treatment and described how inhibitors of GCS may act as chemotherapeutic agents (125). Several classic P-glycoprotein drugs retard the conversion of ceramide to glucosylceramide (72,79,80,126) (Fig. 1Go). Tamoxifen as used in the Dartmouth regimen for the treatment of melanoma (127) and evaluated for the treatment of pancreatic carcinoma and malignant gliomas (128,129) blocks glucosylceramide formation in vinblastine-resistant cells (79). In doxorubicin-resistant cells, clinically relevant concentrations of tamoxifen, verapamil, and cyclosporin A markedly decrease glucosylceramide levels with 50% inhibitory concentrations of 1.0, 0.8, and 2.3 µM, respectively (80). When MCF-7-AdrR cells were supplemented with ceramide in the presence of tamoxifen, decreased viability and apoptosis occurred (72). However, exposure of MCF-7-AdrR cells to the tamoxifen analogue triphenylethylene, which is missing the dimethylethanolamine moiety, does not inhibit ceramide glycosylation or sensitize cells to doxorubicin. These and additional results (130) suggest a structural and a stereochemical requirement of tamoxifen analogues to be effective (e.g., cis-tamoxifen has no chemotherapy-sensitizing activity).

Several other hormone-modulating agents that are capable of altering drug resistance also retard cellular synthesis of glucosylceramide. Toremifene, an antiestrogen that is chemically and pharmacologically similar to tamoxifen, is also effective (126,131) and is being evaluated for reversing multidrug resistance in renal cell cancer patients (132). The antiprogestine mifepristone (RU486) inhibits proliferation and induces apoptosis in breast cancer cells (133). Mifepristone also inhibits glucosylceramide production (126) while sensitizing MCF-7-AdrR cells to the toxic effects of doxorubicin (72). Of interest, the combination of mifepristone and tamoxifen has been shown to elicit greater cell death than either agent alone when tested on MCF-7 breast cancer cells (134). The antifungal ketoconazole, which is structurally similar to tamoxifen, overcomes resistance to doxorubicin and vinblastine (135) and inhibits glucosylceramide synthesis (our unpublished data).

SDZ PSC 833 is a cyclosporin-based multidrug resistance modulator that appears to act via a P-glycoprotein mechanism (136). However, we have also shown that this agent is an effective inducer of ceramide formation (137). Investigators (138) have reported the antiproliferative effects of SDZ PSC 833 in drug-resistant cells, and our group (73,81) demonstrated that this agent's ability to increase cellular ceramide is associated with a progressive decline in cell survival. SDZ PSC 833 is active in the 1- to 5-µM range; in drug-sensitive MCF-7 breast cancer cells, SDZ PSC 833-induced ceramide generation is associated with decreased cell survival (137), independent of the expression of P-glycoprotein (139). SDZ PSC 833 increases vinblastine sensitivity in both P-glycoprotein-rich and P-glycoprotein-poor cancer cells (73). An example of the influence of SDZ PSC 833 on ceramide metabolism is shown in Fig. 3Go, D, with the human ovarian carcinoma cell line SKOV-3. Cells with enhanced capacity for ceramide glycosylation (18) are more resistant to SDZ PSC 833 than wild-type cells (81). A metabolic study (81) shows that drug-resistant cells initially generate ceramide in response to SDZ PSC 833, but this ceramide is converted to glucosylceramide. Studies with fumonisin B1 (73,81,137) and analyses of sphingomyelin decay indicate that SDZ PSC 833 activates de novo synthesis of ceramide in cancer cells. Examination of intracellular ceramide metabolites showed that doxorubicin-resistant MCF-7-AdrR breast cancer cells converted ceramide to glucosylceramide, whereas drug-sensitive MCF-7 cells contained only free ceramide and no detectable glucosylceramide (126).

1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP), and 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) are structural analogues of the natural sphingolipid substrate of GCS and are potent competitive enzyme inhibitors (140). PPMP effectively inhibits glucosylceramide synthesis (50% inhibitory concentrations = 0.9 µM) in MCF-7-AdrR cells (80). A PPMP concentration that maximally inhibits glucosylceramide synthesis has no effect on cell viability but does sensitize cells to doxorubicin (80). Nicholson et al. (141) showed preferential killing of drug-resistant cell lines by PPMP and its analogue PPPP. Spinedi et al. (142) showed that PDMP suppressed glucosylceramide synthesis and markedly potentiated the apoptotic effect of C6-ceramide in CHP-100 neuroepithelioma cells. The authors concluded that activation of glucosylceramide synthesis is a cellular mechanism for escape from ceramide-induced apoptosis.

The imino sugars N-butyldeoxynojirimycin and N-butyldeoxygalactonojirimycin also inhibit GCS (143,144) and are currently in clinical trials for treatment of Gaucher's disease (a glycosphingolipid lysosomal storage disease). The ability to block ceramide glycosylation makes the imino sugars promising therapeutic agents for the treatment strategy shown in Fig. 1Go.

Studies indicate that the coadministration of ceramide metabolism modulators enhances levels of ceramide. Investigators have shown that cytotoxicity of the novel synthetic retinoid 4-HPR can be enhanced by adding modulators of ceramide metabolism (145). Exposure of CHLA90 cells to 4-HPR produced a fivefold increase in ceramide levels, a fourfold increase in glucosylceramide levels, and a 2.5 order of magnitude increase in cell killing (83). When PPMP was added, inhibition of GCS completely prevented the increase in glucosylceramide, yielded a sevenfold increase in ceramide levels, and produced a synergistic multiorder of magnitude increase in cytotoxicity (145). Adding tamoxifen to treatment with SDZ PSC 833 in MCF-7/AdrR breast cancer cells increasingly inhibits the metabolism of ceramide and further diminishes cell survival. When treatment with tamoxifen is combined with doxorubicin, ceramide levels increase fivefold to 26-fold and cell survival drops to zero (81). It appears that drug combinations eliciting the greatest ceramide increase may be the most cytotoxic.

A number of pathways affecting the metabolic fate of ceramide are activated in response to various chemosensitizing agents. Nevertheless, in some cases, the mechanisms by which ceramide metabolism is affected by these agents is not completely clear. In some cells treated with PPMP, ceramide increases (146) as a result of blocking glycosylation. The mode by which agents, such as tamoxifen, cyclosporine A, mifepristone, and verapamil, elevate ceramide is poorly understood. Although these drugs block the formation of glucosylceramide from ceramide (79,80,126,130,131), direct interaction with GCS has not been demonstrated. These agents, therefore, cannot be considered to be glycosylation inhibitors; instead they might induce apoptosis by direct binding to target proteins of ceramide. However, work with 4-HPR suggests direct targeting of the enzymes of de novo ceramide synthesis (83,145). Pre-exposure of intact neuroblastoma cells to 4-HPR elicits time- and dose-dependent increases in serine palmitoyltransferase and ceramide synthase, as measured in in vitro assays with microsomes (our unpublished data).

The role of GCS in regulating cellular response to chemotherapy has also been demonstrated by introducing GCS antisense complementary DNA (cDNA) into doxorubicin-resistant cancer cells (Fig. 4Go). Transfecting drug-resistant MCF-7/AdrR cells with GCS antisense cDNA decreases cellular glycosylation, effectively reversing drug resistance (147,148). Reverse transcription-coupled polymerase chain reaction, western blot, and in vitro assays showed that MCF-7-AdrR/asGCS, the cell line generated, has decreased GCS mRNA, GCS protein, and GCS enzymatic activity and is 28-fold more sensitive to doxorubicin than the parent MCF-7-AdrR cell line. Exposure to doxorubicin causes both time- and dose-dependent increases in ceramide levels, caspase-3 activity, and cell death in MCF-7-AdrR/asGCS cells transfected with GCS antisense when compared with MCF-7/AdrR parent cells. These findings demonstrate that transfection of GCS antisense restores sensitivity to anthracyclines and resumption of ceramide/caspase apoptotic signaling. A reverse scenario also has been demonstrated by transfecting drug-sensitive MCF-7 breast cancer cells with GCS sense cDNA (Fig. 4Go), conferring chemotherapy resistance (21,22).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Influence of sense and antisense glucosylceramide synthase (GCS) transfection on response to chemotherapy. Transfection of wild-type breast cancer cells (MCF-7) with GCS complementary DNA (cDNA) (solid arrow) confers chemotherapy resistance. Transfection of chemotherapy-resistant breast cancer cells (MCF-7-AdrR) with GCS antisense cDNA (open arrow) completely restores doxorubicin sensitivity and reverses resistance to paclitaxel and vinblastine. The biochemical characteristics accompanying cell transfection are listed.

 

    CONCLUSIONS
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 
Cancer cells develop multiple, and often overlapping, mechanisms that allow them to become resistant to chemotherapeutic agents. The dysfunctional metabolism of ceramide is another one of these inherent or acquired mechanisms that contribute to cellular drug resistance. Numerous studies have helped define the ceramide signaling pathways that contribute to cell death. Studies also indicate that alterations in these cell death signaling pathways may contribute to resistance to standard chemotherapeutic agents in several in vitro cancer models, including breast, prostate, and squamous cell cancers. Investigators have demonstrated the efficacy of targeting ceramide synthesis or degradation pharmacologically to enhance the cytotoxic effects of several clinically relevant drugs. In addition, knocking out the GCS enzyme (which converts ceramide to an inactive metabolite) by transfection of cells with GCS antisense cDNA has been shown to completely reverse breast cancer cell resistance to anthracyclines (148). Targeting ceramide metabolic and cell death signaling pathways is an attractive clinical treatment strategy for overcoming drug resistance and continues to be studied actively. A Rapid Access to Preventive Intervention Development grant recently has been issued by the Developmental Therapeutics Program, National Cancer Institute, National Institutes of Health, Bethesda, MD, to support drug-directed toxicology of 4-HPR and safingol, agents that increase ceramide and enhance chemotherapy cytotoxicity in tumor cell lines in vitro (145).


    NOTES
 
Present address: A. Senchenkov, Department of Surgery, Medical College of Ohio, Toledo.

Supported in part by Public Health Service grant CA77632 (to M. C. Cabot) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by grant 737V20037 from the California Cancer Research Program; by the Strauss Foundation, Sandra Krause, Trustee; by the Streisand Foundation; by the Fashion Footwear Association of New York Shoes on SaleTM; by the Associates for Breast and Prostate Cancer Studies, Los Angeles, CA; by the John Wayne Cancer Institute Auxiliary; and by the Leslie & Susan Gonda (Goldschmied) Foundation.

We thank Ms. Weiling Chen, Dhory Baghallian, and Gwen Berry for their assistance.


