Genistein Inhibits Highly Proliferative Hematopoietic Stem Cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Genistein is a soybean-derived isoflavone with antioxidant and anti-inflammatory effects. It also has tyrosine kinase inhibitory properties that attenuates proliferation of both normal and cancerous cells. Patients with severe congenital neutropenia (SCN) require continuous G-CSF treatment throughout their lifetime. Such prolonged treatment increases the incidence of myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) in this population. We hypothesize that genistein could counteract the deleterious effects of excessive hematopoietic stem cell (HSC) proliferation, which can lead to DNA damage, increase in radical oxygen species (ROS) production and DNA instability. To test our hypothesis we choose the p53 heterozygous (+/−) mouse model, once those animals have a constitutive enhancement in HSC proliferation. They also display high levels of ROS and DNA damage in comparison to wild type (WT). We started our experiments treating WT and p53+/− mice 3 times a week with subcutaneous injections of genistein. After treatment, we analyzed the total number of lin-ckit+sca+ (KLS) cells. The number of p53+/− KLS decreased while the WT KLS numbers remained unaffected. KLS cells were then analyzed for the amount of DNA damage via nuclear pH2AX staining. Our results show a 2 fold decrease in DNA damage on the p53+/− cells from treated animals, compared to untreated ones, while the levels of DNA damage remained unaltered on WT. Basal levels of ROS on p53+/− KLS cells were higher than in the WT KLS cells, but after treated with genistein those levels decreased by 10 fold. Interestingly, long term treatment with genistein did not result in a decrease of white blood cells on neither p53+/− nor WT animals. To elucidate the mechanism of action, KLS cells were sorted and cell cycle analyses showed that genistein inhibited cell cycle specifically in the stem cell population. Genistein reduced the percentage of p53+/− KLS cells in cycle, but it had no effect on WT cells. We then tested genistein effects on HSC population in bone marrow competitive repopulation assay. We transplanted lethally irradiated mice with a 50:50 mix of bone marrow from WT and p53+/− mice. After the transplant the animals were treated 3 times a week with subcutaneous injections of genistein or adjuvant. Animals were euthanized after 5 weeks of treatment and blood and bone marrow were analyzed. After genistein treatment, the percentages of p53+/− KLS cells in bone marrow were 60% on treated mice and 80% on untreated mice. This data suggests that genistein preferentially inhibits the highly proliferative stem cell population, indicating its preventive or therapeutic potential against the deleterious effects of excessive HSC proliferation. Understanding this mechanism of action could potentially reveal novel treatments for patients suffering with severe congenital neutropenia, leukemia, or other hematological malignancies with route on excessive HCS proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3489. doi:1538-7445.AM2012-3489