Mechanisms of fenretinide-induced apoptosis - Springer Link

Apoptosis (2006) 11:1677–1694 DOI 10.1007/s10495-006-9289-3

Mechanisms of fenretinide-induced apoptosis N. Hail Jr. · H. J. Kim · R. Lotan

Published online: 15 July 2006 C Springer Science + Business Media, LLC 2006


Abstract Fenretinide, a synthetic retinoid, has emerged as a promising anticancer agent based on numerous in vitro and animal studies, as well as chemoprevention clinical trials. In vitro observations suggest that the anticancer activity of fenretinide may arise from its ability to induce apoptosis in tumor cells. Diverse signaling molecules including reactive oxygen species, ceramide, and ganglioside GD3 can mediate apoptosis induction by fenretinide in transformed, premalignant, and malignant cells. In many cell types, these signaling intermediates appear to be induced by mechanisms that are independent of retinoic acid receptor activation, and ultimately initiate the intrinsic or mitochondrial-mediated pathway of cell elimination. Numerous investigations conducted during the past 10 years have discovered a great deal about the apoptogenic activity of fenretinide. In this review we explore the mechanisms associated with fenretinide-induced apoptosis and highlight certain mechanistic underpinnings of fenretinide-induced cell death that remain poorly understood and thus warrant further characterization. Keywords Apoptosis . Cancer chemoprevention . Fenretinide . 4HPR . Mitochondria . Ceramide . Reactive oxygen species . Review

N. Hail Jr. (
) Department of Clinical Pharmacy, School of Pharmacy, The University of Colorado at Denver and Health Sciences Center, Box C238, Denver, CO 80262, USA e-mail: [email protected] H. J. Kim · R. Lotan Department of Thoracic/Head and Neck Medical Oncology, The University of Texas, MD Anderson Cancer Center, Houston, Texas, USA

The cancer chemoprevention concept Although cancer continues to be one of the major causes of death worldwide and only modest progress has been made in reducing the morbidity and mortality of this dreadful disease, extensive preclinical and clinical research has led to substantial progress in understanding the multi-step nature of the prolonged carcinogenesis process. This understanding has led to the conviction that most human malignancies should be fought on multiple fronts. Thus, in addition to cancer therapy, cancer prevention has become an important means of controlling cancer [1, 2]. Common prevention strategies include avoiding exposure to known cancer-causing agents, enhancement of host defense mechanisms against cancer, life style modifications, and chemoprevention. The phrase “cancer chemoprevention” was introduced by Sporn in 1976 when he referred to the prevention of the development of malignancy by vitamin A and its analogs, known collectively as retinoids. Sporn [3] contended that the process of carcinogenesis had the potential to be controlled by physiological or pharmacological means during its preneoplastic stages, whereby the promotion of precancerous cells could be stabilized, arrested, or reversed. Several thousand agents reportedly have chemopreventive activity, and among them, more than 40 promising agents and agent combinations have been evaluated clinically for cancer chemoprevention [4]. For example, the retinoids all-trans retinoic acid (ATRA) and 13-cis retinoic acid have been effective in arresting or reversing premalignant lesions like bronchial metaplasia, oral leukoplakia, uterine cervical dysplasia, and actinic keratoses [5]. In randomized trials of patients with familial adenomatous polyposis, the non-steroidal anti-inflammatory drugs sulindac and celecoxib inhibited the growth of adenomatous polyps and promoted polyp regression [6]. Furthermore, chemoprevention trials have shown that the antiestrogen Springer

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tamoxifen can reduce the incidence of breast cancer [7], and the antioxidant vitamin E can reduce the incidence of prostate cancer [8] and suppress the progression of bronchial premalignant lesions [5].

Fenretinide and cancer chemoprevention Fenretinide (i.e., N-(4-hydroxyphenyl)retinamide, also known as 4HPR, Fig. 1) is a synthetic analog of ATRA that was first produced by R. W. Johnson Pharmaceuticals in the late 1960’s. The substitution of an amide-linked 4-hydroxyphenyl group for the carboxyl group of ATRA markedly reduced adverse side effects like liver toxicity. Furthermore, fenretinide lacks the ability to induce point mutations or chromosomal aberrations, and is therefore not genotoxic [9]. These qualities suggested that fenretinide could be compatible for long-term use in a chemopreventive modality. Indeed, in animal models, fenretinide has demonstrated chemopreventive efficacy against carcinogenesis of the breast [10], prostate [11], pancreas [11], and skin [11– 13]. Moreover, in a clinical setting, fenretinide slowed the progression of prostate cancer in men diagnosed with an early stage of the disease [14], protected against the development of ovarian cancer and a second breast malignancy in premenopausal women who had been treated to prevent the progression of early-stage breast cancer [15], and prevented relapse and the formation of secondary primary lesions in patients following the surgical removal of oral leukoplakia [16]. Natural retinoids like ATRA and 13-cis retinoic acid often induce differentiation and/or cytostasis in target cells [5, 17, 18]. Conversely, fenretinide triggers distinct biologic effects including the generation of reactive oxygen species (ROS) and lipid second messengers that appear to be involved in apoptosis induction in transformed, premalignant, and malignant cells in vitro (Table 1). Interestingly, like fenretinide,

Fig. 1 Chemical structures of fenretinide and all-trans retinoic acid

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a number of putative chemopreventive agents have also been shown to induce apoptosis in such cells in vivo and/or in vitro, suggesting that apoptosis is a novel target for the chemoprevention of cancer [2]. Furthermore, the apoptogenic activity of fenretinide in vitro appears to be selective in transformed cells while apparently sparing their normal counterparts. This has been documented in normal fibroblast [19], lymphocytes [20, 21], myometrial cells [22], hepatocytes [23], and cervical epithelial cells [24]. While fenretinide has been considered primarily as a cancer chemopreventive agent, several in vitro and animal studies examining neuroblastoma [25], leukemia [26], and lymphoma [27–29] cells suggest that the apoptogenic activity of this synthetic retinoid may also have value in a cancer chemotherapy setting. Before exploring the mechanisms associated with fenretinide-induced apoptosis, we will highlight apoptosis pathways and their regulation.

