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Purpose: There are number of reports available regarding defensins activity against mammalian cells besides their antimicrobial and immune regulatory activities. This study aims to investigate anticancer and apoptosis activity of the purified defensins from leukodepletion filters alone or in synergism with bacterial peptide, nisin, on prostate and colorectal cancer. Methods: Leucoflex LCR-5 filters were backflushed by an optimized elution system. Isolated granulocytes were sonicated and the supernatant treated before further purification by high performance liquid chromatography (HPLC). SDS-PAGE and western blot testing verified the fraction. Cell culture on PC-3 (human prostate adenocarcinoma), and HCT-116 (human colorectal carcinoma) were conducted following by MTT assays in addition to annexin flow cytometry for sole and synergistic effects with peptide nisin. Results: Viable and active neutrophils could recover, and α-defensins were extracted and purified. Combinations of an optimal dose of α-defensins and nisin showed a remarkable synergistic effect on cancer cell lines (over 90% and 70% for PC-3 and HCT-116, respectively). Conclusion: It also observed that less than 40% of both cells could survive after co-treatment with optimal dose. Also, apoptosis was increased after treatment by these peptides together. Annexin Vpositive populations significantly increased in percentage in comparison with control.