Induction Of Cancer Cell Apoptosis And Growth Inhibition By Icaritin Is Associated With Its Ability To Suppress Fatty Acid Synthase Activity

Background:Icariin(MW 676),a principal flavonoid glycoside in Herba Epimedii,can be metabolized to icaritin(MW 368) in vivo.Many studies show that flavonoids such as epigallocatechin gallate(EGCG),luteolin and quercetin,can inhibit the growth of cancer cells.Although there are many reports on icariin about its antitumor activity,the mechanisms reported are limited to its estrogen-like effects.Flavonoids-fatty acid synthase(FAS) has been found to be the common target of the flavonoids mentioned above.FAS is the critical enzyme of the fatty acid synthesis.As a fundamental material of cell membrane,the amount of fatty acid affects the growth of cells. Under normal conditions FAS is highly expressed in liver to synthesize fatty acid while in other organs it is lowly expressed.Many human tumors express elevated levels of FAS to afford sufficient material for their fast growth.The different expression of FAS between normal and cancer cells leads to the hypothesis that the fatty acid synthetic pathway could be exploited as a new target of therapeutic antimetabolites.Another over-expressed protein in tumor cells is human epidermal growth factor receptor-2(HER2).HER2 is a tyrosine kinase receptor and belongs to erbB family.It is involved in the regulation of cell growth and differentiation.HER2 is expressed in many kinds of cancer cells,and its over-expression indicates chemotherapy resistance and bad prognosis.Recent studies show that there is connection between FAS and HER2,which are both over-expressed in cancer cells.Inhibition of FAS suppresses HER2 oncogene over-expression in cancer cells and vice versa.In our study we tried to prove the suppression effect of icaritin on cancer cells and to exploit its value for medical use.We also tried to elucidate whether its target is the FAS/HER2 pathway.We have already found that minute quantity of icaritin can stimulate estrogen receptor (ER) positive cells growth,and ER positive cancer cells are not so sensitive to icaritin.So we chose ER negative cancer cells,breast cancer cell SK-Br-3,prostate cancer cell LnCap and normal breast cell MCF-10a as the subjects. Methods:1 Icaritin induced cancer cell apoptosis and growth inhibition:First we examined the growth inhibition of human breast cancer cell line SK-Br-3, prostate cancer cell line LnCap and normal human breast cell line MCF-10a by icaritin. Incubated with icaritin for 72hr,cells were assayed by MTT method.This test was to elucidate whether icaritin induces cancer cells growth inhibition,and what will happen to normal cells when exposed to icaritin.Apoptosis was tested by flow cytometry(FCM).We also collected cell images at 48hr.2 The mechanism of the icaritin induced cancer cell growth inhibition:The last part had shown the cell toxicity of icaritin,and in this part we tested whether exogenous palmitate can reverse the growth inhibition caused by icaritin.We incubated SK-Br-3 cells with palmitate and then with icaritin.Enzymatic activity of FAS was measured on cellular extracts of SK-Br-3 cells.The cellular extracts was incubated with icaritin for 10min and then monitored at 340nm to measure NADPH oxidation.If the activity of FAS is inhibited,the consumption of NADPH will decline,and the OD at 340nm will increase.In this way we can find out whether icaritin inhibit the activity of FAS.To elucidate the effect of icaritin on the FAS/HER2 pathway,we tested the expression of HER2 and polyoma enhancer activator 3(PEA3) by western blot after treating SK-Br-3 cells with icaritin.It turned out that the expression of HER2 decreased and that of PEA3 increased.PEA3 is a molecule bridging FAS and the HER2 pathway.When FAS is inhibited,PEA3 will accumulate and combine to the promoter of HER2,which will inhibit the expression of HER2.So if icaritin can inhibit the activity of FAS,the expression of PEA3 will increase while that of HER2 will decrease.Results:1 Icaritin induced cancer cell apoptosis and growth inhibition:It turned out that icaritin could Obviously inhibit the growth of SK-Br-3 (IC₅₀=8.6μg/mL(72hr)) and LnCap(IC₅₀=3.08μg/mL(72hr)),However,it had no significant effect on MCF-10a(IC₅₀＞20μg/mL).SK-Br-3 and LnCap showed significant difference compared to untreated group when treated with 5μg/mL icaritin,while MCF-10a was 20μg/mL.Cell apoptosis was analyzed by FCM.The apoptosis rate of cells treated with icaritin(8.6μg/mL) at 36hr is 8.12±1.13%,and 49.55±1.01%at 48hr.All the results showed that icaritin could induce cancer cell growth inhibition in a dose and time dependent manner.2 Icaritin induced cancer cell growth inhibition through the FAS activity inhibitionIn this part we tested whether exogenous palmitate could reverse the growth inhibition caused by icaritin.We incubated SK-Br3 cells with palmitate(7μg/mL) and then with icaritin,and took those cells not pretreated by palmitate as controls.The results showed that the survival rate of tumor cells pretreated with palmitate was raised by 18.30±1.0%,21.87±0.9%and 15.37±2.5%at icaritin concentration of 8μg/mL,5μg/mL and 2.5μg/mL compared with the controls respectively(P＜0.05).Further results showed that compared with the DMSO control group the FAS activity in the icaritin treated ones were suppressed by 15.35%at 5μg/mL,49.26%at 8μg/mL,and 70.11%at 20μg/mL.So we came to the conclusion that FAS was the target of icaritin.3 Icaritin induced cancer cell growth inhibition through the effect on FAS/HER2 pathwayWestern blot showed that compared to the untreated group,cells treated with icaritin showed high PEA3 expression(6.58times) and low HER2 expression(0.63times).So we conclude that the effect on FAS/HER2 pathway is part of the mechanism of the icaritin cytotoxicity.Conclusion:1 Icaritin could induce ER negative cancer cells apoptosis and growth inhibition;2 FAS is the target of icaritin;3 Icaritin could up regulate PEA3 and down regulate.HER2 through the FAS/HER2 pathway.