We have previously shown that a single dose of GT3 greatly reduces postirradiation intestinal, hematological and vascular changes as well as postirradiation lethality in mice with a DRF in excess of 1.3. Because PTX has been shown to enhance the effects of vitamin E when used in the prophylaxis or treatment of delayed effects of radiation in several organ systems, the aim of the present study was to investigate whether PTX could also enhance protection by GT3 against early TBI-induced toxicity and lethality.
PTX is a phosphodiesterase inhibitor developed to modify blood viscosity and to improve blood circulation. It has been on the market as a rheological agent for over 30 years. More recently, the agent’s potential effects on cytokine-induced inflammatory responses, as well as other properties, have been recognized as well. PTX is believed to have beneficial effects in various inflammatory diseases, especially those with a TNF-α-driven inflammatory response.
Multiple studies have been performed to investigate the use of PTX as both a radioprotector and a radiosensitizer. Preclinical studies have shown that PTX might increase radiosensitivity of p53-mutated tumor cell lines by modulating cell cycle progression (19
). Moreover, PTX might suppress DNA double-strand break repair and improve tumor oxygenation (22
). When used as a tumor radiosensitizer, PTX does not appear to increase normal tissue radiation toxicity. In a phase III randomized clinical trial to study the effect of PTX on the radiation response of non-small cell lung cancer, PTX treatment did not increase dysphagia, odynophagia, pulmonary fibrosis or pneumonitis (24
When studied as a radioprotector, PTX has often been used in combination with vitamin E. This combination therapy has been shown to reduce radiation fibrosis in various organ systems (4
). Little is known about the effect of PTX on the acute radiation response. Ward et al.
have shown that in rats PTX does not reduce (semi-) acute dermal and pulmonary radiation injury, assessed 2 months after radiation exposure (25
As shown previously, GT3 reduces postirradiation intestinal injury. The current study shows that prophylactic treatment with PTX in combination with GT3 is more efficient in reducing TBI-induced mortality than treatment with PTX or GT3 alone. Our data suggest that the increased survival with the combination treatment is likely because of accelerated hematopoietic recovery and is not due to further improvement of gastrointestinal or vascular radiation responses. The addition of PTX to GT3 did not improve postirradiation crypt colony survival or intestinal mucosal surface area and failed to potentiate the effect of GT3 on postirradiation vascular peroxynitrite production. Rather, the additional protection conferred by PTX (in combination with GT3) in the ‘‘GI subsyndrome range’’ is assumed to be dependent on GI protection by GT3.
On the other hand, the combination therapy significantly increased both ex vivo bone marrow colony formation at 1 day after radiation exposure and in vivo spleen colony formation at day 10 postirradiation. Whereas combination treatment was shown to have a pronounced effect on postirradiation hematopoietic colony formation, only a modest effect on postirradiation circulating blood cell counts was observed in our experiment. Compared to GT3 alone, GT3 combined with PTX improved only platelet recovery; no effect on leukocytes or erythrocytes was observed over the benefit of GT3 alone. Although the lack of effect on peripheral leukocyte and erythrocyte recovery might seem unexpected considering the observed effect on bone marrow CFUs and spleen colonies, this observation might be explainable. First, GT3 is a potent radioprotector with strong hematopoietic effects. Our current survival data show that GT3 protects against TBI-induced death up to a dose of 11.5 Gy. Combination treatment was shown to be more potent, since it offered full protection against death even at a dose of 12.5 Gy. In the current study, blood cell counts were performed after 8.5 Gy TBI. This dose was chosen to provide sufficient numbers of surviving animals in the vehicle- and PTX-treated groups, but it may not have been optimal for detecting a difference in peripheral blood cell recovery between the GT3 group and the GT3+PTX group. Moreover, the effect of adding PTX might be lineage specific and more pronounced for platelet formation than for the formation of leukocytes and erythrocytes.
To obtain information about the mechanism by which combination treatment modulates the bone marrow microenvironment and thereby the postirradiation hematopoietic response, we studied the effect of the different treatments on bone marrow plasma cytokine/chemokine levels. This technique to study the bone marrow niche was described by Kissel et al.
