BzATP modulates DMBA/TPA skin effects (Experiment-1)
Local administration of DMBA/TPA induced formation of skin lesions, papillomas at weeks 5–12, and squamous spindle-cell carcinomas afterwards (Figs. 1, 2). About one thirds of the papillomas involuted after week 14 and the remaining persisted either as non-cancerous papillomas, or have transformed to cancerous lesions (Figs. 1, 2, 3, 4, 5, 6). All cancerous lesions arose from pre-existing papillomas. None of the animals in the control group had developed skin lesions (Fig. 1A).
Co-treatment with BzATP, applied locally on skin areas exposed to DMBA/TPA altered the incidence and pattern of skin lesions (Figs. 1, 2, 3, 4, 5, 6). To evaluate the effects of BzATP, changes in skin lesions in the DMBA/TPA and DMBA/TPA+BzATP groups were compared relative to the length of treatment. Since formation of papillomas and cancerous lesions was time-related, with a marked cut-off at weeks 13–14 (Fig. 3A), data were analyzed separately for weeks 0–12 and 14–28.
Proportion of animals with lesions
At weeks 0–12 the proportion of living animals with papillomas tended to be lower in the DMBA/TPA+BzATP group than in the DMBA/TPA group, and analysis of the proportion having a papilloma separately gave a significant (p < 0.05) difference at week 5 of treatment (Fig. 3A; 48 ± 12% versus 80 ± 10%, respectively). The proportion of living animals with any skin lesion at weeks 14–21 was similar in the two groups, being initially influenced by the death of two animals with cancers in the DMBA/TPA group at weeks 13–14 (Fig. 3A, filled circles). However, the proportion of living animals with any skin lesion differed significantly among the groups in weeks 22–28 (Fig. 3A). During that period of time the proportion of living animals with non-cancerous lesions (existing and involuting papillomas) decreased in both groups (Fig. 3B). In contrast, the proportion of living animals with cancerous lesions in the DMBA/TPA group increased steadily while in the DMBA/TPA+BzATP group it decreased over time (Fig. 3B). For example, in week 28 the proportions of living animals with cancerous lesions in the DMBA/TPA and the DMBA/TPA+BzATP groups were 100% and 43 ± 9%, respectively (Fig. 3B).
Mean number of lesions
In both groups the mean number of papillomas per living animal increased at weeks 0–12, but the increase in the DMBA/TPA+BzATP group tended to be smaller than in the DMBA/TPA group (Fig. 4A). Independent samples t-test revealed a significant difference at week 10 (2.3 ± 0.5 and 1.2 ± 0.4 papillomas per animal [mean ± SD], respectively, p < 0.04). Also, repeated measures ANOVA yielded a significant time effect (p < 0.01) for the DMBA/TPA and DMBA/TPA+BzATP curves at weeks 0–12 (Fig. 4A). At weeks 14–28 the mean number of total lesions per living animal was not significantly different between the two groups (Fig. 4A). In both groups the mean number of non-cancerous lesions decreased over the 14–28 weeks period (Fig. 4B), while the mean number of cancerous lesions remained the same (Fig. 4C).
Mean lesion size
Animals in both groups were compared relative to the total size of lesions (in mm3) per animal. In both groups the mean total papillomas size per living animal increased at weeks 0–12, but the increase in the DMBA/TPA+BzATP group was smaller than in the DMBA/TPA group (Figs. 1B, C, 5A). Independent samples t-test revealed significant differences at all weeks for mean total papillomas size (p < 0.01–0.03, Fig. 5A). For example, in week 12 mean total papillomas size (in mm3) per animal was 5.8 ± 1.1 versus 3.4 ± 1.0 (mean ± SD), respectively (p < 0.01, Fig. 5A). Likewise, repeated measures ANOVA yielded a significant time effect (p < 0.01); a significant group effect (p < 0.02); and a non-significant time*group interaction effect (p > 0.1), for the DMBA/TPA and DMBA/TPA+BzATP curves (Fig. 5A). The latter analysis indicates non-interacting trends for the two curves.