Apoptosis pathways and their regulatory intermediates Apoptosis is the mechanism utilized by metazoans to regulate tissue homeostasis through the controlled elimination of redundant or potentially deleterious cells. In addition, apoptosis is an important target for anticancer agents because extensive research on cancer development has revealed that cancerous cells are permissive survivors due to acquired mechanisms of apoptosis resistance in addition to uncontrolled proliferative programming. Apoptosis can be broken down into three distinct phases. The initiation phase constitutes a triggering event that is largely dependent on cell type and apoptotic stimulus (e.g., ROS, DNA damage, ion fluctuations, and cytokines). This is followed by an effector phase in which distinct biochemical events systematically activate catabolic hydrolases (i.e., proteases and nucleases) [30]. These enzymes participate in the degradation phase of apoptosis through the cleavage of proteins and DNA [31]. Most of the recent advances in the elucidation of apoptosis pathways have come about through the characterization of the effector mechanisms. There are several components comprising the effector mechanisms of apoptosis, and two effector mechanisms associated with the activation of caspases (i.e., cysteine proteases involved in apoptosis) and/or cellular degradation have been characterized extensively. These include the mitochondrial-mediated effector mechanism (intrinsic) and the death receptor-mediated (e.g., tumor necrosis factor (TNF) receptor and Fas) effector mechanism (extrinsic) [31, 32]. The intrinsic pathway of apoptosis relies on mitochondrial membrane permeabilization (MMP) to release the apoptogenic mitochondrial proteins [AMPs, e.g., cytochrome c [33], endonuclease G [34, 35], second mitochondrial activator of caspases (Smac) [36], Omi/HtrA2 [37],

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Table 1 Apoptosis induction by fenretinide in transformed, premalignant, and malignant cells in vitro, proposed apoptosis pathway, and/or regulatory intermediate(s)a Cell type [Reference]

Apoptosis pathway [Reference] Regulatory intermediate(s) [Reference]

B-cells (Transformed) [93] Bladder Carcinoma [194, 207] Breast Carcinoma [94, 109, 111, 123–126, 138, 172, 192, 208, 209] Breast Epithelial (Transformed) [210] Cervical Carcinoma [24, 93, 102, 106, 193, 211]

Intrinsic [93] N.D. Intrinsic [109, 111, 172, 192]

Colon Carcinoma [93, 138] Embryonal Carcinoma [72, 212] Esophageal Carcinoma [76] Glioma [213, 214] Head and Neck Carcinoma [71, 77, 162, 215] Hepatoma [113, 159, 195] Leukemia [20, 21, 26, 27, 93, 98, 105, 107, 132, 134, 196] Lung Carcinoma [76, 77, 138, 155, 216–218] Lymphoma [27, 29, 154] Melanoma [138, 219, 220] Meningioma [203] Neuroblastoma [25, 79, 80, 95, 97, 99, 101, 110, 131, 157, 221–223] Ovarian Carcinoma [74, 140, 170, 171, 202] Pancreatic Carcinoma [138] Prostate Carcinoma [71, 138, 139, 148, 163, 191, 224, 225] Sarcoma [96, 108] Skin Carcinoma [19, 78, 102–104] Skin Keratinocytes (Premalignant) [102] Skin Keratinocytes (Transformed) [102] T-cells (Transformed) [20] a

N.D. Intrinsic [93, 102, 106] Intrinsic [93] N.D. N.D. N.D. Intrinsic [162] Intrinsic [113, 159, 195] Intrinsic [20, 21, 93] N.D. N.D. N.D. Intrinsic [203] Intrinsic [82, 97, 99, 157]

Intrinsic [170, 171, 202] N.D. Intrinsic [191] Intrinsic [96, 108] Intrinsic [19, 78, 102–104] Intrinsic [102] Intrinsic [102] Intrinsic [20]

ROS and Bcl-2 Family [93] N.D. Ceramide [94, 138], Nitric Oxide [123–126], Oxidative Stress [109], ROS [111, 172], and Bcl-2 Family [192] ROS [210] ROS [24, 93, 102, 106], Bcl-2 Family [93], and BAG-1 [193, 211] Ceramide [138], ROS [93], and Bcl-2 Family [93] N.D. RARs [76] N.D. ROS [71, 77, 162], RARs [71, 77], and JNK [162] ROS [113, 159], Caspase-8 [195] Ceramide [20], ROS [21, 93, 98, 105, 107], and Bcl-2 Family [93] ROS [77], and RARs [76, 77] Bcl-2 Family [154] Ceramide [138] N.D. RARs [79], Ceramide [25, 131], GD3 [80], ROS [79,95], GADD153 [99, 101, 157], JNK [95], and Bcl-2 Family [97, 99] Ceramide [140, 202], RARs [74, 75], and c-Fos [140] Ceramide [138] Ceramide [139], RARs [71], ROS [71], Bcl-2 Family, [191], and JNK [148, 163] Ceramide [96], ROS [96, 108], and p38MAPK [108] ROS [103, 104] and RARs [78] ROS [102] ROS [102] N.D.

Please refer to the text for additional details; N.D., not determined.

apoptosis-inducing factor (AIF) [38], and its homologue AIF-homologous mitochondrion-associated inducer of death (AMID) [39] required for the degradation phase of apoptosis. Bcl-2 family members play a central role in the regulation of MMP and apoptosis. During conditions of cell stress, antiapoptotic Bcl-2 family members (e.g., Bcl-2 and Bcl-XL ) residing in the outer mitochondrial membrane can be destabilized through a decrease in expression, or by the induction of proapoptotic Bcl-2 family members (e.g., Bax, Bad, and Bak). In this scenario, the ratio of proapoptotic family members to antiapoptotic family members becomes greater allowing the formation of proteinaceous outer membrane channels by the proapoptotic Bcl-2 family members [40]. Consequently, MMP is achieved liberating AMPs that can activate the caspase cascade anchored by caspase-9 to induce apoptosis (Fig. 2). The autonomous regulation of MMP is characterized as the mitochondrial permeability transition (MPT). The MPT is a rate-limiting and self-amplifying process that is

influenced by several mitochondrial proteins localized in the inner and outer mitochondrial membranes. Many of these proteins (e.g., voltage-dependant anion channel, adenine nucleotide translocase, hexokinase, peripheral benzodiazepine receptor, and cyclophilin D) are believed to constitute the permeability transition pore complex (PTPC) [41]. Normally, the proteins in the outer and inner mitochondrial membranes that constitute the PTPC are in close proximity to each other and in a closed or low conductance conformation. A host of factors, including pathologic stimuli (e.g., Ca2+ and ROS) as well as various chemical agents, can cause the PTPC to adopt an open conformation [41]. This allows water and solutes up to 1500 Da to infiltrate the mitochondrial matrix, which can cause colloidal osmotic swelling of the mitochondrion [31]. If multiple PTPCs open concurrently and mitochondrial swelling is extensive, AMPs are released to the cytoplasm via the physical rupture of the outer mitochondrial membrane. The MPT is a rate-limiting and self-amplifying process because the release of mitochondrial constituents (e.g., Springer

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Fig. 2 Regulation of the intrinsic and extrinsic pathways of apoptosis. Please refer to the text for details. Abbreviations: MMP, mitochondrial membrane permeabilization; and AMPs, apoptotic mitochondrial proteins

