Background
The current theory of growth of epithelial cells predicts regulation by the concerted effects of mitogenic stimuli and apoptosis [
1,
2]. Apoptosis is a homeostatic process orchestrated by the host's genome of selective cell deletion without stimulating inflammatory response [
3‐
5]. Dysregulation of apoptotic cell-death has been implicated in states of disease and in the neoplastic transformation [
6,
7]. Among the pro-apoptotic systems that operate in epithelia [
8] the P2X
7 is an important mechanism because the receptor is expressed by proliferating cells [
9], and activation of the receptor induces apoptosis that controls directly growth of the epithelial cells [
10].
The P2X
7 receptor is a membrane-bound, ligand-operated channel [
11‐
13]. The natural ligand of the receptor is ATP [
11,
12] which is present in the extracellular fluid of epithelial cells at high nanomolar, low micromolar levels [
14‐
18]. In contrast to other types of ATP receptors, activation of the P2X
7 receptor requires relatively high concentrations of the ligand [
12]. However, studies in epithelial cells of the female reproductive tract showed a threshold effect and activation of P2X
7-mediated apoptosis already by nanomolar concentrations of ATP [
8,
18], suggesting that ATP levels which are present in the extracellular fluid suffice to activate the receptor.
Binding of the ligand to the P2X
7 receptor can activate various cell-specific signaling cascades, including the IL-1β [
19], TNFα – TRAIL [
20], and the p38, JNK/SAPK [
21] and NF-κB cascades [
22]. However, a unique effect of activation of the P2X
7 receptor is formation of pores in the plasma membrane [
12]. In uterine epithelial cells formation of P2X
7 receptor pores induces apoptosis by a mechanism that involves uncontrolled influx of Ca
2+ via P2X
7-pores and activation of the mitochondrial – caspase-9 pathway [
13,
18,
23].
Until recently relatively little was known about the biological role of the P2X
7 in vivo, and particularly in the epidermis. Earlier studies suggested involvement of the P2X
7 receptor in the inflammatory and immune processes since the receptor is expressed in Langerhans and inflammatory dendritic epidermal cells [
24] and in cultured immature dendritic epidermal cells [
25]. Overexpression of P2X
7 was found in lesional skin of psoriasis and atopic dermatitis, where an intense P2X
7 immunoreactivity was confined to the cell membrane of the basal layer [
26]. P2X
7 may also play a role in chemokine secretion by normal keratinocytes but available data are inconsistent. Inoue et al [
27] reported that treatment of cultured normal keratinocytes with the P2X
7 specific agonist 2',3'-0-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) increased IL-6 release, while Pastore et al [
26] described that BzATP down-modulated chemokine secretion.
Studies also suggested a role for P2X
7 in the control of epidermal growth, but most studies were observational. The P2X
7 receptor is expressed in normal [
28], in precancerous epidermal tissues [
29], and in skin cancer cells [
26,
30,
31]. P2X
7 receptors were detected already in 8–11 week-old human fetal epidermis; they colocalized with caspase-3 and with periderm cells positive for transferase-mediated dUTP nick-end-labeling (TUNEL) [
32]. Co-localization of the P2X
7 with apoptosis-related markers was also reported in adult human epidermis [
28], and recent studies reported BzATP-induced cell death in normal and cancer keratinocytes, presumably by augmented apoptosis [
29,
30].
Although existing observational data suggest that the P2X7 may regulate growth of epithelial cells, no previous studies investigated experimentally the biological role of the P2X7 receptor in vivo. The present study tested the hypothesis that P2X7 controls epidermal cell growth in vivo by apoptosis.