    REFERENCES
 Top
 Notes
 Abstract
 Drug Resistance
 Ceramide Formation
 Ceramide Cell Signaling and...
 Ceramide and Response to...
 Ceramide and Response to...
 Ceramide Metabolism and...
 Chemosensitization and Reversal...
 Conclusions
 References
 

1 Volm M, Kastel M, Mattern J, Efferth T. Expression of resistance factors (P-glycoprotein, glutathione S- transferase-pi, and topoisomerase II) and their interrelationship to proto-oncogene products in renal cell carcinomas. Cancer 1993;71:3981–7.[Medline]

2 Gottesman MM. How cancer cells evade chemotherapy: sixteenth Richard and Hinda Rosenthal Foundation Award Lecture. Cancer Res 1993;53:747–54.[Medline]

3 Bernal SD, editor. Drug resistance in oncology. New York (NY): Marcel Dekker; 1997.

4 Kellen JA, editor. Alternative mechanisms of multidrug resistance in cancer. Boston (MA): Birkhaüser; 1995.

5 Ueda K, Cardarelli C, Gottesman MM, Pastan I. Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Natl Acad Sci U S A 1987;84:3004–8.[Abstract]

6 Sikic BI, Fisher GA, Lum BL, Halsey J, Beketic-Oreskovic L, Chen G. Modulation and prevention of multidrug resistance by inhibitors of P-glycoprotein. Cancer Chemother Pharmacol 1997;40(40 Suppl): S13–9.[Medline]

7 Krishnamachary N, Center MS. The MRP gene associated with a non-P-glycoprotein multidrug resistance encodes a 190-kDa membrane bound glycoprotein. Cancer Res 1993;53:3658–61.[Abstract]

8 Zaman GJ, Flens MJ, van Leusden MR, de Haas M, Mulder HS, Lankelma J, et al. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump. Proc Natl Acad Sci U S A 1994;91:8822–6.[Abstract]

9 Grant CE, Valdimarsson G, Hipfner DR, Almquist KC, Cole SP, Deeley RG. Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs. Cancer Res 1994;54:357– 61.[Abstract]

10 Deffie AM, Batra JK, Goldenberg GJ. Direct correlation between DNA topoisomerase II activity and cytotoxicity in adriamycin-sensitive and -resistant P388 leukemia cell lines. Cancer Res 1989;49:58–62.[Abstract]

11 Drake FH, Hofmann GA, Bartus HF, Mattern MR, Crooke ST, Mirabelli CK. Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II. Biochemistry 1989;28:8154–60.[Medline]

12 Morrow CS, Cowan KH. Glutathione S-transferases and drug resistance. Cancer Cells 1990;2:15–22.[Medline]

13 Reed JC. Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance. Curr Opin Oncol 1995;5:541–6.

14 Mueller H, Eppenberger U. The dual role of mutant p53 protein in chemosensitivity in human cancers. Anticancer Res 1996;16:3845–8.[Medline]

15 Zhou G, Kuo MT. Wild-type p53-mediated induction of rat mdr1b expression by the anticancer drug daunorubicin. J Biol Chem 1998;273:15387–94.[Abstract/Free Full Text]

16 Kickhoefer VA, Rajavel KS, Scheffer GL, Dalton WS, Scheper RJ, Rome LH. Vaults are up-regulated in multidrug-resistant cancer cell lines. J Biol Chem 1998;273:8971–4.[Abstract/Free Full Text]

17 Lavie Y, Fiucci G, Liscovitch M. Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells. J Biol Chem 1998;273:32380–3.[Abstract/Free Full Text]

18 Lavie Y, Cao H, Bursten SL, Giuliano AE, Cabot MC. Accumulation of glucosylceramides in multidrug-resistant cancer cells. J Biol Chem 1996;271:19530–6.[Abstract/Free Full Text]

19 Lucci A, Cho WI, Han TY, Giuliano AE, Morton DL, Cabot MC. Glucosylceramide: a marker for multiple-drug resistant cancers. Anticancer Res 1998;18:475–80.[Medline]

20 Cai Z, Bettaieb A, Mahdani NE, Legres LG, Stancou R, Masliah J, et al. Alteration of the sphingomyelin/ceramide pathway is associated with resistance of human breast carcinoma MCF7 cells to tumor necrosis factor-alpha-mediated cytotoxicity. J Biol Chem 1997;272:6918–26.[Abstract/Free Full Text]

21 Liu YY, Han TY, Giuliano AE, Cabot MC. Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells. J Biol Chem 1999;274:1140–6.[Abstract/Free Full Text]

22 Liu YY, Han TY, Giuliano AE, Ichikawa S, Hirabayashi Y, Cabot MC. Glycosylation of ceramide potentiates cellular resistance to tumor necrosis factor-alpha-induced apoptosis. Exp Cell Res 1999;252:464–70.[Medline]

23 Kolesnick RN. 1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells. J Biol Chem 1987;262:16759–62.[Abstract/Free Full Text]

24 Kolesnick RN, Clegg S. 1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase. J Biol Chem 1988;263:6534–7.[Abstract/Free Full Text]

25 Kolesnick RN. Sphingomyelinase action inhibits phorbol ester-induced differentiation of human promyelocytic leukemic (HL-60) cells. J Biol Chem 1989;264:7617–23.[Abstract/Free Full Text]

26 Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, Kolesnick R. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 1995;82:405–14.[Medline]

27 Ito A, Horigome K. Ceramide prevents neuronal programmed cell death induced by nerve growth factor deprivation. J Neurochem 1995;65: 463–6.[Medline]

