Study Of The Oridonin Induce HepG₂ Cancer Cell Apoptosis And The Related Signaling Pathway

1.Background The cancer has become one of the important factors of human death, has been a serious threat to human’s physical and mental health.Compared with the developed countries,the incidence of tumor in our country is higher,and the incidence become a rising trend.Currently, the mainly treatment of the tumor is chemotherapy, radiotherapy and surgery, although these methods have played an important role in treatment of tumor,but chemical drugs to treat cancer easy to cause multidrug-resistant and the increased costs and side effects,so look for high efficiency, strong specificity, side effects of small antineoplastic drugs is particularly important.Recent years, traditional Chinese medicine paid more and more attention because of the advantages of the small side effects, low prices,extensive sources,wide varieties and so on.Oridonin is separation and extraction from labiatae plants,colorless prismatic crystals, colorless, bitter taste,ene two terpenoids kauri natural compounds which is insoluble in water.Oridonin has a significant restrain for a wide variety of tumor cells such as pancreatic cancer, cervical cancer, liver cancer and so on.Although human make a lot of research for the oridonin anticancer and its anticancer mechanism,but there are still many unsolved of its role in the signal path and characteristics of the tumor cells and can not provide a safe and effective guidance for clinical administration.This paper use the modern molecular biology techniques to further reveal the role of oridonin in winter the mechanism of tumor cells, further provide theoretical basis for clinical medication.2.Purpose To research the oridonin induce HepG₂ cancer cell apoptosis; research the relative mechanism of anti-tumor role of the HepG₂ cancer cell.3.Methods3.1 Different concentrations of oridonin from low to high effect on HepG₂ cancer cells after24 h,determined by MTT method to detect inhibition rate and growth curve;Crystal violet to observe drug cell number changes3.2 Different concentration oridonin effect HepG2 cell,optical microscope to observe the morphological changes, and flow cytometry instrument detection of oridonin influence of the cell cycle distribution of the HepG₂ cells.3.3 Western blotting testing ATR and p- ATR, ATM and p- ATM, CHK1 and P- CHK1,Cdk1 and P- Cdk1,, H2 AX and gamma H2 AX, CDC25 C and P- CDC25 C and so on which Caused apoptosis by cell cycle related protein expression after different concentrations of oridonin from low to high effect on HepG₂ cancer cells.4.Results4.1 MTT assay results showed that low concentration of oridonin have a weak inhibitory on HepG₂ cell,high concentration of oridonin have a strong inhibition of the HepG₂ cell,and Inhibition rate are dose dependent;Crystal violet method results showed that the higher drug concentrations of oridonin, crystal violet stain is less, the lower the cell survival rate, the less live cells.4.2 Flow cytometry detection showed that oridonin block HepG2 cancer cells in G2 / M phase, the higher the drug concentration of oridonin,the more the number of cells in G2 / M phase.4.3 Western blotting technology detecting oridonin effect on HepG2 cancer cells related proteins show that after Different concentrations of oridonin treated 24 h, the Western imprinting results indicate that with the increase of oridonin concentration, P-ATM, P- ATR, gamma H2 AX, P53, Phos- P53, P- CDC25 C, P- Cdk1, P- CHK1, P-CHK2 proteins expressed in cells increased, CDC25 C, Cdk1 protein expression gradually reduce, indiscriminate Cyclin B1 protein expression5.Conclusions Oridonin can obviously inhibit of the HepG₂ cell;the cell IC50 is（31.92±1.8）μmol/L; during the oridionin inhibited HepG₂ cancer cell growth process,it closely associated with the cell cycle,oridionin can make the HepG₂ cancer cell block in G2 / M phase,and its mechanism is probably by increasing the cell cycle related proteins such as P-ATM,P-ATR, P-P53 、 P-Cdk1 、 P-CDC25 C and so on to activate the cell cycle checkpoint to affect cell cycle process to inhibited cancer cells.