Ca2+ ) to the cytosol can trigger MPT in vicinal mitochondria [42]. Bioenergetic catastrophe is one feature of the MPT that reinforces the ultimate demise of the dying cell. Besides the loss of AMPs, many of which participate directly in the maintenance of mitochondrial homeostasis [43], the MPT typically promotes the dissipation of the electrochemical gradient across the inner mitochondrial membrane known as the mitochondrial inner transmembrane potential (
m ) and enhances ROS production through the disintegration of electron transport [30 ]. Together, these events progressively shut down mitochondrial oxidative phosphorylation (OXPHOS) [44]. This may explain why inhibiting caspase activity may not necessarily protect a cell form dying following the induction of MPT-induced MMP [45]. The extrinsic pathway of apoptosis is activated at the cell surface through the interaction of specific ligands and their receptors. Upon ligand binding (e.g., TNFα, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand), death receptors (e.g., TNF receptor, TRAIL receptor (DR5), and Fas) cluster in the plasma membrane, which enhances the recruitment of cytosolic adapter proteins [46]. Caspase-8 is the apical caspase in the extrinsic pathway of apoptosis. The zymogen of caspase-8 can interact with a receptor-adapter protein complex to generate the catalytic form of caspase-8. Once active, caspase-8 can process downstream effector caspases like caspase-3 that participate in cellular degradation (Fig. 2). In certain cell systems, the activation of caspase-8 is sufficient to initiate the proteolytic cascade required to induce apoptosis [46, 47]. Caspase-8 can also integrate the extrinsic and intrinsic pathways of apoptosis by cleaving the proapoptotic Bcl-2 family member Bid. The truncated form of Bid can induce MMP following translocation to the outer mitochondrial membrane. This allows AMPs like cytochrome c [48] and endonuclease G [34] to be released from the mitochondria to participate in cellular degradation. It should also be noted that, once activated, effector caspases (e.g., caspase-3 and -6) could initiate a feedback amplificaSpringer

tion loop that is capable of activating apical caspases (e.g., caspase-8). This feedback mechanism is believed to amplify proteolytic processes during the terminal phase of apoptosis [49] (Fig. 2). Proteins such as the X-linked inhibitor of apoptosis protein (XIAP), survivin, or the FLICE-like inhibitory protein (FLIP) can also influence the sensitivity to cell death signaling and/or cellular degradation through the direct or indirect inhibition of caspase activity [50] (Fig. 3). In addition to the mitochondria, growing evidence suggest that the endoplasmic reticulum (ER), Golgi apparatus, and lysosomes also function as major points of integration for damage sensing in the cell, since these organelles can provide signaling intermediates that participate in the regulation of apoptosis pathways [51] (Fig. 3). For instance, the disruption of ER Ca2+ homeostasis can promote the activation of caspase-12, which is believed to be an initiator caspase localized on the cytoplasmic face of the ER [52–54]. The release of ER Ca2+ stores can also trigger the opening of the PTPC resulting in mitochondrial-mediated apoptosis [55], or promote the activation of cytosolic Ca2+ -dependent proteases know as calpains [56, 57]. Furthermore, both the disruption of ER Ca2+ homeostasis and the unfolded protein response (UPR) can promote the nuclear translocation of the ER protein activating transcription factor (ATF)-6. Once in the nucleus, ATF-6 can promote the induction of transcription factors like the growth arrest and DNA damageinducible transcription factor 153 (GADD153) that regulate the intrinsic pathway of apoptosis through the modulation of Bcl-2 family members [58, 59]. The release of the lysosomal lipid second messenger ceramide or resident acidic cysteine proteases known as cathepsins can trigger MMP and mitochondrial-mediated apoptosis [60, 61] through direct mitochondrial disruption in the case of ceramide [60, 62], or indirectly via the activation of proapoptotic Bcl-2 family members (i.e., Bax, Bak, and Bid) by cathepsins [61, 63]. Furthermore, the ceramide metabolite ganglioside GD3 (GD3) apparently targets the mitochondria causing mitochondrial disruption and MPT-induced

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Fig. 3 Regulatory intermediates involved in apoptosis induction. Please refer to the text for details. Abbreviations: TNF, tumor necrosis factor; UPR, unfolded protein response; NF-κB, nuclear factor-kappa B; GADD153, growth arrest and DNA damage-inducible transcription

factor 153; XIAP, X-linked inhibitor of apoptosis protein; FLIP, FLICElike inhibitory protein; ROS, reactive oxygen species; AIF, apoptosisinducing factor; Smac second mitochondrial activator of caspases; and GD3, ganglioside GD3

apoptosis once it is released from the Golgi complex [64– 67]. Therefore, in a manner similar to integration of the extrinsic and intrinsic pathways of apoptosis by caspase-8 and the cleavage of Bid, dispersible regulatory intermediates like ROS, Ca2+ , calpains, cathepsins, ceramide, and GD3 can participate in the intracellular succession and/or amplification of the intrinsic pathway of apoptosis. The sections that follow will examine the proposed mechanisms associated with of fenretinide-induced apoptosis in various cell types. These mechanisms will be discussed with respect to how we believe they relate to the initiation, effector, and degradation phases of apoptosis.

retinoic acid response elements and retinoid X response elements, respectively). In the absence of ligands, the receptor dimmers recruit proteins that act as co-repressors that prevent gene transcription. However, upon ligand binding, the co-repressors dissociate from he complex with receptors and are replaced by co-activators such that the receptors become active transcription factors that regulate the transactivation of target genes [69]. Fenretinide does not bind RARs because it lacks a carboxyl functional group that is believed to be required for this activity [17]. Nonetheless, it has been reported that fenretinide-treated cells in culture show evidence of enhanced transcriptional activity of these receptors [17, 70, 71]. The expression of RARs by cultured cells is reportedly associated with varying degrees of sensitivity to fenretinideinduced apoptosis. For example, RAR knockout cells retained sensitivity to apoptosis induction by fenretinide [72], and there was a concomitant increase in RAR expression in ovarian carcinoma cells made resistant to fenretinideinduced apoptosis by continuous culture with fenretinide [73]. Conversely, cells that constitutively expressed relatively high levels of RARs exhibited more sensitivity to apoptosis induction by fenretinide [74–76], and in certain cell types RAR antagonists suppressed fenretinide-induced apoptosis, at least partly [71, 77–79]. Lovat et al. [80] have proposed that the inhibitory effects of RAR antagonists on apoptosis induction by fenretinide in neuroblastoma cells may be due to the suppression of acidic sphingomyelinase induction through retinoic acid response elements within the acidic sphingomyelinase promoter. Yet, this assumption does not take into account the observation

Mechanisms involved in the initiation of apoptosis by fenretinide The role of retinoic acid receptors Natural retinoids and many of their synthetic analogs modulate cell proliferation and/or differentiation via nuclear retinoid receptor-mediated transactivation of target genes [68]. There are two types of nuclear retinoid receptors known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that belong to the steroid hormone receptor superfamily [69]. Three subtypes of RARs and RXRs are encoded by different genes designated α, β, and γ . The natural ligands for RARs are ATRA and 9-cis retinoic acid, whereas the RXRs bind only 9-cis retinoic acid. These receptors form RXR-RAR heterodimers or RXR-RXR homodimers that bind to DNA sequences called response elements (i.e.,

