Study Of Target Finding For The C₂₁ Steroidal Esters From Marsdenia Tenacissima

Background and ObjectiveMalignant tumor?cancer?is one of the most serious diseases endangering human life and health in the world at present,and because of its difficult treatment,the overall cure survival rate is low and the cost of treatment is expensive,which not only becomes the burden of the patients themselves,but also a major burden for society.The combination of surgery and chemotherapy is the main method for the treatment of cancer.However,cancer cells often develop drug resistance against cancer drugs.Moreover,the development from one drug resistance to cross resistance to many drugs resulted in multi-drug resistance,which resulted in the failure of chemotherapy.The mechanism of drug resistance in tumor cells was very complicated,the drug resistance of tumor cells may caused by drug efflux pump,the over expression of multi-drug resistance related proteins,the enhancement of cell repair ability,the mutation of drug target and the decrease of apoptosis.However,the mechanism of drug resistance has not been clearly clarified.Studies have shown that drug resistance in many tumors may be the result of combination or synergism of multiple mechanisms.The steroid esters of Chinese herbal medicine?M3,M7,BAT,as shown in FIG.1?can enhance the proliferation inhibitory activity of drugs such as paclitaxel,doxorubicin and vincristine on certain tumor cells,but the specific mechanism of action is not clear.Therefore,the study of its target is particularly important for the development of tumor sensitizers.Methods1.Fourteen crystal structure cytochrome P450 enzymes and PXR receptor were selected as target libraries,three C₂₁steroidal compounds were analyzed for target finding based on molecular docking technique.2.On the premise that paclitaxel sensitized by M7 was effective on HepG2cell line,paclitaxel was administered alone,combined with M7 and paclitaxel for proteomic analysis.The proteomics results were analyzed and the expression levels of target protein and P-gp protein were verified by Western Blot method to determine the possible mechanism of M7Results1.Four proteins were selected as possible targets based on molecular docking technique:CYP 3A4,CYP 2C8,CYP2E1 and PXR.2.Proteomics analysis showed that RPN 2(ribosomal binding protein II was identified as the target protein.There was no difference in the expression of RPN2 between control group and M7 group;compared with paclitaxel alone,a1.7-fold decrease in RPN2 expression in combination group;compared with paclitaxel alone,the expression of RPN2 protein was decreased by 12.5 times in combination group.3.Western Blot confirmed the expression of target protein and P-gp:The expression of P-gp protein in control group and M7 group was lower than that in paclitaxel alone group and combined group,and the expression of P-gp in combined group was lower than that in paclitaxel alone group.Conclusion1.Based on the molecular docking analysis,the possible mechanism of increasing taxol sensitivity to paclitaxel:?1?M3?M7?BAT may enhance the anti-tumor effect of paclitaxel by regulating the activity of CYP3A4,CYP2C8,CYP2E1 to reduce its metabolism of paclitaxel;?2?M3?M7?BAT may enhance the anti-tumor effect of paclitaxel by regulating the activity of PXR to regulate the expression of CYP3A4 and P-gp,to reduce the metabolism and efflux of paclitaxel.2.Proteomics analysis suggested that M7 may reduce the glycosylation of P-gp by inhibiting the expression of RPN2 and decreasing the glycosylation of P-gp encoded by MDR1 gene,and affecting the membrane localization of P-gp,reducing the function of“drug pump”,and increasing the concentration of paclitaxel in tumor cells.