The Synthesis Of Rhein Derivative 4L Targeting RAC1 Protein And Analysis Of Inhibitory Effect Of Derivative 4L On The Growth Of Ovarian Cancer Cells

Objective:The rhein derivative 4L is designed and synthesized based on the parent structure of a lead compound rhein as well as considering the potential target RAC1 that inhibits the metastasis and invasiveness of ovarian cancer,the molecular docking software is also taken advantage of during the process of designing.To investigate the effect of the newly synthesized derivative on ovarian cancer cell SKOV3 and its underlying mechanism.Methods:1.We synthesized the rhein derivative 4L after an intermediate compound named X by modifying the structure of rhein based on the target of RAC 1 and the software of the molecules docking.The ultraviolet and visible absorption spectrum,fluorescence excitation spectrum and fluorescence emission spectrum of the compound were measured using an ultraviolet spectrophotometer and a multifunction microplate reader.The distribution of derivative in ovarian cancer cells was observed by a laser confocal microscopy.The intake of the derivative by SKOV3 cells was detected by high presser liquid chromatography.2.The inhibitory effect of the derivative on SKOV3 cells was determined by the MTT assay,the effect of derivative on cell morphology of SKOV3 cells was observed by the HE staining assay,the apoptosis of SKOV3 cells induced by derivative was detected by the flow cytometry,the effect of the derivative on cell migration and invasion of SKOV3 cells was explored by the would-healing assay and transwell assay,respectively.3.The expression of RAC 1 regulated by derivative on the SKOV3 cells was detected by western blot assay.A lentiviral vector contained the RAC1 promoter and the luciferase reporter gene was constructed according to the principle of luciferase reporter gene system,a cell line named SKOV3-RAC1-LUC was obtained after selecting the lentiviral-transfected SKOV3 cells by applying puromycin in culture medium,the activity of RAC 1 regulated by derivative was deteceted by luciferase reporter gene system.Results:1.Rhein derivative 4L is successfully synthesized,both rhein,compound X and derivative 4L can interact with RAC 1 protein,and the binding energy is-13.00 kcal/mol,-10.35 kcal/mol,-16.96 kcal/mol,respectively,indicating the interaction between the derivative 4L and RAC1 is more steady than that of rhein,compound X.The maximum UV wavelength of rhein and derivative 4L is 261nm and 262nm respectively;the maximum excitation wavelength and emission wavelength of rhein is 460nm and 640nm and the maximum excitation wavelength and emission wavelength of rhein derivative 4L is 440nm and 520nm.Derivative 4L can not only bind to cell membranes of SKOV3 cells,but also get into the cells,The drug that enters the cells is mainly distributed in the cytoplasm but less in the nucleus.The intake of rhein and rhein derivative 4L in ovarian cancer SKOV3 cells is 1.34±0.34 μg/mg and 116.14±1.43μg/mg at the same treated concentration,respectively,the intake of derivative 4L was higher than that of rhein(P<0.001).2.After treating with derivative 4L for 48h,the proliferation of ovarian cancer cells is significantly inhibited,and the inhibitory effect increases along with the augment of the drug concentration,the half inhibition rate(IC50)of 4L is 9.977μg/mL,the inhibitory effect is obviously better than rhein of which half IC50 is 17.338μg/mL;the results of HE staining observed by the microscope shows that the number of cells decreases,the ratio of nuclear to cytoplasm increases,the cytomembrane of cells becomes shrinkage along with the increase of drug concentration;SKOV3 cells can be obviously induced the apoptosis after treating with rhein derivative 4L for 48h,and the apoptosis occurs in a dose-dependent manner;derivative 4L can inhibit the migration and invasion of SKOV3 cells.3.Derivative 4L can reduce the expression of RAC 1 protein in SKOV3 cells in a dose-dependent manner;the sequence of the insert in the plasmid was identical to the expected according to the sequence results shows that the lentiviral vector contained the RAC 1 promoter and the luciferase reporter gene was successfully constructed,so does the stably transfected cell line;the luciferase assay shows that the RAC 1 protein of SKOV3 cells can be targeted inhibited by derivative 4L.Conclusion:1.The intake of derivative 4L by the ovarian cancer cell line SKOV3 is higher than that of the lead compound rhein,indicating an effective structural modification of rhein.2.Derivative 4L can inhibit the proliferation and induce the apoptosis of ovarian cancer SKOV3 cells,the metastasis and invasion of ovarian cancer can be inhibited by the derivative 4L.3.RAC1 protein can be targeted inhibited by derivative 4L to inhibit the invasion and metastasis of ovarian cancer SKOV3 cells,which might be a small molecular inhibitor targeting the RAC1.