Effect Of Anthraquinone Derivative C3 On Colon Cancer Cells Through ERK1/2-ERCC1 Signaling Pathway

Extracellular regulated protein kinase cascade(ERK1/2)is a classical intracellular signaling pathway,which plays an important role in the regulation of various cell life activities and the occurrence of cancer.In cascade reactions,ERK1/2 phosphorylation activates various transcription factors and regulates cellular physiological processes.Excision-repair cross-complementation group 1(ERCC1)is one of the downstream transcription factors of ERK1/2,identifying damaged DNA and repairing it.It was found that targeting ERK1/2 with emodin,an anthraquinone derivative,could inhibit the expression of ERCC1 downstream and the deterioration of cancer.Anthraquinone derivatives are natural phenols with 9,10-anthraquinone as skeleton and have good anticancer activity.At present,a variety of anthraquinone derivatives have been used as anticancer drugs in clinical practice and achieved good results.However,anthraquinone derivatives have poor water solubility,limited content in nature and complex extraction procedures.Therefore,new anthraquinone derivatives are designed and synthesized by chemical methods,which provides a new idea for the research and development of anticancer drugs.A water-soluble anthraquinone derivative(1-nitro-2-acylanthraquinone-valine,abbreviated as C3)was synthesized from 2-Methylanthraquinone by a series of chemical reactions.It was found that C3 had inhibitory effect on colon cancer cells,but the specific molecular mechanism was unknown.The effects of C3 on the proliferation and migration of colon cancer cells HCT116 and HT29 were studied in this paper.The main results are as follows:1.C3 inhibits the proliferation of HCT116 and HT29 cells.Firstly,MTT assay was used to detect the effect of C3 on the proliferation of HCT116 and HT29 cells.The results showed that C3 from 60 ?g/m L to 150 ?g/m L inhibited the proliferation of HCT116 and HT29 cells in a dose-dependent manner.At the same time,microscopic observation showed that C3 could reduce the cell morphology and round,reduce the number of cells,shrink the nucleus and uneven distribution of chromatin.Subsequently,flow cytometry and Western blot assay showed that C3 blocked cells at G0/G1 phase by down-regulating cyclin D1 and Cyclin E and cyclin-dependent kinases(CDK2 and CDK4).2.C3 inhibits the migration of HCT116 and HT29 cells.Scratch test was used to analyze the effect of C3 on the migration of HCT116 and HT29 cells.The results showed that the migration rate of HCT116 cells decreased from 100% to 14.04% and that of HT29 cells decreased to 9.96% after treated with 90 ?g/m L C3 for 48 hours.Western blot and immunofluorescence assay were used to detect the effects of C3 on the expression of E-Cadherin and Vimentin,markers of epithelial-mesenchymal transition(EMT).The results showed that C3 decreased the expression of Vimentin and increased the expression of E-Cadherin,indicating that C3 could inhibit the process of EMT in cells.3.C3 inhibits the proliferation and migration of HCT116 and HT29 cells by mediating ERK1/2-ERCC1 signaling pathway.The effect of C3 on the phosphorylation level of ERK1/2 was detected by Western blot experiment.The results showed that C3 decreased the phosphorylation level of ERK1/2 in a dose-and time-dependent manner.The results of q RT-PCR and Western blot showed that C3 destroyed the stability of ERCC1 through 26 s proteasome,promoted its protein degradation,and down-regulated the transcriptional level and protein expression of ERCC1.At the same time,ERCC1 was down-regulated when U0126 inhibited ERK1/2 expression,but ERK1/2 expression did not change significantly after transfection of si-ERCC1 RNA knocked down ERCC1 protein level,which proved that ERK1/2 was an upstream signal molecule of ERCC1.MTT assay and scratch assay were used to analyze the combined effects of U0126 and C3 on colon cancer cells.It was found that both proliferation and migration of colon cancer cells were inhibited.This indicated that C3 could reduce the expression of ERCC1 and inhibit the proliferation and migration of colon cancer cells by inhibiting the activity of ERK1/2.In conclusion,C3 inhibits the proliferation and migration of HCT116 and HT29 cells through ERK1/2-ERCC1 signaling pathway,alters cell and nuclear morphology,and blocks cell cycle progression.This study laid a foundation for the treatment of malignant tumors with new anthraquinone derivatives.