At weeks 14–28 the variability of the lesions sizes among the two groups was large, due to the excessive growth of some lesions unproportionally to others (e.g. Figs. 1D–I). This precluded us from comparing means of lesion size among the two groups. However, since most non-cancerous lesions in both groups tended to be smaller than 10 mm3 and the proportion of animals with non-cancerous lesions of > 10 mm3 was low (< 10%) in both groups (Fig. 5B, triangles), data of the proportion of living animals with cancerous lesions > 10 mm3 were compared among the two groups. Figure 5B shows a significantly smaller proportion of living animals in the DMBA/TPA+BzATP group with cancerous lesions > 10 mm3 after week 23 than in the DMBA/TPA group. For example, in week 28 the proportion of living animals with cancerous lesions > 10 mm3 were 81% ± 8% compared to 16 ± 4% in the DMBA/TPA and the DMBA/TPA+BzATP groups, respectively (Fig. 5B).
Interestingly, five mice in the DMBA/TPA+BzATP group survived despite having developed relatively large cancerous lesions (e.g. Fig. 1I), while maintaining normal weight and exhibiting normal behavior. In contrast, most mice in the DMBA/TPA group with already smaller cancerous lesions (e.g. Fig. 1H) had to be euthanized per protocol due to poor general condition and excessive tumor burden. Analysis of the proportion of living animals with cancerous lesions > 200 mm3 showed a tendency for higher proportion of animals in the DMBA/TPA+BzATP group than in the DMBA/TPA group (Fig. 5C), but the differences did not reach statistical significance.
Summary of the trends of cancer development and survival rates
Using time-to-event data analysis it was found that development of cancerous lesions was significantly slower and lower in the DMBA/TPA+BzATP group than in the DMBA/TPA group (Fig. 6A).
Survival curves for the DMBA/TPA and DMBA/TPA+BzATP groups were generated based on event (death from cancer) and time-to-event (in weeks) for each group. Log-rank test was used to compare the survival curves based on group. The overall survival rates among the two groups did not differ statistically, although there was a tendency of earlier deaths in the DMBA/TPA group compared to the DMBA/TPA+BzATP group (Fig. 6B).
Animals' weights
There were no significant differences in animals' weights among the control or the DMBA/TPA and DMBA/TPA+BzATP treatment groups over the course of 28 weeks (not shown).
Morphological and histological skin changes
There were no significant differences in the morphological (Figs. 1A–I) and histological characteristics (Figs. 2A, B) of the unaffected normal skin in the DMBA/TPA and the DMBA/TPA+BzATP groups. Similarly there were no significant differences in the morphological (Figs. 1B–E) and histological characteristics of papillomas in the DMBA/TPA and DMBA/TPA+BzATP groups (Figs. 2C, D). In both groups after week 14 some papillomas remained intact while other started to involute (Figs. 1D, E, 2E). However, in both groups most papillomas (about two third) underwent cancerous transformation to squamous cell carcinomas with spindle-cell changes (Figs. 2F–I). There were no significant changes in the morphological (Figs. 1F–I) and histological characteristics (not shown) of cancers in the two groups.
P2X7receptor expression is lower in mouse skin cancer tissues
Cellular effects of BzATP are mediated mainly by the P2X7 receptor [11, 12]. The present data showed that mouse normal, papilloma, and cancer skin cells express the P2X7 receptor, but levels of the receptor in cancer tissues were significantly lower than in normal skin or papilloma tissues (Fig. 7). Immunostaining with the anti P2X7 receptor antibody of tissue cross sections containing normal skin revealed intense immunoreactivity that localized predominantly in the epidermis within proliferating keratinocytes and epidermal hair shafts (Figs. 7A, B). In papillomas, P2X7 immunoreactivity was intense (Fig. 7C), similar to normal tissues (Fig. 7A), and it localized predominantly within proliferating keratinocytes at the base of the developing papillomas (Figs. 7C, D). In contrast, P2X7 immunoreactivity in cancer tissues was significantly lesser than in normal epidermal or papilloma tissues (Figs. 7E, F), and data analysis of the P2X7 immunostaining revealed a four fold lesser P2X7 immunoreactivity in cancer than in normal tissues (Fig. 7G).
The immunostaining data were confirmed by Western blot experiments. Assays of the P2X7-specific 75 KDa band revealed a five fold lower density in cancer tissues than in normal tissues (Fig. 7H). Further confirmation was obtained by P2X7 mRNA experiments where qPCR assays revealed a five fold lower P2X7 mRNA/GAPDH mRNA levels in normal tissues than in cancer tissues (Fig. 7I). Collectively, the data in Fig. 7 indicate that P2X7 receptor expression levels in mouse skin cancer tissues are four-five fold lower than in mouse normal skin tissues.
BzATP augments apoptosis in the normal skin (Experiments 2 and 3)
To better understand the cellular effects of BzATP in vivo, experiments investigated the effects of BzATP in the normal mouse on skin morphology and histology; on the immunoreactivities with the P2X7 antibody; and on apoptosis.
In animals of Experiment-2, BzATP was applied twice weekly for 4 weeks on the anterior region of the shaved dorsal skin and each animal served as its own control by having vehicle-containing solution applied twice weekly for 4 weeks on the posterior region of the shaved dorsal skin (Fig. 8A). Treatments with BzATP or the vehicle solution had no visible morphological effect on the skin (Fig. 8A), and histological evaluation showed no differences in cross sections obtained from the BzATP-treated or control skin areas (Figs. 8B, C).
Experiment-2 also studied the effects of local treatment with BzATP on skin apoptosis in vivo by TUNEL staining. The negative control experiment showed minimal auto-fluorescence in cross sections of the mouse skin (Figs. 8D, E). In skin cross sections of non-treated mice, only faint TUNEL staining decorated the epidermis (Fig. 8F). In contrast, in skin cross sections of BzATP-treated mice numerous epidermal basal/parabasal cells and epidermal hair shaft cells stained TUNEL positive (Fig. 8G). Additionally, DAPI stains of cross sections of BzATP-treated skin revealed greater proportion of nuclei at advanced stages of condensation, fragmentation and pyknosis (Fig. 8I) compared to controls (Fig. 8H).
In animals of Experiment-3, BzATP was applied twice weekly for 16 weeks on the entire shaved dorsal skin. The control group included mice that were treated only with the vehicle. Similar to Experiment-2, treatment with BzATP had no significant effect on skin morphology (Figs. 9A, C) and histology (Figs. 9B, D), compared to treatment with the vehicle only. Also, treatment with BzATP had no significant effect on P2X7 immunoreactivity (Figs. 9E, F). However, treatment with BzATP increased the number of TUNEL stained epidermal basal/parabasal and hair shaft cells (Figs. 9G, H), similar to the result in Experiment-2. P2X7-TUNEL co-staining showed that the increased TUNEL staining co-localized with P2X7 immunoreactivity (Figs. 9I, J [low magnification], Figs. 3K, L [higher magnification]).
Collectively, the data in Fig. 9 indicate that treatment with BzATP, applied locally twice a week on the shaved dorsal skin of normal mice, up-regulated apoptosis of proliferating epidermal and hair shaft keratinocytes. However, in the normal mouse skin the BzATP treatment and the augmented apoptosis did not affect morphology or histology of the skin.
Treatments with BzATP for 4 weeks (Experiment-2) or for 16 weeks (Experiment-3) had no significant effects on the behavior of the animals, on their feeding habits, or on their body weight (not shown). In addition, in animals of Experiment-3 mean ALT and AST plasma levels were similar among the BzATP and control groups, and were in the normal range for the mouse (not shown).
Effects of BzATP on P2X7expression and TUNEL in papilloma and cancer tissues
P2X7 immunoreactivities in cross sections of papillomas did not differ in intensity among specimens obtained from DMBA/TPA- or DMBA/TPA+BzATP – treated mice (Figs. 10A, B). The intensity of P2X7 immunoreactivity in cross sections of skin cancers was significantly weaker than in normal (Fig. 7A) and in papilloma tissues (Figs. 10A, B), but it did not differ among the DMBA/TPA and DMBA/TPA+BzATP groups (Figs. 10E, F).
TUNEL staining was weak in cross sections of papillomas (Fig. 10C) and cancer tissues (Fig. 10G) from the DMBA/TPA group, similar to findings in cross sections of normal skin (Figs. 8F, 9G). In contrast, TUNEL staining was more intense in cross sections of papillomas (Fig. 10D) and cancer tissues (Fig. 10H) from the DMBA/TPA+BzATP group. In papillomas obtained from mice of the DMBA/TPA+BzATP group, enhanced TUNEL staining decorated basal/parabasal layers of keratinocytes outgrowing at the base of the developing papilloma (Fig. 10D).
Mechanism of BzATP-augmented apoptosis in mouse keratinocytes
Dependence of BzATP-augmented apoptosis on the expression of the P2X7receptor
To better understand the mechanism of BzATP pro-apoptotic skin effects, experiments utilized cultured primary mouse keratinocytes that were obtained from wild-type mice, and from the P2X7-receptor – deficient P2X7-/-Pf and P2X7-/-GSK mice.
