The Study Of The Effect And Mechanism Of Mitophagy Through PINK1/Parkin Pathway Induced By Warangalone In Breast Cancer Cells

BackgroundBreast cancer is the most common malignancy in women worldwide.Although breast cancer treatment methods are constantly innovating,chemotherapy still occupies the main position.Chemotherapy drugs used clinically often have side ef fects such as neutropenia and aging.Therefore,it is urgent to develop a low-toxic and high-efficiency drug for breast cancer treatment.Warangalone is a flavonoid compound isolated from the fruit of Tuoshu.It has antioxidant,blood pressure lowering and anticancer activities.Cellular autophagy and apoptosis play an important role in the prevention and treatment of tumors as a form of programmed cell death.Does climbing isotopes exert their anti-breast cancer effects by inducing autophagy and/or apoptosis of breast cancer cells?What is the specific mechanism?Not yet clear.PurposeTo explore the anti-breast cancer effect and molecular mechanism of warangalone,and to provide a theoretical basis for the development of warangalone as an anti-breast cancer drug.Methods1.MTT assay and cell cycle kit were used to detect the proliferation activity of warangalone in different types breast cancer cells.2.Hoechst staining,Annexin V-FITC/PI staining and Western blotting were used to detect whether warangalone induced cell apoptosis in breast cancer cells.3.MDC staining,Western blotting and immunofluorescence staining were used to detect whether warangalone induced cell autophagy in breast cancer cells.4.After mitochondria stained with Mito-tracker Red,confocal microscope and transmission electron microscope were used to observe the mitochondrial morphological changes;Mitochondrial membrane potential detection kit and reactive oxygen species detection kit were used to detect mitochondrial membrane potential and reactive oxygen species.5.Mito-tracker Red stained mitochondria,LysoTracker Green stained lysosomes,and immunofluorescence staining of LC3,PINK1 and Parkin,confocal microscope was used to observe the co-localization of mitochondrion and lysosome or LC3,PINK1 and Parkin expression and co-localization;Weste rn blot and PCR were used to detect the mRNA and protein expression of PINK 1 and Parkin.Results1.Warangalone inhibits cell proliferation in breast cancer cellsWarangalone significantly inhibited the proliferation of breast cancer cells(MDA-MB-231,MCF7,ZR75-1 and SKBR3)at 20μM 24h,15 μM 24h,20μM 24h and 15 μM 24h(P<0.001;P<0.001;P<0.001;P<0.001).Warangalone blocked MDA-MB-231 cells at G1 phase(P<0.05)and MCF7 cells at S/G2 phase(P<0.001).2.Warangalone induces apoptosis in breast cancer cells20 μM warangalone significantly increased the apoptosis rate when treated with MDA-MB-231 cells(P<0.01)for 24 h,and 10 μM warangalone significantly increased the apoptosis rate in MCF7 cells(P<0.05)for 24 h.After treated with breast cancer cells(MDA-MB-231 and MCF7)for 24 h by 30 warangalone,PARP expression was significantly down-regulated(P<0.001;P<0.05),and Bax/Bcl-2 expression ratio was up-regulated(P<0.05;P<0.01)3.Warangalone induces autophagy in breast cancer cellsTreated with 20 μM warangalone in breast cancer cells(MDA-MB-231 and MCF7)for 12 h,acid vesicles in the cells increased,autophagy-labeled proteins LC3Ⅱ(P<0.01;P<0.01)and P62(P<0.01;P<0.001)expressions were significantly up-regulated.Treated with 20 μM warangalone in breast cancer cells(MDA-MB-231 and MCF7)for 12 h.the expression of LC3 protein in the cells was up-regulated under a confocal fluorescence microscope.However,after autophagy was inhibited by the autophagy inhibitors chloroquine and 3-methyladenine,the PARP expression in MDA-MB-231 cells was further significantly down-regulated after treated with 20 μM warangalone for 12 hours(P<0.001;P<0.001),and the PARP protein in MCF7 cells did not change significantly4.Warangalone promotes ROS production and mitochondrial damage in breast cancer cellsAfter treated with 20 μM warangalone in MDA-MB-231 cells for 12 h,the mitochondrial morphology changed from filaments to short granules,the mitochondrial ridge damage even disappeared,and the mitochondrial membrane potential decreased.Treated with 20 μM warangalone in MDA-MB-231 cells for 12 h,mitochondrial ROS was significantly up-regulated(P<0.05).In addition,20 μM warangalone significantly increased the mitochondrial mass of MDA-MB-231 cells(P<0.001),while the inhibition of ROS production by the ROS inhibitor N-acetylcysteine significantly reduced the mitochondrial mass(P<0.05).5.Warangalone induces mitochondrial autophagy in breast cancer cellsAfter 20 μM warangalone treated breast cancer cells for 12 h,there was a clear co-localization between mitochondria and lysosomes,mitochondria and LC3 in MDA-MB-231 and MCF7 cells.Transmission electron microscopy showed that there were mitochondria wrapped in double-layer vesicles in the MDA-MB-231 and MCF7 cells treated with warangalone,that is,mitochondrial autophagosomes.6.Warangalone promotes mitochondrial autophagy in breast cancer cells through the PINK 1/Parkin signaling pathwayAfter 30 μM warangalone treated MDA-MB-231 cells for 12h,the expression of PINK1 and Parkin mRNA were significantly up-regulated(P<0.05;P<0.01);Warangalone up-regulated PINK1 and Parkin protein expression in a concentration-dependent manner(P<0.01;P<0.01).After ’20 μM warangalone treated MDA-MB-231 cells for 12h,there was obvious colocalization between PINK1 and Parkin under confocal microscopeConclusions1.Warangalone inhibits cell proliferation,and induces apoptosis and autophagy in breast cancer cells.However,the inhibition of autophagy promotes apoptosis.2.Warangalone leads to mitochondrial damage via ROS,and activates mitophagy.3.The mitophagy through PINK1/Parkin pathway activated by Warangalone participates in anti-cancer activity.