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that RAR antagonists failed to suppress ROS generation by fenretinide [79], which appears to be intimately involved in ceramide-induced, 12-lipoxygenase (12-LOX)-dependent apoptosis in neuroblastoma cells [80, 81]. Furthermore, neither ATRA nor 9-cis retinoic acid promoted ROS generation or apoptosis in neuroblastoma cells [82], suggesting that fenretinide’s cytotoxic effects were predominately independent of RAR activation in these cells. In most of the cell systems examined to date, the apoptogenic activity of fenretinide appears to develop independently of RAR activation [83]. Thus, determining the molecular and/or biochemical basis for the inhibition of fenretinide-induced apoptosis by certain RAR antagonists in neuroblastoma cells [79], as well as skin [78], prostate [71], and lung [77] carcinoma cells deserves further examination. The role of ROS The mitochondria are the primary source of ROS production in most cells [84], and anomalous ROS generation by these organelles can play a pivotal role in apoptosis signaling [85, 86]. For example, excessive mitochondrial ROS generation and/or the disruption of mitochondrial redox homeostasis can promote the oxidation of thiols that regulate the conformation of proteins constituting the PTDC, which can cause MPT induction [87]. Mitochondrial ROS generation can also facilitate the dispersal of cytochrome c from the inner mitochondrial membrane by breaching its electrostatic and/or hydrophobic affiliations with cardiolipin [88]. The membranes constituting the ER, Golgi apparatus, and lysosomes are also sensitive to ROS and/or alterations in cellular redox homeostasis [89]. The redox-regulated release of proapoptotic constituents such as Ca2+ from the ER [55] and cathepsins from lysosomes [61, 63] have been reported. Moreover, aberrant ROS production and/or oxidative stress can activate sphingomyelinases and increase ceramide production [60, 90, 91]. Since the mitochondria and MPT induction appear to be a target of ceramide during apoptosis [62, 91], the coupling between oxidative stress and ceramide generation is probably bi-directional and amplifying in nature, since both process would be expected to ultimately eliminate ROS over-producing cells [92]. Perhaps the most common property of fenretinideinduced apoptosis is its inhibition by antioxidants (e.g., vitamin C, vitamin E, N-acetylcysteine, butylated hydroxyanisole, and pyrrolidine dithiocarbamate), suggesting an essential role for ROS and oxidative stress in fenretinide’s cytotoxicity [24, 25, 71, 77, 93–108]. Cells exposed to fenretinide also reportedly undergo cardiolipin oxidation [21, 109], implying a mitochondrial susceptibility to cytochrome c release. The rapid production of ROS, particularly hydroperoxides, is commonly observed in a variety of tumor cell types following exposure to fenretinide (Table 1). This process is Springer

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inevitably diminished when cells are cultured in hypoxic conditions [25, 96]. Pharmacological inhibition of fenretinideinduced ROS production can be achieved by co-treating cells with antioxidants (e.g., vitamin C and vitamin E) [24, 25, 93, 104, 105], mitochondrial poisons (e.g., rotenone, cyanide, and carbonyl cyanide m-chlorophenylhydrazone) [21, 103, 106], coenzyme Q analogues [103], the PTPC inhibitor cyclosporin A [104], 12-LOX inhibitors [101, 110], and inhibitors of ceramide and GD3 synthesis [80]. Genetic approaches, including the depletion of mitochondrial DNA [103], Bcl-2 overexpression [93], Bax/Bak knockouts [93], galectin-3 overexpression [111], and small interfering RNAs directed against enzymatic processes associated with ceramide or GD3 synthesis [80], have also proven effective in reducing the prooxidant effects of fenretinide in cultured cells. Interestingly, ROS generation by fenretinide was conspicuously increased in cells co-treated with certain synthetic RAR antagonists (i.e., CD2665 and CD2848) [79], center i inhibitors of mitochondrial complex III (i.e., antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide) [103, 106], the ER Ca2+ mobilizing cancer chemopreventive agent 2-cyano3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) [112], or the mitochondrial adenine nucleotide translocase ligand bongkrekic acid [113]. Furthermore, fenretinide reportedly functioned as an antioxidant by reducing 1,1-diphenyl-2picrylhydrazyl radicals [114, 115], suggesting that fenretinide can participate in redox cycling reactions [1, 103, 114, 115]. Collectively, these observations point to the involvement of enzymatic processes, possibly those associated with OXPHOS [103, 106, 116] and/or 12-LOX activity [80, 101], in fenretinide-induced ROS production. Several important questions linger regarding the prooxidant effects of fenretinide. First, can fenretinide trigger ROS production in isolated mitochondria oxidizing complex I and/or complex II substrates [1, 103]? Second, how do Bcl-2 family members and galectin-3 regulate fenretinide-induced ROS generation? Given that: (1) fenretinide’s prooxidant and cytotoxic effects are diminished during hypoxia [25, 96] (a condition that should inhibit OXPHOS [103]), (2) OXPHOS appears to be a target of fenretinide [1, 21, 103, 106], (3) galectin-3 contains the same anti-death motif found in Bcl-2 [111], and (4) several studies have reported that Bcl-2 and its family members can modulate OXPHOS in addition to apoptosis [117–122], the answers to these questions would not only supply a firm rationale for cell sensitivity to fenretinide, but also a mechanistic basis for possible resistance to this agent. Similarly, given that certain RAR antagonists markedly enhanced fenretinide-induced ROS generation in neuroblastoma cells while offering protection against apoptosis induction [79], it would be salient to determine if these effects occur in the presence or absence of 12-LOX activation. This would be an important question to address because

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RARs may regulate ceramide production, and ultimately 12-LOX activation, in neuroblastoma cells by the transcriptional regulation of the acidic sphingomyelinase promoter [80]. Fenretinide-induced apoptosis is reportedly contingent on nitric oxide production in breast cancer cells [123–126]. Therefore, it would also be important to determine if these processes were sensitive to antioxidants, since nitric oxide can react with superoxide to form a peroxynitrite, a very potent oxidizing and nitrating agent [127]. The role of lipid second messengers Ceramide is a sphingolipid second messenger that is generally recognized to promote apoptosis in response to inflammatory cytokines like Fas and TNF, as well as conditions associated with oxidative stress [92, 128]. Ceramide can be generated in cells de novo, or through the hydrolysis of sphingomyelin [25, 80]. During conditions of cell stress, the deregulation of ceramide generating and/or utilizing processes are believed to cause a net increase in cellular ceramide levels that is sufficient to trigger apoptosis induction [129]. In addition to its direct involvement in apoptosis, ceramide can also serve as a carbon source for glycosphingolipid synthesis in the Golgi network. The glycosphingolipid GD3 is a metabolite of ceramide that has also been implicated in apoptosis induction [128, 130]. GD3 is a minor ganglioside in most normal tissues except placenta and thymus [130]. However, like ceramide, cellular levels of GD3 apparently increase in response to apoptotic stimuli [65, 67], and the inhibition of GD3 synthase, the enzyme responsible for GD3 synthesis, can block apoptosis induction by various mechanisms [128, 130]. Fenretinide exposure enhances cellular ceramide levels during apoptosis [20, 25, 80, 94, 96, 131–134] and oncosis [25]. The enhancement of ceramide generation by fenretinide occurs independent of caspase activation [94, 132, 133], as opposed to the caspase-dependent production of ceramide by cells exposed to certain inflammatory cytokines [135–137]. It is believed that ceramide levels increase during fenretinideinduced apoptosis by means of de novo synthesis [25, 94, 131, 133, 138], and/or the hydrolysis of sphingomyelin [80]. Inhibitors of ceramide synthesis and metabolism can modulate fenretinide-induced ceramide production, while promoting variable degrees of sensitivity to apoptosis induction. For example, the ceramide synthase inhibitor fumonisin B1 markedly reduced fenretinide-induced ceramide generation in a variety of cell types [25, 94, 131–134, 138, 139]. Interestingly, fumonisin B1 reportedly enhanced apoptosis induction by fenretinide in neuroblastoma cells [25]; was too toxic alone to be combined with fenretinide when examining apoptosis in leukemia [134], neuroblastoma [138], and sarcoma [96] cells; decreased fenretinide-induced apoptosis in leukemia [132], and endothelial [133] cells, as well as carci-

