The Estrogen-related Effect And Mechanistic Study Of A Genistein Derivative GS14

Background:Genistein is a kind of isoflavone that widely distributed in the plants. It has a17β-estradiol like structure which endows it with natural phyto-estrogenic activity. A number of in vitro and in vivo experiments have proved that genistein has good bioactivities such as anti-tumor activity, anti-osteoporosis activity, estrogenic and anti-estrogenic activity, cardio-protective activity, etc. Its side effect is not that severe comparing to the estrogen. However, there are some disadvantages, for example, genistein has poor lipophicity and hydrophily which cause its bad bioactivity, also, its multiple targets bring weak specificity. So a lot of genistein derivatives were synthesized in the purpose of strengthening its bioactivity. Our lab had synthesized a group of compounds with structure modifications of genistein at7-OH group. In our previous bioactivity assays, the compound named GS14(7-(4-(diethylamino) butoxy)-genistein) demonstrated best bioactivities.Objective:Our experiments were designed to study the activity of a novel genistein derivative GS14, focusing on the estrogenic activity including the estrogenic activity in vitro, anti-osteoporosis effect against OVX rats and simple mechanisms. We were also trying to study its neuro-protective effect and anti-tumor activity in vitro. Methods:Many methods were used here including in vitro and in vivo experiments to study the bioactivity of GS14.A Estrogenic effect of GS141. Using MTT method to compare the cytotoxic effect of GS and genistein against estrogen dependent human breast cancer cell MCF-7; adopting E-Screen Assay to test and compare the in vitro estrogenic activity of GS14, genistein and17β-estradiol;2. Using ovariectomized surgery to build the osteoporosis model in SD female rats, the whole process lasted for12weeks; automatic biochemical instrument tested the biochemical markers of serum and urine, and DXA measured the femur bone density of the SD rats, while H-E staining were used to observe the morphological bone structure change of the1/3distal part of rat femur;3. The proliferative effects of genistein and GS14on the osteoblast MC3T3were tested by MTT method; using realtime-PCR to analyze quantitatively the impact of GS14on the differentiation marker of osteoblasts:runx2and osteocalcin.B Neuro-protective activity of GS141. In vitro inhibitory effect of genistein and GS14against AChE were tested by light absorption spectrometry;2. Ovariectomy surgery was used in ICR mice to create models with impaired learning and memory ability; the learning and memory ability of OVX mice were evaluated by Morris Water maze experiment and Y maze experiment.C In vitro anti-tumor activity of GS141. In vitro cytotoxic effects of GS14against a lot of tumor cell strains were tested by MTT method.Results:A Estrognic effect of GS141. When the concentration of genistein and GS14was over1μM, the two compounds behaved cytotoxic effect against MCF-7while the effect of GS14was stronger, otherwise, genistein and GS14demonstrated proliferative effect and achieved the best effect at the concentration of1μM; in vitro estrogenic effect of GS14was better than genistein, and weaker than17β-estradiol;2. Three months after the OVX surgery, the ratio of uterus and body weight was reduced significantly, the level of serum ALP was increased, the femur density declined to0.295g/cm2comparing to0.316g/cm2of the sham group, the bone structure was deteriorated, suggesting that our model was successful;17β-estradiol and GS14treated groups all increased the BMD to some extent (BMD of1mg/kg GS14treated groups was0.305g/cm2, while BMD of17β-estradiol treated group was0.304g/cm2), reduced the level of serum ALP and improved the bone structure. Also GS14didn’t have an uterotrophic effect. However, the therapy of GS14was not dose-dependent;3. Genistein and GS14didn’t show any significant proliferative effect towards MC3T3; After4days’ treatment,1μM GS14could significantly up-regulate the expression of runx2by23% higher than control, while1nM17β-estradiol increase by67% higher than control, however,1μM genistein did not cause any increase. At day4, addition of1μM GS14could increase the expression of osteocalcin by57%higher than control,1μM genistein increased by32%, while1nM17β-estradiol increased by40%; co-treatment of1μM GS14with1nM ICI182,780significantly abrogated the stimulatory effect on runx2and osteocalcin genes expression.B Neuro-protective activity of GS141. GS14had strong inhibitory effect against AChE in vitro, its IC50was0.182±0.020μM, the effect was stronger than genistein, but weaker than the positive control tacrine of which IC50was0.013±0.002μM;2. In Morris Water Maze experiment, the latent time of OVX model mice was longer than all other groups and its swimming track in1min was the most complex. GS14and17β-estradiol treatment could alleviate this phenomenon. Y maze experiment had similar results.C Anti-tumor activity in vitro of GS141. GS14demonstrated good cytotoxic effect against many tumor cell strains in vitro. Conclusions:1. GS14had stronger estrogenic effect in vitro than genistein. It could allevitate osteoporosis rat to some extent without dose-dependent effect. According to the experiment result, we hypothesized that this effect was partly due to its stimulatory effect of osteoblast differentiation. The effect abrogated by ICI182,780indicated that ER pathways played an important role.2. GS14had fine neuro-protective effect. It had strong inhibitory effect against AChE in vitro, which was much stronger than genistein. It could also alleviate the decline of learning and memory ability of OVX mice.3. GS14had impressive anti-tumor activity in vitro comparing to genistein. However, it was also cytotoxic to the normal cell. So the safety problems were in concern and needed further investigation.