| Cancer is one of the diseases with the highest mortality rate,among which liver cancer ranks second in cancer mortality.Therefore,they have brought heavy harm to human life and health as well as family and society.Clinically,the most effective treatment for liver cancer is surgery,preoperative and postoperative concurrent radiotherapy and chemotherapy.However,surgical resection is prone to recurrence,and radiotherapy and chemotherapy have side effects.Therefore,it is necessary to develop efficient and targeted drugs for the treatment of liver cancer.Doxorubicin hydrochloride(Dox)is a broad-spectrum chemotherapeutic drug widely used to treat a variety of cancers,such as glioma and melanoma.Because Dox is not specific,its side effects on the heart and kidney have become a bottleneck in the treatment of tumors.In order to reduce the side effects of chemical drugs,carrier drug delivery has become a research hotspot.Nucleic acid aptamers can specifically bind to targets,have the advantages of high affinity,easy modification and low immunogenicity,and are emerging drugs for the treatment of tumors.AS1411 is the first nucleic acid aptamer drug used in clinical trials.It can target the overexpressed nucleolin on the surface of tumor cells,internalize into tumor cells,and inhibit a variety of tumor cells.It is a good choice as a targeted drug carrier.Aptamers as drugs or drug carriers usually need to be modified or modified to optimize their structure to improve stability and biological activity.In this study,Dox was used as a drug against Hep G2 cells,and the optimized AS1411 and Dox were coupled to explore the targeting and effectiveness of targeted drugs on Hep G2 and further study the apoptosis mechanism of Hep G2 cells.Firstly,three AS1411 derivatives AS1411-J,AS1411-X1 and AS1411-X2 were obtained by mirroring AS1411,modifying5 k Da polyethylene glycol(PEG)at the 5’end,and replacing the T bases at positions 13 and 24 with 2’-deoxyinosine(2’-d I).The three derivatives were characterized by circular dichroism(CD),nuclear magnetic resonance(NMR),agarose gel electrophoresis,flow cytometry,fluorescence inverted microscope and cytotoxicity test.The results showed that AS1411,AS1411-J,AS1411-X1 and AS1411-X2 had stable G-quadruplex structure,and the stability in serum was8,12,12 and 24 h,respectively.The affinity Kd of AS1411-J,AS1411-X1 and AS1411-X2 with Hep G2 cells was 265.2 ± 38.87,149.9 ±29.55,225.1 ± 83.01 and 267.6±88.43 n M,respectively,which had good specificity,but only AS1411 and AS1411-J had significant antitumor activity.Then AS1411-J and Dox were coupled to successfully synthesize the targeted drug AS1411-J-Dox.The targeting and anti-tumor activity of AS1411-J-Dox were evaluated by cytotoxicity and physiological indicators.The results showed that AS1411-J-Dox could reduce the activity of Hep G2 cells without adverse effects on normal hepatocytes,and had good anti-tumor activity and targeting.In addition,AS1411-J-Dox could also inhibit the enzyme activities of GDH,GOT,GPT,T-GSH and LDH in Hep G2 cells.Finally,the anti-tumor mechanism of AS1411-J-Dox was studied by flow cytometry,fluorescence quantitative PCR and Western blot.The results showed that AS1411-J-Dox could arrest cells in S phase(19%)and G2/M phase(25.9%),and regulate apoptosis by down-regulating Bcl-2 expression and up-regulating p38 and BAX expression.In summary,AS1411-J has good stability,high affinity and specificity and is a good carrier for targeted drugs.AS1411-J-Dox is a targeted antitumor drug with potential application ability,which provides a reference for the treatment of cancer and the study of AS1411 anti-tumor mechanism. |
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