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noma cells from breast [94], ovary [140], and prostate [139]; and reportedly had no effect on either ceramide production or apoptosis induction in neuroblastoma cells [80]. Inhibitors of glucosylceramide synthase (e.g., tamoxifen and d,l-threo-1-phenyl-2 hexadecanoylamino-3morpholino-1-propanol), an enzyme associated with ceramide metabolism, increased ceramide levels in certain cell types exposed to fenretinide [134, 138, 139], and also reportedly enhanced fenretinide-induced apoptosis in neuroblastoma [138], carcinoma [138, 139], and leukemia [134] cells. Conversely, others have reported that the inhibition of glucosylceramide synthase delayed fenretinide-induced apoptosis in neuroblastoma cells [80]. These general inconsistencies may be due to the non-specific activity of the chemical agents used to regulate ceramide production and/or metabolism in fenretinide-treated cells [80]. This potential obstacle was apparently overcome by using small interfering RNAs to knock down the enzymatic activity associated with ceramide or GD3 synthesis. This strategy revealed that fenretinide promoted ceramide and ultimately GD3 production through a pathway involving the hydrolysis of sphingomyelin and the activity of GD3 synthase in neuroblastoma cells [80]. The generation of GD3, rather than ceramide per se, was reportedly associated with apoptosis induction in neuroblastoma cells [80]. While several studies have shown that ceramide [141–145] and GD3 [64–66] target the mitochondria directly to promote ROS production and MPT induction, Lovat et al. [80] demonstrated a direct link between GD3 and 12-LOX activation during apoptosis induction in neuroblastoma cells, since the specific 12-LOX inhibitor baicalein blocked both fenretinide- and exogenous GD3-induced apoptosis. In addition, baicalein inhibited ROS production by fenretinide and exogenous GD3 in neuroblastoma cells, suggesting the involvement of 12-LOX in this process. Fenretinide has increased ceramide levels approximately 5- to 10-fold over controls in a variety of cell types [25, 94, 96, 132–134, 138], including neuroblastoma cells where GD3 was proposed to mediate fenretinide-induced apoptosis [80]. It remains to be determined whether the relationship between ceramide, gangliosides, and 12-LOX activity demonstrated during fenretinide-induced apoptosis of neuroblastoma cells [80, 81] is also applicable to other cancer cell types. The depletion of mitochondrial DNA confers resistance to apoptosis induction by ceramide [91] and fenretinide [103], and inhibitors of mitochondrial respiration delayed GD3-induced cell death [65]. Thus, it would be interesting to examine ceramide and/or GD3 levels in respiration-deficient cells following fenretinide exposure. This should establish if the possible sensitivity to these proapoptotic intermediates is a cause or consequence of diminished OXPHOS in tumor cells. Furthermore, if fenretinide can promote ROS production by isolated mitochondria, it would also be illuminating to examine ceramide generation by these organelles, since Springer

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mitochondrial ceramide production may result from aberrant autochthonous ROS generation [92]. The role of stress-related protein signaling pathways In fenretinide-treated cells, signals like enhanced ROS, ceramide, and/or GD3 may trigger the activation of cellular stress response pathways. Many proteins, including various transcription factors and kinases, are responsive to stressful conditions in cells. Once active, these proteins can determine a cell’s fate through the initiation of apoptosis. For example, the transcription factors p53 [146], GADD153 [58], and nuclear factor-kappa B (NF-κB) [147] have all been implicated in apoptotic regulatory mechanisms associated with oxidative and/or genotoxic stress and in various cell systems. The proapoptotic activity of fenretinide appears to be mediated by NF-κB in prostate carcinoma [148] and neuroblastoma [149], cells since pharmacological or genetic inhibition of this transcription factor decreased fenretinide’s cytotoxicity. Yet, a peptidic inhibitor of the nuclear translocation of NF-κB was unable to modulate the fenretinide-induced loss of
m and cell death in cervical carcinoma cells. Similarly, a B-cell line engineered with an inducible inhibitor of NF-κB (IκB) exhibited the same kinetics of fenretinide-induced
m loss and apoptosis irrespective of IκB expression [93], and hepatoma cells were sensitized to fenretinide-induced apoptosis via the pharmacological inhibition of NF-κB [150]. These results suggest that the involvement of NF-κB in fenretinide-induced apoptosis may be dependent on cell type. The p53 status of tumor cells may dictate their responsiveness to certain types of chemoprevention [151] and therapy [152]. The observation that fenretinide can exert chemopreventive activity in p53-deficient mice [151], or in animals inoculated with p53-deficient tumor cells [153], suggest that p53 was dispensable for these effects. In several cell types p53 expression reportedly remained unchanged during apoptosis induction by fenretinide [22, 25, 138, 154, 155]. In colon carcinoma cells that either expressed p53, or lacked p53 expression because of homologous recombination, there appeared to be no difference in sensitivity to fenretinideinduced apoptosis [93]. Furthermore, two colon carcinoma cell lines that were selected for oxaliplatin resistance were as sensitive as their parental counterparts to fenretinide-induced apoptosis, indicating that fenretinide may overcome cellular resistance to conventional chemotherapy [93]. However, p53 activity may be needed for fenretinide-induced apoptosis in certain cell types as shown for bladder carcinoma cells [156]. Fenretinide treatment has been shown to promote the induction of GADD153 in neuroblastoma [97, 99, 110, 157] and hepatoma [158, 159] cells, as well as carcinoma cells derived from skin, pharynx, and cervix [160]. Both N-acetylcysteine and GADD153 antisense oligonucleotides Springer