Discussion
The main finding of the study was that pharmacological activation of P2X
7-mediated apoptosis, by local skin application of the P2X
7-receptor agonist BzATP, inhibited DMBA/TPA-induced formation of skin papillomas and squamous spindle-cell carcinomas. Since the main cellular effect of BzATP was augmentation of apoptosis, this discovery provides the first direct support for the hypothesis that apoptosis is an important mechanism in vivo which controls the development and progression of neoplasia. The present findings also support the hypothesis that P2X
7 is an important physiological pro-apoptotic system in epithelia, particularly those derived from the ectoderm (skin and breast), the uro-genital sinus (bladder), and the distal paramesonephric duct (uterine cervix and endometrium) [
9,
38,
41], and Li, Qi, Zhou, Fu, Abdul-Karim, MacLennan, and Gorodeski GI: P2X
7 receptor expression is decreased in epithelial cancer cells of ectodermal, uro-genital sinus, and distal paramesonephric-duct origin (submitted, 2009).
Co-treatment with BzATP delayed formation of DMBA/TPA-induced papillomas, and resulted in fewer and smaller papillomas. Some papillomas regressed and involuted spontaneously, as was previously described [
35], and the effect was unrelated to the treatment with BzATP. However, the majority (about two thirds) either progressed into squamous spindle-cell carcinomas or persisted as non-cancerous lesions. The latter trends depended on whether animals were co-treated with BzATP; thus, in mice co-treated with BzATP the proportion of animals with cancers at week 14 was lower than in the DMBA/TPA+BzATP group (50% versus 80%) and remained relatively stable, while in the DMBA/TPA group the proportion of animals with cancers increased steadily, reaching 100% at week 24. These data suggest that local treatment with BzATP inhibits formation of DMBA/TPA-induced skin papilloma, and it can also inhibit papilloma transformation into cancers.
BzATP had little effect on the number of cancerous lesions per animal at weeks 14–28, and on the proportion of animals with cancerous lesions > 10 mm
3 at weeks 14–22. In contrast, after week 23 the proportion of living animals with cancerous lesions > 10 mm
3 increased in the DMBA/TPA group while it had decreased in the DMBA/TPA+BzATP group. These data suggest that local treatment with BzATP exerts an inhibitory effect on the development on skin neoplasia. Interestingly, at weeks 15–24, among animals with cancerous lesions, the proportion of living animals with lesions larger than 200 mm
3 tended to be higher in the DMBA/TPA+BzATP group than in the DMBA/TPA group. This effect cannot be explained by augmented proliferation since BzATP did not stimulate DNA synthesis in cultured normal keratinocytes. Instead, the effect could be explained by comparing the survival curves (Fig.
6B) and the proportions of animals with smaller and larger size cancerous lesions (Figs.
5B, C). Thus, cancer-related deaths in the DMBA/TPA group were associated more often with smaller lesions while cancer-related deaths in the DMBA/TPA+BzATP group were associated with relatively larger lesions. This suggests that treatment with BzATP also prolonged the life of animals with developed cancers.
The data showed that the main targets of BzATP in the normal skin are proliferating keratinocytes of the epidermal basal/parabasal layers and hair shafts. In these P2X
7-receptor – expressing cells BzATP augmented apoptosis without evoking inflammatory changes that potentially could have been induced by activation of the P2X
7 receptor [
11,
12]. Experiments in P2X
7-deficient normal keratinocytes and in normal keratinocytes treated with anti-sense P2X
7 oligonucleotides showed that the P2X
7 receptor is a necessary mediator of the pro-apoptotic effect of BzATP, suggesting that the effect of BzATP is mediated by augmentation of P2X
7-mediated apoptosis.
Similar to the normal skin, the main targets of BzATP in papilloma tissues were P2X
7-receptor expressing proliferating keratinocytes at the base of developing papillomas. The importance of this finding relates to the fact that in the mouse DMBA/TPA model, papillomas at risk for developing into cancer are characterized by rapidly proliferating keratinocytes in the basal and parabasal layers of the papilloma [
35]. Since treatment with BzATP decreased the incidence of DMBA/TPA-induced papillomas and their transformation into cancer, it is likely that the cellular mechanism of BzATP action involved augmented apoptosis of proliferating papilloma keratinocytes bearing the potential of malignant transformation.
