Construction Of Screening Model For C-Myc Gene Expression Modulators And Screening Of C-Myc Modulators From Traditional Chinese Medicine

Cancer, a leading cause of death worldwide, is a serious threat to human health. The traditional therapies such as surgery, chemotherapy and radiotherapy are usually ineffective and poor prognosis. So discovery of new therapeutic drugs is urgently needed.C-Myc is an oncogene, it is activated in various animal and human tumors, it can increase cellular proliferation, transformation, apoptosis, and genetic instability, which make it a cancer therapeutic target for drug discovery.In our research, we firstly established a firefly luciferase reporter driven by the c-Myc gene promoter. We amplified the 1418 bp fragment of c-Myc gene 5’non-coding region from the genome in Hep G2 cells. Then we inserted it into the p GL3-basic vector to construct the luciferase reporter vector p GL3-c-Myc. According to the results of luciferase activity test, we confirmed that the fragment we identified had high promoter activity.Then, we screened the c-Myc expression modulators from the Traditional Chinese Medicine(TCM). Using luciferase reporter assay method, more than 400 natural compounds derived from TCM were screened. At last, we found that compound TI588 can improve and TI101 can inhibit the activities of c-Myc promoter. Further results confermed that TI588 indeed enhanced and TI101 inhibited the expression of c-myc in both m RNA and protein levels by RT-PCR and western blot analysis.Further more, we demonstrated the anti-tumor effect of TI101, and clarified the possible mechanisms. The results of MTT showed that TI101 can inhibit the cell viabilities of both human hepatoma cells(Hep G2) and human normal liver cells(L02). The inhibition rate to Hep G2 is higher than that to L02. Further studies showed that the IC50 value of TI101 to L02 is about 71.101μg/m L, while the IC50 value to Hep G2 is 3.137μg/m L. Therefore, we believe that TI101 has developed value as antitumor drug. Then, we uncovered that the TI101 perform its anti-tumor effect by inducing cell apoptosis and inhibiting cell proliferation.Results from DAPI staining, Flow cytometry and western blot showed that TI101 induces Hep G2 cells apoptosis through the mitochondria apoptosis pathway. In addition, we have found that TI101 can obviously inhibit proliferation of Hep G2 cells by inducing G2/M arrest detected by the Brd U incorporation and Flow cytometry.In summary, our studies have provided an effective model for antitumor drug screening,and successfully screened a natural compound TI101 which observably induce cell apoptosis and inhibit cell proliferation. Our work laid a solid material foundation for anti cancer drug development.