Mechanisms of vanilloid-induced apoptosis - Springer Link

Apoptosis 2003; 8: 251–262 C 2003 Kluwer Academic Publishers

Mechanisms of vanilloid-induced apoptosis Numsen Hail, Jr. Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA

Chemical compounds that contain the vanillyl moiety (4hydroxy-3-methoxybenzyl) are collectively classified as vanilloids. Vanilloid phytochemicals can be found in a variety of sources, some of which are routinely consumed by humans throughout the world. The dietary and/or medicinal use of vanilloids may be effective in inhibiting or reversing carcinogenesis, which has sparked a considerable interest in these compounds as potential chemopreventive or chemotherapeutic agents. Certain vanilloids are also valuable as pharmacological tools for investigating neurobiology, and have been proven effective in alleviating neurogenic pain and inflammation. Recently several vanilloids have demonstrated the ability to induce apoptosis in various cell types. Vanilloids can interact with proteins and membranes to initiate pleiotropic effects, some of which are potentially cytotoxic. Certain vanilloids bind to cation channels on nociceptive sensory neurons to regulate Ca2+ uptake, which can promote neurotoxicity resulting in apoptosis and necrosis. Furthermore, some vanilloids appear to interfere with enzymatic processes in the plasma membrane and the mitochondria by functioning as coenzyme Q antagonist. This can promote reactive oxygen species production and/or the disruption of redox homeostasis resulting in apoptosis. This review will examine the cellular targets, cytotoxic effects, and the downstream effector mechanisms associated with vanilloid-induced apoptosis. Keywords: Apoptosis; mitochondrial respiration; plasma membrane NADH-oxidoreductase; vanilloid receptors; vanilloids.

Introduction Studies of natural products provide opportunities to reveal interesting biology and generate leads pertaining to specific cellular targets, activities, and therapeutic manipulations.1 Several pungent phytochemicals are classified as vanilloids, compounds that contain the vanillyl moiety (4-hydroxy-3-methoxybenzyl). For example, capsaicin (Figure 1), the pungent constituent of hot pepCorrespondence to: N. Hail, Department of Thoracic/Head and Neck Medical Oncology, Box 432, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030-4095, USA. Tel: (713) 792-8950; Fax: (713) 7927976; e-mail: [email protected]

pers of the genus Capsicum, and resiniferatoxin, a complex diterpenoid isolated from the latex of certain succulent African Euphorbias are considered archetypal vanilloids.2 Additional phytochemicals representing this class of compounds include curcumin (a constituent of turmeric (Curcuma longa),3 gingerol (derived from the rhizome of ginger (Zingiber officinale),4 and paradol (isolated from the rhizome of ginger and the seeds of Grains of Paradise (Aframomum melegueta).4 Vanilloid-containing botanical products have been used as medicinal agents since antiquity.2,5 For instance, oral solutions containing red pepper powder are employed as a traditional South African remedy for gastrointestinal illnesses,6 Euphorbia latex has been used topically to remove unwanted cutaneous features like warts and freckles,5 turmeric is used in traditional Indian medicine for a wide range of therapeutic utilities,7 and preparations from the rhizome of ginger have been used in traditional oriental medicine to ameliorate such symptoms as inflammation, rheumatic disorders, and gastrointestinal discomfort.4 In contemporary medicine, capsaicin formulations are used topically to treat a variety of diseases associated with neurogenic pain and inflammation,1 and both capsaicin and resiniferatoxin are currently under evaluation in clinical trials for various pathological conditions.2,5 The ubiquitous human consumption of spices and foods containing vanilloids and related phenolic compounds suggest that these agents may also be important as dietary antioxidants and/or anti-inflammatory mediators.8 Certain vanilloids are potentially useful in inhibiting or reversing of carcinogenesis. Capsaicin apparently protects against experimentally induced mutagenesis and tumorigenesis in various model systems, including benzo[a]pyrene-induced pulmonary adenomas and carcinomas in mice,9 4-nitroquinoline 1-oxide-induced tongue carcinoma in rats,10 and vinyl carbamate-induced skin tumors in mice.11,12 Furthermore, curcumin reportedly exhibits chemopreventive properties against azoxymethane-induced colon tumor formation in rats,13 and 12-O-tetradecanoylphorbol-13-acteate (TPA)-induced skin tumors in mice.14,15 Apoptosis is commonly recognized as the mechanism utilized by metazoans to eliminate redundant or Apoptosis · Vol 8 · No 3 · 2003

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N. Hail Figure 1. Chemical structures of selected vanilloid phytochemicals. The vanillyl moiety of capsaicin is circled.

