High-throughput Screening Of Antitumor Drugs Based On Cell Cycle FUCCI System And Research On Their Pharmacological Activities

Background:Tumor is one of the diseases with the highest mortality in the world,and its’occurrence and development are mainly related to cell cycle.At present,the main methods to detect cell cycle are:acridine orange(AO)method,5-bromo-2-deoxyuridine(BrdU)labeling method,Western blot and flow cytometry.However,these methods have some limitations,such as unable to perform living imaging.To overcome these,we applied a new technology of FUCCI(Fluorescent Ubiquitination-based Cell Cycle Indicator)system to perform the high-throughput screening of antitumor drugs.Objective:By applying the cell cycle FUCCI system,we performed high-throughput screening of antitumor drugs based on the change of fluorescence ratio.After screening,we studied the pharmacological activities and antitumor regulatory mechanisms of these putative candidates.Methods:(1)To observed the cell cycle in living imaging,we constructed the FUCCI-7402 tool by transfecting FUCCI plasmids into Bel-7402 cells.The FUCCI-7402 tool was validated by bleomycin,a classic G2 phase arrested drug;(2)To perform high-throughput screening of anthraquinone compounds library and 444 natural compounds library,we observed the change of fluorescence after drug administration using FUCCI-7402 tool;(3)To detect the cell cycle changes of Bel-7402 cells after rhein treatment,we used flow cytometry method to verify preliminary screening;(4)To detect the effects of rhein on proliferation and apoptosis of lung cancer cell PC9,we used CCK-8(Cell Counting Kit-8)method,flow cytometry and JC-10(mitochondrial membrane potential assay kit with JC-10)for detection;(5)To detect the changes of apoptosis related genes and regulatory pathways,we used RNA-Seq to study the differential expressed genes after rhein treatment in PC9.Results:(1)The FUCCI tool was constructed and validated by bleomycin,a classic G2 phase arrested drug;(2)Evaluation of first-line chemotherapy drugs in clinic:after treated with fluorouracil(30μmol/L)and camptothecin(3μmol/L)in Bel-7402 cells,we found that the fluorescence ratio of FUCCI-7402 increased,indicating that the cell cycle was arrested at G1/S phase.Flow cytometry results showed that the proportions of G1 phase cells increased from 28.74%to 66.87%and 47.88%after treatment with fluorouracil and camptothecin,respectively.Flow cytometry results were consistent with FUCCI fluorescence results;(3)After preliminary screening,we found a series of G1/S arresting drugs,such as rhein,cytochalasin E,gambogic acid,apigenin and puromycin from anthraquinone compounds library and 444 natural compounds library by FUCCI-7402 tool;We confirmed the changes of cell cycle after rhein treatment(100μmol/L)in Bel-7402 cells by flow cytometry.The proportion of cells in G1 phase increased from 37.78%to65.15%compared with the control,which indicated that rhein arrested the cell cycle of Bel-7402cells at G1 phase;(4)Rhein(200μmol/L)significantly inhibited the proliferation of lung cancer PC9 cells,and the survival rate of PC9 was about 50%.The apoptosis rate increased from 28.24%to62.59%,and the mitochondrial membrane potential decreased in the presence of rhein;(5)By transcriptome analysis,we found that rhein induced apoptosis of PC9 mainly through p53 signaling pathway,MAPK signaling pathway,m TOR signaling pathway and cell cycle related pathways.Conclusion:We constructed a high-throughput screening tool based on cell cycle FUCCI system,and evaluated some clinical first-line chemotherapy drugs.The tool was used to screen the anthraquinone compounds library and 444 natural compounds library.It was found that rhein,cytochalasin E,gambogic acid,apigenin and puromycin could arrest the cell cycle at G1/S phase.From pharmacological studies,we found that rhein inhibits PC9 proliferation and induces apoptosis mainly through p53 signaling pathway,MAPK signaling pathway,m TOR signaling pathway and cell cycle related pathways.