Molecular Mechanisms of Fenretinide-Induced Apoptosis of Neuroblastoma Cells

PENNY E. LOVAT,a MARCO CORAZZARI,b BOJIDAR GORANOV,a MAURO PIACENTINI,b AND CHRISTOPHER P. F. REDFERNa
aNorthern Institute for Cancer Research, University of Newcastle upon Tyne,
Newcastle upon Tyne, NE2 4HH, UK
bINMI-IRCC Lazzaro Spallanzani, Rome, 00149, Italy

ABSTRACT: Synthetic retinoids such as fenretinide [N-(4-hydroxyphenyl)reti- namide] induce apoptosis of neuroblastoma cells, act synergistically with che- motherapeutic drugs, and may provide opportunities for novel approaches to neuroblastoma therapy. Fenretinide-induced cell death of neuroblastoma cells is caspase dependent and results in the release of cytochrome c from mitochon- dria independently of changes in permeability transition. This is mediated by a signaling pathway characterized by the generation of reactive oxygen species (ROS) via 12-lipoxygenase (12-LOX), and an oxidative-stress-dependent in- duction of the transcription factor, GADD153 and the BCL2-related protein BAK. Upstream events of fenretinide-induced signaling involve increased levels of ceramide as a result of increased sphingomyelinase activity, and the subse- quent metabolism of ceramide to gangliosides via glucosylceramide synthase and GD3 synthase. These gangliosides may be involved in the regulation of 12- LOX leading to oxidative stress and apoptosis via the induction of GADD153 and BAK. The targeting of sphingomyelinases or downstream effectors such as 12-LOX or GADD153 may present novel approaches for the development of more effective and selective drugs for neuroblastoma therapy.

KEYWORDS: fenretinide; apoptosis; neuroblastoma

INTRODUCTION

High-dose chemotherapy and autologous bone marrow transplantation are the mainstay of therapy for neuroblastoma, the commonest extracranial solid tumor of childhood.1 The variable biological behavior of these tumors and the fact that spon- taneous regression can occur are features of the disease suggesting that further ad- vances in treatment may come from a greater understanding of neuroblastoma biology and the cellular mechanisms controlling differentiation and cell death. Ret- inoic acid induces differentiation of neuroblastoma cells, and 13-cis retinoic acid now forms an important element of treatment for residual disease of stage 4 neuro- blastoma after chemotherapy.2 However, the refinements of retinoic acid therapy are limited by observations that retinoic acid–induced differentiation of neuroblastoma cells may render them resistant to chemotherapy.

In contrast to retinoic acid, some synthetic derivatives of retinoic acid such as fen- retinide [N-(4-hydroxyphenyl) retinamide] are able to induce apoptosis rather than differentiation,4,5 and, unlike 13-cis retinoic acid, show synergistic responses with chemotherapeutic drugs in a range of different cell types.6–9 Therefore, fenretinide or similar compounds may provide opportunities for novel approaches to neuroblas- toma therapy. To develop the potential of fenretinide for neuroblastoma therapy, it is important to elucidate the molecular mechanisms by which fenretinide induces apo-
ptosis and its synergistic effects with chemotherapeutic agents in this cell type. Stud- ies in this laboratory have been carried out using the human neuroblastoma cell line SH-SY5Y as a model system, with other cell lines such as SK-N-Be, HTLA230, and LAN-5 being used in parallel studies to confirm the main results.8,10–14 Although questions remain, there has been substantial progress towards elucidating the mech- anisms of action of fenretinide, and new potential drug targets are emerging.