potentially deleterious cells. Thus, apoptosis is considered an essential process for regulating tissue homeostasis. Apoptosis induction is arguably the most potent defense against cancer making it a desirable end point for both chemoprevention16 and chemotherapy.17 It is generally accepted that most antineoplastic agents inhibit the proliferation of transformed cells by inducing apoptosis.17–21 Recently, several vanilloids including capsaicin,22–35 resiniferatoxin,26,30,32,33 gingerol,4 paradol,4 and curcumin7,36–45 have been shown to induce apoptosis in various transformed cell types in vitro. Furthermore, both capsaicin10 and curcumin13,46 reportedly promote apoptosis in vivo as the mechanism of tumor cell elimination in animal models for carcinogenesis. These observations continue to fuel the interest in vanilloids as potential anticancer agents. In addition to their role as pharmacological tools for investigating neurobiology, certain vanilloids (e.g., capsaicin and resiniferatoxin) are also potent neurotoxins,2,47,48 which can promote apoptosis49–51 and necrosis51 in primary sensory neurons when injected subcutaneously. This activity may ultimately provide a therapeutic benefit in the management of certain types of neurogenic pain and inflammation.47,52,53 Capsaicin also caused a marked reduction in thymus weight and inhibited thymocyte differentiation by triggering apoptosis when administered subcutaneously to neonatal rats.54 Apoptosis is a relatively linear process. It is triggered by an initiation phase that is highly varied depending on 252 Apoptosis · Vol 8 · No 3 · 2003

cell type and apoptogenic stimuli (e.g., oxidative stress, DNA damage, ion fluctuations, and cytokines). This is followed by an effector phase where the cell undergoes distinct biochemical changes that result in the systematic activation catabolic enzymes (proteases and nucleases) that ultimately participate in the cleavage of proteins and DNA characteristic of the degradation phase.55–58 It is generally held that caspase (cysteine proteases involved in apoptosis) and nuclease activation is indispensable for the acquisition of the full apoptotic morphology. Furthermore, several elegant studies have established that a variety of different apoptogenic stimuli can activate the effector phase of apoptosis, at least in part, via the permeabilization of mitochondrial membranes.18,59 This can be accomplished by the pore forming activity of proapoptotic Bcl-2 family members (e.g., Bid and Bax) on the outer mitochondrial membrane,60–63 and/or through the induction of mitochondrial permeability transition (MPT), a process associated with the opening of nonspecific proteinaceous pores that increase the permeability of an otherwise intrinsically impermeable inner mitochondrial membrane.59,64,65 These events can disrupt mitochondrial membranes and release soluble mitochondrial proteins like cytochrome c,66 apoptosis-inducing factor (AIF),67 and endonuclease G68 that participate in the biochemical reactions involved in cellular degradation. The loss of cytochrome c from the mitochondria and/or the induction of MPT would be expected to result in bioenergetic catastrophe in respiring cells.55,69,70 Consequently, the events associated with the permeabilization of mitochondrial membranes can be considered the decisive regulatory point in cell death signaling.18,59 An extensive evaluation of the literature and previous experimental observations26 demonstrate that there are potentially several cellular targets for vanilloids in various cell types, many of which can trigger acute cytotoxic effects capable of inducing apoptosis. This review will examine the cellular targets of vanilloids, the cytotoxic effects associated with the interactions of vanilloids at these targets, and the potential downstream effectors mechanisms that contribute to apoptosis induction.

The role of vanilloid receptors in vanilloid-induced apoptosis Many of the acute cellular and physiologic effects associated with vanilloid exposure occur via the interactions between vanilloids and vanilloid receptors, specific membrane recognition sites that act as voltage-independent, relatively non-selective cation channels. Vanilloid receptors are expressed almost exclusively by primary sensory neurons associated with nociception and neurogenic inflammation. It is generally accepted that vanilloid