28 Testi R. Sphingomyelin breakdown and cell fate. Trends Biochem Sci 1996;21:468–71.[Medline]

29 Spiegel S, Merrill AH Jr. Sphingolipid metabolism and cell growth regulation. FASEB J 1996;10:1388–97.[Abstract/Free Full Text]

30 Dbaibo GS, Obeid LM, Hannun YA. Tumor necrosis factor-alpha (TNF-alpha) signal transduction through ceramide. Dissociation of growth inhibitory effects of TNF-alpha from activation of nuclear factor-kappa B. J Biol Chem 1993;268:17762–6.[Abstract/Free Full Text]

31 Kim MY, Linardic C, Obeid L, Hannun Y. Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation. J Biol Chem 1991;266:484–9.[Abstract/Free Full Text]

32 Mathias S, Dressler KA, Kolesnick RN. Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha. Proc Natl Acad Sci U S A 1991;88:10009–13.[Abstract]

33 Ballou LR, Chao CP, Holness MA, Barker SC, Raghow R. Interleukin-1-mediated PGE2 production and sphingomyelin metabolism. Evidence for the regulation of cyclooxygenase gene expression by sphingosine and ceramide. J Biol Chem 1992;267:20044–50.[Abstract/Free Full Text]

34 Santana P, Pena LA, Haimovitz-Friedman A, Martin S, Green D, McLoughlin M, et al. Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 1996;86:189–99.[Medline]

35 Haimovitz-Friedman A. Radiation-induced signal transduction and stress response. Radiat Res 1998;150(5 Suppl):S102–8.[Medline]

36 Chang Y, Abe A, Shayman JA. Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription. Proc Natl Acad Sci U S A 1995;92:12275–9.[Abstract]

37 Strum JC, Small GW, Pauig SB, Daniel LW. 1-beta-D-Arabinofuranosylcytosine stimulates ceramide and diglyceride formation in HL-60 cells. J Biol Chem 1994;269:15493–7.[Abstract/Free Full Text]

38 Jarvis WD, Grant S. The role of ceramide in the cellular response to cytotoxic agents. Curr Opin Oncol 1998;10:552–9.[Medline]

39 Malisan F, Testi R. Lipid signaling in CD95-mediated apoptosis. FEBS Lett 1999;452:100–3.[Medline]

40 Basu S, Kolesnick R. Stress signals for apoptosis: ceramide and c-Jun kinase. Oncogene 1998;17:3277–85.[Medline]

41 Hannun YA. Functions of ceramide in coordinating cellular responses to stress. Science 1996;274:1855–9.[Abstract/Free Full Text]

42 Merrill AH Jr, Schmelz EM, Wang E, Schroeder JJ, Dillehay DL, Riley RT. Role of dietary sphingolipids and inhibitors of sphingolipid metabolism in cancer and other diseases. J Nutr 1995;125(6 Suppl):1677S–82S.[Medline]

43 Okazaki T, Bell RM, Hannun YA. Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation. J Biol Chem 1989;264:19076–80.[Abstract/Free Full Text]

44 Kolesnick RN, Kronke M. Regulation of ceramide production and apoptosis. Annu Rev Physiol 1998;60:643–65.[Medline]

45 Hannun YA. The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem 1994;269:3125–8.[Free Full Text]

46 Okazaki T, Bielawska A, Bell RM, Hannun YA. Role of ceramide as a lipid mediator of 1 alpha,25-dihydroxyvitamin D3- induced HL-60 cell differentiation. J Biol Chem 1990;265:15823–31.[Abstract/Free Full Text]

47 Haimovitz-Friedman A, Kolesnick RN, Fuks Z. Ceramide signaling in apoptosis. Br Med Bull 1997;53:539–53.[Abstract]

48 Okazaki T, Kondo T, Kitano T, Tashima M. Diversity and complexity of ceramide signalling in apoptosis. Cell Signal 1998;10:685–92.[Medline]

49 Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1955;24;270:1326–31.

50 Liu J, Mathias S, Yang Z, Kolesnick RN. Renaturation and tumor necrosis factor-alpha stimulation of a 97-kDa ceramide-activated protein kinase. J Biol Chem 1994;269:3047–52.[Abstract/Free Full Text]

51 Karasavvas N, Erukulla RK, Bittman R, Lockshin R, Zakeri Z. Sterospecific induction of apoptosis in U937 cells by N-octanoyl-sphingosine steroisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis. Eur J Biochem 1996;236:729–37.[Abstract]

52 Wolff RA, Dobrowsky RT, Bielawska A, Obeid LM, Hannun YA. Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction. J Biol Chem 1994;269:19605–9.[Abstract/Free Full Text]

53 Dobrowsky RT, Kamibayashi C, Mumby MC, Hannun YA. Ceramide activates heterotrimeric protein phosphatase 2A. J Biol Chem 1993;268:15523–30.[Abstract/Free Full Text]

54 Yao B, Zhang Y, Delikat S, Mathias S, Basu S, Kolesnick R. Phosphorylation of Raf by ceramide-activated protein kinase. Nature 1995;378:307–10.[Medline]

55 Bird TA, Kyriakis J, Tyshler L, Gayle M, Milne A, Virca GD. Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase. J Biol Chem 1994;269:31836–44.[Abstract/Free Full Text]

56 Coroneos E, Wang Y, Panuska JR, Templeton DJ, Kester M. Sphingolipid metabolites differentially regulate extracellular signal-regulated kinase and stress-activated protein kinase cascades. Biochem J 1996;316(Pt 1):13–7.[Medline]

