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The Journal of Immunology, 2004, 172: 6330-6335.
Copyright © 2004 by The American Association of Immunologists

Augmentation of Chemotherapy-Induced Cytokine Production by Expression of the Platelet-Activating Factor Receptor in a Human Epithelial Carcinoma Cell Line1

Marc Darst*, Mohammed Al-Hassani*, Tao Li*,{dagger}, Qiaofang Yi*, John M. Travers*,{dagger}, Davina A. Lewis* and Jeffrey B. Travers2,*,{dagger},{ddagger}

Departments of * Dermatology and {dagger} Pediatrics, H. B. Wells Center for Pediatric Research, and {ddagger} Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In addition to their known cytotoxic effects, chemotherapeutic agents can trigger cytokine production in tumor cells. Moreover, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors (PAF-R) linked to cytokine production, it is not known whether PAF is involved in chemotherapeutic agent-induced cytokine production. These studies examined the role of the PAF system in chemotherapy-mediated cytokine production using a model system created by retroviral-mediated transduction of the PAF-R-negative human epidermal carcinoma cell line KB with the human PAF-R. The presence of the PAF-R in KB cells resulted in augmentation of the production of cytokines IL-8 and TNF-{alpha} induced by the chemotherapeutic agents etoposide and mitomycin C. These effects were specific for the PAF-R, as expression of the G protein-coupled receptor for fMLP did not affect chemotherapeutic agent-induced cytokine production. Moreover, ablation of the native PAF-R in the epithelial cell line HaCaT using an inducible antisense PAF-R strategy inhibited etoposide-induced cytokine production. Oxidative stress and the transcription factor NF-{kappa}B were found to be involved in this augmentative effect, because it was mimicked by the oxidant tert-butyl-hydroperoxide, which was blocked both by antioxidants and by inhibition of the NF{kappa}B pathway using a super-repressor I{kappa}BM mutant. These studies provide evidence for a novel pathway by which the epidermal PAF-R can augment chemotherapy-induced cytokine production through an NF-{kappa}B-dependent process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recent studies have indicated that in addition to their known cytotoxic effects, chemotherapeutic agents can trigger cytokine production in a variety of cell types in vitro (1, 2). The significance of tumor cell-derived cytokine production in the therapeutic effectiveness or side effect profile of chemotherapeutic agents is unclear. However, significant levels of cytokines, including IL-8, TNF-{alpha}, and others, can be found in patients undergoing chemotherapy for a variety of tumors (3, 4).

Accumulating evidence suggests that platelet-activating factor (PAF;3 1-alkyl-2-acetyl glycerophosphocholine)-mediated pathways are involved in cutaneous inflammation and epithelial cell stress responses. PAF is a glycerophosphocholine-derived lipid mediator implicated in numerous inflammatory processes (reviewed in Refs. 5 and 6). Epithelial cells synthesize PAF and related 1-acyl-PAF-like species in response to various stimuli, including ionophores, growth factors, PAF agonists, the pro-oxidative stressor tert-butyl-hydroperoxide, UV light irradiation, and acute thermal damage (7, 8, 9, 10, 11, 12). Although PAF can be metabolized to other biologically active lipids, the majority of PAF effects appear to be mediated by interaction with a seven-transmembrane G protein-coupled receptor (GPCR), the PAF receptor (PAF-R) (reviewed in Ref. 13). In addition to producing PAF, keratinocytes and many carcinoma cell lines also express the PAF-R (14, 15). Activation of the epidermal PAF-R leads to the production and release of PAF, IL-6, IL-8, IL-10, TNF-{alpha}, and eicosanoids (12, 16, 17).

In addition to a tightly coupled enzymatic pathway for PAF synthesis involving the subsequent actions of phospholipase A2 and acetyltransferase, PAF and PAF-like lipids can be produced via direct free-radical mediated cleavage of glycerophosphocholines (GPC) containing unsaturated fatty acids (e.g., arachidonate) at the sn-2 position (18). Recent studies have suggested that the PAF-R is a target for UV B radiation in part through this nonenzymatic pathway of PAF agonist formation (12, 17, 19, 20). For example, the presence of the PAF-R augments the production of cytokines IL-8 and TNF-{alpha} as well as the apoptotic response of the known pro-oxidative stressor UV-B radiation in carcinoma cell lines, processes inhibited by antioxidants (12, 19, 20).