Mechanisms of vanilloid-induced apoptosis

receptors mediate the effects of certain vanilloids in these cells. These effects are manifested by excitation followed by desensitization due to elevations in intracellular free Ca2+ ([Ca2+ ]i ).2 Vanilloid receptors can become active cation channels once certain vanilloids diffuse across the plasma membrane and bind to the cytoplasmic domains of these receptors.71,72 In addition to the archetypal vanilloids capsaicin and resiniferatoxin,2 gingerols have also demonstrated the ability to increase [Ca2+ ]i through the activation of vanilloid receptors.73 It is commonly known that excessive Ca2+ influx triggered by vanilloid exposure can disrupt Ca2+ homeostasis and promote excitotoxicity in nociceptive neurons.48,52,74–77 Depending on the concentration, duration of exposure, and route of administration, certain vanilloids (e.g., capsaicin and resiniferatoxin) may either desensitize the nociceptive nerve ending or completely delete the neuron itself.47,53,76 Elevations in [Ca2+ ]i can mediate apoptotic cell death in various cell systems via the activation of Ca2+ -dependent enzymes like phospholipases, proteases, and endonucleases.78 The subcutaneous administration of capsaicin has been shown to promote DNA fragmentation indicative of apoptosis in nociceptive sensory neurons.49–51 Furthermore, capsaicin reportedly increased [Ca2+ ]i causing the activation of Ca2+ sensitive proteases in cultured rat dorsal root ganglion cells, which was required for cell death.77 Vanilloid-mediated excitotoxicity in nociceptive sensory neurons is also believed to disrupt mitochondrial function.48,79 The mitochondria possess an exceptional capacity to sequester [Ca2+ ]i as a means of reestablishing Ca2+ homeostasis.80 However, this buffering capacity is typically achieved at the expense of mitochondrial respiration; and may initiate MPT following excessive mitochondrial Ca2+ loads. Among the various stimuli that cause MPT, Ca2+ has been investigated extensively and, thus, is considered to be a prototypical inducing agent.18,81,82 MPT allows water and solutes up to 1500 Da to infiltrate the mitochondrial matrix. This results in colloidal osmotic swelling of the matrix,57,83 which can cause the physical rupture of the outer mitochondrial membrane.59,64,65 Several studies have documented mitochondrial swelling in primary sensory neurons treated with capsaicin47,49,50,84,85 and resiniferatoxin.53,86 Furthermore, the Ca2+ influx triggered by capsaicin exposure has been shown to enhance mitochondrial Ca2+ accumulation,79 and dissipate mitochondrial inner transmembrane potential (m )48 in cultured rat dorsal root ganglion cells. Both events could conceivably trigger MPT.83,87,88 Together, these observations illustrate that [Ca2+ ]i can orchestrate events associated with vanilloidinduced neuronal cell degradation. These events may include the activation of Ca2+ -sensitive proteases, MPT induction, and DNA fragmentation that are characteristic of apoptosis.

Excessive Ca2+ influx triggered by vanilloid exposure can also promote necrotic cell death in primary sensory neurons in vivo51 and in vitro,53 and in non-excitable cells transfected to express vanilloid receptors.32,52,53 In these studies, necrotic cell death apparently occurred due to rapid cell swelling and lysis of the plasma membrane.32,51–53 The ability of capsaicin to promote apoptosis or necrosis in primary sensory neurons in vivo may be due to the species-specific expression and sensitivity of vanilloid receptors.51 Indeed, the nociceptive neurons of many mammalian species exhibit marked variability in vanilloid receptor expression as well as the vanilloid binding affinity of these receptors.2 Thus, in addition to vanilloid concentration, duration of exposure, and route of administration, it is likely that the manifestation of apoptosis or necrosis in primary sensory neurons, or in cells transfected to express vanilloid receptors, would ultimately depend on the integrity of the plasma membrane. This may be influenced by the quantity of vanilloid receptors expressed at the plasma membrane, their sensitivity to vanilloid-induced activation, the extent of Ca2+ influx, and the ability of endogenous mechanisms to buffer [Ca2+ ]i . The conflicting reports in the literature implicating apoptosis and/or necrosis in vanilloid-induced cell death, particularly in primary sensory neurons, warrant additional mechanistic studies. These studies may seek to extend the existing data by investigating the role of apoptotic determinants like caspase activation, mitochondrial cytochrome c release, Bcl-2 family members, and/or MPT in vanilloid/vanilloid receptor-mediated cell death. These studies may decisively establish apoptosis as an end point of neurotoxicity in nociceptive neurons, and should provide valuable information regarding the potential pharmacological impact of certain vanilloids in neurobiology and medicine.

Effects of vanilloids on biomembranes All of the vanilloids discussed thus far can partition within the lipid bilayers of biomembranes by virtue of their lipophilicity. This activity has the potential to alter membrane fluidity, redox state, and lipid-protein interactions. For example, capsaicin and resiniferatoxin reportedly promote ion pseudochannel formation in planar lipid bilayers,89 and capsaicin can alter plasma membrane structure and fluidity in a concentration-dependent manner.90–92 The effects of capsaicin on plasma membrane fluidity in intact cells was evidently cell type specific and required a non-lipid extractable plasma membrane component.90 Capsaicin also promoted lipid peroxidation in cultured human endothelial cells, which was associated with cell death.93 Furthermore, curcumin apparently promoted membrane thiol oxidation in isolated Apoptosis · Vol 8 · No 3 · 2003