In wild-type mouse keratinocytes BzATP augmented apoptosis in a dose-related manner; effects began at BzATP levels as low as 50 nM, reaching maximal effect at 100–250 μM with an estimated BzATP EC50 of about 10 μM (Fig. 11A).
Pre-treatment with P2X7-receptor anti-sense oligonucleotide decreased expression of the P2X7-receptor (Fig. 11B, insert); it also inhibited baseline apoptosis (which most likely is induced paracrinologically by ATP secreted by the cells [8]), and blocked the pro-apoptotic effect of BzATP (Fig. 11B). Pre-treatment with random-control oligonucleotides had no effect on P2X7-receptor expression (Fig. 11B, insert), or on baseline apoptosis and the apoptosis induced by BzATP (Fig. 11B).
The dependence of the pro-apoptotic effect of BzATP on the expression of the P2X7 receptor was further demonstrated in experiments using keratinocytes obtained from P2X7-receptor – deficient mice. Compared to wild-type mouse keratinocytes, in both the P2X7-/-Pf and P2X7-/-GSK keratinocytes treatment with 100 μM BzATP failed to induce apoptosis (Fig. 11C).
Formation of P2X7pores
In uterine epithelial cells [23], as well as in other types of cells [11–13], P2X7-receptor – dependent apoptosis involves agonist-induced acute calcium influx via P2X7 pores. To understand whether BzATP-induced apoptosis in mouse keratinocytes involves formation of P2X7 pores, experiments compared activation by BzATP of the P2X7 receptor (in terms of BzATP-induced increase in cytosolic calcium [23]), and the formation of P2X7 pores (in terms of BzATP-induced increase in the influx of ethidium bromide [23]).
In mouse wild-type keratinocytes treatment with 100 μM BzATP induced acute increase in cytosolic calcium, which lasted at least 6 min (Fig. 12A). In cells bathed in low calcium BzATP induced only spiked increase in cytosolic calcium, while the prolonged sustained increase in cytosolic calcium was abolished (Fig. 12A). The spiked, short-term increase in cytosolic calcium most likely represents calcium release from intracellular stores [8]. The lack of prolonged increase in cytosolic calcium in cells bathed in extracellular medium low in calcium indicates that the BzATP-induced prolonged increase in cytosolic calcium involves calcium influx.
Experiments using mouse wild-type keratinocytes also revealed that treatment with BzATP induced an acute increase in the influx of ethidium bromide (Fig. 12B) with a time-course similar to the increase in cytosolic calcium (Figs. 12A, B). Both effects had similar dose-dependence for BzATP (Fig. 12C), and they resembled the dose-dependence of apoptosis on BzATP with threshold effects at 50–100 nM and pre-maximal responses at 100–250 μM (Figs. 11A, 12C).
BzATP-induced increases in cytosolic calcium and influx of ethidium bromide depend on the expression of the P2X7receptor
Similar to the effects of BzATP on apoptosis, pre-treatment with the P2X7-receptor anti-sense oligonucleotide blocked the BzATP-induced increase in cytosolic calcium (Fig. 13A) and the BzATP-induced increase in ethidium bromide (Fig. 13B). Pre-treatment with the random-control oligonucleotides had no effect on the responses to BzATP (Fig. 13).
The BzATP-augmented apoptosis depends on extracellular calcium
In mouse wild-type keratinocytes lowering extracellular calcium attenuated baseline apoptosis and blocked the BzATP-induced apoptosis in a dose-related manner (Fig. 14A).
The BzATP-augmented apoptosis involves caspase-9 and caspase-3
Treatment of mouse wild-type keratinocytes with the caspase-9 inhibitor LEHD-FMK blocked BzATP-induced apoptosis while the caspase-8 inhibitor IETD-FMK did not have a significant effect (Fig. 14B). The positive controls were DEVD-FMK (specific inhibitor of the terminal caspase-3) and zVAD-FMK (non-specific pan-caspase inhibitor) which similarly blocked the BzATP-induced apoptosis (Fig. 14B).
Treatment with BzATP did not induce cell proliferation
To determine if the development of the large cancerous lesions in some animals in the DMBA/TPA+BzATP group was the result of a pro-mitogenic effect of BzATP, rates of DNA synthesis (in terms of [3H]thymidine incorporation) in response to BzATP were measured in mouse wild-type normal keratinocytes. Pre-treatments with the P2X7-receptor anti-sense P2X7 oligonucleotides or the random-control oligonucleotides, and treatments with BzATP had no significant effect on [3H]thymidine incorporation (Fig. 15).