Molecular Mechanism Of Delphinidin Inducing Cell Cycle Arrest And Apoptosis In HER-2 Positive Breast Cancer Cells

Previous studies have found that black rice anthocyanidins?BRACs?exert a proliferation effect on HER-2 positive breast cancer cells,and tumor control effects due to their strong antioxidant properties.Acting as the major active component of anthocyanins,Delphinidin?Dp?can inhibit the proliferation of HER-2 positive breast cancer cells.However,the mechanism of action continues to be unknown.This study aims to further confirm the growth and proliferation effects of Dp against HER-2positive breast cancer,clarify the molecular mechanism of Dp against HER-2 positive breast cancer,and provide new evidence for the prevention and treatment of Dp on HER-2 positive breast cancer.ObjectiveThe experiment intends to investigate the effect of Dp on the growth and proliferation of HER-2 positive breast cancer cells and its mechanism of action,and provide experimental data for the prevention and treatment of HER-2 positive breast cancer by flavonoids.MethodsHER-2 positive breast cancer cells MDA-MB-453?HER-2⁺,ER^-,PR^-,HER-2⁺?,BT-474?Luminal B,ER⁺,PR⁺,HER-2⁺?and human normal breast epithelium cells MCF-10A were chosen as the research object.Cells were treated with different concentrations of Dp?10,20,40,80 and 160?mol/L?:?1?CCK8 method was used to detect cell proliferation;?2?HE staining was used to observe cell morphology changes.?3?PI staining method was used to detect cell cycle distribution.?4?JC-10 staining was used to analyse the mitochondrial membrane potential changes.?5?TUNEL method was deployed for detecting DNA fragmentation level.?6?Annexin V-FITC/PI double staining was used for detecting apoptosis level.?7?Western blot was used to detect the expression levels of cell cycle regulatory proteins(Cyclin B1,Cdk1,p21 ^Waf1/Cip1),apoptosis-related proteins?Bax,Bcl-2?,MAPK?c-Raf,Phospho-c-Raf,MEK1/2,Phospho-MEK1/2,Erk1/2,Phospho-Erk1/2,SAPK/JNK,Phospho-SAPK/JNK?and NF-?B?PKC?,IKK?,IKK?,Phospho-IKK?/?,I?B?,Phospho-I?B?,Phospho-NF-?B/p65?signaling pathway.?8?Immunofluorescence was used to detect the nuclear translocation of NF-?B/p65.Results?1?Dp inhibited the proliferation of MDA-MB-453 and BT-474 cells in a dose-dependent manner.?2?Some cells fell off and lysed,while cell volume decreased in the Dp treatment group of MDA-MB-453 and BT-474 cells.?3?Dp inhibited the expression of Cyclin B1 and Cdk1 in MDA-MB-453 and BT-474 cells,while inducing cell cycle arrest in G₂/M phase.Dp also inhibited the expression of Cdk1 in MCF-10A cells,and induced cell cycle arrest in G₂/M phase.?4?Dp down-regulated Bcl-2protein expression,up-regulated Bax protein expression and decreases mitochondrial membrane potential in MDA-MB-453 and BT-474 cells.?5?Dp down-regulated the phosphorylation levels of c-Raf,MEK1/2 and ERK1/2 protein in ERK pathway,up-regulated the phosphorylation level of JNK protein in MDA-MB-453 and BT-474cells.?6?Dp down-regulated the phosphorylation levels of NF-?B/p65?I?B??IKK?/??PKC?in MDA-MB-453 and BT-474 cells.?7?Dp inhibited MDA-MB-453 and BT-474 cell NF-?B/p65 nuclear translocation.ConclusionFirstly,Dp induces the cell cycle arrest in G₂/M phase by inhibiting the expression levels of cyclin B1 and Cdk1 in MDA-MB-453 and BT-474 cells.Secondly,Dp induces mitochondrial pathway apoptosis by regulating the expression levels of Bcl-2 and Bax protein.Thirdly,Dp induces apoptosis by inhibiting the NF-?B signaling pathway,and activating the JNK signaling pathway.In addition,high concentration of Dp?80?mol/L?induces the cell cycle arrest in G₂/M phase by inhibiting the expression of Cdk1,but does not cause apoptosis,and does'nt inhibit cell proliferation in MCF-10A cells,indicating that Dp only plays an anti-proliferative and pro-apoptotic role in the MDA-MB-453 and BT-474 HER-2 positive breast cancer cells.