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rat liver mitochondria resulting in MPT induction,94 and caused nonspecific, apoptosis-independent, alterations in plasma membrane dynamics in human erythrocytes.95 Capsaicin treatment initiated a rapid increase in [Ca2+ ]i , and caused apoptosis in transformed human T cells (i.e., Jurkat cells),31 mitogen-activated human T cells,31 and human glioblastoma cells.23 The increase in [Ca2+ ]i did not appear to be mediated by vanilloid receptors in glioblastoma cells because capsazepine, a prototypical vanilloid receptor antagonist,2 was unable to block this effect.23 Furthermore, [Ca2+ ]i was not believed to be the stimulus that triggered apoptosis in T cells or glioblastoma cells.23,31 It is possible the rapid increase [Ca2+ ]i resulted from a nonspecific disruption of the plasma membrane because the concentrations of capsaicin examined in T cells and glioblastoma cells (200 µM and 250 µM, respectively) were within the concentration range (approximately 100 to 300 µM) reportedly required for capsaicinmediated physical changes in the plasma membranes of rabbit platelets, human erythrocytes, and rat mast cells.90 Capsaicin was evidently unable to increase [Ca2+ ]i at concentrations (100 and 200 µM) that caused marked apoptosis in cells from two human cutaneous squamous cell carcinoma (SCC) cell lines,26 and exposure to 100 µM capsaicin was apparently unable to increase [Ca2+ ]i in human leukemia cells (i.e., HL-60 cells).96 Together, these findings suggests that the Ca2+ mobilizing effect of capsaicin observed in T cells and glioblastoma cells may have been concentration-dependent and/or cell-type specific.

The role of the plasma membrane NADH-oxidoreductase in vanilloid-induced apoptosis Proteins associated with electron transport are consolidated at specific sites in mammalian cells that are involved in bioenergetic processes and the maintenance of redox homeostasis. The plasma membrane NADHoxidoreductase electron transport chain (PMOR) is cyanide-insensitive,22 and believed to comprise a cytoplasmic NADH-coenzyme Q reductase, the lipophilic electron carrier coenzyme Q, and a terminal coenzyme Q oxidase located on the external plasma membrane surface.97 The activity of the PMOR appears to be growth factor- and hormone-responsive in normal cells, whereas the PMOR in tumor cells is constitutively activated.22 The PMOR is reportedly involved in the control of cell growth and proliferation by maintaining the optimal proportions of oxidized and reduced pyridine nucleotides in cells that utilize anaerobic glycolysis to produce ATP (e.g., cells deficient in mitochondrial respiration).97 This mechanism of redox homeostasis is implemented by shuttling electrons from cellular NADH to an extracellular elec254 Apoptosis · Vol 8 · No 3 · 2003

tron acceptor, presumably O2 , resulting in superoxide production.97,98 The induction of PMOR activity in malignant and spontaneously immortalized human cells reportedly caused the reduction of extracellular cytochrome c indicating superoxide generation,97 and there is evidence of an O2 -binding site in the terminal coenzyme Q oxidase located on the external plasma membrane surface of human cervical carcinoma cells (i.e., HeLa cells).99 However, there is no direct evidence supporting extracellular superoxide production by anaerobic cells in vivo.98 The PMOR exhibits two separate enzymatic activities, NADH-ferricyanide reductase activity where extracellular ferricyanide functions as an artificial electron acceptor, and NADH-oxidase activity for which O2 is believed to be the natural extracellular electron acceptor.22,33 The NADH-oxidase activity of plasma membranes isolated from various transformed cells was reportedly inhibited by capsaicin in a concentration-dependent manner.22,29 Capsaicin, dihydrocapsaicin, and resiniferatoxin also inhibited the NADH-oxidase activity of plasma membranes derived from rat liver in a concentration-dependent manner.33,100 These observations suggested that certain vanilloids could impede the NADH-oxidase activity of the PMOR by function as coenzyme Q antagonist in the plasma membrane.22,29,33 In addition to revealing a novel cellular target for capsaicin, dihydrocapsaicin, and resiniferatoxin, these early studies were also instrumental in demonstrating that these agents could initiate apoptosis in transformed cells. The inhibition of the PMOR by capsaicin, dihydrocapsaicin, and resiniferatoxin is thought to redirect the normal electron flow in the plasma membrane. This can promote intracellular reactive oxygen species (ROS, e.g., superoxide, hydroxyl radicals, and hydroperoxide) production, oxidative stress, and redox changes in the plasma membrane that regulate Ca2+ influx.33 The possible involvement of ROS in vanilloid-induced apoptosis has been examined in several elegant studies by Macho, et al.30–32 These studies revealed that Jurkat cells exposed to capsaicin, resiniferatoxin, or the synthetic vanilloid phorbol 12-phenylacetate 13-acetate 20-homovanillate displayed a loss of nuclear DNA, which was preceded by intracellular superoxide production and the dissipation of m .30–32 Both superoxide production and the dissipation of m are invariant biochemical hallmarks of early apoptosis.55 However, given the sequence of these events, that is marked ROS production before a conspicuous dissipation of m indicative of MPT, it appeared that vanilloid exposure initiated ROS production at the PMOR resulting in oxidative stress, which triggered MPT-induced apoptosis.30 Capsaicin treatment reportedly reduced the basal level of ROS generation and lipid peroxidation in human glioblastoma cells prior to apoptosis induction.23 The reduction in basal ROS generation, presumably due to the inhibition of the PMOR,


