INTRODUCTION

Radiotherapy is widely used in cancer treatment. Although current radiotherapy protocols can be highly effective, not all cancer cells may be killed, and damage to surrounding normal tissue is common. Damage to normal tissue can result in short and long-term complications (1, 2). Prostate irradiation can cause rectal damage leading to pain or bleeding in the short term, while the long-term effects may include incontinence and impotence. Significant research has been expended identifying potential radioprotector molecules to reduce the short and long-term effects of radiotherapy including amifostine (3, 4), Tempol (5), N-acetylcysteine (6, 7), MnSOD (8) and antioxidant vitamins (9, 10) all of which are involved in free radical scavenging, modulation of DNA repair, apoptosis and the immune system. Results of efficacy have been mixed, with problems of toxicity and lack of differential radiosensitivity between normal and tumor tissue. New radioprotectors that sensitize tumor tissue at the same time as protecting normal tissue would be ideal, thus increasing cancer kill rate and reducing unwanted side effects to normal tissue. Increasing evidence demonstrates that certain mild pro-oxidant compounds derived from natural herbal medicines might enhance radiotherapy by modulating the redox state of cancer cells to high pro-oxidant levels (11, 12). One such compound is Parthenolide (PTL), a major active ingredient derived from the traditional anti-inflammatory medicinal plant feverfew (Tanacetum parthenium) which belongs to the family of sesquiterpene lactones containing an α-methylene-γ-lactone moiety and an epoxide group (13). In addition to its anti-inflammatory effect, PTL has been shown to be highly toxic in a variety of cancer cell lines (14–18). Mechanistically, PTL has been shown to increase apoptosis in cancer cells through inhibition of multiple pro-survival pathways, such as NF-κB and PI3K-AKT (11, 19). One of the most interesting discoveries about PTL's anti-cancer mechanism has been its ability to increase radiosensitivity in tumor tissues while at the same time protecting normal cells from radiation effects. PTL can preferentially inhibit growth and induce apoptosis of prostate cancer cell lines compared to normal prostate cells in vitro and can inhibit prostate tumor initiating cells in mouse xenografts (20). Recently this differential radioprotection was shown to be mediated by a novel redox mediated modification of KEAP1 (Kelch-like ECH-associated protein 1) and Nrf2 [Nuclear factor (erythroid-derived 2)-like 2] (18). PTL activates NADPH oxidase in prostate cancer cell lines but not in normal cells, thus mediating intense oxidative stress in prostate cancer cells by increasing reactive oxidative species (ROS) generation and decreasing antioxidant defense capacity (17). Because prostate cancer cells are under higher endogenous levels of oxidative stress it is hypothesized that additional exposure to the ROS induced by PTL pushes cancer cells toward death, whereas normal cells maintain redox homeostasis through KEAP1/Nrf2 mediated adaptive oxidative responses. These results demonstrate that PTL has the potential to reduce the radiation-induced side-effects of radiotherapy while killing more tumor cells and facilitating increased radiation doses in radiotherapy.

During radiotherapy, patients are not only exposed to high therapeutic doses of radiation but can also receive low doses of radiation during CT imaging protocols. This is becoming increasingly common with the use of image guided radiotherapy. Low doses of radiation (generally below 100 mGy) given before a high dose are known to “condition” cells resulting in protection from a proportion of the high-dose-induced damage. This has been termed the radio-adaptive response [reviewed in (21, 22)]. The conditioning dose can result in reduced genetic damage (23, 24) and apoptosis (25–27) in normal cells, decreased tumor incidence and increased tumor latency (28–30) compared to the effects of the high dose alone. The exact mechanism of the radio-adaptive response is not fully understood, but it is known that p53 (31), ROS (31, 32), NFĸB (33, 34), MnSOD (35) and several pathways in the immune system (36, 37) play important roles. This raises the possibility that image doses of radiation, which are usually less than 100 mGy may therefore act as a radioprotector of normal tissue. The low-dose radio-adaptive response has conventionally been considered to only be active in normal cells and that tumor cells are no longer responsive to low ‘adapting' doses of radiation (38), however, it has been recently demonstrated that low doses of radiation can elicit an adaptive response in tumor cells as well involving increased expression of survivin, an apoptosis inhibiting protein (39). Importantly, it has been shown that PTL suppresses survivin (40). The timing between the image and radiotherapy dose is likely to be important in the ability of the image dose to protect tumor cells. This creates a dilemma for image-guided radiotherapy where the image dose may be protecting normal tissue and may therefore be reducing damaging side effects to normal tissue but at the same time be protecting the tumor and therefore reducing the efficacy of radiotherapy. Here we investigated if a low dose of radiation, in the range of conventional imaging doses, could contribute to protecting normal and tumor tissue from high-dose-induced damage, in the presence and absence of PTL and a more bioavailable analogue of PTL, dimethylaminoparthenolide (DMAPT) (41).

PTL has not previously been studied in mice with a normal immune system which is important for the radioadaptive response or in an autochthanous cancer model which more closely approximates cancer formation and progression in humans. The autochthanous TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model of prostate cancer (42) was used as a proof-of-principle to test whether intrinsic differences in cellular redox conditions can be used to kill tumor cells while protecting healthy cells from unwanted side effects of radiation in vivo.

MATERIALS AND METHODS

Treatment with Parthenolide/DMAPT and Radiation

All studies were performed on 16-week-old C57BL/6J or TRAMP mice. At this age TRAMP mice are in early stages of prostate tumor development and most prostate tissues have moderate to high levels of PIN. Mice were administered 40 mg/kg of PTL, 100 mg/kg DMAPT or vehicle controls (10% ethanol in saline or sterile water, respectively) by oral gavage 3× per week for one week. A dose of 100 mg/kg of DMAPT was selected as it is the highest safe dose tested in vivo (43). Due to the low solubility of PTL a dose of 100 mg/kg was deemed unfeasible for this study; therefore, 40 mg/kg was selected due to previous in vivo use (20). Twenty-four h after the final PTL or DMAPT treatment, mice were exposed to whole-body X irradiation. Mice were restrained in individual compartments of a 6-mm thick Perspex holder during the irradiation. Mice received either 6 Gy alone (Fig. 1A and C), 10 mGy followed by 6 Gy irradiation, sham irradiation followed by 6 Gy, or 10 mGy followed by sham irradiation, with a 3 h interval between irradiations (Fig. 1B). Irradiation was performed using either a 6 MV X-ray beam from a Varian 600CD Linear Accelerator (Varian Medical Systems, Inc., Palo Alto, CA) or 300 kV X-ray beam from a X-RAD 320 Cabinet irradiator (Precision X-Ray Inc., North Branford, CT) for experiments where mice were administered 6 Gy. Irradiation with 10 mGy was carried out using the X-RAD 320 Cabinet irradiator. The dose calibration of the Varian 600CD Linear Accelerator was made according to the International Atomic Energy Agency's technical report on Absorbed Dose Determination in External Beam Radiotherapy (series no. 398) (44). The dose calibration of the orthovoltage 300 kV X-ray beam produced by the X-RAD 320 was performed according to the Institute of Physics and Engineering in Medicine and Biology (IPEMB) protocol (45, 46). Control sham-irradiated mice underwent the same procedures as irradiated mice; however, the X-ray machines were not turned on. At the appropriate time point after X irradiation, mice were euthanized using CO₂ asphyxiation. At necropsy, full tissue panels were taken from all mice and snap frozen on dry ice in OCT cryoprotectant medium (Tissue-tek, Japan). Prior to analysis, prostates were thawed and micro-dissected in ice cold phosphate buffered saline (PBS), then immediately re-embedded in OCT. Tissues were stored at –80°C until required for analysis.

FIG. 1.

Schematic overview of the PTL/DMAPT treatment and irradiation protocols. In all experiments mice received 3 doses of PTL or DMAPT on day 1, 4 and 7, and were irradiated 24 h after the last dose. Panel A: TRAMP and C57BL/6J mice received either a 6 Gy dose or sham irradiation and analysis was performed 6 h postirradiation. Panel B: TRAMP and C57BL/6J mice received either a 10 mGy dose or sham irradiation, followed by a 6 Gy dose or sham irradiation 3 h later, and tissues were analyzed 6 h postirradiation. Panel C: TRAMP mice received a 6 Gy dose or sham irradiation and analysis was performed at 6, 24 or 72 h postirradiation.

RESULTS

Parthenolide Protects Normal Tissues from Radiation-Induced Damage when Delivered Alone or In Combination with a Conditioning Low Dose of Radiation

C57BL/6J and TRAMP mice were treated as per the protocol described in Fig. 1A to determine if PTL could protect from apoptosis induced by a high dose of radiation in normal tissues, and to determine if normal tissues responded differently in the different strains of mice. Across all experiments baseline apoptosis frequency in normal tissues did not differ significantly between C57BL/6J and TRAMP strains (n = 12–26); mean baseline apoptosis frequency in both mouse strains was 0.0041 (± 0.0023 SD) in normal dorsolateral prostate, and 0.0074 (± 0.0036 SD) in normal spleen, with mean baseline of 0.53 (± 0.17 SD) apoptotic cells per crypt in normal colorectal tissues. When analyzed 6 h after exposure to 6 Gy, the mean apoptosis frequency increased 31-fold to 0.13 (± 0.035 SD) in normal dorsolateral prostate (n = 23), and 29-fold to 0.21 (± 0.027 SD) in normal spleen (n = 67), with mean apoptotic cells per crypt increasing 11-fold to 5.81 (± 0.54 SD) in normal colorectal tissue of C57BL/6J mice (n = 67). PTL (40 mg/kg) induced a partial protection from 6 Gy radiation-induced apoptosis in normal C57BL/6J dorsolateral prostate (34.5% reduction, P = 0.002), and spleen (17.3% reduction, P = 0.041) tissues and in normal TRAMP spleen tissues (31.4% reduction, P = 0.01) 6 h postirradiation (Fig. 2). Radiation-induced apoptosis was not significantly reduced by PTL in normal colorectal tissues of either mouse strain.

FIG. 2.

Mean apoptosis (±1 SD) in (panel A) normal C57BL/6J dorsolateral prostate, and normal C57BL/6J and TRAMP (panel B) spleen and (panel C) colorectal tissue after 3× 40 mg/kg PTL or vehicle control treatments over 1 week, with exposure to 6 Gy whole-body X irradiation 24 h after the final treatment, with analysis at 6 h postirradiation. *P < 0.05, n = 5–15.

C57BL/6J mice were treated as per the protocol described in Fig. 1B to determine if a 10 mGy-conditioning dose of radiation could add to PTL-induced reduction of apoptosis in normal tissues after 6 Gy (Fig. 3). A dose of 10 mGy alone induced a significant increase in apoptosis (489.5% increase, P = 0.017) in spleen (Fig. 3B); but not in prostate or colorectal tissues (Fig. 3A and C) when analyzed 9 h postirradiation. In the absence of PTL, the 10 mGy-conditioning dose induced a radio-adaptive response when delivered 3 h before a 6 Gy high dose in the dorsolateral prostate (28.4% reduction in apoptosis, P = 0.045), compared to the sham-conditioning dose plus 6 Gy exposure (Fig. 3A). There was also a trend towards an adaptive response in normal spleen tissue (15.8% reduction in apoptosis, P = 0.084) (Fig. 3B). PTL decreased apoptosis in sham-treated dorsolateral prostate (28.7% reduction, P = 0.048) and reduced 6 Gy-induced apoptosis in the presence and absence of a 10 mGy-conditioning dose in spleen and prostate (P < 0.05), however a 10 mGy dose did not significantly add to PTL-induced protection from apoptosis (P > 0.05).

FIG. 3.

Mean apoptosis (±1 SD) in normal C57BL/6J mouse (panel A) dorsolateral prostate, (panel B) spleen and (panel C) colorectal tissue after 3× 40 mg/kg PTL or vehicle control treatments over 1 week, with combinations of exposure to sham, 10 mGy and 6 Gy whole-body X irradiation 24 h after the final PTL treatment, with analysis at 6 h postirradiation. *P < 0.05, n = 6.

Comparison of Radioprotection using PTL and DMAPT

The efficacy of DMAPT (a PTL analogue with increased solubility and bioavailability) was compared with PTL in C57BL/6J mice using the protocol shown in Fig. 1A. DMAPT increased the magnitude of radioprotection from 6 Gy-induced apoptosis in normal C57BL/6J mouse dorsolateral prostate (71.7% reduction, P = 0.026), spleen (48.2% reduction, P = 0.0001) and colorectal tissues (38.0% reduction, P = 0.0002), compared to the radioprotection provided by PTL (dorsolateral prostate: 41.4% reduction, P = 0.045; spleen: 29.5% reduction, P = 0.011; colorectal: 15.7% reduction, P = 0.067) (Fig. 4). Compared to PTL, DMAPT increased radioprotection in dorsolateral prostate, spleen and colorectal tissues by a further 1.6–2.4-fold.

FIG. 4.

Mean apoptosis (±1 SD) in C57BL/6J mouse (panel A) dorsolateral prostate, (panel B) spleen and (panel C) colorectal tissues after 3× 40 mg/kg PTL, 100 mg/kg DMAPT or vehicle-control treatments over 1 week, after 6 Gy whole-body X-irradiation 24 h after the final drug treatment, with analysis at 6 h postirradiation. *P < 0.05, n = 6.

DMAPT Radioprotects Normal Tissues while Sensitizing Tumors to Radiation-Induced Damage

TRAMP mice were treated as per the protocol shown in Fig. 1B. TRAMP mice were treated with DMAPT and X irradiated with 6 Gy in the presence or absence of a 10 mGy-conditioning dose (Fig. 5). In the absence of DMAPT, a 10 mGy-conditioning dose delivered 3 h prior to the high-dose-reduced 6 Gy-induced apoptosis in normal TRAMP spleen (13.6% reduction, P = 0.047) (Fig. 5B), compared to animals that received a sham-conditioning dose. In normal TRAMP spleen and colorectal tissues, DMAPT reduced 6 Gy-induced apoptosis in the presence and absence of a 10 mGy conditioning dose, compared to vehicle-control-treated mice (Fig. 5B and C). In the TRAMP model, most prostates are in the mid-stages of tumor development by 16 weeks of age. Across all experiments (n = 26 mice), mean apoptosis frequency in TRAMP dorsolateral prostate was 0.012 (± 0.0046 SD) at baseline and increased 11-fold to 0.1344 (± 0.053 SD) after 6 Gy irradiation (n = 51 mice). DMAPT increased radiation-induced apoptosis in TRAMP prostate tissues in the absence and presence of a 10 mGy conditioning dose (81.9% increase, P = 0.02; 135.0% increase, P = 0.0024, respectively) (Fig. 5A). In the absence of DMAPT, there was no significant difference in apoptosis in TRAMP prostate PIN tissue with or without a 10 mGy conditioning dose.

FIG. 5.

Mean apoptosis (±1 SD) in TRAMP (panel A) dorsolateral prostate, (panel B) spleen and (panel C) colorectal tissues after 3× 100 mg/kg DMAPT or vehicle-control treatments over 1 week, after sham, 10 mGy or 6 Gy whole-body X irradiation (alone or in combination) 24 h after the final DMAPT treatment, with analysis at 6 h postirradiation. *P < 0.05, n = 11–14.

Differential Radiosensitization from DMAPT in TRAMP Tissues Persists beyond the Initial Phase of Apoptosis

In temporal studies, TRAMP mice were treated as per the protocol described in Fig. 1C. In the absence of radiation, apoptosis in spleen, colorectal and dorsolateral prostate tissue was not significantly different between treatment groups and did not significantly alter across the different time points (P > 0.05, ANOVA with Turkey's multiple comparisons test) (Fig. 6). In TRAMP dorsolateral prostate (Fig. 6A), significant augmentation of 6 Gy-induced apoptosis was observed in the DMAPT treated mice at 6 h postirradiation (119.4% increase, P = 0.0018) compared to vehicle-treated mice. An increase in apoptosis was still present 18 hours later in the DMAPT-treated group (107.2% increase, P = 0.048) and apoptosis levels returned to baseline frequency by 72 h. In colorectal tissues (Fig. 6B), a reduction in 6 Gy-induced apoptosis was observed 6 and 24 h postirradiation (34.6% reduction, P = 0.0034 and 55.6% reduction, P = 0.0002), respectively, compared to the vehicle-control-treated mice. By 72 h, apoptosis returned to baseline levels. In TRAMP spleen (Fig. 6C-i), radioprotection was observed in DMAPT treated mice 6 h (40.1% reduction, P = 0.021) and 72 h (64.3% reduction, P = 0.0001) postirradiation. The apoptosis observed at 6 h was largely follicular in both the DMAPT and vehicle-control-treated groups exposed to 6 Gy, and had returned to baseline levels by 24 h. At 72 h there was an increase in apoptosis in the 6 Gy-vehicle-treated mice, but not in DMAPT-treated mice. In vehicle-control-treated spleens, this second wave of apoptosis was again largely follicular (Fig. 6C-ii), however apoptosis in DMAPT-treated spleen was mostly observed in extra-follicular red pulp regions.

FIG. 6.

Mean apoptosis (±1 SD) in TRAMP (panel A) dorsolateral prostate, (panel B) colorectal and (panel C-i) spleen tissues after 3× 100 mg/kg DMAPT treatments over 1 week, with exposure to a 6 Gy or sham irradiation, and tissues analyzed at 6, 24 and 72 h postirradiation. *P < 0.05, n = 5-15. Panel C-ii: Fluorescently labeled apoptotic (TUNEL) cells in normal TRAMP spleen at 6, 24 and 72 h postirradiation. Scale bars 50 μm. At 6 h postirradiation the apoptosis was largely identified in the follicles in both DMAPT and vehicle control treatment groups; at 72 h, the majority of apoptosis in vehicle-control-treated spleen was still in follicular regions, however apoptosis in DMAPT-treated spleen was mostly observed in extra-follicular red pulp regions.

Enhanced Radiosensitivity of TRAMP Prostate Tumor Tissue by DMAPT is Greater with Increasing PIN grade

TRAMP dorsolateral prostates were scored for PIN grade using the method proposed by Berman-Booty et al. (48) (Fig. 7A–C). This method assigns tissues a score based on both the most common lesions and the most severe lesions. In TRAMP dorsolateral prostate tissues most tissues have developed moderate to high-grade PIN. Although there was no significant increase in apoptosis in the PTL group compared to the vehicle-control group (Fig 7D), there was a significant correlation between the amount of apoptosis induced by PTL in the presence of 6 Gy irradiation and increasing PIN grade (R² = 0.4037, P = 0.015) (Fig. 7E). There was no significant correlation in apoptosis with increasing PIN grade (R² = 0.0104, P = 0.77) in the vehicle-treated group. Radiation-induced apoptosis doubled in dorsolateral prostates of TRAMP mice when they were treated with the more bioavailable DMAPT (101.3% increase, P = 0.039) (Fig. 7F). As was the case with PTL, TRAMP dorsolateral prostates with high-grade PIN development were preferentially sensitized to radiation-induced apoptosis when mice were pre-treated with DMAPT (R² = 0.7909, P = 0.0001), while tissues with lower grades of PIN displayed the same apoptosis frequency as those of vehicle control-treated TRAMP mice (R² = 0.0242, P = 0.3) (Fig. 7G).

FIG. 7.

Representative morphology of TRAMP-dosolaterale-prostate lobes. Panel A: Low-grade PIN, with a few short papillary proliferations of hyperplastic epithelium projecting into the lumen; panel B: moderate-grade PIN, with more prominent papillary proliferations of hyperplastic epithelial cells that project into the lumen; and panel C: high-grade PIN, where the mass of proliferating epithelial cells form a cribriform pattern completely filling the lumen of the gland. Haematoxylin stain, 40× magnification, scale bars 50 μm. Mean apoptosis (±1 SD) in irradiated-TRAMP-dorsolateral prostates after (panel D) 3× 40 mg/kg PTL or vehicle control treatments over 1 week, or (panel F) 3 × 100 mg/kg DMAPT or vehicle control. *P < 0.05, n = 11–15. Apoptosis in dorsolateral prostate tissue from TRAMP mice treated with (panel E) PTL or (panel G) DMAPT plotted against PIN tumor grade. TRAMP tissues scored using the method described by Berman-Booty et al. (48)

DISCUSSION

A major limiting factor of radiotherapy is normal tissue toxicity. Prostate cancer radiotherapy can result in short-term side effects including colorectal injury which can induce diarrhea and bleeding, while the long-term effects may include incontinence, impotence and infertility. Here we investigated the ability of the naturally occurring anti-inflammatory compound, PTL and the water soluble PTL analogue, DMAPT, to protect normal tissues and sensitize prostate tumor tissues to radiation-induced apoptosis. Previous studies have shown that the increased hydrophilicity of DMAPT provides greater bioavailability compared with PTL (41, 43) and that DMAPT exerts anti-cancer effects in several cancer types in vivo (43, 49–52). The increased bioavailability of DMAPT makes it the drug of choice for future human clinical trials. In vivo, DMAPT has been shown to selectively radiosensitize human prostate cancer PC3 cell derived tumors in nude mice (18); however, these preclinical investigations have been limited to xenograft studies using immune compromised animals. Studying the TRAMP model here with an intact immune system and where the tumor progression within the prostate more closely mimics human prostate cancer progression provides the opportunity to gain a better understanding of the therapeutic efficacy of PTL. In this study, we observed simultaneous protection of normal tissues and radiosensitization of TRAMP prostate tumor tissues to 6 Gy irradiation, demonstrating the potential for DMAPT to be a therapeutic for prostate cancer, not only to decrease radiotherapy side effects but potentially to increase cure rate.

Significant radiosensitivity was observed in TRAMP dorsolateral prostate with moderate to high-grade PIN lesions, with DMAPT doubling the frequency of radiation-induced apoptosis in these tissues. Although the TRAMP mice used were born within 4 days of each other, TRAMP prostate tumor development progresses at different rates between individual mice, and there is a relatively wide range of PIN development between animals. This results in large variation in apoptosis frequency between animals, however it allows the effects of PTL to be observed in different stages of TRAMP prostate tumor development. Both PTL and DMAPT preferentially increased radiosensitivity in tissues with higher grade PIN, compared to tissues with lower grades of PIN (based on lesion severity) with DMAPT showing a stronger correlation, possibly due to the higher tissue exposure with DMAPT compared to PTL. High-oxidative-stress levels are a hallmark of prostate cancer, and ROS levels are particularly elevated in aggressive prostate tumors compared to localized disease (53, 54). Oxidative stress has been shown to increase with increasing PIN grade in TRAMP mice (55) and our data suggests that PTL/DMAPT may have the greatest effect in the presence of higher ROS levels. PIN grade in the TRAMP model does not directly correlate with Gleason grading of human prostate cancer, however the enhanced radiosensitization of high-grade-TRAMP PIN tissue may be an indicator that DMAPT could be highly effective in high-grade Gleason tumors and potentially for targeted treatment of metastatic prostate cancer, which often occurs from high-grade Gleason tumors and is incurable (56).