57 Sawai H, Okazaki T, Yamamoto H, Okano H, Takeda Y, Tashima M, et al. Requirement of AP-1 for ceramide-induced apoptosis in human leukemia HL-60 cells. J Biol Chem 1995;270:27326–31.[Abstract/Free Full Text]

58 Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S. et al. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 1996;380:75–9.[Medline]

59 Kolesnick R, Golde DW. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 1994;77:325–8.[Medline]

60 Cifone MG, De Maria R, Roncaioli P, Rippo MR, Azuma M, Lanier LL, et al. Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase. J Exp Med 1994;180:1547–52.[Abstract]

61 Herr I, Wilhelm D, Bohler T, Angel P, Debatin KM. Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis. EMBO J 1997;16:6200–8.[Abstract/Free Full Text]

62 Weigmann K, Schutze S, Machleidt T, Witte D, Kronke M. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 1994;78:1005–15.[Medline]

63 Fadeel B, Zhivotovsky B, Orrenius S. All along the watchtower: on the regulation of apoptosis regulators. FASEB J 1999;13:1647–57.[Abstract/Free Full Text]

64 Dbaibo GS, Perry DK, Gamard CJ, Platt R, Poirier GG, Obeid LM, et al. Cytokine response modifier A (Crm A) inhibits ceramide formation in response to tumor necrosis factor (TNF)-{alpha}: CrmA and Bcl-2 target distinct components in the apoptotic pathway. J Exp Med 1997;185:481–90.[Abstract/Free Full Text]

65 Zhang J, Alter N, Reed JC, Borner C, Obeid LM, Hannun YA. Bcl-2 interrupts the ceramide-mediated pathway of cell death. Proc Natl Acad Sci U S A 1996;93:5325–8.[Abstract/Free Full Text]

66 Schwander R, Wiegmann K, Bernardo K, Kreder D, Kronke M. TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase. J Biol Chem 1998;273:5916–22.[Abstract/Free Full Text]

67 Mizushima N, Koike R, Kohsaka H, Kushi Y, Hand S, Yagita H, et al. Ceramide induces apoptosis via CPP32 activation. FEBS Lett 1996;395:267–71.[Medline]

68 Wang E, Norred WP, Bacon CW, Riley RT, Merrill AH Jr. Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J Biol Chem 1991;266: 14486–90.[Abstract/Free Full Text]

69 Jaffrezou JP, Levade T, Bettaieb A, Andrieu N, Bezombes C, Maestre N, et al. Daunorubicin-induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis. EMBO J 1996;15:2417–24.[Abstract]

70 Turnbull KJ, Brown BL, Dobson PR. Caspase-3-like activity is necessary but not sufficient for daunorubicin-induced apoptosis in Jurkat human lymphoblastic leukemia cells. Leukemia 1999;13:1056–61.[Medline]

71 Cabot MC, Giuliano AE. Apoptosis—a cell mechanism important for cytotoxic response to adriamycin and a lipid metabolic pathway that facilitates escape. Breast Cancer Res Treat 1997;46:71.

72 Lucci A, Han TY, Liu YY, Giuliano AE, Cabot MC. Modification of ceramide metabolism increases cancer cell sensitivity to cytotoxics. Int J Oncol 1999;15:541–6.[Medline]

73 Cabot MC, Giuliano AE, Han TY, Liu YY. SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells. Cancer Res 1999;59:880–5.[Abstract/Free Full Text]

74 Bhalla K, Ibrado AM, Tourkina E, Tang C, Mahoney ME, Huang Y. Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells. Leukemia 1993;7:563–8.[Medline]

75 Myrick D, Blackinton D, Klostergaard J, Kouttab N, Maizel A, Wanebo H, et al. Paclitaxel-induced apoptosis in Jurkat, a leukemic T cell line, is enhanced by ceramide. Leuk Res 1999;23:569–78.[Medline]

76 McCloskey DE, Kaufmann SH, Prestigiacomo LJ, Davidson NE. Paclitaxel induces programmed cell death in MDA-MB-468 human breast cancer cells. Clin Cancer Res 1996;2:847–54.[Abstract]

77 Mehta S, Blackington D, Omar I, Kouttab N, Myrick D, Klostergaard J, et al. Combined cytotoxic action of paclitaxel and ceramide against the human Tu138 head and neck squamous carcinoma cell line. Cancer Chemother Pharmacol 2000;46:85–92.[Medline]

78 Charles AG, Han TY, Liu YY, Hansen N, Giuliano AE, Cabot MC. Taxol-induced ceramide generation and apoptosis in human breast cancer cells. Cancer Chemother. Pharmacol. In press 2001.

79 Cabot MC, Giuliano AE, Volner A, Han TY. Tamoxifen retards glycosphingolipid metabolism in human cancer cells. FEBS Lett 1996;394:129–31.[Medline]

80 Lavie Y, Cao HT, Volner A, Lucci A, Han TY, Geffen V, et al. Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin A, block glycosphingolipid metabolism by inhibiting ceramide glycosylation in human cancer cells. J Biol Chem 1997;272:1682–7.[Abstract/Free Full Text]

81 Lucci A, Han TY, Liu YY, Giuliano AE, Cabot MC. Multidrug resistance modulators and doxorubicin synergize to elevate ceramide levels and elicit apoptosis in drug-resistant cancer cells. Cancer 1999;86:300–11.[Medline]

82 Sun SY, Yue P, Lotan R. Induction of apoptosis by N-(4-hydroxyphenyl)retinamide and its association with reactive oxygen species, nuclear retinoic acid receptors, and apoptosis-related genes in human prostate carcinoma cells. Mol Pharmacol 1999;55:403–10.[Abstract/Free Full Text]

83 Maurer BJ, Metelitsa LS, Seeger RC, Cabot MC, Reynolds CP. Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)-retinamide in neuroblastoma cell lines. J Natl Cancer Inst 1999;91:1138–46.[Abstract/Free Full Text]

84 DiPietrantonio AM, Hsieh TC, Olson SC, Wu JM. Regulation of G1/S transition and induction of apoptosis in HL-60 leukemia cells by fenretinide (4HPR). Int J Cancer 1998;78:53–61.[Medline]

85 Hall PD, Kreitman RJ, Willingham MC, Frankel AE. Toxicology and pharmacokinetics of DT388-GM-CSF, a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human granulocyte–macrophage colony-stimulating factor (GM-CSF) in C57BL/6 mice. Toxicol Appl Pharmacol 1998;150:91–7.[Medline]

86 Frankel AE, Lilly M, Kreitman R, Hogge D, Beran M, Freedman MH, et al. Diphtheria toxin fused to granulocyte–macrophage colony-stimulating factor is toxic to blasts from patients with juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia. Blood 1998;92:4279–86.[Abstract/Free Full Text]

87 Frankel AE, Hall PD, McLain C, Safa AR, Tagge EP, Kreitman RJ. Cell-specific modulation of drug resistance in acute myeloid leukemic blasts by diphtheria fusion toxin, DT388-GMCSF. Bioconjug Chem 1998;9:490–6.[Medline]

88 Senchenkov A, Han TY, Frankel AE, Kottke TJ, Kaufmann SH, Cabot MC. Enhanced ceramide generation and induction of apoptosis in human leukemia cells exposed to DT DT388-GM-CSF, a truncated diphtheria toxin fused to human granulocyte-macrophage colony-stimulating factor. Blood. In press 2001.

89 Fruehauf JP, Myers CE, Sinha BK. Synergistic activity of suramin with tumor necrosis factor alpha and doxorubicin on human prostate cancer cell lines. J Natl Cancer Inst 1990;82:1206–9.[Abstract]

90 Tu SM, McConnell K, Marin MC, Campbell ML, Fernandez A, von Eschenbach AC, et al. Combination adriamycin and suramin induces apoptosis in bcl-2 expressing prostate carcinoma cells. Cancer Lett 1995;93:147–55.[Medline]

91 Gill JS, Windebank AJ. Role of ceramide in suramin-induced cancer cell death. Cancer Lett 1997;119:169–76.[Medline]

92 Church D, Zhang Y, Rago R, Wilding G. Efficacy of suramin against human prostate carcinoma DU145 xenografts in nude mice. Cancer Chemother Pharmacol 1999;43:198–204.[Medline]

93 Lawrence JB, Conover CA, Haddad TC, Ingle JN, Reid JM, Ames MM, et al. Evaluation of continuous infusion suramin in metastatic breast cancer: impact on plasma levels of insulin-like growth factors (IGFs) and IGF-binding proteins. Clin Cancer Res 1997;3:1713–20.[Abstract]

94 Beedassy A, Cardi G. Chemotherapy in advanced prostate cancer. Semin Oncol 1999;26:428–38.[Medline]

95 Smith DC. Chemotherapy for hormone refractory prostate cancer. Urol Clin North Am 1999;26:323–31.[Medline]

96 Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. Ordering of ceramide formation, caspase activation, and mitochondrial changes during CD95- and DNA damage-induced apoptosis. J Clin Invest 1999;103:971–8.[Abstract/Free Full Text]

97 Perry DK, Carton J, Shah AK, Meredith F, Uhlinger DJ, Hannun YA. Serine palmitoyltransferase regulated de novo ceramide generation during etoposide-induced apoptosis. J Biol Chem 2000;275:9078–84.[Abstract/Free Full Text]

98 Laethem RM, Hannun YA, Jayadev S, Sexton CJ, Strum JC, Sundseth R, et al. Increases in neutral, Mg2+-dependent and acidic, Mg2+-independent sphingomyelinase activities precede commitment to apoptosis and are not a consequence of caspase 3-like activity in Molt-4 cells in response to thymidylate synthase inhibition by GW1843. Blood 1998;91:4350–60.[Abstract/Free Full Text]

99 Geilen CC, Bektas M, Wieder T, Orfanos CE. The vitamin D3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes. FEBS Lett 1996;378:88–92.[Medline]

100 Getzenberg RH, Light BW, Lapco PE, Konety BR, Nangia AK, Acierno JS, et al. Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat prostate model system. Urology 1997;50:999–1006.[Medline]

101 Mengubas K, Riordan FA, Bravery CA, Lewin J, Owens DL, Mehta AB, et al. Ceramide-induced killing of normal and malignant human lymphocytes is by a non-apoptotic mechanism. Oncogene 1999;18:2499–506.[Medline]

102 Suzuki A, Iwasaki M, Kato M, Wagai N. Sequential operation of ceramide synthesis and ICE cascade in CPT-11-initiated apoptotic death signaling. Exp Cell Res 1997;233:41–7.[Medline]