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inhibited the induction of GADD153, and protected hepatoma cells from fenretinide-induced apoptosis [159]. Similarly, the induction of GADD153 was blocked by vitamin C and 12-LOX inhibitors in neuroblastoma cells, which also suppressed fenretinide-induced apoptosis [101]. These results suggested that the induction of GADD153 was dependent on 12-LOX-induced ROS production in fenretinidetreated neuroblastoma cells. Once activated, GADD153 caused MMP and apoptosis via the induction of Bak [97, 99]. In neuroblastoma cells, this mechanism of apoptosis induction by fenretinide was believed to be unique because it contrasted form those triggered by conventional chemotherapeutic drugs like cisplatin, carboplatin, or etoposide [157]. The c-Jun N-terminal kinase (JNK) and p38 mitogenactivated protein kinase (p38MAPK ) are activated by a wide range of cellular stresses including ROS, UV irradiation, cytokines, osmotic shock, mechanical injury, and chemical agents [161]. Fenretinide caused the activation of JNK and p38MAPK in neuroblastoma cells, which was suppressed by pre-treating these cells with vitamin C [95]. We have also observed a similar phenomenon in fenretinide-treated head and neck carcinoma cells [162]. In prostate cancer cells, JNK activation also followed fenretinide treatment [148, 163]. However, Chen et al. [163] showed this process was not antioxidant sensitive, implying an ROS-independent mechanism of JNK activation by fenretinide. Fenretinide-induced apoptosis in prostate cancer cells was decreased concomitant with the expression of dominant negative JNK [148, 164]. We have also observed the suppression of fenretinide-induced apoptosis in head and neck carcinoma cells by down regulating JNK or p38MAPK activity using chemical inhibitors or small interfering RNAs [162]. The suppression of fenretinide-induced apoptosis in sarcoma cells was also observed concomitant with the pharmacological inhibition of p38MAPK [108]. A kinetic analysis of JNK activation in skin carcinoma cells revealed a maximal threefold increase over controls after a 2-hour exposure to fenretinide, which subsequently declined to basal levels after 5- and 10-hour exposures. The activity of JNK was biphasic, and increased again by two-fold over control after a 20-hour exposure to fenretinide. Based on these findings, Ulukaya et al. [78] concluded that JNK activation may not contribute to the apoptotic response triggered by fenretinide in skin carcinoma cells. Considering that only 50% of these cells were viable following a 24-hour exposure to fenretinide [78], it could be argued that JNK activation was associated with cell survival rather than apoptosis induction. The activation of the extracellular signal-regulated kinase (ERK) occurs in association with oxidative stress-mediated cell death [165, 166]. ERK activation was required for the testosterone-induced sensitization of prostate carcinoma cells to fenretinide-induced apoptosis [167]. Our work with

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head and neck carcinoma cells has revealed that fenretinide treatment also promoted ERK activation, which occurred with kinetics similar to JNK and p38MAPK activation. The activation of ERK in head and neck carcinoma cells was diminished by co-treatment with antioxidants, suggesting that ROS production was associated with this process. We also found that the pharmacological inhibition of ERK activity resulted in a decrease in the fenretinide-induced cleavage of procaspase-9 and poly(ADP-ribose) polymerase (PARP, a substrate of caspase-3) in head and neck carcinoma cells [162]. These findings suggest that ERK activation is required for fenretinide-induced apoptosis in prostate and head and neck carcinoma cells. Conversely, constitutive ERK activation conferred resistance to fenretinide-induced apoptosis in prostate cancer cells, and the pharmacological inhibition of ERK activity sensitized these cells to fenretinide-induced apoptosis [148]. The signal transduction pathway regulated by both JNK and p38MAPK terminates with the activation of the transcription factor activator protein-1 (AP-1). AP-1 regulates various cellular processes, including cellular proliferation, transformation, and cell death, by the activation of target genes [168]. Shimada et al. [167] has shown that the testosteroneinduced sensitization of prostate cancer cells to fenretinideinduced apoptosis requires AP-1 activity and the induction of c-Jun, since antisense oligonucleotides of c-Jun blocked both the process of sensitization and apoptosis in these cells. The AP-1 constituent c-Fos has also been found to regulate apoptosis induction by fenretinide in ovarian cancer cells [140]. Both fumonisin B1 and the expression of dominant negative c-Fos caused a decrease in fenretinide-induced AP-1 activation and apoptosis, suggesting that AP-1 mediated a stress response associated with apoptosis induction in these cells [140]. Collectively, these observations suggest that certain stress response pathways may be essential for fenretinide-induced apoptosis in some cells. Yet, the activation of said pathways might also confer resistance to fenretinide-induced apoptosis in other cells. Little has been reported with regard to how these stress response mediators regulate the effector and/or degradation phases of fenretinide-induced apoptosis. Thus, further investigations are warranted to examine the possible roles of these pathways in the regulation of cell survival or apoptosis in response to fenretinide.

The regulation of MMP during fenretinide-induced apoptosis The role of the MPT The dissipation of
m is typically measured in intact cells by the decreased retention of lipophilic cationic fluorescent

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dyes like rhodamine 123 or 3,3 -dihexyloxacarbocyanine iodide [104 , 169]. Exposure to fenretinide causes the rapid dissipation of
m , indicative of MPT induction, before caspase activation in various cell types [93, 102, 103, 106, 109, 170, 171]. Although, in neuroblastoma [79] and skin carcinoma [78] cells caspase activation and apoptosis by fenretinide apparently occurred without the marked dissipation of
m . In prostate and breast cancer cells, fenretinide caused a conspicuous decrease in mitochondrial cyclophilin D expression prior to apoptosis induction [172 ]. As observed with ROS generation, antioxidants suppressed the rapid dissipation of
m [93 , 102–104, 106, 109, 170, 171] and the loss of cyclophilin D expression [172] following fenretinide exposure, suggesting that oxidative stress was responsible for these effects. It would be prudent to further examine the role of the PTPC constituent cyclophilin D in the process of fenretinide-induced MPT and apoptosis, since the loss of this matrix protein provided mitochondrial protection against MPT, and diminished cell death during Ca2+ and ROS-induced stress [173]. Furthermore, the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, which binds to the adenine nucleotide translocase to regulate the sensitivity of the PTPC [174], was effective in inhibiting the loss of
m and preserving the viability of cervical carcinoma cells exposed to fenretinide [93 ], suggesting a role for the PTPC in fenretinideinduced MPP. Interestingly, T-cells infected with the T-cell lymphotropic virus type I exhibited resistance to the loss of
m and ceramide production triggered by fenretinide [20], implying that a viral protein may be regulating the sensitivity of MPT induction in these cells. Furthermore, lymphocytes infected with the T-cell lymphotropic virus type I also reportedly overexpress galectin-3 [175], which may confer resistance to MMP and mitochondrial-mediated apoptosis [111]. Pharmacological inhibition of the PTPC promoted variable responses when examined in fenretinide-induced MPT and apoptosis. Antagonists of the MPT (i.e., cyclosporin A and bongkrekic acid) delayed the loss of
m and apoptosis in fenretinide-treated skin [102, 104] and ovarian [171] carcinoma cells. In breast cancer cells, cyclosporin A had no effect on [109], or enhanced [123], apoptosis induction by fenretinide. Furthermore, bongkrekic acid enhanced, and the PTPC agonist atractyloside inhibited, fenretinide-induced MPT and apoptosis in hepatoma cells [113]. Inhibitors like cyclosporin A and bongkrekic acid typically offer only transient protection from MPT induction by various stimuli in isolated mitochondria [176], and long-term exposure to modulators of the PTPC can promote toxicity in intact cells. [177] For instance, cyclosporin A is commonly recognized as an inhibitor of OXPHOS in vivo [178]. This activity may develop because of the impedance of mitochondrial ADP/ATP exchange by cyclosporin A [179], which may explain why this Springer

