EDITORIALS

Fenretinide: the Death of a Tumor Cell

John C. Reed

Correspondence to: John C. Reed, M.D., Ph.D., The Burnham Institute, La Jolla, CA 92037 (e-mail: jreed{at}burnham-inst.org).

Synthetic retinoids hold great promise as new agents for the prevention and treatment of cancer. Attempts by chemists to generate novel retinoid-like structures have resulted in several compounds with potent antitumor activity [reviewed in (1)]. However, not all of these synthetic retinoid-like compounds bind known members of the retinoic acid receptor (RAR; RXR [i.e., retinoid X receptor]) or nuclear receptor families of transcriptional regulators. The compound N-(4-hydroxylphenyl)retinamide (4-HPR; fenretinide) represents a synthetic "retinoid" for which the primary cellular target is unidentified. Although 4-HPR can induce expression of RARß, it remains controversial whether the antitumor effects of this compound can be explained by binding to retinoid receptors (2,3). 4-HPR has been demonstrated to induce apoptosis at concentrations typically in the range of 1-5 µM in a wide variety of human cancer cell lines, including retinoid-resistant tumor cells (2). In preclinical animal studies, 4-HPR has demonstrated activity against established tumors and exhibits potent chemopreventive properties in transgenic mouse models involving oncogene-driven tumorigenesis. Trials are also presently under way with 4-HPR in humans. But how does 4-HPR work?

In this issue of the Journal, Maurer et al. (4) suggest that 4-HPR may induce apoptosis of neuroblastoma cell lines through generation of the lipid second messenger ceramide. Production of ceramide has been associated with apoptosis induction in a wide variety of settings, although this is certainly not its only function [reviewed in (5)]. Moreover, addition of synthetic ceramide-containing lipids to cultured tumor cell lines can induce apoptosis in many instances. How ceramide induces apoptosis, however, still remains questionable. The known targets of this lipid include specific ceramide-binding protein kinases and phosphatases, but these ceramide-activated enzymes have not been directly implicated in apoptosis (5).

Recently, evidence of a direct effect of ceramide on mitochondria has been reported (6-8). Addition of ceramide to isolated mitochondria induces an interruption in electron-chain transport, resulting in production of reactive oxygen species (ROS). It is intriguing that 4-HPR has been documented to trigger intracellular increases in ROS, and it has been suggested that ROS generation may be among its chief mechanisms of action where apoptosis induction is concerned (9,10). In support of this idea, Maurer et al. (4) show that neuroblastomas cultured under low oxygen tension are more resistant to 4-HPR, shifting the concentration of 4-HPR required for 1-log kill from approximately 5 µM to approximately 10 mM—which may put the effective antitumor dose beyond the range that is practical clinically. Since mitochondrial ROS generation induced by ceramide can occur under virtually anaerobic conditions (8), one would predict that 4-HPR-induced ROS generation would proceed unabated despite hypoxia, if ceramide is the relevant stimulus. Unfortunately, Maurer et al. did not examine whether low-oxygen conditions reduce the 4-HPR-induced generation of intracellular ROS; thus, the mechanism responsible for the decreased efficacy of 4-HPR in the setting of hypoxia remains unclear.

Which comes first in 4-HPR-treated tumor cells—ceramide production or ROS generation? Not only can ceramide and ROS induce apoptosis, but also they are commonly produced as a consequence of apoptosis. For example, ceramide can be generated as a downstream consequence of activation of caspase-family cell death proteases—the ultimate effectors of apoptosis (11,12). Similarly, most apoptotic stimuli trigger the phenomenon of mitochondrial permeability transition as either an early or a late event, which results in production of ROS by these damaged organelles [reviewed in (13)]. Induction of permeability transition also results in release of mitochondrial cytochrome c into the cytosol, where it binds to a caspase-activating protein (Apaf-1) and triggers a cascade of proteolytic activation of additional downstream caspases—which in turn can induce ceramide production [reviewed in (13-15)]. Thus, proving that either ceramide or ROS is causative in cell death induction is not trivial.

Maurer et al. (4) used synthetic compounds that inhibit caspases to show that 4-HPR still retains some cytotoxicity, through induction of necrosis, when apoptosis is denied to cells as the preferred modus of committing suicide. This observation that caspase inhibitors block apoptosis but do not prevent cell death is typical of most anticancer drugs [reviewed in (15)] and generally is taken as evidence that mitochondrial damage was likely a primary or at least an early event. The explanation for the phenomenon is that, while the consequences of cytochrome c release in terms of caspase activation were suppressed, preventing apoptosis, cells without mitochondrial cytochrome c cannot maintain electron-chain transport and thus ultimately perish by necrosis for lack of adenosine triphosphate production through oxidative phosphorylation [reviewed in (14)]. Thus, we assume that 4-HPR induces caspase-independent damage to tumor cells that can be lethal, but whether both ceramide production and ROS generation occurred upstream of caspase activation was not addressed (4). Moreover, whether mitochondria are an important target of 4-HPR's action is left to speculation, since the effects of 4-HPR on mitochondrial membrane potential and cytochrome c release have not been examined.

What other mechanisms might 4-HPR use to kill tumor cells? Previous investigations of this compound in leukemia cell lines (9) have demonstrated effects on the expression of certain bcl-2 family genes. The proteins encoded by these genes play a central role in apoptosis regulation, functioning as either inducers or blockers of cell death [reviewed in (16)]. Most Bcl-2 family proteins reside in mitochondrial membranes, and anti-apoptotic members such as Bcl-2 can prevent cell death induced by ROS, ceramide, and anticancer drugs. Whereas caspase inhibitors do not prevent cell death induction by either 4-HPR (4) or ceramide (12), enforced expression of Bcl-2 reportedly does (11,12). When taken together with observations that 4-HPR decreases the expression of bcl-2, at least in leukemia cell lines (9), these observations argue that 4-HPR might induce apoptosis through its actions as a regulator of the expression of bcl-2 family genes. Unlike classical retinoids that appear to directly decrease the expression of anti-apoptotic bcl-2 family genes through their binding to RAR- or RXR-family transcription factors, it remains unknown whether the effects of 4-HPR on gene expression represent a primary action versus a secondary consequence—e.g., as a result of ROS generation that can alter the activity of NF-{kappa}B and some other transcription factors.

Knowing the primary cellular target of 4-HPR would certainly assist chemists with generation of additional 4-HPR analogues having greater potency and possibly fewer side effects. More research, therefore, is needed to improve understanding of the mechanisms by which 4-HPR influences pathways involved in cell death regulation in cancers. To be sure, however, lack of knowledge about the cytotoxic mechanisms of compounds with potential antitumor activity has provided little deterrence to clinical application in the past. Results of clinical trials will, therefore, be the ultimate arbiter of whether anyone cares how 4-HPR works. Wherever possible, however, clinicians and translational researchers are urged to incorporate informative laboratory studies into their clinical trials involving 4-HPR, so that more can be understood about the molecular effects of this compound on human tumor cells in vivo. Signal transduction pathways are extremely cell context dependent; thus, while in vitro cell culture experiments and animal models are useful, they are not always representative of the clinical situation in humans. Many a drug has succumbed to "pharmacatosis" for lack of insights as to how best to exploit it. Intelligent incorporation into clinical trials of laboratory studies that seek to identify surrogate biomarkers of response in tumor cells in patients and that attempt to reveal in vivo mechanisms is needed if we are to get the most out of 4-HPR and other promising anticancer drugs.

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