was believed to disrupt redox homeostasis in these cells causing apoptosis.23,34 Thus, the PMOR-mediated disruption of redox homeostasis, either through the increase or suppression of ROS production, appears to be intimately associated with vanilloid-induced apoptosis in certain transformed cell types. Preincubation of human lymphoblastoid cells with ferric transferrin, an activator of the PMOR, reportedly inhibited capsaicin- and resiniferatoxin-induced apoptosis.33 However, a related study demonstrated that capsaicininduced apoptosis could be enhanced if Jurkat cells were pretreated with the PMOR activator ferricyanide.31 Though conflicting, these observations suggest the involvement of the PMOR in apoptosis induction in these cells. Coenzyme Q pretreatment also protected human lymphoblastoid cells from capsaicin- and resiniferatoxininduced apoptosis implying that these vanilloids were functioning as coenzyme Q antagonists in the PMOR.33 In addition to its role as a lipophilic electron carrier, growing evidence suggest that coenzyme Q can function as an antioxidant during conditions of oxidative stress,101,102 and coenzyme Q may also regulate the sensitivity of MPT during mitochondrial stress.81 Capsaicin and resiniferatoxin appear to cause ROS generation and/or oxidative stress in various transformed cells,3,23,26,30–32,93 which is believed to activate MPT-induced apoptosis.26,30–32 Therefore, it is possible coenzyme Q diminished vanilloidinduced apoptosis in lymphoblastoid cells, at least in part, by functioning as an antioxidant and/or an MPT inhibitor. Certain immunosuppressant drugs (e.g., cyclosporin A and FK-506) reportedly reduced capsaicin-induced apoptosis in human lymphoblastoid cells evidently by blocking the activation of the apoptotic mediator calcineurin.33 However, this study did not determine if capsaicin treatment caused an increase [Ca2+ ]i in these cells. This would have been an important mechanistic clue considering that Ca2+ influx is believed to regulate the activation of calcineurin and other calmodulin-dependent enzymes.103 Preincubation of Jurkat cells with cyclosporin A also blocked capsaicin-induced apoptosis. This activity was attributed directly to the inhibition of MPT31 because, in addition to its immunosuppressant properties,103 cyclosporin A is also a potent MPT inhibitor.87,104,105 Mouse myeloid cells expressing Bcl-2,33 and human leukemia cells over-expressing Bcl-230 were reportedly resistant to capsaicin-induced apoptosis. The presence of Bcl-2 was believed to protect against mitochondrial damage caused by PMOR-mediated oxidative stress.30,33 These findings indicate that certain vanilloids can disrupt the PMOR activity of various transformed cells by functioning as a coenzyme Q antagonists. Oxidative stress appears to activate downstream effectors like calcineurin33 and MPT30–32 resulting in apoptosis. The ability of Bcl-2 and cyclosporin A to oppose capsaicin-induced apoptosis

in several cell types suggests that the mitochondria are important downstream regulators of cell death. The suppression of PMOR activity by certain vanilloids appears to be an important mechanism for inducing apoptosis in several transformed cell types, and may be an effective means of inhibiting or reversing carcinogenesis.22,29,31,33 Revealing the primary regulatory function of the PMOR in tumor cell growth and proliferation is likely to be a focus of future investigations. A cell surface coenzyme Q oxidase has been indentified in HeLa cells that is believed to be a constituent of the PMOR. This protein has been characterized and cloned.99 Molecular techniques should provide a way to knock out or suppress the expression of this protein in HeLa cells to determine if it is required for PMOR activity and/or cell viability. If this experiment does not result in cell death, it would be interesting to determine if the potential loss of PMOR activity could provide a mechanism of resistance against capsaicin-induced apoptosis in these cells.

The role of mitochondrial respiration in vanilloid-induced apoptosis In addition to its inhibitory effects on PMOR activity, capsaicin can also block electron transfer at the NADH: coenzyme Q oxidoreductase (i.e., EC 1.6.99.3, complex I) of the mitochondrial electron transport chain presumably by acting as a coenzyme Q antagonist.106–110 A structureactivity relationship exists among complex I inhibitors (e.g., capsaicin, rotenone, and stigmatellin) because these agents act at or close to the coenzyme Q binding site of the enzyme111–113 much in the same manner predicted for the inhibitors of the PMOR (e.g., capsaicin, dihydrocapsaicin, and resiniferatoxin).22,10 Thus, the functional similarities shared by certain components of the PMOR and the mitochondrial electron transport chain could render them equally vulnerable to inhibition by capsaicin and perhaps other vanilloids. Mitochondria constitute 15 to 50% of the total cytoplasmic volume in most mammalian cells, and they participate in more metabolic functions, especially those involved in cellular ATP production, than any other organelles.114 The mitochondria also consume approximately 90% of cellular oxygen and are a biologically significant source of ROS generation.115,116 The endosymbiont hypothesis of apoptosis evolution proposes that certain proteins, situated in the primitive endosymbiot/host interface, would have played a strategic role in the establishment of endosymbiosis giving rise to mitochondria. Eucaryotic cells would evolve to exploit the bioenergetic windfall associated with endosymbiosis and, with the caveat of nuclear control (e.g., p53 and pro-apoptotic Bcl-2 family members), would also be provided with an intrinsic mechanism for initiating apoptosis through the release of soluble apoptogenic mitochondrial proteins to Apoptosis · Vol 8 · No 3 · 2003