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agent also inhibits ADP-stimulated respiration in isolated mitochondria [180] and suppresses oxygen consumption by intact cells [103,177]. Consequently, the use of pharmacological modulators of the PTPC in intact cells for experiments exceeding a few hours in duration may not necessarily provide sufficient protection against MPT induction and the loss of viability. Interestingly, fenretinide was unable to promote mitochondrial swelling in freshly-isolated BALB/c mouse liver mitochondria that were oxidizing succinate (rotenone was included in the reaction buffer to prevent reverse electron flow through complex I), or trigger the permeabilization of PTPC-containing proteoliposomes [93]. These observations suggested that fenretinide’s mitochondrial effects were indirectly mediated via ROS generation in intact cells [93]. Nevertheless, fenretinide-induced ROS production could be reduced in intact cells co-treated with rotenone [21, 103] or certain coenzyme Q analogues [103], implying that the turnover of complex I may contribute to the prooxidant activity of fenretinide [103]. Experimental methodologies using isolated mitochondria, submitochondrial particles, and/or proteoliposomes containing purified mitochondrial proteins are undoubtedly state-of-the-art with respect to establishing the direct mitochondriotoxicity of agents like fenretinide. Still, there are limitations for many of theses assays that potentially require additional validation of reputed mitochondrial effects in a cellular context. Consequently, the direct and/or indirect mitochondrial effects of fenretinide may be challenging to elucidate fully [1]. The role of Bcl-2 family members In certain cell systems, the release of AMPs like cytochrome c appears to act in an all-or-nothing fashion with respect to caspase activation and apoptosis induction [181–184]. Furthermore, in addition to MMP, a distinct reorganization of the mitochondrial cristae [185] and/or the oxidation of cardiolipin [88] may also be required to liberate sufficient cytochrome c to trigger caspase activation and induce apoptosis [185]. The regulation of MMP by the MPT or Bcl-2 family members constitutes two distinct processes that potentially have a common origin. We have previously discussed the role of ROS generation in the induction of the MPT. Similarly, the modulation of MMP by Bcl-2 family members has been linked to enhanced ROS generation and oxidative stress in several cell systems [147, 186–190]. Hence, during apoptosis induction by certain mechanisms, ROS generation may be rate limiting in the regulation of MMP by both the MPT and Bcl-2 family members. Furthermore, we would content that the two principal players in MMP could theoretically co-participate in the induction

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of apoptosis during conditions of oxidative stress, since MMP and ROS can potentially participate in a feed-forward amplification loop where an increase in ROS causes MPT in a subset of mitochondria, which leads to additional ROS production and further modulation of MMP by redoxrelated changes in proapoptotic Bcl-2 family members [93]. In this regard, fenretinide-induced ROS production was instrumental for the loss of
m and apoptosis induction in cervical cancer cells. All of the apoptogenic effects of fenretinide, including ROS generation, were suppressed in these cells concurrent with the overexpression of Bcl-2 [93 ]. Similarly, in transformed mouse embryo fibroblasts that were null for Bax and Bak, the prooxidant and proapoptotic effects of fenretinide were diminished. These observations suggested that ROS caused proapoptotic conformational changes in both Bax and Bak, and the Bcl-2-sensitive translocation of Bax to the mitochondria resulting in MMP [93]. However, there is a conundrum regarding how the aforementioned Bcl-2 family members regulated both fenretinide-induced ROS generation and MMP in certain cell types, which perhaps will be resolved in the future [93]. It may well be determined that there is a causal link between certain Bcl-2 family members and the regulation of OXPHOS, discussed previously, which is required for fenretinideinduced ROS production and apoptosis in certain cell types. The ROS-dependent, antioxidant sensitive induction of Bak was required for fenretinide-induced apoptosis in neuroblastoma cells [97, 99]. This process was blocked by Bak antisense oligonucleotides implying a critical role for Bak in fenretinide-induced apoptosis [97]. The antioxidant sensitive suppression of Bcl-2 expression and induction of Bax have been observed in prostate carcinoma cells exposed to fenretinide [191]. The fenretinide-induced loss of Bcl-2 expression has also been reported in breast [111, 192], cervical [193], and bladder [194] carcinoma cells, and Bcl-XL expression was decreased during fenretinide-induced apoptosis in hepatoma cells [195]. Furthermore, Bcl-XL overexpression diminished the synergistic effect of fenretinide on TRAIL-induced apoptosis in ovarian carcinoma cells [170]. On the other hand, the modulation of Bcl-2 family members was evidently not associated with apoptosis induction by fenretinide in leukemia cells [21, 154, 196], as well as carcinoma cells derived from the cervix [24] and lung [155]. Perhaps in certain cell types were Bcl-2 family members predominate in the regulation of fenretinide-induced MMP and apoptosis, Bcl-2 inhibitors like the natural product tetrocarcin A [197], the synthetic molecule HA14-1 [198], or Bcl2 antisense oligonucleotides (e.g., Genasense [199]) may be effective in sensitizing these cells to the cytotoxicity of fenretinide.

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The regulation of cellular degradation during fenretinide-induced apoptosis The role of caspases Caspases are constitutively present in most mammalian cells, and they reside in the cytosol as single chain proenzymes or zymogens. Over a dozen caspases have been identified in mammalian cells, and approximately two-thirds of these have been suggested to function in apoptosis [200]. In most reports, the evidence for caspase activity in fenretinidetreated cells is indirect (e.g., the cleave of PARP, nucleosomal DNA fragmentation, and/or procaspase-3 processing) demonstrating that the cells could activate caspases during apoptosis rather than proving caspase activity was required for the process. Exposure to fenretinide promoted the cleavage of the zymogen of caspase-3 to yield the active form of caspase-3 in head and neck [201], lung [201], ovarian [171], and cervical [106] carcinoma cells, to name a few. The processing of caspase-3 has been reported to promote the subsequent cleavage of PARP in leukemia [26] and breast cancer cells [111]. In skin cancer [103] and leukemia [93] cells, exposure to fenretinide also promoted the cleavage of a fluorogenic caspase substrate that was presumably mediated by caspase-3 activity. Furthermore, in leukemia cells fenretinide caused an increase in caspase-3 activity that was apparently due to enhanced zymogen stability. This process was believed to be associated with a mechanism unrelated to ROS production or oxidative stress [107]. In many cell types, the fenretinide-induced activation of caspase-3 is reportedly accompanied or preceded by the activation of caspase-9, implying that cell death was mediated, at least in part, via the intrinsic pathway of apoptosis [78, 170, 171]. In this regard, fenretinide enhanced TRAIL-mediated apoptosis in ovarian cancer cells through the recruitment of a mitochondrial-dependent amplification loop involving the participation of caspase-9 [170]. Furthermore, caspase-8 activation was observed in human T-cell lymphotropic virus type I-transformed T-cells, which was attributed to a feedback activation mechanism regulated by caspase-3 and/or caspase-9 [20]. Interestingly, fenretinide treatment promoted the activation of caspase-8 in ovarian cancer cells that was believed to occur through enhanced ceramide production, and independent of death receptor activation. Yet, neither a cell permeant peptidic caspase-8 inhibitor (i.e., z-IETD-fluoromethylketone) nor the expression of the viral caspase-8 inhibitor cytokine response modifier A (CrmA) was able to suppress fenretinide-induced apoptosis in these cells [202]. The activation of caspase-8 was also observed during fenretinide-induced apoptosis in Fasdefective hepatoma cells [195]. Caspase-8 appeared to regulate cytochrome c release from the mitochondria, caspase-9 activation, and apoptosis following fenretinide exposure. All

