| 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 160μmol/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-κB（PKCα,IKKα,IKKβ,Phospho-IKKα/β,IκBα,Phospho-IκBα,Phospho-NF-κB/p65）signaling pathway.（8）Immunofluorescence was used to detect the nuclear translocation of NF-κB/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-κB/p65、IκBα、IKKα/β、PKCαin MDA-MB-453 and BT-474 cells.（7）Dp inhibited MDA-MB-453 and BT-474 cell NF-κB/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-κB signaling pathway,and activating the JNK signaling pathway.In addition,high concentration of Dp（80μmol/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. |
