An Icaritin Derivative IC2 Induced Autophagy And Apoptosis In Breast Cancer Cells By Inhibiting SCD-1

Objectives: Targeting key enzymes of lipid metabolism to develop breast cancer anticancer drugs has become a hot spot in breast cancer pharmacology research in recent years.Stearoyl-CoA desaturase 1 is a key enzyme that catalyzes the production of monounsaturated fatty acids from saturated fatty acids.It is closely related to the progression and prognosis of breast cancer and is a potential therapeutic target for breast cancer.Present SCD-1 inhibitors such as A939572 and MF-438 are still in the pre-clinical research stage due to their relatively large side effects.The objectives of this study:（1）to develop SCD-1targeted drugs originate from icaritin,an active ingredient of Chinese herbal medicine with less toxic and side effects;（2）to detect the effect of this active compound on breast cancer cells;（3）to explore the relevant mechanism of the effect.Methods: Simulative modification of the ICT group and molecular docking with SCD-1were conducted with the computer-aided drug design to obtain potential SCD-1 inhibitors.ICT derivatives were synthesized in the laboratory and then tested with MTT to detect the effects on MCF-7,SK-BR-3,MDA-MB-231 breast cancer cells.Western blotting and GCMS were used to verify the expression and enzyme activity of SCD-1 in MCF-7 cells when treated with the ICT derivatives.ERa expression was also detected with western blotting.（2）DAPI staining and LIVE/DEAD^TM dead cell staining were used to detect the death of MCF-7cells after treated with different concentrations of ICT derivatives.Cells were transfected with Ad-m Cherry-GFP-LC3 B adenovirus to observe the process of autophagy.Western blotting was used to detect the expression of LC3B Ⅱ,a marker protein of autophagy,and the phosphorylation level of upstream molecule AMPK.Flow cytometry was used to detect cell apoptosis and ROS generation,JC-1 staining to detect the mitochondrial membrane potential and western blotting to detect apoptosis-related proteins.The relationship between autophagy and apoptosis and the role of MAPK family in the process of apoptosis was analyzed by adding AMPK inhibitor and autophagy inhibitor.（3）Exogenous oleic acid was added and then the cell activity was tested.Oil red O and BODIPY staining were used to observe the formation of intracellular lipid droplets.The change of cardiolipin on mitochondrial membranes was detected with NAO staining.The effect of ICT derivatives on the energy metabolism in mitochondria was analyzed through ATP detection kit.Results: According to CADD,ICT derivatives,including IC1,IC2,and IC3,possessed better SCD-1 inhibitory effects than ICT.IC2 combined with SCD-1 produced the lowest binding energy,suggesting it is a potential SCD-1 inhibitor.Experiments showed that IC2dose-dependently inhibited the growth of three types of breast cancer cells and decreased the expression and enzyme activity of SCD-1,confirming that IC2 is a novel SCD-1 inhibitor with anticancer effect on breast cancer.In addition,low-dose IC2 can slightly promote the proliferation of MCF-7 cells,while 20μM IC2 promoted cell death,accompanied by decreased expression of ERa.（2）DAPI staining and dead cell staining reflected that high concentrations of IC2 significantly accelerated cell death.The level of autophagy and apoptosis increased,consistent with up-regulation of LC3B Ⅱ expression and AMPK phosphorylation,activation of PARP and down-regulation of Bcl-2,reduction of mitochondrial membrane potential and generation of abundant ROS.AMPK inhibitor and autophagy inhibitor regulated autophagy and apoptosis,and simultaneously influenced the phosphorylation levels of MAPK family,P38,ERK,and JNK.（3）Exogenous oleic acid rescued the cell viability inhibited by IC2 and promoted the formation of lipid droplets.IC2 hardly affected cardiolipin on mitochondrial membrane or inhibited ATP production.On the contrast,20 μM IC2 stimulated ATP production.Conclusions:（1）IC2 was a novel SCD-1 inhibitor that can inhibited the proliferation of MCF-7,SK-BR-3,and MDA-MB-231 cells.（2）IC2 activated AMPK to trigger autophagy and induced mitochondrial-dependent apoptosis in MCF-7 cells.AMPK was involved in the regulation between autophagy and apoptosis when treated with IC2,and the MAPK family played an important role in the apoptosis.（3）IC2 reduced monounsaturated fatty acid production to produce anticancer activity by inhibiting SCD-1.