FENRETINIDE AND OXIDATIVE STRESS

The elimination of tumor cells by apoptosis is the main mechanism of action of chemotherapeutic drugs.15 In broad terms, apoptosis is mediated by three different pathways: an extrinsic or cell-surface death receptor pathway and two intrinsic path- ways involving damage or stress within mitochondria or the endoplasmic reticulum, respectively.16 Activation of one or more of these pathways results in downstream caspase activation and cell death. Combining drugs that act through different path- ways has the potential to produce synergistic responses. Furthermore, preferential targeting of tumor cells may result if such cells are differentially sensitive to the ac- tivation of one or more of these pathways compared with normal, nontumor cells.
Apoptosis in response to fenretinide has been confirmed in SH-SY5Y cells using a variety of techniques.10 However, it has also been reported that fenretinide at high doses will induce necrosis as well as apoptosis in some human neuroblastoma cell lines.5 In SH-SY5Y cells, fenretinide-induced apoptosis is mediated by caspase- dependent pathways activated by the release of cytochrome c from mitochondria.10 The inhibition of apoptosis by antioxidants and retinoic acid receptor (RAR) antag- onists suggests that signaling pathways involving reactive oxygen species (ROS) and RARs are both required for fenretinide-induced apoptosis.10 However, the ability of fenretinide to induce ROS may be the key to its apoptosis-inducing properties since all-trans, 9-cis, or 13-cis retinoic acid do not induce ROS in these cells.8 As yet, the evidence for an involvement of RARs is based solely on the ability of RAR antago- nists to block fenretinide-induced apoptosis.

In an attempt to identify the enzyme systems responsible for oxidative stress in response to fenretinide, we carried out studies using a range of chemical inhibitors with SH-SY5Y and HTLA230 cells. Only inhibitors of phospholipase A2 (PLA2) and general lipoxygenases (LOX) blocked fenretinide-induced apoptosis and the production of ROS. Further experiments, including the use of specific LOX inhibi- tors such as baicalein, suggested that 12-LOX was the mediator of fenretinide-in- duced ROS and apoptosis in these cells and that PLA2 was necessary to generate the arachidonic acid substrate for 12-LOX (FIG.1).11,13 We were unable to find evidence for 15-LOX expression in SH-SY5Y cells, and the data suggested that SH-SY5Y cells contain an active 12-LOX with little contribution of 5-LOX to cellular LOX ac- tivity. 12-LOX activity results in the production of the eicosatetraenoids 12-HpETE (12- hydroperoxyeicosatetraenoic acid) and 12-HETE (12-hydroxyeicosatetraenoic acid), and the effects of 12-LOX inhibitors in blocking fenretinide-induced apoptosis can be reversed by adding these eicosatetraenoids to the neuroblastoma cells at the time of treatment with fenretinide and LOX inhibitor.11, 13 However, 12-HETE and 12-HpETE do not induce apoptosis when added to SH-SY5Y cells on their own, and this suggests that some other function of fenretinide, possibly the activation of RARs, or of 12-LOX, is required to induce apoptosis. This question needs further investigation. Nevertheless, the fact that 12-LOX inhibitors block fenretinide- induced ROS suggests that increased 12-LOX activity is responsible for oxidative stress.

FIGURE 1. Fenretinide-induced apoptosis: the generation of ROS resulting from in- creased 12-LOX activity. Arachidonic acid (AA), released from membrane phospholipids via phospholipase A2 (PLA2) activity, is a substrate for 12-lipoxygenase (12-LOX). The ste- reospecific oxygenation of AA by 12-LOX to form 12-HpETE and 12-HETE results in the accumulation of reactive oxygen species (ROS). ROS production then triggers a cascade of events involving the mitochondrial release of cytochrome c and activation of caspases lead- ing to apoptosis.

LOX enzymes have been implicated in modulating apoptosis in other cell sys- tems, either through inhibition or activation. Although inhibiting 12-LOX activity in some cell types induces apoptosis,17 in fibroblasts 12-LOX activation leads to apoptosis,18 and cellular LOX activity has a pro-apoptotic effect in CHP100 neuroepi- thelioma cells.19,20 Evidence from other studies points to the importance of 12-LOX in the biology of neuronal cells, since 12-HETE and its derivatives are the main LOX products in the mammalian brain21 and may be involved in aspects of synaptic transmission22 as well as nerve cell death.23

DOWNSTREAM EFFECTORS OF OXIDATIVE STRESS

The growth arrest and DNA damage (GADD)–inducible transcription factor, GADD153, was identified as a fenretinide-inducible gene by screening a cDNA ar- ray of apoptosis-related cDNAs. The rapid and sustained induction of GADD153, at both the mRNA and protein level, is a key event in response to fenretinide in SH- SY5Y cells and results from a fenretinide-dependent increase in ROS and 12-LOX activity.11 In other cell types, GADD153 is induced by cellular stress, including that resulting from treatment with alkylating agents and UV light, nutrient stress, the generation of ROS, and endoplasmic reticulum stress.24–26 Since RAR antagonists do not block GADD153 induction, this represents an RAR-independent effect of fenretinide necessary for apoptosis in these neuroblastoma cells (FIG. 2). GADD153 is a key control point in apoptosis of other cell types,27–29 and since it is a transcrip- tion factor, GADD153 may be responsible for the induction of other genes required for fenretinide-induced apoptosis in neuroblastoma cells. The necessity for GADD153 in fenretinide-induced apoptosis has been confirmed by inserting GADD153 cDNA in sense or antisense orientation into a tetracycline-inducible ex- pression vector and producing stably transfected SH-SY5Y cell derivatives. Overex- pression of GADD153 results in an increased level of apoptosis in the absence of fenretinide and an increased apoptotic response to fenretinide; conversely, expres- sion of antisense GADD153 cDNA blocks the apoptotic response to fenretinide.11,14 The cDNA array screen also identified the pro-apoptotic BCL2-family protein BAK as a fenretinide-inducible gene in SH-SY5Y cells,11 and this is confirmed by immunofluorescence flow cytometry and Western blot data.12 As with GADD153, there is no detectable induction of BAK in response to the chemotherapeutic drugs cisplatin, etoposide, or carboplatin in these neuroblastoma cells, and the induction of BAK in response to fenretinide is blocked by antioxidants.12 The function of BAK as a pro-apoptotic protein in SH-SY5Y cells has been confirmed by preparing stably transfected SH-SY5Y derivatives in which expression of BAK sense or antisense cDNA is inducible in response to tetracyclines. Under these conditions, the induc- tion of BAK increases the levels of apoptosis, both with and without fenretinide treatment, whereas the expression of antisense BAK abrogates fenretinide-induced apoptosis.12 The hypothesis that GADD153 is responsible for the induction of BAK was tested by comparing the level of BAK expression in SH-SY5Y cells stably trans- fected with tetracycline-inducible sense- or antisense-GADD153 cDNA.11 These experiments showed that the induction of GADD153 independently of fenretinide led to the induction of BAK, which suggests that GADD153 directly regulates BAK expression (FIG. 2). However, the expression of the antisense-GADD153 construct did not completely abolish BAK induction in response to fenretinide,12 and it is pos- sible that GADD153-independent mechanisms induced by fenretinide contribute to BAK induction in these cells.

Studies on other cell types have shown that BAK can induce the release of cyto- chrome c from mitochondria, independently of mitochondrial permeability transi- tion, in combination with BH3 domain–only members of the BCL2 family.30 Therefore, the induction of BAK may be an event downstream of GADD153 induc- tion leading to cytochrome c release and subsequent apoptosis in fenretinide-treated neuroblastoma cells.

Although the treatment of some cell types with chemotherapeutic drugs results in GADD153 induction,27,31,32 a sustained induction of GADD153,comparable to that obtained with fenretinide, does not result from the treatment of SH-SY5Y neuroblastoma cells with the chemotherapeutic drugs cisplatin, etoposide, or carboplatin.Conversely, these drugs induce the expression of the p53-regulated gene p21/WAF1, a gene that is not induced in response to fenretinide. Although p21/WAF1 can be reg- ulated independently of p53, other evidence for SH-SY5Y cells indicates that p53 mediates apoptosis in response to these chemotherapeutic drugs.33 Therefore, it is likely that fenretinide and chemotherapeutic drugs induce apoptosis by p53- independent and -dependent mechanisms, respectively.

