Effects Of Low Dose Levistolide A On The Senescence And DNA Damage Of HepG2 Hepatoma Cells

Chuanxiong is a traditional natural product of medicinal and food homology.Levistolide A(LA)is a monomer compound extracted from the root of Ligusticum Chuanxiong.At present,the results showed that LA can significantly inhibit the proliferation and promote the apoptosis of HCT116 colon cancer cells in a concentration and time-dependent manner,and the anti-colon cancer effect of LA is not dependent on p53.According to the reports,LA can inhibit the proliferation of NSCLC cells and induce apoptosis;the injection of H1299 cells into nude mice showed better antitumor activity than cisplatin.However,whether LA has anti-hepatoma effect and whether LA has anti-aging effect on tumor cells has not been reported,therefore needs to be revealed by further research.To clarify whether LA can inhibit the growth of hepatoma cells and the effect of LA on the senescence of hepatoma cells,Hep G2 cells were used in this study.MTT assay was used to detect the growth inhibition of LA on hepatoma cells.?-galactosidase staining and morphological observation were used to detect the effect of low dose of LA on the senescence of Hep G2 cells.Quantitative real-time PCR were used to detect the regulation of aging marker genes in Hep G2 cells by LA.Finally,Western blotting was applied to detect the protein expression of DNA damage related genes regulated by LA.The results are as follows:1.LA inhibits the growth of Hep G2 cells,and LA induces the senescence of Hep G2 cells in a low dosage.The effect of LA on the proliferation of Hep G2 cells was detected by MTT.The results showed that LA significantly inhibited the cell viability of Hep G2 cells in a dose-dependent effect.The result of ?-galactosidase staining was positive.Therefore,the above results showed that low dose of LA induced aging process in Hep G2 cell in a dose-dependent emanner.2.Low-dose LA induced the expression of senescence-related genes andDNA damage-related proteins in liver cancer cells.After low-dose Levistolide A(6.25 ?M,12.5 ?M)treatment of Hep G2 cells for 48 hours,the total RNA of the treated cells was extracted by Trizol method,semi-quantitative PCR and real-time quantitative PCR experiments were used to detect aging-related genes p53,p21 and IL-1?.The results showed that the expression of these genes in the treated group was significantly higher than that in the control group.The expression of DNA damage-related proteins?-H2 AX,p-ATM and p-Chk2 was further detected by Western blot.The results showed that the expression levels of the above proteins in the cells of the treatment group were significantly higher than those of the control group in a concentration-dependent manner.The above results indicate that low-dose Levistolide A activates the DNA damage response pathway after 48 hours of action on Hep G2 cells,enhances p53 activity,and regulates the downstream cyclin kinase inhibitor p21 to induce cell senescence.In conclusion,LA can not only inhibit Hep G2 cells,but also mediate the senescence of Hep G2 cells through DNA damage pathway.Through the research of this paper,we hope to provide new ideas for the treatment of liver cancer in clinic,and also lay the foundation and theoretical basis for the development of new anti-tumor drugs.