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initiate apoptosis.117 Bioenergetic processes can produce excessive ROS under certain conditions that are potentially deleterious to mitochondrial and other cellular functions. Thus, it has been also suggested that the intrinsic pathway of apoptosis, regulated by MPT, serves a teleolonomic function in metazoans by deleting aberrant ROSproducing cells to facilitate the maintenance of tissue homeostasis.70 The interruption of electron flow at complex I can cause nonenzymatic ROS production due to redox cycling of reduced electron carriers upstream of the inhibition site.116,118 Capsaicin, resiniferatoxin, and several synthetic vanilloids reportedly triggered cyanide-sensitive ROS production in rat leukemia cells suggesting that these agents were interacting at the mitochondrial electron transport chain.3 The initial exposure to capsaicin and resiniferatoxin caused a rapid increase in hydroperoxide production and decreased oxygen consumption by cells from two human cutaneous SCC cell lines (i.e., COLO 16 and SRB-12 cells) well before the apparent manifestations of MPT induction (i.e., a marked dissipation of m , superoxide generation, and caspase activation) and DNA fragmentation indicative of apoptosis.26 2 ,7 -Dichlorofluorescin, the probe used to detect hydroperoxide generation in the SCC cells, can serve as a peroxidase substrate during the dismutation of hydrogen peroxide, and its rate of oxidation to the fluorescent product 2 ,7 -dichlorofluorescein is greatly enhanced by peroxidase activity.119 Thus, vanilloid-induced hydroperoxide production appeared to denote a condition of oxidative stress in SCC cells, which may have been initially modulated, to some degree, by cellular antioxidant enzymes such as superoxide dismutases and peroxidases. If the initial prooxidant effects of capsaicin and resiniferatoxin were uniquely associated with the inhibition of the PMOR, the ensuing oxidative stress could have decreased oxygen consumption by the SCC cells because hydrogen peroxide and other ROS can interfere with electron transport and disrupt mitochondrial respiration.116,120 Conversely, if these vanilloids were directly inhibiting mitochondrial respiration, presumably by blocking complex I activity, similar effects would also be expected. Thus, the question remained as to whether capsaicin and resiniferatoxin triggered oxidative stress and apoptosis in SCC cells by blocking PMOR activity, complex I activity, or both. To directly address the question regarding the possible cause of vanilloid-induced hydroperoxide production, respiration-deficient clones lacking mitochondrial DNA (ρ 0 ) derived from COLO 16 and SRB-12 cells were examined to determine if capsaicin and resiniferatoxin could trigger hydroperoxide production in these cells.26 Mitochondrial DNA encodes specific subunits of the electron transport chain enzymes (including complex I) and the mitochondrial ATP synthase that are required for mi-

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tochondrial respiration.121 Consequently, ρ 0 cells derive their ATP exclusively from glycolysis.122 ρ 0 clones derived from human B-type lymphoblastoid cells have been reported to increase the activity of the PMOR by approximately fourfold to maintain redox homeostasis following the loss of mitochondrial electron transport.123 If this were also the case in the ρ 0 clones derived from COLO 16 and SRB-12 cells, and if the inhibition of the PMOR was uniquely associated with the initial hydroperoxide production observed in the parental SCC cells, it seemed highly possible that capsaicin or resiniferatoxin would stimulate more hydroperoxide generation in the ρ 0 clones than they did in the parental cells. However, this was evidently not the case. The ρ 0 clones that were exposed to capsaicin or resiniferatoxin displayed only a slight, yet discernable and reproducible, increase (approximately 25%) in hydroperoxide generation relative to that observed in controls, which was considerably less that the approximately threefold increase observed during a similar exposure in the parental cells. These results indicated that most of the initial hydroperoxide produced in COLO 16 and SRB-12 cells exposed to capsaicin or resiniferatoxin was of mitochondrial origin and was probably associated with the inhibition of mitochondrial respiration. Furthermore, the ρ 0 clones also exhibited resistance to capsaicin- and resiniferatoxin-induced MPT and apoptosis suggesting that mitochondrial respiration was intimately associated with vanilloid-induced apoptosis in the parental SCC cells.26 Further studies are warranted to determine whether the inhibition of mitochondrial respiration is uniquely associated with the apoptogenic effects of capsaicin and resiniferatoxin in other cell types. It would also be worthwhile to compare the activities of complex I in SCC cells and in normal human epidermal keratinocytes, as well as the relative sensitivities of these cells to vanilloid-induced cytotoxicity.124 ROS can promote divergent cellular effects depending on the extent of their production and the enzymatic or nonenzymatic mechanisms available for their dismutation in a given cell type. Thus, ROS can serve as mitogenic stimuli, senescence promoters, or cell death mediators.125 Even in the absence of mitochondrial respiration, capsaicin and resiniferatoxin still caused some hydroperoxide generation in the ρ 0 SCC cells. Capsaicin and resiniferatoxin also inhibited the proliferation of these cells by promoting G1 arrest. The disruption of PMOR activity may have been responsible for these effects in the ρ 0 SCC cells.26 This was assumed because the PMOR has been implicated in intracellular ROS generation30 and may be required for the proliferation of cells with impaired mitochondrial function.97,123 Additional studies are needed to ascertain the role of the PMOR in the control of cell growth and proliferation in ρ 0 cells and/or cells with recognized defects in mitochondrial respiration.


