Time course of apoptotic tumor response after a single dose of chemotherapy: comparison with 99mTc-annexin V uptake and histologic findings in an experimental model.

UNLABELLED: In tumors the process of apoptosis occurs over an interval of time after chemotherapy. To determine the best timing for detecting apoptosis in vivo with (99m)Tc-annexin V after chemotherapy, we examined the changes in (99m)Tc-annexin V accumulation over time in comparison with those of caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) expression level after cyclophosphamide treatment in an experimental model. METHODS: Hydrazinonicotinamide (HYNIC)-annexin V was labeled with (99m)Tc ((99m)Tc-annexin V). Rats were inoculated with allogenic hepatoma cells (KDH-8) into the left calf muscle. Eleven days after the inoculation, the rats were randomly divided into the group receiving a single dose of cyclophosphamide (150 mg/kg intraperitoneally) and the control group. (99m)Tc-Annexin V (18.5 MBq [0.5 mCi] per rat) was injected intravenously in the rats 4, 12, and 20 h after the treatment and also to the control rats (n = 5 in each group). Radioactivity in tissues was determined 6 h after (99m)Tc-annexin V injection. Immunostaining of caspase-3 and TUNEL were performed to detect apoptosis, and the rates of positively stained cells were calculated. RESULTS: (99m)Tc-Annexin V accumulation in tumors significantly increased at 20 h (0.077 +/- 0.007 [%ID/g] x kg, where %ID/g = percentage injected dose per gram) but not at 4 or 12 h (0.048 +/- 0.008 and 0.052 +/- 0.014 [%ID/g] x kg, respectively) after cyclophosphamide treatment. (99m)Tc-Annexin V accumulation in tumors and the rate of apoptotic cells determined by caspase-3 immunostaining and TUNEL were significantly higher in treated rats 20 h after cyclophosphamide treatment as compared with control rats. CONCLUSION: The effective detection of apoptotic tumor response with (99m)Tc-annexin V required 20 h after cyclophosphamide treatment in an experimental model. The present results provide an important basis for determining the best timing of annexin V imaging after the start of chemotherapy in a clinical setting.