103 Takeda Y, Tashima M, Takahashi A, Uchiyama T, Okazaki T. Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3. J Biol Chem 1999;274:10654–60.[Abstract/Free Full Text]

104 Haimovitz-Friedman A, Kan CC, Ehleiter D, Persaud RS, McLoughlin M, Fuks Z, et al. Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J Exp Med 1994;180:525–35.[Abstract]

105 Michael JM, Lavin MF, Watters DJ. Resistance to radiation-induced apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling. Cancer Res 1997;57:3600–5.[Abstract]

106 Chmura SJ, Nodzenski E, Beckett MA, Kufe DW, Quintans J, Weichselbaum RR. Loss of ceramide production confers resistance to radiation-induced apoptosis. Cancer Res 1997;57:1270–5.[Abstract]

107 Hama-Inaba H, Wang B, Mori M, Matsushima T, Saitoh T, Takusagawa M, et al. Radio-sensitive murine thymoma cell line 3SB: characterization of its apoptosis-resistant variants induced by repeated X-irradiation. Mutat Res 1998;403:85–94.[Medline]

108 Martin SJ, Newmeyer DD, Mathias S, Farschon DM, Wang HG, Reed JC, et al. Cell-free reconstitution of Fas-, UV radiation- and ceramide-induced apoptosis. EMBO J 1995;14:5191–200.[Abstract]

109 Kolesnick R, Hannun YA. Ceramide and apoptosis [letter]. Trends Biochem Sci 1999;24:224–5; discussion 227.[Medline]

110 Hofmann K, Dixit VM. Ceramide in apoptosis—does it really matter? Trends Biochem Sci 1998;23:374–7.[Medline]

111 Fisher DE. Apoptosis in cancer therapy: crossing the threshold. Cell 1994;78:539–42.[Medline]

112 Green DR, Bissonnette RP, Cotter TG. Apoptosis and cancer. Important Adv Oncol 1994;37–52.

113 Haslam RH, Lamborn KR, Becker LE, Israel MA. Tumor cell apoptosis present at diagnosis may predict treatment outcome for patients with medulloblastoma. J Pediatr Hematol Oncol 1998;20:520–7.[Medline]

114 Bourteele S, Hausser A, Doppler H, Horn-Muller J, Ropke C, Schwarzmann G, et al. Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in apoptotic Kym-1 cells. J Biol Chem 1998;273:31245–51.[Abstract/Free Full Text]

115 Seelan RS, Qian C, Yokomizo A, Bostwick DG, Smith DI, Liu W. Human acid ceramidase is overexpressed but not mutated in prostate cancer. Genes Chromosomes Cancer 2000;29:137–46.[Medline]

116 Strelow A, Bernardo K, Adam-Klages S, Linke T, Sandhoff K, Kronke M, et al. Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death. J Exp Med 2000;192:601–12.[Abstract/Free Full Text]

117 Tsuruo T, Iida H, Yamashiro M, Tsukagoshi S, Sakurai Y. Enhancement of vincristine- and adriamycin-induced cytotoxicity by verapamil in P388 leukemia and its sublines resistant to vincristine and adriamycin. Biochem Pharmacol 1982;31:3138–40.[Medline]

118 Germann UA, Shlyakhter D, Mason VS, Zelle RE, Duffy JP, Galullo V, et al. Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance in vitro. Anticancer Drugs 1997;8:125–40.[Medline]

119 Sandor V, Fojo T, Bates SE. Future perspectives for the development of P-glycoprotein modulators. Drug Resistance Updates 1998;1:190–200.

120 Slater LM, Sweet P, Stupecky M, Gupta S. Cyclosporin A reverses vincristine and daunorubicin resistance in acute lymphatic leukemia in vitro. J Clin Invest 1986;77:1405–8.[Medline]

121 List AF, Spier C, Greer J, Wolff S, Hutter J, Dorr R, et al. Phase I/II trial of cyclosporine as a chemotherapy-resistance modifier in acute leukemia. J Clin Oncol 1993;11:1652–60.[Abstract]

122 Radin NS. Rationales for cancer chemotherapy with PDMP, a specific inhibitor of glucosylceramide synthase. Mol Chem Neuropathol 1994;21:111–27.[Medline]

123 Inokuchi J, Mason I, Radin NS. Antitumor activity via inhibition of glycosphingolipid biosynthesis. Cancer Lett 1987;38:23–30.[Medline]

124 Radin NS, Inokuchi J. Glucosphingolipids as sites of action in the chemotherapy of cancer. Biochem Pharmacol 1988;37:2879–86.[Medline]

125 Radin NS. Chemotherapy by slowing glucosphingolipid synthesis. Biochem Pharmacol 1999;57:589–95.[Medline]

126 Lucci A, Giuliano AE, Han TY, Dinur T, Liu YY, Senchenkov A, et al. Ceramide toxicity and metabolism differ in wild-type and multidrug-resistant cancer cells. Int J Oncol 1999;15:535–40.[Medline]

127 Del Prete SA, Maurer LH, O'Donnell J, Forcier RJ, LeMarbre P. Combination chemotherapy with cisplatin, carmustine, dacarbazine, and tamoxifen in metastatic melanoma. Cancer Treat Rep 1984;68:1403–5.[Medline]

128 Gelmann EP. Tamoxifen for the treatment of malignancies other than breast and endometrial carcinoma. Semin Oncol 1997;24(1 Suppl 1): S1–65–S1–70.[Medline]

129 Couldwell WT, Weiss MH, DeGiorgio CM, Weiner LP, Hinton DR, Ehresmann GR, et al. Clinical and radiographic response in a minority of patients with recurrent malignant gliomas treated with high-dose tamoxifen. Neurosurgery 1993;32:485–9; discussion 489–90.[Medline]

130 Jones RC, Cho WI, Han TY, Jarman M, Hardcastle IR, Cabot MC. Altering glycolipid metabolism in MDR cells, new ideas in cancer treatment [abstract]. Proc Am Assoc Cancer Res 1997;38:593.