The role of caspases in vanilloid-induced apoptosis Caspases are believed to be the terminal effectors of apoptosis, which are activated through cell-surface death receptors (e.g., Fas, tumor necrosis factor receptor (TNFR) and/or mitochondrial mediators (e.g., cytochrome c). Caspases are constitutively present in most mammalian cells, and reside in the cytosol as single chain pro-enzymes. Over a dozen caspases have been identified in mammalian cells, and approximately two-thirds of these have been suggested to function in apoptosis.126,127 There are two types of caspases, upstream caspases called initiator caspases (e.g., caspases 8, 9, and 10), and their downstream targets known as effector or executioner caspases (e.g., caspases 3, 6, and 7).127 Caspase activity appears to be associated with vanilloid-induced apoptosis in various cell types. For example, an increase in procaspase-3 cleavage was observed during capsaicin-induced apoptosis in human hepatocarcinoma cells,25 and an increase in caspase-3 activity reportedly occurred in the course of capsaicin-induced apoptosis in H-ras transformed human breast epithelial cells.28 In both cell types, a specific caspase-3 inhibitor, acetylAsp-Glu-Val-Asp-aledhyde (Ac-DEVD-CHO), blocked the antiproliferative effects of capsaicin.25,28 Capsaicin treatment also promoted a decrease in Bcl-2 expression and an increase in Bax expression in hepatocarcinoma cells suggesting that caspase activation was mediated via a mitochondrial pathway.25 Exposure to capsaicin and resiniferatoxin caused DEVDase-like caspase activity in COLO 16 and SRB-12 cells, which was believed to be a consequence of MPT.26 Furthermore, the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-chloromethylkeytone (Ac-DEVD-cmk) blocked DNA fragmentation in Jurkat cells exposed to the synthetic vanilloid phorbol 12-phenylacetate 13-acetate 20-homovanillate. However, the inhibition of DNA fragmentation did not prevent vanilloid-induced cell death in these cells.32 Curcumin triggered apoptosis in Ehrlich’s ascites carcinoma cells43 and HL-60 cells44 apparently by increasing Bax expression, which caused the release of cytochrome c from the mitochondria and the activation of caspase-3. Curcumin reportedly initiated ROS production and MPT in rat histiocytoma cells resulting in caspase-dependent apoptosis,7 which could be blocked by Ac-DEVDCHO.40 Curcumin induced large scale DNA fragmentation in human T cells, which was attributed to the activity of AIF and caspase-3.39 Curcumin induced apoptosis in HL-60 cells by way of caspase-8 activation, Bid cleavage, cytochrome c release from the mitochondria, and the activation of caspase-3.36 In human melanoma cells, curcumin reportedly promoted Fas receptor aggregation in a Fas ligand-independent manner resulting in apoptosis presumably through the activation of caspase-8 and caspase3. A caspase-8 inhibitor and broad-based caspase inhibitor

diminished curcumin-induced cell death in these cells.37 Furthermore, the activation of caspase-8 and caspase-3 has been implicated in curcumin-induced apoptosis in certain human gastric and colon cancer cells.42 However, a novel apoptosis-like pathway, independent of mitochondria and caspases, was seemingly triggered by curcumin in Jurkat cells.45 Further studies are required to establish the upstream targets and singnaling events involved in the intitation of curcumin-induced apoptosis. This information may assist in clarifying the role of Bcl-2 family members, mitochondrial mediators, and death receptors in curcumin-induced caspase activation and apoptosis.

