Effect Of IR-783 On Proliferation/Migration Of Breast Cancer Cells And Its Mechanism

Objective:This study aimed to observe the effect of IR-783 on the proliferation and migration in human breast cancer cells and explore its extact mechanism,which can provide mechanistic basis for the application of IR-783 in the treatment of breast cancer.Methods:Breast cancer cell line MDA-MB-231 and MCF-7 were treated with different concentrations of IR-783.The cell viability and proliferation ability were assessed by MTT、colony formation assay and Edu assay.Flow cytometry was used to detect the cell cycle.The migration and invasion ability of cells were assessed using wound healing assay and Transwell assay.The cellular ATP levels were measured with an ATP Assay Kit.Fluorescence microscopy and Transmission electron microscopy were used to observe the morphology of mitochondria and F-actin.Western blotting was used to detect the expression levels of CyclinD1、CyclinE、CDK2、MMP-2、MMP-9、OPA1、Mfn1、Mfn2、MFF、Fis1、Drp1.Results:1.After treatment with various concentrations of IR-783(0 μM/L、20 μM/L、40 μM/L、60 μM/L、80 μM/L、100 μM/L、120 μM/L、140 μM/L、160 μM/L)for 24 h or with 80 μM IR-783 for different time intervals(0 h,3 h,6 h,12 h,24 h,36 h,48 h and 72 h),we reported that IR-783 treatment significantly decreased cell proliferation in a dose-and time-dependent manner compared with the control.Colony formation assay indicated that the number of colonies formed in IR-783-treated group was significantly reduced in MDA-MB-231 and MCF-7 breast cancer cells compared with the control.Edu assay revealed significant fewer EdU-positive cells following treatment with IR-783 compared with the control.2.After treatment with IR-783(0,80 and 160 μM)for 24 h,IR-783 induced MDA-MB-231 and MCF-7 cell cycle arrest at the G0/G1 phase.Transwell assay and wound healing assay results indicated that IR-783 inhibits MDA-MB-231 and MCF-7 cell migration.MDA-MB-231 and MCF-7 cellular ATP levels decreased in a dose-dependent manner in response to the IR-783.A laser-scanning confocal microscope suggested that IR-783 inhibits filopodia formation by reducing F-actin formation.Transmission electron microscopy results revealed that IR-783 induced mitochondrial injury in MDA-MB-231 cells,and confocal laser scanning microscopy results revealed that IR-783 treatment promoted mitochondrial fission,as observed by a marked increase in number of small,punctate mitochondria in IR-783 treated cells compared with control cells,which exhibited stable and elongated structures.3.Western blotting results revealed that IR-783 reduced the expression of Cyclin D1,Cyclin E,CDK2,MMP-2 and MMP-9 in MDA-MB-231 cell.Meanwhile,IR-783 treatment resulted in a significant decrease in the expression of OPA1,Mfn1 and Mfn2,and increased the levels of MFF,Fis1 and Drp1 compared with the control.ConclusionIR-783 inhibited the proliferation and migration of MDA-MB-231 and MCF-7 cells by inducing mitochondrial fission and subsequently decreasing ATP levels,resulting in cell cycle arrest and filopodia formation suppression.