The molecular mechanisms involved in chemotherapeutic agent-mediated cytokine production are unknown. However, inasmuch as other proapoptotic mediators, including chemotherapeutic agents, are also potent pro-oxidative stressors, the objective of these studies was to assess whether the epidermal PAF-R could augment cytokine production induced by chemotherapeutic agents. Using the PAF-R-negative human carcinoma cell line KB transduced with the PAF-R, these studies demonstrate that the PAF-R can augment cytokine production due to etoposide and mitomycin C. The mechanism of how PAF-R activation could augment chemotherapy-induced cytokine production was also examined in these studies and was found to be dependent on activation of NF-{kappa}B proteins, sequence-specific transcription factors induced in response to inflammatory and other stressful stimuli (reviewed in Ref. 21). The current findings describe a putative mechanism by which this GPCR can augment the cytokine-producing effects of chemotherapeutic agents in epithelial carcinomas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise.

Cell culture

The human epidermoid cell line KB was grown in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS (HyClone, Logan, UT). A KB PAF-R model system was created by transduction of PAF-R-negative KB cells with the MSCV2.1 retrovirus encoding the human leukocyte PAF-R as described previously (16). KB cells transduced with the PAF-R (KBP) or with control MSCV2.1 retrovirus (KBM) were characterized by Southern and Northern blot analyses and by radioligand binding and calcium mobilization studies to demonstrate that the PAF-R was functional (16). Similarly, to create a KB cell line expressing the fMLP receptor (KBF), the fMLP-R cDNA was cloned into the MSCV2.1 retroviral vector. The presence of a functional fMLP-R in KBF cells was confirmed by Northern blotting to demonstrate fMLP-R mRNA and a positive intracellular calcium mobilization response to exogenous fMLP using the fluorescent dye fura-2-acetoxymethyl ester as previously described (22). All experiments were replicated with at least two separate KBM, KBP, and KBF clones.

Generation of HaCaT cells expressing an inducible PAF-R antisense

HaCaT as-PAF-R model system was established using the RevTet-on system (Clontech Laboratories, Palo Alto, CA) as previously described (22). PAF-R cDNA was cloned in a reversed orientation into the HindIII site of the response retroviral vector pRevTRE. The insert orientation was confirmed by restriction mapping and sequencing. To generate the retroviruses, amphotropic packaging cell line phoenix 293 was transfected with either the retroviral Tet-on regulator, the pRevTRE-as-PAF-R, or the pRevTRE back bone using FuGene 6 (Roche, Indianapolis, IN), and transient supernatants containing infectious amphotropic retrovirus were collected 48 h later. In the first round of infection, the infectious supernatant made from the retroviral Tet-on regulator was used to infect the parental HaCaT cells and cells resistant to 1 mg/ml G418 were subject to infection with the infectious supernatant made from the pRevTRE-as-PAF-R or the viral backbone pRevTRE and selected with 600 µg/ml hygromycin. To increase the number of gene copies in the cells, we performed a second round of infection, in which the transduced HaCaT cells that were double resistant to G418 and hygromycin were infected with both viruses again. After a 48-h induction with 10 mg/ml doxycycline, calcium mobilization studies using HaCaTpRevTRE-as-PAF-R and HaCaTpRevTRE cells were conducted to confirm lack of intracellular calcium flux to 1-hexadecyl-2-N-methylcarbamoyl-glycerophosphocholine (CPAF), but normal responses to bradykinin in the HaCaTpRevTRE-as-PAF-R cells.

Generation of KBP cells expressing the super-repressor I {kappa} B {alpha}

The I {kappa} BM containing S32A and S36A mutation of I {kappa} B {alpha} was cloned into the retroviral DNA vector MIEG3, which uses enhanced green fluorescent protein as the selectable marker, and KBM and KBP cells stably expressing I {kappa} BM created and characterized as previously described (22, 23).