Additional mechanisms of vanilloid-induced apoptosis Several studies have alluded to additional mechanism through which certain vanilloids promote antitumor activity in vivo and in vitro. For example, topical application of capsaicin11,128,129 or curcumin129 can suppress the TPA-induced activation of nuclear factor-kappa B (NF-κB)/Rel and activator protein 1 (AP-1) transcription factors in mouse epidermis. The suppression of these transcription factors by capsaicin and curcumin may account for their chemopreventive effects on both mouse skin tumorigenesis and inflammation.11,12,128,129 Both capsaicin10 and curcumin13,46 reportedly promote apoptosis in vivo as the mechanism of tumor cell elimination in animal models for carcinogenesis. Future studies are warranted to ascertain if the suppression of NFκB and AP-1 activity can be linked to the regulation of capsaicin- or curcumin-induced apoptosis in tumor cells in vivo. Capsaicin treatment markedly activated c-Jun N-terminal protein kinase (JNK)-1 and p38 mitogenactivated protein kinase (MAPK) in H-ras transformed human breast epithelial cells. This activity was believed to mediate capsaicin-induced apoptosis because certain kinase inhibitors, or the overexpression of dominant-negative forms of MAPKs, blocked cell death.28 Exposure to capsaicin reportedly caused the disruption of redox homeostasis, the down-regulation of cyclooxygenase (COX) expression, and apoptosis in human neuroblastoma cells. The down-regulation of COX expression was believed to regulate capsaicin-induced apoptosis because cell death could be enhanced if these cells were pretreated with NS398, a specific COX-2 inhibitor, or indomethacin, a nonspecific COX inhibitor.34 Capsaicin, curcumin, and many naturally occurring phenolic and polyphenolic compounds are believed to function as dietary antioxidants and/or anti-inflammatory mediators.8 However, most of these agents have relatively low redox potentials that could potentially allow them to function as pro-oxidants through the formation of intracellular phenoxyl radicals. This could promote cytotoxic Apoptosis · Vol 8 · No 3 · 2003

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effects via the co-oxidization of GSH and NADH, and the production of secondary ROS.130–132 It is worth mentioning that NF-κB,133–136 AP-1,136,137 JNK-1,138,139 p38,140 and cyclooxygenases141,142 have all been implicated in regulatory mechanisms associated with oxidative stress and apoptosis. Several vanilloids appear to promote ROS production via their interactions with the PMOR and the mitochondrial electron transport chain, or potentially by undergoing redox cycling. Therefore, it is interesting to speculate that most of the additional cellular and molecular effects reportedly associated with vanilloid exposure in vivo and in vitro resulted, at least in part, via the disruption of redox homeostasis.

Figure 2. Mechanisms proposed for capsaicin-induced apoptosis in transformed cells. Please refer to the text for additional details (PMOR, plasma membrane NADH-oxidoreductase electron transport chain; ROS, reactive oxygen species; MPT, mitochondrial permeability transition; COX, cyclooxygenase).

Conclusions and future directions Vanilloids can initiate pleiotropic effects in various biological systems. During the past 25 years, numerous studies have described many important cellular and molecular aspects of vanilloid activity. The elucidation of the mechanisms associated with vanilloid-induced apoptosis is a relatively new and biologically significant focal point of vanilloid research. Capsaicin and resiniferatoxin appear to be useful in managing certain types of neurogenic pain and inflammation via their interactions with vanilloid receptors. Further studies are required to determine if this activity results primarily from the desensitization of nociceptive sensory neurons or through the deletion of these cells by an apoptotic or necrotic mechanism. Capsaicin and curcumin can modulate the process of carcinogenesis in various model systems apparently by triggering apoptosis in tumor cells, and all of the vanilloids depicted in Figure 1 have demonstrated the ability to promote apoptosis in several transformed cell types in vitro. These observations suggest that these agents may be useful in preventing or treating cancer. A structure-activity relationship appears to exist among vanilloids that could account for many of their apoptogenic effects in transformed cells. Capsaicin is perhaps the most extensively investigated vanilloid with respect to its ability to promote apoptosis in various transformed cell types. Several discrete signaling intermediates have been indentified in the pro-apoptotic activity of capsaicin, many of which may well be dependent on cell type. The pathways constituting these signaling intermediates are schematically represented in Figure 2. The gaps in these pathways, denoted by question marks, suggest that there are other upstream and/or downstream factors involved in the process of capsaicin-induced apoptosis. Future studies should endeavor to provide additional mechanistic information to help fill these gaps, which could ultimately determine the clinical relevance of each pathway in the prevention or treatment of cancer. Continued research on the mechanisms of vanilloid-induced apoptosis is anticipated to provide further support for the use of 258 Apoptosis · Vol 8 · No 3 · 2003

these agents in the prevention or treatment of cancer, the alleviation of certain neurologic conditions, and perhaps the mitigation of other pathologic conditions.

Acknowledgment

This work was supported in part by a Cancer Prevention Fellowship sponsored by the NCI Grant R25 CA57780.

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