Normal tissue toxicity markedly reduces the therapeutic index of genotoxic anti-cancer agents including ionizing radiation. Countermeasures against tissue damage caused by radiation are limited by their potential to also protect malignant cells and tissues. Here we tested a panel of signal transduction modifiers for selective radioprotection of normal but not tumor tissues. These included three inhibitors of GSK3 (LiCl, SB216763 and SB415286) and two inhibitors of NF-κB (ethyl pyruvate and RTA 408). Among these, the thiol reactive triterpenoid RTA 408 emerged as a robust and effective protector of multiple organ systems (gastrointestinal, skin and hemopoietic) against lethal doses of radiation. RTA 408 preserved survival and proliferation of crypt cells in lethally irradiated small intestines while reducing apoptosis incidence in crypts and villi. In contrast, RTA 408 uniformly inhibited growth of established CWR-22Rv1, LNCaP/C4-2B, PC3 and DU145 xenografts either alone or combined with radiation. Anti-tumor effects in vivo were associated with reduced proliferation and intratumoral apoptosis and with inhibition of NF-κB-dependent transcription in PC3 cells. Selective protection of normal tissue compartments by RTA 408 critically depended on tissue context and could not be replicated in vitro. Collectively, these data highlight the potential of RTA 408 as a cytoprotective agent that may be safely used in chemoradiation approaches.

<p>Supplementary Figure 4: Effects of RTA 408 on radiation sensitivity of prostate cancer cell lines as determined by clonogenic survival assays. Cells were treated at 24 and 1 h with RTA 408 prior to IR exposure as indicated. Differences between curves were analyzed by two-way ANOVA, designating drug treatment and IR as variables. P value summaries of pair-wise comparisons between drug and vehicle treatment curves are shown.</p>

<p>Supplementary Figure 1: Reduction in apoptosis incidence in the skin of irradiated mice. Topical radiation (30 Gy single dose) was applied to the hindlegs of mice with shielding protecting the rest of the body. RTA 408 (17.5 mg/kg) was administered 1 d prior, 1 h prior and 1 d post IR; skins were harvested 2 d post IR. Apoptotic cells were detected by TUNEL staining in tissue samples obtained 24 h after radiation exposure. The number of apoptotic cells in RTA 408-treated mice was statistically significantly reduced compared to IR + vehicle-treated mice (Student's t-test; p<0.05). DP = dermal papilla; hf = hair follicle; e = epidermis; d = dermis; m{plus minus}SD of at least six fields were evaluated.</p>

<p>Supplementary Figure 4: Effects of RTA 408 on radiation sensitivity of prostate cancer cell lines as determined by clonogenic survival assays. Cells were treated at 24 and 1 h with RTA 408 prior to IR exposure as indicated. Differences between curves were analyzed by two-way ANOVA, designating drug treatment and IR as variables. P value summaries of pair-wise comparisons between drug and vehicle treatment curves are shown.</p>

<p>Supplementary Figure 3: Effects of RTA 408 on prostate cancer cell survival in vitro. Cells were treated for 24 h with RTA 408 at different concentrations. Protein extracts were prepared from attached and/or detached cells to capture the full extent of apoptosis. Protein extracts from detached cells were prepared at RTA 408 concentrations {greater than or equal to}0.5 μM; solid lines above protein concentrations indicate detached cells as a source of proteins. Levels of cleaved caspase 3, cleaved PARP-1, cleaved cytokeratin-18 (M30) and cleaved caspase-8 were determined by immunoblot analyses.</p>