The early supra-additive apoptotic response of R3327-G prostate tumors to androgen ablation and radiation is not sustained with multiple fractions

Purpose: The treatment of R3327-G tumor-bearing rats with androgen ablation (AA) via castration results in a supra-additive increase in apoptosis when 2-8 Gy γ-irradiation (RT) is given as a single dose 3-14 days afterwards. We report here the dose response and effect of multiple fractions on this supra-additive apoptotic response. Materials and Methods: Dunning R3327-G tumors were grown in the flanks of Copenhagen rats and the experiments were initiated at a tumor volume of 1.0-1.5 cc. Androgen ablation was achieved by castration 3 days prior to γ-irradiation. Apoptosis was measured with a terminal deoxynucleotidyl transferase dUTP-biotin nick end-labeling assay 6-h after RT, unless otherwise specified. Results: The dose response of the supra-additive apoptotic response was assessed by irradiating castrated animals with single doses of 2, 4, 8, or 16 Gy (n = 5 per group); tumor cell apoptosis at 6-h following irradiation was 2.4% ± 0.7% (± SEM), 4.2% ± 0.8%, 6.5% ± 1.4%, and 1.6% ± 0.3%, respectively. The RT only and AA only controls had < 1% apoptosis. The effect of fractionated RT on apoptosis was investigated to determine if the supra-additive apoptotic response was sustained with repeated 2-8 Gy fractions. When tumor-bearing animals were treated with repeated daily 2-Gy fractions, there was a reduction in the level of the supra-additive apoptotic response. After five 2-Gy fractions at 24-h intervals, apoptosis in the combined treated tumors was at levels seen in the AA controls. This raised the possibility that more than 24 h are required for recovery of the high supra-additive apoptotic levels seen after one fraction. When the interfraction interval was extended to 96 h, there was no significant increase in apoptosis over the additive effect of AA and RT. Although there was a decline in supra-additive apoptosis with repeated fractions, a dose response for tumor growth delay was evident for RT alone using 2.5-Gy fractions. Moreover, the combination of AA + fractionated RT resulted in a supra-additive enhancement in tumor growth delay to 5 cc. Conclusion: The early supra-additive apoptotic response from AA and single fraction radiation is not seen at high single fraction doses and is not sustained with repeated fractions. Therefore, the classical apoptotic response that occurs within 24 h of irradiation is not likely to be the main mechanism responsible for any clinical benefit seen with this combination