Mechanism Of Icaritin Derivative IC2 Induces Apoptosis In Prostate Cancer Cells By Inhibiting SCD1

Objectives: Prostate cancer(PCA)has become the leading cause of urinary system tumors incidence in China.Although the treatment of surgery,hormone therapy,radiation therapy,etc has been broadly employed in PCA therapy,the curative effects still decline with PCA developing into castration-resistant prostate cancer(CRPC).In recent years,the development of drugs targeting lipid metabolism-related molecules has become a hot spot in PCA pharmacological research.The lipogenic enzyme stearoyl-Co A desaturase-1(SCD1),which can convert Saturated fatty acids(SFA)into Monounsaturated fatty acids(MUFA),is closely related to the progress of PCA and has been considered a potential target for PCA treatment.A variety of small molecule inhibitors targeting SCD1 have been developed with significant antitumor effects,however,obvious toxic effects have been observed after SCD1 inhibitor treatment in vivo,thereby preventing SCD1 inhibitors from entering clinical trials as anticancer drugs.Therefore,the focus of current pharmacological research is on how to solve the toxic and side effects and have significant inhibitory effects on SCD1.The objectives of this research:(1)To explore whether its derivatives,which are based on the safe active ingredient Icaritin(ICT)in traditional Chinese medicine,can be used as lead compounds targeting SCD1;(2)To detect the biological effect of the lead compound on PCA tumor and explore its mechanism of action.Methods:(1)Immunohistochemical detection of SCD1 expression in adjacent tissue and PCA tissue;(2)Pharmacological effects and mechanism detection of IC2: 1)Proliferation inhibitory effects: MTT detects the inhibitory effect of ICT and its derivatives on PC3 and LNcap cells;cell clone formation assay detects the effect of lead compound IC2 on PC3 cell proliferation;2)Apoptosis-inducing effects and mechanism: DAPI staining,Annexin VFITC/PI double staining,Western Blottig protein expression to detect PC3 cell apoptosis,flow cytometry to detect intracellular ROS level;q RT-PCR to detect m RNA expression,Western Blottig protein expression was used to detect endoplasmic reticulum(ER)stress in PC3 cells;3)Targeting SCD1 effects: q RT-PCR,Western Blottig and GC-MS were used to detect the expression of SCD1 m RNA,protein and enzyme activity in PC3 cells respectively;The PC3 cell line overexpressing SCD1 was constructed by lentivirus transfection,the expression of SCD1 in cells was detected by Western Blottig,and the inhibitory effect of IC2 on cell growth was detected by MTT;OA and PA were added exogenously,and the effect of IC2 on cell viability was detected by MTT;Cell colony formation assay and DAPI staining were used to observe the proliferation and apoptosis of PC3 cells overexpressing SCD1,and Western Blottig was used to detect the expression of apoptotic proteins after overexpressing SCD1 and OA treatment,and flow cytometry was used to measure the level of intracellular ROS in overexpressed cells;Western Blottig detected the expression of ER stress proteins after overexpression of SCD1 and OA treatment;(3)Research on related phenomena and mechanisms induced by IC2: Western Blottig to detect intracellular AR protein expressi on,q RT-PCR to detect SREBP1 m RNA expression;Oil red staining to detect intracellular lipid droplet generation;q RT-PCR was used to detect the m RNA expression of lipid droplet formation-related enzymes in PC3 and LNcap cells;Overexpression of SCD1 and the addition of exogenous OA increased the expression of SCD1 in cells and reversed the IC2-induced PC3 ER stress and apoptosis.Results:(1)Immunohistochemical results showed that the expression level of SCD1 in PCA tissue was significantly higher than that in adjacent tissue,which proved that SCD1 was related to the development of PCA;Compared with several other ICT derivatives,IC2 had the most pronounced dose-dependent inhibitory effects on both PCA cells and inhibited PC3 cell proliferation;IC2 inhibited the expression of SCD1 protein,m RNA and enzymatic activity in PC3 cells,confirming the inhibitory effects on SCD1.(2)IC2 induces the apoptosis of PC3 cells by down-regulating the expression of Bcl-2,which proves the apoptosis-inducing effect of IC2;IC2 can induce the expression of intracellular endoplasmic reticulum(ER)stress marker molecules BIP and CHOP;The treatment of PC3 with the addition of an inhibitor of ER stress alleviated IC2-induced apoptosis;Overexpression of SCD1 and addition of exogenous OA can increase the expression of SCD1 in cells and reverse the IC2-induced PC3 ER stress and apoptosis.(3)IC2 can induce the increase of AR expression and the decrease of SREBP1 expression in PC3 cells;IC2 can induce lipid droplet formation in PC3 and LNcap cells,and affect the expression of lipid droplet formation-related proteins in cells.Conclusion: The Icaritin derivative,IC2,inhibits the SCD1 activity of prostate cancer cells,thereby inducing ER stress and promoting apoptosis.