Aqueous extracts of Rhizopus oryzae induce apoptosis in human promyelocytic leukemia cell line HL-60

Protection against in vivo infection with Salmonella and enhancement of in vitro superoxide production by peripheral blood neutrophils are two reported effects of treatment with aqueous extracts of the fungus Rhizopus oryzae U-1 (Aq-ROU). Here, we report that Aq-ROU also has antiproliferative activity and can induce apoptosis in a human promyelocytic leukemia cell line, HL-60. During Aq-ROU-induced apoptosis, HL-60 cells undergo genomic DNA fragmentation characteristic of apoptosis after just 6 hr of treatment. Using phosphatidylserine (PS) as an indicator of apoptosis, we also found that the proportion of apoptotic cells increased significantly after 9 hr of treatment. Indeed, induction of apoptosis by Aq-ROU reached 43.3% after 24 hr of treatment, which is comparable to the effects of a known apoptosis-inducer, actinomycin D. Moreover, the activities of caspase-3, -8, and -9 increased in parallel with Aq-ROU treatment, with a peak of activity 9 hr after the initial treatment. Taken together, the results suggest that R. oryzae contains one or more water-soluble factor that can reliably and efficiently induce apoptosis in human cells via activation of caspase-3.