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of these effects appeared to develop independently of Bid processing or the induction of Bax in hepatoma cells. Furthermore, the suppression of fenretinide-induced apoptosis was achieved by the expression of CrmA or the dominantnegative form of FLICE, implying that fenretinide may be able to overcome apoptosis resistance in certain cells through the direct activation of caspase-8 [195]. Interestingly, fenretinide also promoted an increase in DR5 expression in primary meningioma cells besides triggering the MPT. This would suggest that death receptor/caspase-8 activation could be one mechanism through which fenretinide encourages cellular degradation in meningioma cells [203]. In light of these results, it would be interesting to examine fenretinideinduced apoptosis in Jurkat cells and their caspse-8-deficient derivatives [204] to determine if caspse-8 per se can directly determine sensitivity to fenretinide. The role of caspases-independent degradation Certain degradative processes associated with fenretinideinduced apoptosis appear to be independent of caspase activity. As mentioned previously, ceramide and GD3 appear to be responsible for caspase-independent MMP in certain cell systems. Fenretinide exposure can trigger the caspaseindependent release of cytochrome c from the mitochondria in neuroblastoma [79] and leukemia [21] cells, as well as carcinoma cells derived from skin [78], cervix [93, 106, 193], breast [111], and ovary [170]. Similarly, the MPT appears to be initial caspase-independent degradative process associated with fenretinide-induced apoptosis in skin [102–104], cervical [93, 106], colon [93], and ovarian [171] carcinoma cells and transformed B-cells [93]. Furthermore, using electron microscopy we have observed mitochondrial swelling, nuclear envelope collapse, and peripheral nuclear chromatin condensation in skin carcinoma cells following short-term (i.e., two hours) expose to 10 µM fenretinide (N. Hail, Jr. and R. Lotan, unpublished observations) or other putative chemopreventive agents [112, 205, 206] well before marked caspase activation, suggesting that caspase-independent processes contribute to apoptotic degradation in these cells. Several AMPs can promote degradation of cellular molecules in the absence of caspase activation. For instance, the flavoproteins AIF [38] and AMID [39] can trigger chromatin condensation and large-scale DNA fragmentation in a caspase-independent manner once they are liberated form the mitochondria [39]. These proteins can also promote MMP and the release of caspase-activating factors like cytochrome c in a feed-forward amplification loop that is believed to accelerate apoptosis [38]. Endonuclease G, a matrix protein normally involved in mitochondrial DNA synthesis, can also cause caspase-independent large-scale DNA fragmentation when released from the mitochondria [34]. Since the degradation phase of fenretinide-induced apoptosis may be Springer

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mediated to some extent by caspase-independent processes, it would be salient to examine the possible kinetics and consequences of the release of AIF, AMID, and/or endonuclease G following fenretinide-induced MMP. This could be compared and contrasted with cytochrome c release and/or caspase-9 activation to determine the relative contribution of each of these regulatory intermediates in fenretinide-induced apoptosis.

Conclusions and future perspectives If apoptosis can be considered an important target of cancer chemoprevention or chemotherapy, it is imperative to advance the characterization of apoptosis mechanisms. As the understanding of these processes increases, so to will come the opportunity to develop novel chemicals that can selectively manipulate them. In this regard, fenretinide is a potent synthetic retinoid because of its anticancer activity, which may be largely dependent on its ability to engage apoptosis pathways in transformed cells, and its relative lack of adverse side effects in vivo. Many of the apoptogenic pathways triggered by fenretinide are almost certainly cell type dependent. This may be especially true in neuroblastoma cells [81]. We believe the continued characterization of the mechanism(s) associated with fenretinide-induced apoptosis is relevant considering the possible usefulness of fenretinide as a chemopreventive or chemotherapeutic agent. This activity may well contribute to the further development of mechanism-based strategies for the chemoprevention of cancer. Furthermore, given the 170 or so reports of fenretinide’s proapoptotic activity in cultured cells that have been published in the past 10 years, it would seem that this agent could serve as a model for the study of cell death in vitro much in the same way that staurosporine has. As we have discussed in this review, several discrete signaling intermediates have been identified in the proapoptotic activity of fenretinide, many of which may well depend on cell type. The pathways constituting these signaling intermediates are diagrammed in Fig. 4. If we were to take a reductionist approach, ROS production and MMP could reasonably be predicted to be involved, at least in part, in apoptosis induction by fenretinide in most cell systems. However, this would probably oversimplify fenretinide-induced apoptosis given what we know the apparent complexity of this process today. The question marks indicated in the pathways depicted in Fig. 4 suggest that there are upstream and/or downstream factors besides the potential key intermediates of ROS and MMP that remain to be elucidated for apoptosis induction by fenretinide. Expectantly, we have provided some rationale for addressing these questions in this review. Future studies should endeavor to offer additional mechanistic information to help fill these gaps, which could ultimately determine Springer

Fig. 4 Signaling pathways proposed for fenretinide-induced apoptosis in transformed, premalignant, and malignant cells in vitro. Please refer to the text for details. Abbreviations: OXPHOS, oxidative phosphorylation; RARs, retinoic acid receptors; ROS, reactive oxygen species; GD3, ganglioside GD3; 12-LOX, 12-lipoxygenase; GADD153, growth arrest and DNA damage-inducible transcription factor 153; and MMP, mitochondrial membrane permeabilization

the clinical relevance of each pathway in the prevention or treatment of cancer. This information should also contribute to future studies of possible synergism between fenretinide and other candidate chemopreventive agents (e.g., tamoxifen [138, 139] and CDDO [112]) with complementary mechanisms of action, and provide further support for the use of fenretinide to oppose cancer. References 1. Hail N Jr (2005) Mitochondria: a novel target for the chemoprevention of cancer. Apoptosis 10:687–705 2. Sun SY, Hail N Jr, Lotan R (2004) Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst 96:662–672 3. Sporn MB (1976) Approaches to prevention of epithelial cancer during the preneoplastic period. Cancer Res 36:2699–2702 4. Kelloff GJ, Crowell JA, Steele VE et al (1999) Progress in cancer chemoprevention. Ann NY Acad Sci 889:1–13 5. Hong WK, Sporn MB (1997) Recent advances in chemoprevention of cancer. Science 278:1073–1077 6. Thun MJ, Henley SJ, Patrono C (2002) Nonsteroidal antiinflammatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst 20:252– 266 7. Fabian CJ, Kimler BF (2001) Beyond tamoxifen new endpoints for breast cancer chemoprevention, new drugs for breast cancer prevention. Ann NY Acad Sci 952:44–59

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