Activation of P2X(7)-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice.
BACKGROUND
The study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X(7) receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X(7) system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X(7) action.


METHODS
Skin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas) were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X(7)-null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA). Changes in cytosolic calcium or in ethidium bromide influx (P2X(7) pore formation) were determined by confocal laser microscopy.


RESULTS
(a) Co-application on the skin of the P2X7 specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28) the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b) In the normal skin BzATP affected mainly P2X(7)-receptor - expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c) In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d) Levels of P2X(7) receptor, protein and mRNA were 4-5 fold lower in cancer tissues than in normal mouse tissues. (e) In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f) The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for BzATP. (g) Pore formation and the augmented calcium influx were depended on the expression of the P2X(7) receptor, while the BzATP-induced apoptosis depended on calcium influx. (h) The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8.


CONCLUSIONS
(a) P2X(7)-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X(7)-dependent apoptosis is mediated by calcium influx via P2X(7) pores, and involves the caspase-9 (mitochondrial) pathway. (c) The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X(7) receptor. (d) Activation of P2X(7)-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.
Fu
Wen
W
Department of Reproductive Biology, Case Western Reserve University, Cleveland, OH, USA. wen.fu@case.edu
McCormick
Tom
T
Qi
Xiaoping
X
Luo
Liping
L
Zhou
Lingyin
L
Li
Xin
X
Wang
Bing-Cheng
BC
Gibbons
Heidi E
HE
Abdul-Karim
Fadi W
FW
Gorodeski
George I
GI
D015127:9,10-Dimethyl-1,2-benzanthracene; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009842:Oligopeptides / Q000494:pharmacology; D010212:Papilloma / Q000139:chemically induced / Q000378:metabolism* / Q000517:prevention & control; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000139:chemically induced / Q000378:metabolism* / Q000517:prevention & control; D016019:Survival Analysis; D013755:Tetradecanoylphorbol Acetate
D061945:Caspase Inhibitors; D009842:Oligopeptides; C546469:P2rx7 protein, mouse; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D015127:9,10-Dimethyl-1,2-benzanthracene; D000255:Adenosine Triphosphate; D053148:Caspase 3; D053453:Caspase 9; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium