The Toxic Effect Of Resveratrol On Breast Cancer 4T1 Cells And The Following Transcriptomic Analysis

Cancer is one of the most serious threat to human life and health,among which breast cancer is one of the highest incidence of malignant tumors in women.Chemotherapy plays an important role in the preoperative and postoperative treatment of cancer.With the rapid development of drug discovery in recent years,many kinds of drugs have been developed for the comprehensive treatment of cancer,which shows significant efficacy.Resveratrol(RSV),a natural polyterpenoid,can scavenge reactive oxygen species in vivo to carry out the functions of anti-oxidation and anti-aging.Drugs from natural plants are often used as synergistic drugs in cancer therapy because of its low toxicity and side effects.Resveratrol's anti-cancer capability has attracted widespread attention,but its molecular mechanism has not been systematically explained.In order to get a more comprehensive and mechanistic understanding of the toxic effect of resveratrol on cancer cells,the morphology,viability,proliferation,apoptosis,necrosis and cell cycle of 4T1 cells were detected from cellular and molecular levels.The main results of this study are as follows:(1)Effects of RSV on proliferation and viability of different cell lines: Different concentrations of RSV(0,50,100,150,200,and 250 ?M)were used to treat two types of normal cell lines(HK-2 and L02)and two types of tumor cell lines(HepG2 and 4T1)for 24 h,48 h, and 72 h,respectively.CCK-8 assay was used to detect cell viability.Low concentration and short time treatment of RSV had a slight promoting effect on normal cell lines.With the increase of treatment time and treatment concentration,RSV has obvious inhibitory effect on both normal cell lines and cancer cell lines.By comparison,it was found that RSV had a more obvious toxic effect on cancer cells compared to normal cells,especially on 4T1 cells.Therefore,the 4T1 cell line was chosen for further study.The viability of 4T1 cells decreased with the increase of dose and time.According to the dose-dependent viability curve,the IC50 of RSV on 4T1 cells at 48 h was calculated to be 93 ?M.(2)Effects of RSV on cell morphology of 4T1 cells: Different concentrations of RSV(0,50,100,150,200,and 250 ?M)were used to treat 4T1 cells for 24 h,48 h and 72 h,respectively.The changes of cell morphology observed under the inverted microscope were shown that cell adherent was obvious,and the cell growth was nice under the condition of control and low concentration;With the increase of treatment concentration,the cell becomes swollen with blurred edges,disrupted membranes,and decreased density,and there are fewer contact areas between cells,resulting in worse growth environment and accelerated cell death.Furthermore,with the increase of treatment time,the area of most observed cells gradually decreased at each concentration,except for several swollen and ruptured cells.In summary,according to the death morphology,it was speculated that the death of 4T1 cells was induced by apoptosis.(3)Effects of RSV on apoptosis and necrosis of 4T1 cells: After the treatment of RSV,AO/EB kit compared with fluorescence inverted microscope were used to detect the morphological apoptosis.The results of apoptotic rate verified that at around 100 ?M,RSV can induce half of the cells to undergo apoptosis,which is consistent with the previous IC50 results detected by CCK-8.According to the detection of Annexin V/PI kit and flow cytometry,RSV induced apoptosis and death of 4T1 cells in a time-and dose-dependent manner.(4)Effects of RSV on gene expression of 4T1 cells: Through large-scale transcriptome sequencing,a total of 330 significantly differentially expressed genes(DEGs)were identified between Mock group and RSV-treated group,among which 103 genes were upregulated and 227 genes were downregulated.The results of GO annotation and KEGG pathway enrichment showed that DEGs mainly enriched in cell cycle,cell division,cell adhesion and other related pathways.The verification results of qRT-PCR showed that the expression trend of most DEGs was consistent with the results of RNA-seq.RSV may exert toxic effects on cancer cells mainly by regulating the expression of cyclically related genes.(5)Effects of RSV on cell cycle of 4T1 cells: By constructing the protein-protein interaction network of the targets of RSV,it was found that proteins related to cycle,such as Cdc,Cdk,and Ccnd,as well as proteins related to apoptosis,such as Brca,Bcl,and Bax,were key targets in the network;The changes of cell cycle phases at different time points after treating with RSV were further detected,and it was found that the cells were arrested in the S phase because of the percentage of cells in S phase increased and cells in G1/G0 phase decreased.In conclusion,RSV may inhibit the proliferation of 4T1 cancer cells by inhibiting cell cycle and inducing apoptosis.