Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

<p>Abstract</p> <p>Background</p> <p>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 P2X₇receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X₇system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X₇action.</p> <p>Methods</p> <p>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 P2X₇-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 (P2X₇pore formation) were determined by confocal laser microscopy.</p> <p>Results</p> <p>(a) Co-application on the skin of the P2X₇specific 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 P2X₇-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 P2X₇receptor, 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 P2X₇receptor, 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.</p> <p>Conclusion</p> <p>(a) P2X₇-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X₇-dependent apoptosis is mediated by calcium influx via P2X₇pores, 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 P2X₇receptor. (d) Activation of P2X₇-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.</p

Regulation of P2X7 gene transcription

The pro-apoptotic P2X7 receptor regulates growth of epithelial cells. The objectives of the study were to understand P2X7 gene transcription; to identify the active promoter and the transcription initiation site (TpIS); and to begin understanding regulation of P2X7 gene transcription. Experiments in vitro utilized normal and cancerous cultured human uterine cervical epithelial cells, and HEK293 cells overexpressing P2X7-luciferase reporters. Experiments in vivo used surgical specimen of normal and cancerous uterine cervix. Assays involved DNA, RNA, and protein techniques. (a) The P2X7 TpIS was localized to adenine (+1) at nt 1683 of the human P2X7 gene [GenBank Y12851]), with a TTAAA sequence at nt −32/−28 and an active promoter region within nt −158/+32. (b) P2X7 transcription was found to be regulated by two enhancers located at nt + 222/+232 and +401/+573 regions downstream of the active P2X7 promoter. (c) The putative enhancer regions formed four DNA–protein complexes. (d) P2X7 transcription was found to be controlled by hypermethylated cytosines at cytosine-phosphodiester-guanosines (CpG) that cluster or co-localize with the enhancers’ sites. (e) We identified nine CpGs as inhibitory cis elements, and three CpG sites that are hypermethylated in cultured cervical epithelial cells and in cervix epithelia in vivo. (f) In cancer cervical cells, the degree of hypermethylation of the CpG sites was greater than in the normal cervical cells. Expression of the P2X7 receptor is controlled by hypermethylated CpGs that flank transcription enhancers located within a 547-nt region downstream of the promoter

P2X7 receptor expression is decreased in epithelial cancer cells of ectodermal, uro-genital sinus, and distal paramesonephric duct origin

The P2X7 receptor is an important regulator of epithelial cell growth. The aim of the present study was to better understand the biological significance of P2X7 receptor expression in normal and cancer human epithelial tissues. P2X7 receptor and messenger RNA (mRNA) levels were determined in human tissues containing epithelial dysplastic, pre- or early cancerous, and cancer cells, and the levels were compared to those in the corresponding normal epithelial cells within the same tissue of the same case. P2X7 receptor levels were determined by quantification of immunoreactivity specific to the functional (full-length) P2X7 receptor, and P2X7 mRNA levels were determined by real-time polymerase chain reaction. P2X7 receptor levels in cancer cells were similar (colon adenocarcinoma) or greater (thyroid papillary carcinoma) than those in the corresponding normal cells. In contrast, in cancer cells of the ectocervix (squamous), endocervix and endometrium (adenocarcinoma), urinary bladder (transitional cell carcinoma), and breast (ductal and lobular adenocarcinomas), P2X7 receptor levels were lower by about twofold than those in the corresponding normal epithelial cells. Similarly, P2X7 mRNA levels were lower in uterine, bladder, and breast cancer epithelial tissues by about fourfold than those in the corresponding normal tissues. In addition, P2X7 receptor levels were decreased already in dysplastic ectocervical cells and pre- or early cancerous endometrial and bladder cells. The data suggest that in epithelia originating from the ectoderm, the uro-genital sinus, and the distal paramesonephric duct, decreased expression of the P2X7 receptor precedes or coincides with neoplastic changes in those tissues