One of the differences between BzATP effects in the normal skin and in papilloma tissues was the lack of macroscopic effects in the former, while inhibiting the development and growth of papillomas. Thus, treatment with BzATP for 16 weeks in normal mice augmented apoptosis of proliferating keratinocytes but it did not produce thinning or ulceration of the skin, as would be expected of a potent pro-apoptotic drug. Similarly, there were no significant differences in the morphological and histological characteristics of the unaffected normal skin between animals in the DMBA/TPA+BzATP group (BzATP treatment for 30 weeks) and the DMBA/TPA group. However, in the DMBA/TPA+BzATP group the enhanced apoptosis was associated with inhibition of papilloma development. The disparity between BzATP effects in normal and papilloma tissues could be related to differences in the growth rate of the respective keratinocytes. Normal skin cells are slow growing and their overall growth rate is apparently not affected by BzATP; in contrast, in the fast growing papilloma keratinocytes BzATP-induced apoptosis slows and inhibits growth.
The data in normal mice also showed that local treatment with BzATP had no adverse systemic effects, suggesting a relatively safe profile for the drug when applied locally on the skin. These data indicate that BzATP is absorbed from the skin into the basal/parabasal epidermal regions and hair shafts. The data also suggest that the predominant effect of BzATP is induction of apoptosis at the site of application, targeting rapidly growing proliferating keratinocytes.
In contrast to papillomas, the expression level of P2X
7 receptors in DMBA/TPA-induced cancer cells was low, as was evident by three assays: in-situ immunoreactivity, Western blots, and qPCR. These findings are similar to those reported in non-melanoma skin cancer cells [
30] and in uterine, bladder and breast epithelial cancers [
9,
38,
41], and Li, Qi, Zhou, Fu, Abdul-Karim, MacLennan, and Gorodeski GI: P2X
7 receptor expression is decreased in epithelial cancer cells of ectodermal, uro-genital sinus, and distal paramesonephric-duct origin (submitted, 2009). The findings suggest that the rapid proliferation of cancer cells could be in part due to the low expression of the P2X
7 receptor and to attenuated P2X
7-mediated apoptosis. Treatment with BzATP augmented apoptosis even in cancer cells expressing low levels of the receptor, but the effect was smaller than in normal or papilloma cells. The significance of this effect is at present unclear although it could have modified the biological behavior of the cancers and have contributed to the prolongation of life in the affected animals, as was discussed above.
Until recently little was known about the mechanisms of P2X
7-receptor – apoptosis in the skin, and one of the objectives of the present study was to begin to understand the signaling pathways and molecular mechanisms that are involved in BzATP action in keratinocytes. The data suggest that, similar to uterine epithelial cells [
8,
18], the P2X
7-receptor – apoptosis in keratinocytes depends on enhanced calcium influx via P2X
7 pores, and is mediated by the caspase-9 – mitochondrial pathway. The following experimental findings in the present study support this hypothesis: (a) Treatment with BzATP induced formation of pores and enhanced calcium influx; (b) the BzATP-induced apoptosis, pore formation and the augmented and prolonged calcium influx were critically dependent on the expression of the P2X
7 receptor; (c) the BzATP-induced apoptosis, pore formation and the augmented calcium influx had similar dose-dependence on BzATP; (d) the BzATP-induced pore formation and the augmented calcium influx began shortly (30–60 seconds) after adding BzATP. In contrast, the BzATP-induced apoptosis required hours of treatment with BzATP, commensurate with a gene-mediated effect; (e) the BzATP-induced apoptosis depended on the presence of extracellular calcium at a physiological concentration of 1.2 mM, and on calcium influx; (f) the BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8. Since caspase-3 is a terminal step in the caspase cascade [
5,
6], a possible interpretation of the present results is that P2X
7-receptor – apoptosis is mediated by the caspase-9 (mitochondrial) pathway. Collectively the data in mouse keratinocytes suggest that BzATP-dependent activation of the P2X
7 receptor involves formation of pores in the plasma membrane, and that facilitated uncontrolled influx of Ca
2+ via the P2X
7 pores stimulates apoptosis by the mitochondrial – caspase-9 pathway.
P2X
7 pores are believed to be formed of channels composed of pannexins [
42,
43] and ectodomains of the P2X
7 molecule [
23,
42]. However, the ability of agonists to induce apoptosis via the P2X
7 pore mechanism is determined primarily by the cellular expression of the P2X
7 receptor [
13,
23]. The present study showed that papilloma keratinocytes express the P2X
7 receptor; therefore, the high expression levels of the receptor in papilloma cells and the significant apoptotic effects in response to BzATP could explain the inhibitory effect of BzATP on papilloma development. In contrast, the lesser effect of BzATP in skin cancer cells could be explained by the low expression level of the P2X
7 receptor in the cancer cells.
At present little is known whether the neoplastic transformation induces lesser expression of the P2X
7-receptor, or whether the neoplastic transformation is triggered preferentially in cells expressing low levels of the receptor. The former possibility is supported by data in endometrial and bladder cells where low expression of the P2X
7 receptor was found already in pre-cancerous and early cancerous cells but not in hyperplastic benign cells [
38]. Accordingly, the carcinogenic process could have induced lesser expression of the P2X
7 already at early stages of cancer development. On the other hand the possibility that the neoplastic transformation is triggered preferentially in cells expressing low levels of the receptor is supported by data as well, and it could be more fundamental to the understanding of epithelial-cell carcinogenesis. Thus, in uterine cervical epithelia low expression of the P2X
7 receptor was found already in dysplastic cells [
9]. Since only a small fraction of cervical dysplasia cases progresses to cancer [
44‐
46], it is possible that low expression of the P2X
7 receptor in the cervix precedes the neoplastic transformation. Accordingly, abrogation of P2X
7-mediated apoptosis could be responsible for the preservation of genetically aberrant cells that are susceptible to carcinogenic stimuli, favoring neoplastic transformation [
47].
The present data showed only partial inhibition (by about 50%) of papilloma and cancer formation in BzATP-treated mice. The experiments used the relatively low dose of 1 μg/cm2 BzATP, based on the 100 μM concentration used in experiments with cultured cells. The study was not designed to test higher doses and different frequencies of drug administration, and it is possible that higher doses and/or more frequent applications could produce greater inhibition papillomas and cancers. Additional studies are needed to test this possibility.
In addition to improving our understanding of the biogenesis of skin cancers and possibly other types of epithelial cancers where the P2X
7 controls cell growth, the present results provide a basis for continued research of novel chemotherapeutic growth-preventive modalities through regulation of apoptosis. The rationale is that epithelial cancers usually develop from premalignant lesions, e.g. papilloma, and the cancer risk of premalignant epithelial lesions may vary from 0.1% to 20% [
48‐
50]. The present results in the mouse model showed that local treatment with P2X
7-receptor agonists could inhibit the development of papillomas and inhibit the transformation of papillomas into cancers. BzATP appears to be a candidate chemotherapeutic growth-preventive drug for skin papillomas, with an apparent low risk profile of adverse events when administered locally on the skin. However, more studies are needed to test whether BzATP could be used in humans.
Competing interests
CytoCore Inc. funded a small part of the study but it has no financial interest in the study. Dr. Gorodeski was paid consultant to CytoCore Inc. and he holds restricted stocks of CytoCore. The ties between Dr. Gorodeski and CytoCore were severed in March 2008 and Dr. Gorodeski has no financial interest in the study. None of the other authors had any ties with CytoCore. Neither Dr. Gorodeski, nor any of the other authors have other financial or non-financial competing interests. University Hospital CASE Medical Center and Case Western Reserve University have a financial interest in the study
Authors' contributions
WF carried out the animals' experiments. TM supervised the animals' experiments. XQ carried out the immunostaining assays. LL assisted with the animals' experiments. LZ and XL carried out the cell culture assays. BCW participated in its design of the animals experiments. HG participated in its design of the study and carried out the data analysis. FWAK evaluated the pathology results. GIG conceived the study; participated in its design and coordination; and drafted the manuscript. All authors read and approved the final manuscript.