Cytokine measurements

Total RNA of IL-8 and the housekeeping gene GADPH were measured by Northern blot analysis exactly as previously described (16, 20). Human glyceraldehyde-3-phosphate dehydrogenase and IL-8 cDNA probes were obtained from American Type Culture Collection (Manassas, VA). Levels of TNF-{alpha} and IL-8 protein released into the supernatants were measured by ELISA as described previously with minor modifications (16). Briefly, cells were plated at 200,000/ml on 24-well plates for 24 h, then exposed to media with or without drugs. In experiments in which CPAF was used, cells were treated with CPAF or ethanol vehicle (0.1%). For experiments involving antioxidants, cells were preincubated with drug, ethanol, or DMSO vehicle (0.5%) for 30 min, and medium was replaced with prewarmed medium before addition of drugs. The medium was collected at various times, and cytokines were assayed using Quantikine ELISA kits (R&D, Minneapolis, MN). Similarly treated cells were trypsinized and counted (Coulter, Hialeah, FL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The KB PAF-R model system

As PAF may have both receptor-dependent and -independent effects (secondary to the formation of biologically active metabolites), our laboratory has previously created a model system by transduction of the PAF-R into a PAF-R-deficient epidermal cell line to study the role of the PAF-R in epithelial cell biology. The human epidermal carcinoma cell line KB does not express functional PAF-Rs, unlike normal human keratinocytes and the human keratinocyte-derived carcinoma cell line HaCaT (15). A PAF-R-positive KB cell line, KBP, was created by transducing KB cells with a replication-deficient MSCV2.1 retrovirus containing the human PAF-R cDNA. KB cells were also transduced with the retrovirus backbone alone to establish a vector control cell line, KBM. Expression of the PAF-R protein was verified by binding studies using radiolabeled PAF-R antagonist WEB-2086 (16). Calcium mobilization studies demonstrated that the KB PAF-R was functionally active (16). Therefore, this in vitro epidermoid system consists of both PAF-R-negative (KBM) and -positive (KBP) cells.

Effects of chemotherapeutic agents on cytokine production in KB cells

The first studies assessed the ability of the chemotherapeutic agents etoposide and mitomycin C to induce the production of IL-8 in KB cells. As shown in Fig. 1, treatment of KB cells with the PAF-R agonist CPAF resulted in an accumulation of IL-8 mRNA selectively in PAF-R-positive KBP, but not control KBM cells. However, PMA treatment resulted in similar levels of IL-8 mRNA in KBP (not shown) and KBM cells (Fig. 1). Treatment with the chemotherapeutic agents etoposide and mitomycin C resulted in increased levels of IL-8 mRNA, which was more apparent in KBP cells (Fig. 1). It should be noted that unlike the PAF-R ligand CPAF, which induced IL-8 mRNA by 1 h, chemotherapeutic agent-induced effects were not seen until several hours. Consistent with the Northern blotting data, etoposide and mitomycin C treatment of KBP cells resulted in an increased release of IL-8 protein compared with KBM cells (Fig. 2). PMA-induced IL-8 protein release was similar in KBM and KBP cells (Fig. 2). As shown in Fig. 3, TNF-{alpha} production was also selectively enhanced in KBP cells treated with etoposide. Similarly, mitomycin C induced more TNF-{alpha} protein release in KBP over KBM cells (data not shown). These studies indicate that expression of the PAF-R in a carcinoid cell line results in enhanced cytokine production in response to etoposide and mitomycin C.



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  		FIGURE 1. The effects of chemotherapeutic agents on IL-8 mRNA levels in KB cells. KBP and KBM cells were incubated with 100 nM CPAF, 1 µM PMA, or the chemotherapeutic agents etoposide (6 µg/ml) and mitomycin C (5 µg/ml). RNA was extracted at various times and subjected to Northern blot analysis using probes for IL-8 or GAPDH.

 


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  		FIGURE 2. Effect of etoposide on IL-8 protein release in KBM vs KBP cells. KBP and KBM cells were incubated with 100 nM CPAF, 1 µM PMA, 6 µg/ml etoposide, or 5 µg/ml mitomycin C. The supernatants were removed at 8 h and assayed for immunoreactive IL-8 protein using a specific ELISA. The results shown are the mean ± SD of duplicate samples of a representative experiment (of three) and are typical of at least two separate KBM or KBP cells. *, p < 0.05 (higher levels of IL-8 measured in CPAF-, etoposide-, and mitomycin C-treated KBP vs KBM cells).

 


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  		FIGURE 3. Effect of etoposide on TNF-{alpha} protein release in KBM vs KBP cells. KBP and KBM cells were incubated with 100 nM CPAF, 1 µM PMA, or 6 µg/ml etoposide. The supernatants were removed at 8 h and assayed for immunoreactive TNF-{alpha} protein using a specific ELISA. The results shown are the mean ± SD of duplicate samples of a representative experiment (of three) and are typical of at least two separate KBM or KBP cells. *, p < 0.05 (higher levels of TNF-{alpha} measured in CPAF- and etoposide-treated KBP vs KBM cells).

 
To assess whether the PAF-R-mediated enhanced cytokine production in response to chemotherapeutic agents is a general characteristic of GPCR, KB cells were transduced with the fMLP-R. This GPCR was chosen as a control because epithelial cells, unlike bacteria, do not produce the peptide ligand fMLP. Calcium mobilization assays with fura-2-loaded KBF cells resulted in intracellular calcium responses to 100 nM fMLP confirmed that the fMLP-R was functional (data not shown). As shown in Fig. 4, treatment of KBF cells with fMLP resulted in increased release of IL-8. Consistent with the idea that chemotherapeutic agents are not activating GPCRs in a ligand-independent fashion, transducing KB cells with the fMLP-R did not affect responses to etoposide (Fig. 4).



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  		FIGURE 4. Effect of etoposide on IL-8 protein release in KBM and KBP vs KBF cells. KBM, KBP, and KBF cells were incubated with 100 nM CPAF, 100 nM fMLP, 1 µM PMA, or 6 µg/ml etoposide. The supernatants were removed at 8 h and assayed for immunoreactive IL-8 protein using a specific ELISA. The results shown are the mean ± SEM percentage of control IL-8 release from three separate experiments using duplicate samples. *, p < 0.05 (higher levels of IL-8 measured in etoposide-treated KBP vs KBM/KBF cells).

 
Effects of the endogenous PAF-R on etoposide-induced IL-8 production

The next studies were designed to assess whether the levels of PAF-R expression found on the epithelial cell line HaCaT (15) could modulate chemotherapeutic agent-induced cytokine production. To address this question, HaCaT keratinocytes that express native PAF-Rs were transduced with a retrovirus from which an antisense RNA corresponding to the PAF-R mRNA could be induced (HaCaTpRevTRE-as-PAF-R). This model system has been previously characterized, and measurement of intracellular calcium flux or IL-8 production in response to CPAF was used to confirm that treatment of HaCaTpRevTRE-as-PAF-R cells with doxycycline resulted in ablation of endogenous PAF-R expression (22). As shown in Fig. 5, doxycycline (10 mg/ml) treatment of HaCaTpRevTRE-as-PAF-R cells inhibited CPAF-induced, but not PMA-induced, IL-8 production in HaCaTpRevTRE-as-PAF-R cells. It should be noted that doxycycline pretreatment did not affect Ca2+ mobilization responses (not shown) or IL-8 production by the nonmetabolizable PAF-R agonist CPAF in control HaCaTpRevTRE cells (Fig. 5).



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  		FIGURE 5. Effect of ablation of the endogenous PAF-R on etoposide-mediated IL-8 protein release in HaCaT cells. HaCaTpRevTRE and HaCaTpRevTRE-as-PAF-R cells were treated with 10 mg/ml doxycycline for 48 h to allow induction of as-PAF-R construct. After induction, HaCaT cells were incubated with 100 nM CPAF, 6 µg/ml etoposide, or 1 µM PMA. The supernatants were removed at 8 h and assayed for immunoreactive IL-8 protein using a specific ELISA. The results shown are the mean ± SD of duplicate samples from a representative experiment (of three). *, p < 0.05 (lower levels of IL-8 measured in HaCaTpRevTRE-as-PAF-R cells treated with etoposide and CPAF vs HaCaTpRevTRE cells).

 
Ablation of the endogenous PAF-R in HaCaT cells resulted in a diminishment of IL-8 production due to the chemotherapeutic agent etoposide. It should be noted that the modest decrease in etoposide-induced IL-8 production in HaCaTpRevTRE-as-PAF-R cells compared with HaCaTpRevTRE (Fig. 5) was less than the differences between KBP and KBM cells (Fig. 2). This fits with the decreased magnitude of IL-8 production induced by CPAF in HaCaT vs KBP cells and is probably due to the decreased levels of PAF-R expression found in HaCaT compared with KBP cells (16). These studies indicate that endogenous levels of PAF-R expression in carcinomas are adequate to modulate chemotherapeutic agent-induced cytokine production.

Role of oxidative stress on PAF-R augmentation of chemotherapeutic agent-induced cytokine production

Our previous studies using a fluorescent H2O2-sensitive dye demonstrated that etoposide and mitomycin C treatment of KB carcinoma cells results in increased levels of intracellular H2O2 (22). The next studies used an antioxidant to confirm the involvement of oxidative stress in the ability of chemotherapeutic agents to activate the epidermal PAF-R and thus modulate IL-8 production. Pretreatment of KB cells with the antioxidant trolox blunted etoposide-mediated IL-8 release in KBP, but not KBM, cells and tended to negate the effect of the PAF-R in KBP cells (Fig. 6). However, trolox did not affect CPAF-induced IL-8 release in KBP cells. Similarly, pretreatment of KB cells with 10 mM of the antioxidant reservatrol resulted in effects similar to those of trolox (not shown). Consistent with the idea that oxidative stress could induce cytokines via activation of the epidermal PAF-R, exposure of KB cells to the potent pro-oxidative stressor tert-butyl-hydroperoxide resulted in cytokine production only in KBP cells (Fig. 7). These effects of tert-butyl-hydroperoxide were inhibited by preincubation with the antioxidants trolox (Fig. 7) and reservatrol (not shown). Together these studies provide solid support for the hypothesis that oxidative stress induced by chemotherapeutic agents results in PAF-R activation, which can augment the cytokine-producing effects of these agents.



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  		FIGURE 6. Effect of the antioxidant trolox on etoposide vs CPAF-induced IL-8 protein release in KB cells. KB cells were preincubated with 10 mM trolox for 1 h before addition of 100 nM CPAF or 6 µg/ml etoposide. The supernatants were removed 8 h later and assayed for immunoreactive IL-8 protein using a specific ELISA. The results shown are the mean ± SD of duplicate samples from a representative experiment (of three) and are typical of at least two separate KBM and KBP cell clones. *, p < 0.05 (lower levels of IL-8 measured in trolox- and etoposide-treated KBP vs etoposide alone-treated cells).

 


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  		FIGURE 7. Effect of tert-butyl-hydroperoxide on IL-8 protein release in KBM vs KBP cells. KBP and KBM cells were preincubated with 10 mM trolox for 1 h before addition of 100 µM tert-butyl-hydroperoxide (TBH), 100 nM CPAF, or 1 µM PMA. The supernatants were removed at 8 h and assayed for immunoreactive IL-8 protein using a specific ELISA. The results shown are the mean ± SD of duplicate samples from a representative experiment (of three) and are typical of at least two separate KBM or KBP cells. *, p < 0.05 (higher levels of IL-8 measured in CPAF- and tert-butyl-hydroperoxide-treated KBP vs KBM cells).

 
Involvement of the NF-{kappa}B system in PAF-R-mediated augmentation of chemotherapeutic agent-induced cytokine production

The following studies were designed to assess the mechanism by which the PAF-R could augment chemotherapeutic agent-induced cytokine production. Indeed, activation of the epidermal PAF-R is linked to numerous signal transduction systems that could be responsible for IL-8 production. It should be noted that, like a diverse group of stimuli including pro-oxidative stressors, the epidermal PAF-R induces the transcription factor NF-{kappa}B, and activation of this same pathway has also been reported to induce IL-8 production (24).

To test whether PAF-R-mediated activation of the NF-{kappa}B pathway was involved in the augmentation of chemotherapy-mediated IL-8 production, we examined whether a dominant negative inhibitor of NF-{kappa}B could affect PAF-R-mediated augmentation of cytokine production in response to these chemotherapeutic agents. To that end, KBP cells were transduced with a super-repressor I{kappa}B protein (KBP-MIEG-I{kappa}BM) or vector control (KBP-MIEG). We have previously shown that increased I{kappa}B{alpha} degradation and increased NF-{kappa}B binding activity induced by PAF-R agonist CPAF or IL-1{beta} were blocked in these cells transduced with this mutant nondegradable I{kappa}B protein (22, 23). As shown in Fig. 8, blockade of the NF-{kappa}B pathway inhibited the IL-8 production induced by the PAF-R agonist CPAF as well as that induced by etoposide and mitomycin C. However, IL-8 production induced by the protein kinase C agonist PMA was essentially equal in KBP-MIEG and KBP-IKBM cells. These findings suggest that PMA-mediated IL-8 production in KB cells is independent of the NF-{kappa}B pathway. It should also be noted that our previous studies have indicated that PMA-induced IL-6 and IL-8 production is similar in KBM and KBP cells, indicating that PMA effects are independent of the PAF-R (16). These findings fit with previous reports that phorbol esters do not induce PAF biosynthesis (25). The present studies indicate that activation of the NF-{kappa}B pathway is responsible for PAF-R augmentation of chemotherapeutic agent-induced cytokine production (Fig. 8).



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  		FIGURE 8. Effect of inhibition of the NF-{kappa}B pathway on PAF-R augmentation of chemotherapeutic agent-induced IL-8 protein release in KBP cells. KBP cells transduced with I{kappa}BM or MIEG vector control retrovirus were incubated with 100 nM CPAF, 1 µM PMA, 6 µg/ml etoposide, or 5 µg/ml mitomycin C. The supernatants were removed at 8 h and assayed for immunoreactive IL-8 protein using a specific ELISA. The results shown are the mean ± SD of duplicate samples of a representative experiment (of three). *, p < 0.05 (lower levels of IL-8 measured in KBP-I{kappa}BM cells treated with etoposide and CPAF vs KBP-MIEG cells).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies provide evidence that in epithelial carcinoma cells, the PAF-R can augment cytokine production induced by chemotherapeutic agents via an NF-{kappa}B-dependent process. The augmentation of chemotherapeutic agent-induced cytokine production was mediated through the PAF-R, as it was only seen in PAF-R-expressing KBP carcinoma cells. Our results describe a novel function for a GPCR, namely, the induction of an NF-{kappa}B-dependent pathway by which the PAF-R can augment the cytokine production of chemotherapeutic agents.

Recent studies have shown that oxidative stress can trigger the production of lipids with PAF-R agonist activity. For example, chemical oxidation of low density lipoproteins results in the production of a PAF-R activity that has been shown to consist of fragmented alkyl GPC including 1-hexadecyl-2-butanoyl-GPC and 1-hexadecyl-2-butenoyl-GPC, along with trace levels of authentic PAF (18). Of significance, systemic exposure to the strong oxidative stress of tobacco smoke has been shown to induce the production of these PAF-R agonists in hamsters in vivo (26). Other pro-oxidative stressors, such as UV-B, can also stimulate the PAF-R through production of PAF and PAF-like lipids (13). Several lines of evidence suggest that the chemotherapeutic agents etoposide and mitomycin C are activating the PAF-R through the production of PAF/PAF-like species. First, these chemotherapeutic agents are known pro-oxidative stressors (22, 27, 28). Second, pretreatment with antioxidants blocked the augmentation of chemotherapy-mediated cytokine production found in PAF-R-expressing KBP cells. The finding that expression of the GPCR for fMLP in KB cells (KBF) did not affect chemotherapy-induced IL-8 production suggests that these agents were not nonspecifically activating the GPCR in a ligand-independent fashion.

The mechanism by which chemotherapeutic agents can activate the PAF-R is most likely via production of PAF-R agonistic activity, rather than acting directly as PAF-R agonists. Consistent with this, etoposide and mitomycin C are unable to trigger an intracellular Ca2+ mobilization response in fura-2-loaded KBP cells (data not shown). This is in contrast to a PAF-R agonist such as CPAF, which induces an almost immediate Ca2+ flux (16). The lag time in IL-8 mRNA accumulation in response to etoposide vs the more rapid response seen with CPAF in KBP cells (Fig. 1) also fits with the idea that chemotherapeutic agents are inducing the biosynthesis of a PAF-R agonistic activity rather than acting as direct PAF-R agonists. Thus, the data presented in these studies support the hypothesis that these chemotherapeutic agents have the ability to induce the production of PAF/PAF-like species. Ongoing studies are attempting to define the structural identity of chemotherapy-induced PAF-R agonistic activity.

The current studies also begin to define mechanistically how PAF-R activation can augment cytokine production by chemotherapeutic agents. GPCR, including the PAF-R, have been shown to activate the NF-{kappa}B system (22, 23). Consistent with the involvement of NF-{kappa}B in the augmentation of chemotherapy-induced cytokine production, we have recently reported that etoposide and mitomycin C treatment of KBP cells resulted in much greater levels of NF-{kappa}B binding activity than those in KBM cells (22). Blocking NF-{kappa}B activation with a super-repressor I{kappa}B mutant decreased the augmentative effect of PAF-R expression on chemotherapy-induced IL-8 production (Fig. 7).

Although expression of the fMLP-R in KB cells did not affect chemotherapy-induced IL-8 production, this receptor was found to have in common with the PAF-R the ability to activate the NF-{kappa}B pathway. Indeed, fMLP treatment of KBF cells resulted in enhanced NF-{kappa}B binding by gel shift assays (22). Transfection of KBF cells with an NF-{kappa}B-luciferase reporter plasmid to measure NF-{kappa}B activity also revealed increased levels in KBF cells in response to fMLP (22). The ability of the fMLP-R to stimulate the NF-{kappa}B system, yet not affect chemotherapy-induced responses of NF-{kappa}B activation or cytokine production, supports the contention that these pro-oxidative chemotherapeutic agents activate the epidermal PAF-R via production of endogenous ligands.

The present studies using antioxidants also suggest that chemotherapeutic agent-induced cytokine production does not appear to depend upon the pro-oxidative effects of the agents. Indeed, incubation of KBM cells with trolox did not have a significant effect on etoposide-induced IL-8 production (Fig. 6), yet ablated that induced by tert-butyl-hydroperoxide (Fig. 7). That and the significant difference in IL-8 production between the potent oxidant tert-butyl-hydroperoxide vs the weaker oxidant etoposide fit with a less important role of oxidative stress in chemotherapy-mediated cytokine production. However, the presence of the PAF-R allows the cells to respond to chemotherapeutic agent-generated oxidative stress to augment cytokine production.

The significance of chemotherapy-induced cytokine production is unclear. However, the ability of populations to degrade PAF and PAF-like lipids differs greatly, with both acquired and inherited deficiencies of PAF acetylhydrolases described (29). Thus, it is possible that pro-oxidative chemotherapeutic agent-induced PAF activity might be handled differently in different populations, which could result in either increased or possibly decreased tumor cell killing or increased chemotherapy-induced side effects.

In summary, the present studies demonstrate that chemotherapeutic agents such as etoposide and mitomycin C can induce the production of the cytokines IL-8 and TNF-{alpha} in a human epithelial carcinoid cell line. Expression of the epidermal PAF-R results in an augmentation of chemotherapeutic agent-mediated cytokine production via a pro-oxidative process involving the production of PAF-R agonists. We describe a novel pathway by which these pro-oxidative stressors induce PAF/PAF-like species, which then activate the NF-{kappa}B pathway through the PAF-R. The biological significance of this pathway is not clear, but could be the impetus for further studies to define whether the presence of the PAF-R could modulate chemotherapeutic responses in vivo. Finding that this novel pathway is an important determinant in the effectiveness and/or side effect profile of certain chemotherapeutic agents would be useful for planning chemotherapeutic strategies in PAF-R-expressing tumor cells.


   Acknowledgments
 
We thank Drs. Dan Spandau and Raymond Konger for their critical reading of this manuscript.


   Footnotes
 
1 This work was supported in part by grants from The Showalter Memorial Foundation and The Riley Memorial Association, and National Institutes of Health Grants AR01993 and HL62996. Back

2 Address correspondence and reprint requests to Dr. Jeffrey B. Travers, H. B. Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, Room 2659, Indiana University School of Medicine, 702 Barnhill Drive, Indianapolis, IN 46202. E-mail address: jtravers{at}iupui.edu Back

3 Abbreviations used in this paper: PAF, platelet-activating factor; CPAF; 1-hexadecyl-2-N-methylcarbamoyl-glycerophosphocholine; GPC, glycerophosphocholine; GPCR, G protein-coupled receptor; PAF-R, PAF receptor. Back

Received for publication November 14, 2003. Accepted for publication March 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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