Genistein Protects Against DNA Damage in Hematopoietic Stem Cells,
Authors: Souza Liliana , Silva Erica , Calloway Elissa , Kucuk Omer , McLemore Morgan L. 
Published: October 2019

Abstract
Abstract 3437
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. Extensive epidemiological, In vitro, and animal studies have been performed, which suggest that genistein may have beneficial effects on a multitude of human conditions, including cancers. 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, cause an increase in radical oxygen species (ROS) production and DNA instability. This can lead to the impairment of stem cell function or leukemogenesis. We utilized the p53 heterozygous (+/−) mouse model to test our hypothesis. Previous studies have shown that the p53 tumor suppressor gene regulates several functions of hematopoietic cell proliferation, differentiation, apoptosis, and aging. In response to DNA damage, p53 can either elicit cell-cycle arrest or apoptosis. Additionally, p53 deletions and mutations have been found at high frequency in blast crisis chronic myelogenous leukemia and with some frequency in acute leukemia and MDS. p53+/− animals have a constitutive enhancement in HSC proliferation with fewer quiescent cells present when compared to wild type (WT) animals. They also display high levels of ROS and DNA damage. WT and p53+/− mice were treated 3 times a week with subcutaneous injections of genistein. After treatment, the number of lin-ckit+sca+ (KLS) cells in p53+/− decreased while WT numbers remain unaffected by genistein. These KLS cell populations were then analyzed for the amount of DNA double strand breaks (DSB) via the presence of nuclear pH2AX. Our results show that there is a 2 fold decrease in the DSB in the p53+/− KLS cells when compared to untreated p53+/− KLS cells. Initially p53+/− basal levels of ROS in KLS cells are higher than in the WT KLS cells, but when they are treated with genistein our results show that the basal levels of ROS are decreased by 10 fold. Results also reveal that long term treatment with genistein alone did not result in a decrease of white blood cells. 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 had no effect on WT cells. We then evaluated the effects of concurrent treatments with genistein and G-CSF. WT mice were treated 3 times a week with subcutaneous injections of genistein and G-CSF. After the treatment, the KLS cell population in the bone marrow increased 3 fold in WT mice treated with G-CSF. However, the animals treated with G-CSF and genistein presented only a 2 fold increase. Our results also showed that there is a 2 fold increase in the DSB in WT KLS cells from animals treated with G-CSF. Surprisingly, there is no significant increase in DSB in cells from animals treated with G-CSF and genistein. Interestingly enough the animals simultaneously treated with genistein and G-CSF had a similar amount of neutrophils as the animals treated with G-CSF only. Therefore, our data suggests that genistein protects against the deleterious effects of G-CSF in HSC excessive proliferation and at the same time permits the desired increase in the neutrophil population. Understanding this mechanism of action could potentially reveal novel treatment strategies for patients with severe congenital neutropenia or other hematological malignancies.

Disclosures:
No relevant conflicts of interest to declare.