Apoptosis 2004; 9: 291–298 C 2004 Kluwer Academic Publishers
Immune modulation and apoptosis induction: Two sides of the antitumoral activity of imiquimod M. P. Schon ¨ and M. Schon ¨ Rudolf-Virchow-Center, DFG Research Center for Experimental Biomedicine and Department of Dermatology and Venereology, University of Wurzburg, ¨ Germany
Imiquimod, the first member of the imidazoquinoline family of immune response modifiers, has proven good clinical efficacy against basal cell carcinomas and actinic keratoses in several independent studies. In addition, there is recent evidence that imiquimod is also efficacious against other tumors such as cutaneous metastases of malignant melanoma or vascular tumors. Imiquimod exerts its antitumoral effect, at least in part, through binding to TLR-7 and TLR-8 on dendritic cells followed by secretion of a multitude of proinflammatory cytokines. The net result of this proinflammatory activity is a profound tumor-directed cellular immune response. However, recent experimental and clinical data indicate that imiquimod also possesses considerable direct proapoptotic activity against tumor cells both in vitro and in vivo. This novel mode of action appears to be independent of membrane bound death receptors, but involves caspase activation. Induction of apoptosis by imiquimod is, at least in part, presumably mediated through Bcl-2dependent release of mitochondrial cytochrome c and subsequent activation of caspase-9. The structural analogue, resiquimod, exhibited very limited, if any, such proapoptotic activity, possibly due to its lacking ability to enter the cell. Bypassing molecular mechanisms of apoptosis deficiency by a topical compound may be of great utility for treating certain cutaneous tumors. Keywords: apoptosis; bcl-2; imiquimod; immune response modifier; skin cancer.
Introduction Various molecular alterations of cells contribute to the pathogenesis of malignant tumors. In case of epithelial This work was supported in part by a grant from the Deutsche Krebshilfe/Dr.-Mildred-Scheel-Stiftung to MPS and a research grant from the state of Sachsen-Anhalt to MS.
Correspondence to: Michael P. Schon, ¨ Rudolf-Virchow-Center, DFG Research Center for Experimental Biomedicine and Department of Dermatology and Venereology, Julius-MaximiliansUniversity, Versbacher Str. 9, 97078 Wurzburg, ¨ Germany. Tel: +49-931-201-48977; e-mail:
[email protected]
skin cancers, namely basal cell carcinomas (BCC) and squamous cell carcinomas (SCC), UV irradiation, various chemicals, defective genes, point mutations (such as those identified in the patched (PTCH) gene or the p53 tumor suppressor gene), or polymorphisms of carcinogenmetabolizing enzymes have been recognized as etiological factors.1–7 Genetic alterations in these tumors may result in apoptosis deficiency allowing the tumors to grow uncontrolled.7–11 In addition, experimental and clinical evidence is accumulating indicating that impaired T lymphocyte-associated immune surveillance may also significantly contribute to the pathogenesis of both types of skin cancer.12 This evidence includes the rapid development of SCC and/or BCC in some patients with drug-induced immunosuppression,13 CD4+ lymphocytopenia,14 AIDS,15 or hairy cell leukemia.16 In addition, both BCC and SCC respond to therapies modulating cellular immune mechanisms .17–22 Based on the recognition of immunological dysfunctions as contributing factors in the pathogenesis of skin tumors, searches for compounds have been conducted in order to overcome the immune evasion of such tumors. Imiquimod (1-(2methylpropyl)-1H -imidazo{4,5-c }quinolin-4-amine) is an imidazoquinoline family member of Mr = 240.3. This small-molecule compound is highly efficatious in the treatment of BCC23− 27 and intraepidermal keratinocyte neoplasias, the so-called actinic keratoses.27–28 In addition, imiquimod applied systemically in animal experiments has proven efficacy in a variety of transplantable tumors including colon carcinomas, melanomas, lung sarcomas, mammary carcinomas, and bladder carcinomas.29 It has been thought that imiquimod exerts its antitumoral activity primarily through induction of a profound cellular tumor-directed immune response.30 Recent experimental and clinical data, however, have demonstrated that the mode of action of this interesting compound clearly extends far beyond its known function as an immune response modifier inasmuch as imiquimod also confers direct pro-apoptotic activity against malignant and benign tumors of different origin.31–34 We will focus here Apoptosis · Vol 9 · No 3 · 2004
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on the molecular mode of action of imiquimod which underlies its antitumoral activity.
Immunomodulatory effects of imiquimod in skin cancer treatment The immunological aspects of imiquimod are summarized in Figure 1A. Dendritic cells (DC) are considered the primary responsive cell population in the skin, a notion that is supported by observations that DC respond to lower concentrations of imiquimod in vitro as compared to several other cell types.35–39 Recent studies demonstrated that members of the imidazoquinoline family are recognized independently by Toll Like Receptor-(TLR)-7 and TLR-8, thereby activating the signal transduction cascade downstream of these receptors.40,41 The activity of imiquimod is then mediated, at least in part, through intracellular activation of the transcription factor NFκB, which upon activation migrates to the nucleus and upregulates transcription of various cytokines including TNFα, IL-2, IL-6, IL-8, IL-12, G-CSF, GM-CSF, IFNγ , and IFNα, as well as chemokines such as MIP-1α, MIP1β, and MCP-1.35–38,42,43 The net result of these cytokine effects is a profound stimulation of a tumor-directed cellular immune response. As exemplified in Figure 1B, such a tumor-directed immune response is also seen in human skin tumors following exposure to topical imiquimod.33 In addition, imiquimod activates dendritic cells (resident Langerhans cells in the skin) possibly resulting in prolonged protective Th1-skewed immunity against viral infections and malignant tumors.44–47
Induction of tumor cell apoptosis by imiquimod in vitro and in vivo It was thought that neither imiquimod nor resiquimod, a related compound of Mr 314.4 inducing even more pronounced cytokine secretion, macrophage activation and enhancement of cellular immunity as compared to imiquimod,42,45,48–52 exhibit direct antineoplastic activities. It was, therefore, a surprise when it was found that tumor cells cultured in the absence of immune cells consistently exibited reduced cell numbers when they were exposed to imiquimod (25–50 µg/ml; i.e., concentrations approximately 3 logs lower than the marketed formulation) as compared to otherwise identical cultures without imiquimod.31 In contrast, resiquimod showed very little, if any, such direct activity in the absence of immune cells. Further investigations revealed that this effect was due to marked induction of apoptosis by imiquimod, but not resiquimod (Figure 2A). The pro-apoptotic activity was dose-dependent and affected transformed keratinocytes (i.e., tumor cells) stronger than normal keratinocytes. When the generation of histone-bound DNA 292 Apoptosis · Vol 9 · No 3 · 2004
fragments was assessed as a parameter of apoptosis in cultured cells, it was found that in most cases imiquimod induced apoptosis in the cell cultures by 200–700% after 24 h of incubation, in some experiments to an even higher degree. Pro-apoptotic activity of imiquimod was also observed when superficial BCC of three patients were treated topically with the marketed formulation of imiquimod (Aldara 5% cream) for 4 days, although it could not be ruled out that indirect mechanisms of apoptosis induction contributed under in vivo conditions where many different cell types including dendritic cells and other immune cells were present. In any case, given that imiquimod causes apoptosis in concentrations approximately 1000fold below the marketed formulation, it is reasonable to assume that the apoptosis observed in vivo is at least in part a direct effect. In subsequent studies, we and others have confirmed the pro-apoptotic activity of imiquimod in vivo in other tumors such as some cutaneous metastases of human malignant melanoma34 (Figure 2B) or a murine hemangioendothelioma model.32
Imiquimod-induced apoptosis is death receptor-independent, but involves caspase activation Two major routes have been identified through which cytostatic drugs induce apoptosis. The first involves activation of membrane-bound death receptors, such as the CD95 (Fas/APO-1), TNF or TRAIL receptor systems, and the other is dependent on direct mitochondrial cytochrome c release, both resulting in subsequent cell death.11,53–55 Thus, at least three major hypotheses can be delineated (which are not mutually exclusive) concerning the molecular mechanisms involved in imiquimod-induced apoptosis of tumor cells: First, imiquimod could act directly on membrane-bound death receptors, thus initiating the apoptotic signal transduction cascade triggered by CD95 or other death receptors. Second, as described for ceramides,56 imiquimod could affect downstream molecular interactions triggering the caspase cascade and subsequent cell death, thus bypassing the membrane-bound receptors. Third, imiquimod could influence complementary (intrinsic) pathways of apoptosis in the tumor cells such as the Bcl-2-dependent mitochondrial cytochrome c release leading to activation of caspase9 and subsequent cell death. Several surface-bound death receptor systems and their ligands have been described thus far. The best-known example is the TNF receptor family member CD95 (Fas/APO-1) which plays important roles for immune evasion and apoptosis deficiency of many malignant tumors (reviewed in).57,58 Another, more recently identified receptor system of the TNF receptor family is the TRAIL (TNF-related apoptosis inducing ligand) system.59 While
Inflammation and apoptosis by imiquimod Figure 1. Mechanisms of immune modulation by imiquimod. (A) Schematic representation of steps involved in the tumor directed immune response induced by imiquimod. The chemical structure of imiquimod is depicted in the lower left corner. (B) Paraffin-embedded biopsy specimens of an untreated superficial BCC (left panel) and a similar tumor treated for 4 days with topical imiquimod (Aldara 5% cream) have been stained with hematoxylin & eosin. A marked lymphocytic infiltration can be observed in the imiquimod-treated tumor (examples of infiltrating mononuclear cells indicated by white arrows).
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M. P. Sch¨on and M. Sch¨on Figure 2. Pro-apoptotic activity of imiquimod in vitro and in vivo. (A) Squamous cell carcinoma cells (cell line SCL-1) were cultured for 24 h in the presence of the indicated concentrations of imiquimod (left panel) or resiquimod (right panel). Apoptosis in these cell cultures was detected by a modified TUNEL assay (Terminal Desoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling assay). Apoptotic cells are visualized by blue staining. Scale bars = 20 µm. (B) Paraffin-embedded biopsy specimens of an untreated cutaneous metastasis of a malignant melanoma (left panel) and a similar tumor treated for 4 days with topical imiquimod (Aldara 5% cream) have been stained using a modified TUNEL assay. Again, apoptotic cells are visualized by an intense blue staining. Scale bars = 20 µm. Please note the presence of apoptotic tumor cells both in vitro and in vivo.
the ligand, TRAIL, binds to all four known receptors, only TRAIL-R1 (DR4) and TRAIL-R2 (DR5) confer intracellular proapoptotic signals via activation of instigator caspases (e.g. caspase-8,60 ). In contrast, TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) have been termed decoy receptors which do not transmit pro-apoptotic signals and may even have anti-apoptotic effects due to lacking or nonfunctional cytoplasmic death domains.61,62 The expression of CD95 (Fas/APO-1), TNF receptors 1 and 2 as well as TRAIL receptors 1–4 was not detectably elevated by imiquimod in a variety of keratinocytederived and melanocytic tumor lines, thus largely excluding an imiquimod-induced quantitative shift in these receptors.31,34 To test whether imiquimod might activate these receptors without altering expression, apoptosis induced by imiquimod was compared to apoptosis induced by the respective receptors (triggered through a stimulating monoclonal antibody in case of CD95 and through natural ligands in case of the other receptors). It was found that apoptosis of squamous carcinoma cells and 294 Apoptosis · Vol 9 · No 3 · 2004
melanoma cells was similarly increased by imiquimod and the respective receptor-specific stimuli. However, when the cells were incubated with death receptor-directed function-blocking antibodies, receptor-mediated apoptosis was completely abrogated, while the pro-apoptotic effect of imiquimod was not affected (Figure 3A depicts a representative experiment assessing CD95). Overall, blocking the functions of several death receptors expressed by different tumor cell lines did not affect the pro-apoptotic activity of imiquimod, but prevented apoptosis induced by specific stimulation of the respective receptors. Thus, it appeared that imiquimod bypassed the signal transduction pathways initiated by membranebound death receptors, at least in the keratinocyte-derived tumor cell lines and susceptible melanoma cells studied thus far. While blocking of death receptor functions did not detectably influence imiquimod-induced apoptosis, caspasemediated downstream signal transduction cascades were apparently involved. This notion was indicated by the
Inflammation and apoptosis by imiquimod Figure 3. Imiquimod-induced apoptosis in tumor cells is independent of membrane-bound death receptors, but involves caspase activation. (A) The tumor cell line SCL-1 was cultured in triplicate in normal medium (open bar), in medium containing imiquimod (50 µg/ml), in medium containing the Fas-(CD95)-stimulating monoclonal antibody CH11, or combinations of these conditions with the Fas-(CD95) blocking antibody ZB4 as indicated. Apoptosis in the cultures was determined by assessing the generation of histone-bound DNA fragments (Cell Death Detection ELISA, Roche Diagnostics, Mannheim, Germany). Please note that functional blockade of CD95 results in abrogation of CD95-induced apoptosis, but not of imiquimod-induced apoptosis. (B) SCL-1 tumor cells were cultured in triplicate in normal medium (open bar), in medium containing imiquimod (50 µg/ml, black bar), or in medium containing imiquimod and specific oligopeptide inhibitors of caspases as indicated (shaded bars). Again, apoptosis was determined by assessing the generation of histone-bound DNA fragments. Please note that inhibition of several caspases abrogates or diminishes imiquimod-induced apoptosis.
complete abrogation of imiquimod-induced apoptosis in epithelial and melanocytic tumor lines following pancaspase inhibition by zVAD-fmk, a fluoromethylketoneconjugated oligopeptide which binds to a common functional motif of all known caspases thereby irreversibly inhibiting these enzymes. Another line of evidence indicating caspase involvement comes from the observation that activation (i.e., cleavage) of the terminal caspase-3 was readily detected in tumor cells treated with imiquimod, but not the analogue, resiquimod.31,34 When distinct caspases were inhibited by specific fmk-conjugated functionblocking oligopeptides it was found that imiquimodinduced apoptosis could be completely abrogated by inhibition of caspases-3, -8, -9 and -10, while blocking of caspases-4 and -6 resulted in complete inhibition of apoptosis in some and partial inhibition in other cell lines. In contrast, treatment of the cells with oligopeptide inhibitors directed against caspases-1, -2 or –13 did not affect imiquimod-induced apoptosis in either of the cell lines studied. A representative experiment depicting the role of caspases in a SCC line is shown in Figure 3B.
Activation of the mitochondrial pathway of apoptosis by imiquimod and the role of Bcl-2 Many small-molecule pro-apoptotic stimuli induce the Bcl-2-family-regulated release of cytochrome c from the mitochondria into the cytosol, where it binds to APAF-1 and pro-caspase-9, forming the apoptosome. This results in activation of caspase-9 and, consecutively, caspase3.55,60 The aforementioned observation that functional inhibition of caspase-9 resulted in significantly diminished pro-apoptotic activity of imiquimod was consistent with the hypothesis that the caspase-9-mediated mitochondrial (intrinsic) pathway was involved in imiquimod-induced apoptosis. Indeed, when keratinocytic or melanocytic tumor cell lines were exposed to imiquimod, but not resiquimod, a marked translocation of mitochondrial cytochrome c into the cytosol was detected. The kinetics of this translocation nicely matched the activation (i.e., cleavage) of caspase-3 and fragmentation of cellular DNA.31,34 Given that the release of mitochondrial Apoptosis · Vol 9 · No 3 · 2004
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M. P. Sch¨on and M. Sch¨on Figure 4. Mechanisms of apoptosis induction by imiquimod. Schematic representation of our current notion of cellular and molecular events underlying the pro-apoptotic activity of imiquimod.
cytochrome c is governed by the relative amounts and associations of pro-apoptotic (e.g. Bax, Bak, Bid) and anti-apoptotic (e.g. Bcl-2, Bcl-XL ) members of the Bcl-2 protein family,63 it was conceivable that imiquimod affected this regulatory process. Indeed, when melanoma cells susceptible to imiquimod-induced apoptosis were investigated, a marked shift of the Bcl-2/Bax-ratio towards the pro-apoptotic protein Bax was observed.34 Furthermore, overexpression of the anti-apoptotic protein Bcl-2 in cell lines susceptible to imiquimod-induced apoptosis, i.e., immortalized keratinocytes (HaCaT cells) or melanoma lines, resulted in marked suppression of imiquimod-induced apoptosis. In addition, melanoma cells which constitutively expressed high levels of endogenous Bcl-2 proved rather resistant against imiquimodinduced apoptosis.34 These findings strongly suggested that imiquimod activates the mitochondrial pathway of apoptosis. In addition, imiquimod-induced apoptosis is dependent on proteins of the Bcl-2 family, and imiquimod alone cannot overcome the “Bcl-2 checkpoint” of apoptosis deficiency, at least in the tumor lines tested thus far. Its pro-apoptotic activity may, however, be a valuable addition to the therapeutic armory currently in use to treat some frequent malignant tumors. Our current notion of molecular events underlying the imiquimodinduced apoptosis in tumor cells is schematically depicted in Figure 4.
Conclusions and perspectives Induction of apoptosis in tumors by a topical compound represents an exciting new therapeutic concept that may greatly benefit patients with different kinds of epithelial 296 Apoptosis · Vol 9 · No 3 · 2004
skin tumors, such as BCC and SCC, the most frequent malignancies in humans in the northern hemisphere.31 Some cases of endothelial tumors may also respond to such treatment strategies in the future,32 and even selected patients suffering from cutaneous metastases of malignant melanoma may be successfully treated with topical imiquimod.34,64,65 The striking difference regarding proapoptotic activity between imiquimod and resiquimod may be due either to their differential ability to penetrate the cell membrane or to different structural motifs required for apoptosis induction. In any case, one may speculate that in the future minimal structural requirements will be unraveled which allow the generation of compounds with enhanced pro-inflammatory or pro-apoptotic activity.
References 1. Cleaver JE, Bootsma D. Xeroderma pigmentosum: Biochemical and genetic characteristics. Annu Rev Genet 1975; 9: 19–38. 2. Clendenning WE, Block JB, Radde JG. Basal cell nevus syndrome. Arch Dermatol 1964; 90: 38–53. 3. Gailani MR, Stahle-B¨ackdahl M, Leffell DJ. The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas. Nat Genet 1996; 14: 78–81. 4. Hahn H, Wicking C, Zaphiropoulos PG. Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 1996; 85: 841–851. 5. Lear JT, Heagerty AHM, Smith A. Multiple cutaneous basal cell carcinomas: Glutathione S-transferase (GSTM1, GSTT1) and cytochrome P450 (CYP2D6, CYP1A1) polymorphisms influence tumor numbers and accural. Carcinogenesis 1996; 17: 1891–1896. 6. Wolter M, Reifenberger J, Sommer C. Mutations in the human homologue of the Drosophila segment polarity gene
Inflammation and apoptosis by imiquimod
7. 8.
9. 10.
11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21.
22. 23.
24. 25.
patched (PTCH) in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system. Cancer Res 1997; 57: 2581–2585. Ziegler A, Jonason AS, Leffell DJ, et al. Sunburn and p53 in the onset of skin cancer. Nature 1994; 372: 773–776. Dazard JE, Gal H, Amariglio N, Rechavi G, Domany E, Givol D. Genome-wide comparison of human keratinocyte and squamous cell carcinoma responses to UVB irradiation: implications for skin and epithelial cancer. Oncogene 2003; 22: 2993–3006. McKee RM. Epidermal skin tumors. In: Breathnach SM, ed. Textbook of Dermatology. New York: Blackwell Science, 1998: 1656–1687. Popp S, Waltering S, Herbst C, Moll I, Boukamp P. UV-B-type mutations and chromosomal imbalances indicate common pathways for the development of Merkel and skin squamous cell carcinomas. Int J Cancer 2002; 99: 352– 360. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 1456–1462. Callen JP. Possible precursors to epidermal malignancies. In Baker D, ed. Cancer of the Skin. New York: Saunders, 1991: 27–30. Walder BK, Robertson MR, Jeremy DR. Skin cancer and immunosuppression. Lancet 1971; ii: 1282–1283. Kurwa HA, Marks R. Protracted cutaneous disorders in association with low CD4+ lymphocyte counts. Br J Dermatol 1995; 133: 625–629. Sitz KV, Keppen M, Johnson DF. Metastatic basal cell carcinoma in acquired immunodeficiency syndrome-related complex. JAMA 1987; 257: 340–343. Sch¨on MP, Reifenberger J, von Schmiedeberg S, et al. Multiple basal cell carcinomas associated with hairy cell leukemia. Br J Dermatol 1999; 140: 150–153. Cornell RC, Greenway HT, Tucker SB. Intralesional interferon therapy for basal cell carcinoma. J Am Acad Dermatol 1990; 23: 694–700. Edwards L, Whiting D, Rogers D, Luck K, Smiles KA. The effect of intralesional interferon gamma on basal cell carcinoma. J Am Acad Dermatol 1990; 22: 496–500. Kooy AJW, Tank B, Vuzevski VD, van Joost T, Prens EP. Expression of interferon-gamma receptors and interferongamma-induced up-regulation of intercellular adhesion molecule-1 in basal cell carcinoma; decreased expression of IFN-γ R and shedding of ICAM-1 as means to escape immune surveillance. J Pathol 1998; 184: 169–176. Telfer NR, Colver GB, Bowers PW. Guidelines for the management of basal cell carcinoma. British Association of Dermatologists. Br J Dermatol 1999; 141: 415–423. van der Straaten M, Lee P, Weitzul S, Cockerell CJ, Tyring SK. Advances in the treatment of basal cell carcinoma: The promise of pharmacologic therapy. Adv Dermatol 2000; 16: 299–318. Wennberg AM. Basal cell carcinoma—new aspects of diagnosis and treatment. Acta Der Venereol Suppl 2000; 209: 5–25. Beutner KR, Geisse JK, Helman D, Fox TL, Ginkel A, Owens ML. Therapeutic response of basal cell carcinoma to the immune response modifier imiquimod 5% cream. J Am Acad Dermatol 1999; 41: 1002–1007. Hannuksela-Svahn A, Nordal E, Christensen OB. Treatment of multiple basal cell carcinomas in the scalp with imiquimod 5% cream. Acta Derm Venereol 2000; 80: 381–382. Kagy MK, Amonette R. The use of imiquimod 5% cream for the treatment of superficial basal cell carcinomas in a basal cell
26.
27. 28.
29.
30. 31. 32. 33.
34.
35.
36.
37. 38. 39.
40.
41. 42.
43.
nevus syndrome patient. Dermatol Surg 2000; 26: 577–578; discussion 578–579. Marks R, Gebauer K, Shumack S, et al. Imiquimod 5% cream in the treatment of superficial basal cell carcinoma: Results of a multicenter 6-week dose-response trial. J Am Acad Dermatol 2001; 44: 807–813. Tyring S. Imiquimod applied topically: A novel immune response modifier. Skin Therapy Lett 2001; 6: 1–4. Stockfleth E, Meyer T, Benninghoff B, Christophers E. Successful treatment of actinic keratosis with imiquimod cream 5%: A report of six cases. Br J Dermatol 2001; 144: 1050– 1053. Sidky YA, Borden EC, Weeks CE, Reiter MJ, Hatcher JF, Bryan GT. Inhibition of murine tumor growth by an interferon-inducing imidazoquinoline. Cancer Res 1992; 52: 3528–3533. Stanley MA. Mechanism of action of imiquimod. Papillomavirus Rep 1999; 10: 23–29. Sch¨on M, Bong AB, Drewniok C, et al. Tumor-selective induction of apoptosis and the small-molecule immune response modifier imiquimod. J Natl Cancer Inst 2003; 95: 1138–1149. Sidbury R, Neuschler N, Neuschler E, et al. Topically applied imiquimod inhibits vascular tumor growth in vivo. J Invest Dermatol 2003; 121: 1205–1209. Sullivan TP, Dearaujo T, Vincek V, Berman B. Evaluation of superficial basal cell carcinomas after treatment with imiquimod 5% cream or vehicle for apoptosis and lymphocyte phenotyping. Dermatol Surg 2003; 29: 1181–1186. Sch¨on MP, Wienrich BG, Drewniok C, et al. Death receptorindependent apoptosis in malignant melanoma induced by the small-molecule immune response modifier imiquimod. J Invest Dermatol 2004; 122: (in press). Gibson SJ, Imbertson LM, Wagner TL. Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609. J Interferon Cytokine Res 1995; 15: 537–545. Megyeri K, Au W-C, Rosztoczy I. Stimulation of interferon and cytokine gene expression by imiquimod and stimulation by sendai virus utilize similar signal transduction pathways. Mol Cell Biol 1995; 15: 2207–2218. Miller RL, Birmachu W, GS. Cytokine induction by imiquimod, preclinical results and pharmacology. Chemother J 1994; 4: 148–149. Reiter MJ, Testerman TL, Miller RL, Weeks CE, Tomai MA. Cytokine induction in mice by the immunomodulator imiquimod. J Leukocyte Biol 1994; 55: 234–240. Gibson SJ, Lindh JM, Riter TR, et al. Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cell Immunol 2002; 218: 74–86. Hemmi H, Kaisho T, Takeuchi O, et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88dependent signaling pathway. Nat Immunol 2002; 3: 196– 200. Jurk M, Heil F, Vollmer J, et al. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nature Immunol 2002; 3: 499. Wagner TL, Horton VL, Carlson GL. Induction of cytokines in Cynomolgus monkeys by the immune response modifiers, imiquimod, S-27609 and S-28463. Cytokine 1997; 9: 837– 845. Weeks CE, Gibson SJ. Induction of interferon and other cytokines by imiquimod and its hydroxylated metabolite R-842 in human blood cells in vitro. J Interferon Cytokine Res 1994; 14: 81–85.
Apoptosis · Vol 9 · No 3 · 2004
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M. P. Sch¨on and M. Sch¨on 44. Suzuki H, Wang B, Shivji GM, et al. Imiquimod, a topical immune response modifier, induces migration of Langerhans cells. J Invest Dermatol 2000; 114: 135–141. 45. Burns R, Ferbel B, Tomai M, Miller R, Gaspari A. The imidazoquinolines, imiquimod and R-848, induce functional, but not phenotypic, maturation of human epidermal Langerhans cells. Clin Immunol 2000; 94: 13–23. 46. Harrison CJ, Miller RL, Bernstein DI. Posttherapy suppression of genital herpes simplex virus (HSV) recurrences and enhancement of HSV-specific T-cell memory by imiquimod in guinea pigs. Antimicrob Agents Chemother 1994; 38: 2059– 2064. 47. Bernstein DI, Harrison CJ, Tepe ER, Shahwan A, Miller RL. Effect of imiquimod as an adjuvant for immunotherapy of genital HSV in guinea pigs. Vaccine 1995; 13: 72–76. 48. Bernstein DI, Harrison CJ, Tomai MA, Miller RL. Daily or weekly therapy with resiquimod (R848) reduces genital recurrences in herpes simplex virus-infected guinea pigs during and after treatment. J Infect Dis 2000; 183: 844–849. 49. Wagner TL, Ahonen CL, Couture AM, et al. Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod. Cell Immunol 1999; 191: 10–19. 50. Imbertson LM, Beaurline JM, Couture AM, et al. Cytokine induction in hairless mouse and rat skin after topical application of the immune response modifiers imiquimod and S-28463. J Invest Dermatol 1998; 110: 734–739. 51. Tomai MA, Gibson SJ, Imbertson LM. Immunomodulating and antiviral activities of the imidazoquinoline S-28463. Antiviral Res 1995; 28: 253–264. 52. Spruance SL, Tyring SK, Smith MH, Meng TC. Application of a topically-applied immune response modifier, resiquimod gel, to modify the recurrence rate of genital herpes: A pilot study. J Infect Dis 2001; 184: 196–200. 53. Dirsch VM, Stuppner H, Vollmar AM. Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-XL or Bcl-2. Cancer Res 2001; 61: 5817–5823.
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54. Kaufmann SH, Earnshaw WC. Induction of apoptosis by cancer chemotherapy. Exp Cell Res 2000; 256: 42–49. 55. Sun XM, MacFarlane M, Zhuang J, Wolf BB, Green DR, Cohen GM. Distinct caspase cascades are initiated in receptormediated and chemical-induced apoptosis. J Biol Chem 1999; 274: 5053–5060. ¨ 56. Muller-Wieprecht V, Riebeling C, Stooss A, Orfanos CE, Geilen CC. Bcl-2 transfected HaCaT keratinocytes resist apoptotic signals of ceramides, tumor necrosis factor alpha, and 1-alpha, 25-dihydroxyvitamin D(3). Arch Dermatol Res 2000; 292: 455–462. 57. Ivanov VN, Bhoumik A, Ronai Z. Death receptors and melanoma resistance to apoptosis. Oncogene 2003; 22: 3152– 3161. ¨ oren N, El-Deiry WS. Cell surface death receptor signaling 58. Oz¨ in normal and cancer cells. Semin Cancer Biol 2003; 13: 135– 147. 59. MacFarlane M. TRAIL-induced signalling and apoptosis. Toxicol Lett 2003; 139: 89–97. 60. Cryns V, Yuan J. Proteases to die for. Genes Dev 1999; 12: 1551–1570. 61. Abe K, Kurakin A, Mohseni-Maybodi M, Kay B. Khosravi-Far R, The complexity of TNF-related apoptosisinducing ligand. Ann N Y Acad Sci 2000; 926: 52– 63. 62. Sheikh MS, Fornace AJ. Death and decoy receptors and p53mediated apoptosis. Leukemia 2000; 14: 1509–1513. 63. Raisova M, Hossini AM, Eberle J, et al. The bax/bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J Invest Dermatol 2001; 117: 333–340. 64. Bong AB, Bonnekoh B, Franke I, Sch¨on MP, Ulrich J, Gollnick H. Imiquimod, a novel immune response modifier, in the treatment of cutaneous metastases of malignant melanoma Dermatology 2002; 205: 135–138. 65. Steinmann A, Funk JO, Schuler G, von den Driesch P. Topical imiquimod treatment of a cutaneous melanoma metastasis. J Am Acad Dermatol 2000; 43: 555–556.
