Studies Of Nucleolin Aptamer-modified Triptolide’s Anticancer Effect On Gemcitabine-resistant Human Pancreatic Cancer And Its Mechanism In Vitro

Objective:To evaluate the anticancer effect of Nucleolin Aptamer-modified Triptolide(Conjugate)on Gemcitabine-resistant human pancreatic cancer cell line MIA PaCa-2,and explore its mechanism preliminary.Method:(1) We take Gemcitabine-resistant pancreatic cancer cell lines-MIA PaCa-2, as a cell model.CCK-8assay was applied to detect the inhibitive effect of different concentration of Conjugate,Triptolide, Nucleolin Aptameron the proliferation of MIA PaCa-2cell lines at different time (24,48h). And compare the effect of Conjugate, Triptolide.(2) CCK-8assay was applied to detect the inhibitive effect of different concentrations of Conjugate and Gemcitabine on the proliferation of MIA PaCa-2cell line at different time (24,48,72h), then calculate the IC50value.(3) To detect wether Conjugate could induce the apoptosis of MIA PaCa-2,Annexin V/PI double staining and Caspase-3activity assay were applied.(4) After treatment with Conjugate at predetermined concentration gradients (12.5to200nM) for12,24h, Western blotting assay was applied to detect the expression of HSP70in the MIA PaCa-2.Results:(1) Conjugate, Triptolide, Nucleolin Aptamercouldinhibit the proliferation of MIA PaCa-2in an time-and dose-dependent manner.After48h,the inhibition rate for200nM Nucleolin Aptamer reached38.19%.At the same concentration, the inhibition rates of Conjugate on MIA PaCa-2were lower than Triptolide’s(24,48h), when drug concentrations reached25nM, the difference was statistically significant, P<0.05.(2) Conjugate and Gemcitabine both inhibited the proliferation of MIA PaCa-2, in an time-and dose-dependent manner.After72h’s treatment, the IC50for the drugs above were as follows:97.86±12.52nM,142.5±31.26nM,respectively.(3)Annexin V/PI double staining test results indicate that,after the treatment of different concentrations of conjugate for different time(12,24h), the early apoptosis rate were10.4%to18.9%; Caspase-3activity assay results show that,after incubation of12or24h’s,the Caspase-3activity was significantly increased approximately9.24fold of the control.(4) After12,24h’s incubation, Western blotting results showed,50-200nM Conjugate signifficantly decreased the expression of HSP70in MIA PaCa-2.Conclusion:Structural modification decreased Conjugate’s cytotoxicity.(2)In terms of the inhibitory effect on Gemcitabine-resistant pancreatic cancer cell lines-MIA PaCa-2,Conjugate was greater than Gemcitabine, In vitro.(3)Conjugate induce MIA PaCa-2apoptosis effectively,(4) Conjugate’s pro-apoptotic mechanismwas Caspase-dependent,and may be achieved by reducing the expression of HSP70.Conjugate show great advantages to be developed into drug for pancreatic cancer therapy.