Study On Effects Of 1-nitro-2-acylanthraquinone-phenylalanine On Breast Cancer MCF-7 Cells

Anthraquinones were found in many plants,such as polygonum cuspidatum,rheum officinal,barbados aloe and other herbs.Anthraquinone compounds have many biological activities,such as antibacterial,antioxidant,anti-tumor,reducing blood pressure and so on.So far some anthraquinone drugs have been applicated in clinical and acquired some effects.However,anthraquinone compounds have some disadvantages,such as low content in natural plant and difficult extraction process,difficult separation and purification.At the same time,anthraquinone compounds have cardiac toxicity and other side effects.Therefore,synthesis of anthraquinone derivatives with high efficiency and low toxicity by chemical modification method is an important way to develop new anthraquinone drugs.According to the mechanism of anti-cancer drugs and the structure-activity relationship of anthraquinone derivatives,an anthraquinone derivative1-nitro-2-acylanthraquinone-phenylalanine named C7 had been designed and synthesized in our study.The study found that anthraquinone amide derivatives could inhibit the proliferation and migration of human breast cancer cell line MCF-7,meanwhile,could block the cell cycle procession and promote cell apoptosis.However,the mechanism and signal pathway of1-nitro-2-acylanthraquinone-phenylalanine to inhibit the proliferation and migration as well as arrest cell cycle and promote apoptosis of tumor cells are still not clear.In this paper,we investigated the effect and mechanism of the1-nitro-2-acylanthraquinone-phenylalanine(C7)on human breast cancer cell line MCF-7,the main research contents are as follows:1.MTT assay results showed that high concentration(60~100 ?g/mL)of1-nitro-2-acylanthraquinone-phenylalanine could obviously inhibit MCF-7cell proliferation in a dose dependent manner.While,the wound-healing assay suggested that low concentration(20~40 ?g/mL)of1-nitro-2-acylanthraquinone-phenylalanine could decrease the migration rateof MCF-7 cells.When MCF-7 cells were treated with different concentration of 1-nitro-2-acylanthraquinone-phenylalanine,optical microscope was used to examine the morphology of MCF-7 cells.It showed that the morphology of MCF-7 cells changed significantly,the distance between cells increased as well as cells shrinkage and turned round,meanwhile,the ability to adhere to the wall was reduced and some cells were floating in the medium.The changes of nuclear morphology were observed by DAPI staining,it was found that the appearance of nucleus shrunk and chromosome condensation.The result indicated that with the 1-nitro-2-acylanthraquinone-phenylalanine concentration increased,the chromatin of the nucleus was highly condensed and marginalized.q RT-PCR and Western blot were used to detect the expression of cycle factors p21,p53,CDK2,CDK4,CDK7 and Cyclin D1 as well as cell apoptosis factors Bax and Bcl-2 at mRNA and protein level,the results showed that pro-apoptotic genes were increased,whereas the anti-apoptosis genes were significantly reduced.2.Western blot and q RT-PCR were used to detect the expression of MMP-2/9 and I?B? of NF-?B/p65 pathway at mRNA and protein level,meanwhile,the immunofluorescence assay was used to detect the nuclear translocation of NF-?B/p65.The results showed that the phosphorylation of I?B? was obviously inhibited and the p65 protein gradually decreased in the nucleus.It indicated that with the phosphorylation level of I?B? was reduced,the nuclear translocation of p65 protein was relatively reduced,and the NF-?B/p65 signaling pathway was inhibited.Meanwhile,the expression of MMP-2 and MMP-9 were significantly decreased,suggested that the expression of matrix metalloproteinase was related to NF-?B/p65 signaling pathway.As the result,1-nitro-2-acylanthraquinone-phenylalanine could inhibit the phosphorylation of I?B?,block the nuclear translocation of NF-?B/p65 protein,thereby reducing the expression of MMP-2 and MMP-9.In summary,1-nitro-2-acylanthraquinone-phenylalanine could arrest cell cycle and promote apoptosis in MCF-7 cells.Meanwhile1-nitro-2-acylanthraquinone-phenylalanine could inhibit the NF-?B signaling pathway,thus inhibiting the MCF-7 cell migration.These results will lay the foundation for the study of anti breast cancer effect of anthraquinone amide derivatives and provide a new therapeutic strategy for the treatment of breast cancer.