Mechanism Of Rhein Derivative 4F Regulating Rac1 Protein Expression And Inducing Non-apoptotic Programmed Cell Death In Breast Cancer Cells

Objective:Based on targeting Rac1 protein,molecular docking software was used to modify the side chain structure of the leading compound Rhein,we synthesized a novel Rhein derivative 4F（short for 4F in this article）.This research is aiming to explore its anti-tumor activity and verifing the mechanism of inducing non-apoptotic programmed death on breast cacner cells.Methods:1.Molecular docking software MOE.2008 was used to simulate the interaction mode of Rhein and its derivatives 4F with Rac1 protein.The Fluorescence excitation spectra and emission spectra of Rhein and its derivative 4F were detected by multi-functional enzyme label instrument.The uptake and distribution of Rhein and its derivative 4F in cells were detected by laser confocal microscopy.CCK-8 was used to compare the anti-tumor activity and cytotoxicity between Rhein and its derivative 4F among tumor cells and its normal counterpart.Western Blot analysis was used to test the target effect of Rhein and its derivative 4F on Rac1 protein level.Luciferase reporter gene assay was used to verify the regulatory transcription effect of Rhein and its derivative 4F on Rac1 expression in cellular level.2.Breast cancer MDA-MB-231 cells,which contained high expression of Rac1protein,and low-expressed Rac1 MCF-7 cells were chosen for advanced reasearches.CCK-8 assay,coloney formation and growth curve assay were performed to detect cell activity and cell proliferation ability after being exposed to different concentration of Rhein and its derivatives 4F.We applied Transwell Chambers with or without coating Matrigel glue to detect cell migration and invasion ability.The effect of Rhein and 4F on apoptotic rate and the cell cycle distribution was detected by flow cytometry.HE staining,F-actin staining,lysosomal fluorescent probe,mitochondrial fluorescent probe and DAPI staining were used to observe the changes of cell morphology,cytoskeleton and organelle caused by Rhein and 4F.3.TEM were used to observed the ultrastructure of cell samples.The intracellular localization and relative expression of LC3 were tested by immunofluorescence assay.Western Blot analysis were performed to detect the expression levels of autophagy related protein LC3,p62 and Beclin-1,apoptosis related proteins caspase-3,caspase-7,caspase-9,Apaf-1,XIAP,endoplasmic reticulum stress protein Grp78.mRFP-GFP-LC3 virus were used to monitoring cell autophagy flux in cell samples and to verify the mechanism of 4F-induced non-apoptotic progarmmed cell death.Results:1.The docking ASE score of Rhein and its derivative 4F with RAC1 protein were-15.4729 Kcal/mol and-25.3146 Kcal/mol,respectively.The lower the score,the stronger the binding ability with Rac1 protein.Rhein derivative 4F with spontaneous fluorescence and maximum emission was shown at 525 nm.The derivative 4F can be taken into cells easily and mainly distributed around the cytoplasm and nucleus.4F inhibited cell proliferation as a concentration and time dependent manner,and its IC₅₀0 value towards ovarian cancer,liver cancer,nasopharyngeal cancer,osteosarcoma and breast cancer cells was lower than 15μmol/L,and it showed lower level than the leading compound Rhein.4F with lower cytotoxicity was illustrated on the normal breast cell line MCF-10A when compared to positive drug doxorubicin.Western Blot results showed that Rac1 protein was expressed in all MDA-MB-231,MCF-7 and MCF-10A cell lines,wherein MDA-MB-231 demonstrated the highest expression level.Compared to control cells,4F downgrated the Rac1 protein expression levels both in MDA-Mb-231 and MCF-7 cell lines.We successfully constructed a Rac1-promoter-LUC 2 cell model with verification of Luciferase reporter gene experiments.These results proved 4F had targeting ability to Rac1,as well as downgrading Rac1 activity in the gene ranscriptional level of cell samples.2.The IC₅₀0 values of 4F on MDA-MB-231 and MCF-7 cells were（12.80±0.83）μmol/L and（7.54±1.25）μmol/L,respectively,when treated it with 48 h.Both4F and Rhein reduced the number of colonies.Besides,cell growth curve results showed that the doubling time of MDA-MB-231 and MCF-7 cells in the4F treatment group was 119.72 h and 79.4 h,respectively,which was lower than that in the control group.Furthermore,MDA-MB-231 cells with stronger migration and invasion ability than that of MCF-7 cells but this phenomenon could be inhibited by 4F.However,Rhein and 4F hardly affected cell cycle distribution and apoptotic rate either in the MDA-MB-231 or MCF-7 cells.4F changed cell morphology,causing decreased cell volume but increasing the volume and quantity of lysosome.Particularly,vacuoles,a symbol of non-apoptotic cell death morphological changes,appeared during observing the cell samples.3.The results of electron microscopy showed that MDA-MB-231 and MCF-7cells remained complete cell structure,but broken and dissolved nuclei,swollen endoplasmicreticulumandmitochondriawhentreatedwith4F.Autophagosomes and autophagolysosomes were observed in MDA-MB-231cells,while a large number of cytoplasmic vacuoles were observed in MCF-7cells.Immunofluorescence and Western Blot results showed that 4F increased the expression levels of LC3 II in both cells.MDA-MB-231 cells showed an increasing Beclin-1 expression level and decrease p62 expression,but the caspase-3 expression level was maintained stably.mRFP-GFP-LC3 monitoring autophagic flux showed that yellow and free red fluorescent puncta were observed like the positive drug rapamycin appeared after treatment with 4F for6 h,suggesting the formation of early autophagosomes.However,3-MA impeded the formation of yellow and free red puncta in MDA-MB-231 cells.Maintenance of 4F for 12 h,green and yellow fluorescent puncta were significantly reduced,indicating autophagosomes gradually developed into autolysosomes.Differently,4F increased Grp78 and p62 expression in MCF-7cells,but had no significant effect on the expression levels of caspase-7,caspase-9,XIAP and Apaf-1 proteins.Conclusions:1.Rhein derivative 4F,designed to target Rac1 protein,is a novel anthraquinone compound that is easy to be uptake by cells with lower citotoxicity to normal breast cells and excellent anti-tumor activity than the leading compound Rhein.4F reduced the expression of Rac1 in transcription levels and protein levels revel that 4F may be a small molecule targeting inhibitior of Rac1 protein in MDA-MB-231 and MCF-7 cells.2.4F may induced a non-apoptotic programmed cell death in breast cancer MDA-MB-231 and MCF-7 cell lines.3.Rhein derivative 4F induced non-apoptotic programmed death both in MDA-MB-231 and MCF-7 cells,the machanism of which in MDA-MB-231may via autophagic cell death,while in MCF-7 cells linked to paraptosis.