FIGURE 2. The ROS-dependent induction of GADD153 and BAK mediates apoptosis in response to fenretinide. Fenretinide-induced oxidative stress results in induction of the growth and DNA damage (GADD)–inducible transcription factor GADD153 and the pro- apoptotic BCL2-related protein BAK. GADD153 may be responsible for the induction of BAK, and the induction of both genes may be fundamental events linking oxidative stress to cytochrome c release from mitochondria and caspase activation leading to apoptosis.

LINKING THE FENRETINIDE SIGNAL WITH LIPOXYGENASE ACTIVATION

So far in this review, we have shown how PLA2 and 12-LOX are involved in the induction of oxidative stress in response to fenretinide in neuroblastoma cells, and we suggest that oxidative stress is linked to the release of cytochrome c from mito- chondria, and subsequent downstream events of apoptosis, via the transcription fac- tor GADD153 and the induction of the pro-apoptotic protein BAK. Although there is still a great deal that needs to be learned about the regulation of each of these steps in detail, particularly the mechanisms of GADD153 and BAK induction, our under- standing of events further upstream is less well advanced.

FIGURE 3. The production and metabolism of ceramide links fenretinide treatment to the increase in 12-LOX activity. Increased intracellular levels of the lipid secondary mes- senger ceramide results from the metabolism of membrane lipids by acidic sphingomyeli- nase (ASMase), which is induced by fenretinide treatment. Ceramide is then metabolized to the ganglioside GD3 via glucosylceramide synthase and GD3 synthase. GD3 itself, or other gangliosides derived from it, may be responsible for the activation of 12-LOX and the sub- sequent triggering of downstream events of fenretinide-induced apoptosis.39

Studies in other laboratories have suggested that fenretinide increases intracellu- lar levels of the lipid secondary messenger ceramide in neuroblastoma and other cell types.5,34–36 Ceramide is a lipid signaling molecule derived by de novo synthesis via serine palmitoyl transferase and ceramide synthase, or from the metabolism of mem- brane lipids by sphingomyelinases.37,38 Although it has been suggested that fenre- tinide predominantly increases ceramide levels by de novo synthesis,5,34–36 we have been unable to block fenretinide-induced apoptosis of SH-SY5Y cells with fumoni- sin B1, an inhibitor of ceramide synthase. Conversely, we have found that ceramide levels increase in response to fenretinide treatment as a result of acidic sphingomy- elinase activity.39 We have been able to show, using RNA interference techniques, that acid sphingomyelinase is necessary for fenretinide-induced apoptosis. Furthermore, once generated by acidic sphingomyelinase activity, ceramide is subsequently metabolized to ganglioside GD3 via glucosylceramide synthase and GD3 synthase (FIG. 3).39 GD3 itself, or other gangliosides derived from it, may be directly respon- sible for the activation of 12-LOX (FIG. 3).

CONCLUSIONS

The signaling pathways of fenretinide-induced apoptosis can be considered in four sections: (1) events downstream of mitochondria in which caspase activation mechanisms common to the major pathways of apoptosis come into play; (2) the linking of oxidative stress to the initiation of these downstream mitochondrial events, and in which GADD153 and BAK are clearly major players; (3) the produc- tion of oxidative stress, which in neuroblastoma cells involves arachidonic acid and 12-LOX activity; and (4) the cellular mechanisms transducing the fenretinide signal to oxidative stress that involve ceramide and ganglioside synthesis. There is still a great deal to be learned about fenretinide-induced apoptosis; elucidating the mech- anisms of oxidative stress and how this is induced by fenretinide is likely to lead to substantial advances in our understanding of neuroblastoma biology. Furthermore, identifying the components of the apoptotic pathway(s) induced by fenretinide will lead to new targets for drug development. Fenretinide itself, or drugs designed to tar- get novel elements of these apoptotic pathways, can be used to increase the efficacy of neuroblastoma therapy by working synergistically with existing cytotoxic drugs.

ACKNOWLEDGMENTS

Research in the authors’ laboratories was funded in the UK by Cancer and Leu- kaemia in Childhood (CLIC), The North of England Children’s Cancer Research Fund, and The Neuroblastoma Society; and in Italy by AICR, MIUR, and EU (QLG1-1999-00739).

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