131 Cabot MC, Giuliano AE, Han TY, Liu YY, Senchenkov A. The impact of toremifene, a novel antiestrogen and adriamycin on ceramide metabolism and modulation of multidrug resistance (MDR) [abstract]. Breast Cancer Res Treat 1998;50:291.

132 Braybrooke JP, Vallis KA, Houlbrook S, Rockett H, Ellmen J, Anttila M, et al. Evaluation of toremifene for reversal of multidrug resistance in renal cell cancer patients treated with vinblastine. Cancer Chemother Pharmacol 2000;46:27–34.[Medline]

133 Seysouthone V, Patel P, Mufti S, Thomas T. Growth inhibitory and apoptotic effects of RU486 on MCF-7 and T-47D breast cancer cells. Endocr Soc 1994;7:391.

134 El Etreby MF, Liang Y, Wrenn RW, Schoenlein PV. Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells. Breast Cancer Res Treat 1998;51:149–68.[Medline]

135 Siegsmund MJ, Cardarelli C, Aksentijevich I, Sugimoto Y, Pastan I, Gottesman MM. Ketoconazole effectively reverses multidrug resistance in highly resistant KB cells. J Urol 1994;151:485–91.[Medline]

136 Archinal-Mattheis A, Rzepka RW, Watanabe T, Kokubu N, Itoh Y, Combates NJ, et al. Analysis of the interactions of SDZ PSC 833 ([3'-keto-Bmt1]-Val2]- Cyclosporine), a multidrug resistance modulator, with P-glycoprotein. Oncol Res 1995;7:603–10.[Medline]

137 Cabot MC, Han TY, Giuliano AE. The multidrug resistance modulator SDZ PSC 833 is a potent activator of cellular ceramide formation. FEBS Lett 1998;431:185–8.[Medline]

138 Lehne G, Rugstad HE. Cytotoxic effect of the cyclosporin PSC 833 in multidrug-resistant leukaemia cells with increased expression of P-glycoprotein. Br J Cancer 1998;78:593–600.[Medline]

139 Goulding CW, Giuliano AE, Cabot MC. SDZ PSC 833 the drug resistance modulator activates cellular ceramide formation by a pathway independent of P-glycoprotein. Cancer Lett 2000;149:143–51.[Medline]

140 Abe A, Inokuchi J, Jimbo M, Shimeno H, Nagamatsu A, Shayman JA, et al. Improved inhibitors of glucosylceramide synthase. J Biochem (Tokyo) 1992;111:191–6.[Abstract]

141 Nicholson KM, Quinn DM, Kellett GL, Warr JR. Preferential killing of multidrug-resistant KB cells by inhibitors of glucosylceramide synthase. Br J Cancer 1999;81:423–30.[Medline]

142 Spinedi A, Bartolomeo SD, Piacentini M. Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis. Cell Death Differ 1998;5:785–91.[Medline]

143 Platt FM, Neises GR, Dwek RA, Butters TD. N-Butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis. J Biol Chem 1994;269:8362–5.[Abstract/Free Full Text]

144 Andersson U, Butters TD, Dwek RA, Platt FM. N-Butyldeoxygalactonojirimycin: a more selective inhibitor of glycosphingolipid biosynthesis than N-butyldeoxynojirimycin, in vitro and in vivo. Biochem Pharmacol 2000;59:821–9.[Medline]

145 Maurer BJ, Melton L, Billups C, Cabot MC, Reynolds CP. Synergistic cytotoxicity in solid tumor cell lines between N-(4-hydroxyphenyl)retinamide and modulators of ceramide metabolism. J Natl Cancer Inst 2000;92:1897–909.[Abstract/Free Full Text]

146 Sietsma H, Veldman RJ, Kolk D, Ausema B, Nijhof W, Kamps W, et al. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitizes neuroblastoma cells for taxol and vincristine. Clin Cancer Res 2000;6:942–8.[Abstract/Free Full Text]

147 Liu YY, Han TY, Giuliano AE, Hansen N, Cabot MC. Uncoupling ceramide glycosylation by transfection of glucosylceramide synthase antisense reverses adriamycin resistance. J Biol Chem 2000;275; 7138–43.[Abstract/Free Full Text]

148 Liu YY, Han TY, Giuliano AE, Cabot MC. Ceramide glycosylation potentiates cellular multidrug resistance. FASEB J. In press 2001.

149 Spinedi A, Di Bartolomeo S, Di Sano F, Rodolfo C, Ambrosino A, Piacentini M. Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid. Cell Death Differ 1999;6:618–23.[Medline]

150 Separovic D, He J, Oleinick NL. Ceramide generation in response to photodynamic treatment of L5178Y mouse lymphoma cells. Cancer Res 1997;57:1717–21.[Abstract]

Manuscript received February 10, 2000; revised November 30, 2000; accepted December 29, 2000.


This article has been cited by other articles in HighWire Press-hosted journals:


             
Copyright © 2001 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement