Tumor-selective induction of apoptosis and the small-molecule immune response modifier imiquimod.
BACKGROUND
The incidence of nonmelanoma skin cancer, basal cell carcinomas (BCCs), and squamous cell carcinomas (SCCs) is increasing, representing a major medical and economic problem. Imiquimod, a topical small-molecule immune response modifier, has shown efficacy toward BCC and actinic keratoses in clinical trials. Imiquimod acts both indirectly, via cytokine-mediated stimulation of cellular immune responses, and directly, through unknown mechanisms against tumor cells. We examined the mechanism by which imiquimod induces apoptosis in cancer cells.


METHODS
Apoptosis was assessed by enzyme-linked immunosorbent assay, western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays in five SCC cell lines, HaCaT cells (a spontaneously immortalized human keratinocyte cell line), and normal keratinocytes treated with imiquimod, with its analog resiquimod, or with neither. Expression of death receptors, caspases, and cytochrome c in the apoptotic signaling cascade was analyzed using western blot and flow cytometric analyses. The functional relevance of imiquimod-induced cytochrome c release was assessed by transfection of HaCaT cells with Bcl-2. Apoptosis in BCCs in vivo was assessed by TUNEL assays of imiquimod-treated and untreated tumors from three patients. Differences between treated and untreated cells and tumors were determined using a two-tailed Student's t test.


RESULTS
Imiquimod, but not resiquimod, induced apoptosis in all SCC cell lines and HaCaT cells. This induction involved activation of several caspases and Bcl-2-dependent cytosolic translocation of cytochrome c but was independent of the membrane-bound death receptors Fas, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R1-R4 receptors, and tumor necrosis factor-R1 and -R2 receptors. Topical application of imiquimod to BCC tumors in vivo induced apoptosis.


CONCLUSIONS
Imiquimod has the potential to induce apoptosis in skin cancer cells, possibly by circumventing mechanisms developed by malignant tumors to resist apoptotic signals.
Schön
Margarete
M
Department of Dermatology, Otto-von-Guericke-University, Magdeburg, Germany. schoenmargret@web.de
Bong
Anne B
AB
Drewniok
Claudia
C
Herz
Jeannine
J
Geilen
Christoph C
CC
Reifenberger
Julia
J
Benninghoff
Bernd
B
Slade
Herbert B
HB
Gollnick
Harald
H
Schön
Michael P
MP
D000634:Aminoquinolines / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology*; D015153:Blotting, Western; D002280:Basal Cell Carcinoma / Q000188:drug therapy / Q000276:immunology; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000276:immunology* / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects; D003574:Cytochrome c Group / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D000077271:Imiquimod; D007155:Immunologic Factors / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D015603:Keratinocytes / Q000187:drug effects / Q000276:immunology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects
D000634:Aminoquinolines; D000970:Antineoplastic Agents; D003574:Cytochrome c Group; D007093:Imidazoles; D007155:Immunologic Factors; D019253:Proto-Oncogene Proteins c-bcl-2; D018124:Receptors, Tumor Necrosis Factor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D000077271:Imiquimod; C402365:resiquimod