). Cytokine/chemokine levels were expressed per mg bone marrow plasma protein. Notably, differences between treatment groups do not appear to be caused by differences in bone marrow cellularity. When cytokine/chemokines levels were expressed per 107
present bone marrow cells, similar results were observed as when expressed per mg protein. When interpreting the bone marrow plasma data, it has to be borne in mind that PTX was administered only 30 min before radiation exposure and that the effect of PTX on the day 0 data might therefore be limited. Both GT3 and GT3 with PTX caused an increase bone marrow plasma G-CSF levels. Singh et al.
have shown that GT3 treatment increases circulating G-CSF levels (27
). They hypothesized that GT3 improves postirradiation hematopoietic recovery by stimulating the bone marrow with G-CSF. The fact the GT3 increases G-CSF levels in the bone marrow microenvironment may support this theory. However, because GT3 combined with PTX did not improve G-CSF levels compared with GT3 alone, the effects of combination treatment on the bone marrow niche appear to be regulated by factors different from G-CSF. On the other hand, since the experiments in the present study were performed at a sublethal radiation dose to permit serial measurements in control mice, it is also possible that the level of injury in GT3-treated animals was not sufficient to give rise to an increase in G-CSF levels.
Combined treatment with GT3 and PTX caused an increase in bone marrow plasma IL-1α, IL-6 and IL-9. All three interleukins are known to stimulate hematopoiesis (28
). Administration of IL-6 has been shown to accelerate recovery from radiation-induced hematopoietic depression (29
). Even though IL-6 is generally considered to be a multilineage stimulant, it may have a more pronounced effect on platelet recovery. For example, when administered after 5-fluorouracil treatment, IL-6 improves the recovery of circulating platelets, whereas no effect on circulating neutrophils is observed (30
). IL-9 has been shown to enhance in vitro
growth of both early erythroid and megakaryocytic progenitor cell colonies (31
). Considering the cytokine profile induced by GT3 with PTX and the observation that GT3 with PTX only improves postirradiation platelet recovery in the current experiment, it might be possible that the hematopoietic stimuli induced by GT3 with PTX are especially efficient in promoting megakaryocytic proliferation and platelet formation. While our data suggest that treatment with GT3 in combination with PTX affects hematopoietic recovery by the induction of hematopoietic stimuli like IL-1α, IL-6 and IL-9, further research is needed to study the effect of combined treatment on hematopoietic inhibitors. Lysofylline, a phosphodiesterase inhibitor like PTX, has been shown to suppress the release of hematopoietic inhibitors after treatment with cancer chemotherapeutic agents (33
In the present study, bone marrow plasma studies were performed only until day 14 after TBI. Therefore, it is not possible to predict how long the observed increase in cytokines would persist or to what extent similar changes would be present systemically and influence the development of chronic changes in bone marrow or other organs.
It was somewhat surprising that the protection against lethality from these drugs did not require the presence of eNOS. Many, if not all, pleiotropic effects of HMG-CoA reductase inhibitors are mediated through the eNOS pathway (10
), and eNOS, through cAMP, also seems to play a prominent role in the mechanism of action of PTX. For example, both HMG-CoA (34
) and PTX (35
) strongly upregulate thrombomodulin (TM), a potent natural anticoagulant on the endothelial cell surface, which appears to be involved in the regulation of normal tissue radiation responses (37
). The finding that eNOS is not required for protection against lethality with either GT3 or PTX may suggest that the protection by these drugs is largely due to their properties as antioxidants and/or cytokine stimulators. On the other hand, it is also conceivable that HMG-CoA reductase is involved in the protection against lethality by non-eNOS-dependent mechanisms. Yet another possibility is that the role of eNOS after TBI is altered because of radiation-induced uncoupling, a process during which eNOS produces superoxide, rather than nitric oxide (38
). Further research is clearly needed to investigate the role of eNOS and microvascular oxidative/nitrosative stress in response to exposure to different radiation doses where radiation protectors such as GT3 do not confer complete protection. Such studies will allow for demonstration of partial eNOS dependence rather than an absolute requirement for this enzyme.
In conclusion, we have demonstrated that radio-prophylaxis with GT3 in combination with PTX is significantly more effective in improving survival after TBI than prophylaxis with GT3 alone. Our data suggest that administration of GT3 together with PTX may modulate the hematopoietic radiation response by the induction of hematopoietic stimuli. Combination therapy did not reduce postirradiation intestinal injury or vascular peroxynitrite production compared to treatment with GT3, and the protective effect does not appear to depend on eNOS.