While PTL and DMAPT enhanced radiation-induced apoptosis in TRAMP prostate tumor tissue they also protected from high-dose-radiation-induced apoptosis in normal tissues with DMAPT showing superior protection in normal prostate, spleen and colorectal tissue. It was surprising that the response observed in DMAPT-treated mice was not more pronounced given both the increased bioavailability of DMAPT and that a 2.5-fold higher concentration of DMAPT was used here compared to PTL. The poor solubility of PTL restricts dosing of mice to a maximum of 40 mg/kg, providing a maximum plasma concentration of less than 1 μM, which is well below ideal therapeutic plasma concentrations of 5–10 μM (57). DMAPT dosed at 100 mg/kg results in a maximum plasma concentration above 10 μM (43). Although the increased dose of DMAPT did not result in a proportionately greater increase in radioprotection, it still provided a greater therapeutic effect than PTL. The ideal plasma concentration for maximum radioprotection may be somewhere between 1 and 10 μM. These results suggest that DMAPT could be a useful clinical tool for alleviating radiotherapy induced damage to normal tissue, while radiosensitizing tumor tissue. Amifostine is the only radioprotector currently approved for protection of normal tissues during radiotherapy (for treatment of head and neck cancers) (58), however the effects of the drug are short lived. More than 90% of amifostine is cleared within 6 min of administration and it has been shown that if radiotherapy is delivered more than 30 min after amifostine is administered, there may be little clinical benefit (59). The reported levels of radioprotection in response to amifostine if delivered within the restrictive timeframe (59–61) are similar to our results with DMAPT, however DMAPT may be a more appropriate radioprotector for clinical use, as here we show that DMAPT can be delivered at least 24 h before X-ray exposure. There have also been reported problems with amifostine-induced toxicity and lack of differential radiosensitivity between normal and tumor tissue (61, 62), whereas DMAPT has been shown to be highly tolerated with minimal side effects in human clinical studies (63, 64). The DMAPT-induced protection lasted up to 24 h postirradiation in normal colorectal tissues and to 72 h in normal spleen. If these effects can be replicated in a clinical setting it may allow particularly vulnerable tissues to be spared from damage while having the added benefit of increasing tumor killing efficacy. Further in vivo analysis of late-stage-radiation damage to rectum and bladder, such as fibrosis, would help to determine whether long-term protection of these particularly vulnerable tissues is possible using DMAPT. Long-term studies would also allow observation of tumor progression after DMAPT treatment and high-dose irradiation.

In all experiments, endogenous apoptosis frequencies observed in TRAMP and C57BL/6J prostate, spleen and colorectal tissues were all in the range described in the literature (60, 65, 66). In the absence of radiation, PTL and DMAPT did not modulate endogenous apoptosis in the normal tissues except for in one experiment with PTL where a significant reduction in apoptosis was observed in the dorsolateral prostate of C57BL/6J mice, but not in spleen or colon, 30 h after the final drug treatment was delivered. PTL may be protecting a small number of cells, which would normally proceed to apoptosis by inducing antioxidant responses. Given that apoptosis is a normal homeostatic mechanism that acts to maintain healthy cell populations, the clinical significance of this reduction below baseline apoptosis frequency is unclear. The same dose of PTL that was delivered to C57BL/6J mice in this study has previously been delivered to NOD/SCID mice thrice weekly for up to 106 days without any reported negative health effects (20). Further studies are required to determine if PTL is reducing endogenous apoptosis, as it would have been expected that DMAPT would induce a similar or superior reduction, and this was not observed in the experiments here.

In addition to high-therapeutic-radiation doses, low doses of radiation are commonly received by patients prior to and during radiotherapy in the form of imaging CT scans. Low-conditioning doses of radiation in the dose range of imaging CT scans have been shown to induce protection from effects of subsequent high doses of radiation for a wide range of biological endpoints including apoptosis in vivo (25–27). In the absence of PTL/DMAPT, a 10 mGy-dose induced a radioadaptive response in normal prostate and spleen in some experiments, with trends towards an adaptive response in others. A dose of 10 mGy alone induced a significant increase in apoptosis in C57BL/6J spleen but not in TRAMP spleen, although in both cases 10 mGy induced a protection from high-dose-radiation-induced apoptosis, indicating a radioadaptive response. There has only been one previous report of increased apoptosis after exposure to 10 mGy alone and that was at 6 h after in utero exposure of C57BL/6 embryos (67). When 10 mGy was combined with PTL/DMAPT, the magnitude of radioprotection from 6 Gy-induced apoptosis was greater. However, 10 mGy did not result in a significant difference in apoptosis frequency in the presence of PTL/DMAPT and 6 Gy. If there is an additive protective effect, it is small.

Beneficial radioadaptive effects in normal tissues in the context of radiotherapy have been well described in the literature, however there are very few studies describing tumor responses to low-dose radiation. Recently radioadaptive protection of tumors has been reported in vivo (39). If low doses of radiation induce a radioadaptive response in tumors, the efficacy of radiotherapy could be compromised in an image guided radiotherapy scenario. Here, a 10 mGy dose of X-irradiation did not induce an adaptive response in TRAMP prostate PIN tissue, nor did the low-conditioning dose affect the ability of DMAPT to radiosensitize the TRAMP prostate PIN tissue. Further studies are required to determine if the radiation doses and timing of the conditioning image dose and the radiotherapy dose play a role in the ability to radioprotect tumor tissue. Transgene-induced prostate pathology in the TRAMP model is driven by androgen dependent expression of SV40, which contains the large and small T-antigens. The large T-antigen in the transgene interferes with the action of p53 by directly binding its DNA binding domain, silencing p53's transcriptional activity (42). The radioadaptive response is known to be p53 dependent (26) and therefore may not be observable in TRAMP prostate tissues. Adaptive response studies in a model with functioning p53 are required to determine if PTL/DMAPT can counteract any potential radioadaptive responses in tumors which possess functional p53.

To ensure that the DMAPT-induced radioprotection from apoptosis observed 6 h postirradiation in normal tissues was not simply due to a delay in radiation-induced apoptosis, temporal studies were performed on TRAMP mice treated with DMAPT and 6 Gy of X radiation. There was no delayed induction of apoptosis in DMAPT-treated spleen and colorectal tissue where protection compared to control-treated mice was not observed, and radioprotection was maintained up to 24 h in colorectal tissue and 72 h in spleen. This suggests that DMAPT is providing protection in the normal tissues, rather than delaying the apoptotic response. In normal TRAMP spleen, by 24 h postirradiation, apoptotic frequency was reduced to almost baseline levels both in the presence and absence of DMAPT. Unexpectedly, a second wave of apoptosis was observed at 72 h postirradiation in the vehicle-control mice, which was significantly inhibited in the DMAPT-treated mice. There are reports in the literature of delayed waves of radiation-induced apoptosis without significant investigation into the underlying mechanism; however several reports are correlated with reduced p53 expression within tissues (68, 69). Komarova et al. (68) showed that in gamma-irradiated spleen, DNA-damage-induced apoptosis begins in regions with high levels of p53 mRNA expression, while a slower induction of radiation-induced apoptosis was observed in regions with lower p53 mRNA expression. Regardless of the underlying mechanism of action, our results indicate that DMAPT protects normal tissues from a late induction of apoptosis. Importantly, in TRAMP dorsolateral prostate DMAPT-induced radiosensitisation was observed up to 24 h postirradiation, with apoptosis levels returning to baseline frequency by 72 h. This demonstrates that the radiosensitizing effects of DMAPT lasts for a significant period of time postirradiation, which is likely to prove useful in a clinical setting if radiosensitization of tumors continues to occur up to 24 h after a radiotherapy dose has been delivered.

In the current study, we used single whole-body doses of radiation as a proof of principle to determine the potential of PTL/DMAPT as a radioprotector of normal cells whilst radiosensitizing tumor cells. Future studies using multiple fractionated doses targeted to the region of the tumor will more closely mirror current clinical radiotherapy protocols for prostate cancer.

In summary, we have shown that DMAPT holds significant promise for use in conjunction with radiotherapy for prostate cancer. DMAPT reduced the level of radiation-induced apoptosis observed in normal tissues of C57BL/6J and TRAMP mice while doubling the efficacy of tumor cell killing. DMAPT preferentially radiosensitized regions of high-PIN grade within TRAMP prostates; this suggests that DMAPT may be particularly able to target regions of higher oxidative stress, as is often observed in high-grade-metastatic prostate cancers. We have also shown that low doses of radiation may be able to augment the radioprotective effects of DMAPT in normal tissues. Radiotherapy is used to treat a wide range of different cancers. The redox pathways involved in the action of DMAPT are not specific to prostate cancer (70–72) and therefore there is also the potential to utilize DMAPT as a differential radioprotector in conjunction with radiotherapy for other cancer types, as well as prostate cancer.