Study On Icaritin Suppress Hematological Malignancies Through Inducing Production Of Reactive Oxygen Species

Objective:Icaritin,a hydrolytic product of icariin,is extracted from traditional Chinese medicine herb Epimedium Genus.Recent years,icaritin have drawn attention about its extensive inhibition on various neoplasms including hematological malignancies and solid tumor such as leukemiammultiple myeloma,breast cancer and hepatocelluar carcinoma,etc.Investigations revealed that icaritin possess the antiproliferative effect,inducing apoptosis and cell cycle arrest,which is associated with regulation on significant signaling pathway MAPK/ERK/JNK.However,It can’t be explained that the effect of icartin acting on MAPKs differ in different tumor cell lines.Then we come to the hypothesis that MAPKs may be not the primary target of icaritin in tumor cell lines,the regulation of icaritin on MAPKs may be dependent on the upstream regulator ROS.To verify this speculation,chronic myelogenous leukemia cell line K562 and multiple myeloma cell line U266 was used to explore the aititumor mechanism of icaritin by molecular biology methods such as flow cytometry,RT-PCR and Western blot.Materials and methods:K562 cells and U266 cells were incubated with various concentrations of icaritin(0,2,4,8,16,32μΜ)in 24 h,48h and 72 h.Flow cytometry was exerted to identify the production of reactive oxygen species.After combined administration of icaritin and N-Acetyl-L-cysteine,production of reactive oxygen species was examined for comparison.The proportion of apoptotic cells was examined after combined administration of icaritin and N-Acetyl-L-cysteine,as well as the expression level of apoptosis-relative markers.Expression level of Nox4 mRNA was examined by RT-PCR.Phosphorylated JNK-c-jun expression level was detected by Western Blot.Results:1.Icaritin induced the production of reactive oxygen species,which was dose-dependent2.When combined administration of icaritin and N-Acetyl-L-cysteine was used,the production of reactive oxygen species decreased compared with treated with icaritin alone.3.After Icaritin was co-treated with N-Acetyl-L-cysteine,the proportion of apoptotic cells was lower.Meanwhile the expression level of significant molecular markers PARP and Cyt-c for apoptosis also decreased.4.Icaritin promoted the expression level of Nox4 mRNA.5 When combined administration of icaritin and N-Acetyl-L-cysteine is treated with cells,the expression level of activated phosphorylated JNK and phosphorylated c-jun decreased compared with icaritin alone.Conclusions:1.Icaritin induced the production of reactive oxygen species by dose-dependent way,which is in accord with the antiproliferative effect of icaritin on hematological malignancy cells.2.Blocked production of reactive oxygen species reversed the antiproliferative effect of icaritin on cancer cells.The results demonstrated that the suppression of icaritin on cancer cells was dependent on the induced production of reactive oxygen species.3.Blocked production of reactive oxygen species reversed the promoted activation of JNK-cjun.The results demonstrated that the ROS-JNK-c-jun served as the common pathway of icaritin inhibiting proliferation of different hematological malignancy cells.