Production Of NO And Regulation Of Sensitivity To ADM And 5-Fu In Human Breast Cancer Cell Line

ObjectiveThis experiment aimed at evaluating the effects of NO mediated regulation of chemosensitivity in human breast cancer cell MCF-7 with the induction of IL-1βin vitro,elucidating the possibility that endogenous NO attenuated resistance to chemotherapeutic agents,approaching possible mechanism and feasibility to adjunctive chemotherapy of breast cancer.MethodsSelecting human breast cancer cell line MCF-7,and the cells were cultured as monolayer,incubated with cytokine IL-1β.Using NO Assay Kit to detect the production of NO among different IL-1βconcentration groups(10^-13M,10^-12M,10^-11M,10 ^-10M,10^-9M,10^-8M)and different time groups(2h,4h,8h,16h,24h).Western blot analysis was used to measure iNOS protein levels.then choose proper drug concentration and action time.According to the above-mentioned results,establishing control group and experimental group,selecting classic chemotherapeutic agents ADM and 5-Fu,and then treated with chemotherapeutic agents.Using MTT assay to detect the chemosensitivity to ADM and 5-Fu.The control group and experimental group were treated with NOS inhibitor L-NMMA and NO synthesis raw materials L-Arg,then detect the chemosensitivity to ADM and 5-Fu by MTT assay.ResultsThe levels of NO were increased significantly(95.216μM)when MCF-7 cells were incubated with IL-1βfor 4h,and reached to the peak(148.661μM)at 8h,and then decreased gradually.However,there was no obvious change in the control group during the cultrue process compared with the experiment group(P＜0.05).IL-1β,as a cytokine,induced MCF-7 cells to generate endogenous NO.There was a relation of dose-dependence between NO production and IL-1βconcentration.Using MTT assay to detect the survival rate of control group and experimental group.Incubating MCF-7 cells with 0.5μM or 1μM ADM,the survival rate of experiment group can remarkablely decrease(P＜0.05);with the increasing of ADM concentration,the survival rate was not notable between two groups.5-Fu group has the same result.Treatment of MCF-7 cells with NOS inhibitor L-NMMA significantly increased survival rate of experiment group (P＜0.05);conversely,replacement of NO activity by using L-Arg decreased survival rate of experiment group(P＜0.05);however,treatment of MCF-7 cells with L-NMMA and L-Arg simultaneously,survival rate of experiment group didn't significantly decrease(P＜0.05).Western blot analysis showed that:the iNOS wasn't expressed without IL-1β;MCF-7 cell expressed plenty of iNOS by induction of IL-1β.Meanwhile,there was no difference on iNOS whether L-NMMA and L-Arg existed or not.ConclusionIncubated with IL-1βunder proper concentration and proper time,MCF-7 cells can produce plenty of endogenous NO,through inducing iNOS.Plenty of endogenous NO can increase chemosensitivity to ADM and 5-Fu.By administering L-NMMA and L-Arg,it was obvious that NO can mediate the regulation of chemosensitivity.IL-1βcan produce NO by inducing iNOS,moreover quantity of iNOS didn't change with the existence of L-NMMA or L-Arg.This research revealed that endogenous NO which MCF-7 cells produced may raise chemosensitivity of MCF-7 cells with induction of IL-1β,that is similar to the action of exogenous NO compared with previous study,which provided evidence for raising chemosensitivity of breast cancer.