Study On Antitumor Activity,Liver Targeting And Glutathione Responsiveness Of β-elemene Derivatives

Liver cancer was the sixth leading cancer in the world and the second leading cause of cancer-related deaths.According to epidemiological statistics in2015,there were 466,000 cases of liver cancer in China and 422,000 deaths,both accounting for half of the world.β-elemene has been used clinically in China,but the therapeutic effect is limited due to factors such as poor selectivity and low water solubility.Based on the current status of liver cancer in China,our research group carried out structural modification of the anti-tumor drugβ-elemene,synthesizedβ-elemene derivative LFY-105,and used this as the pharmacophore for the synthesis of galactosyl disulfide bond modification compound LFY-1-5,LFY-2-9,LFY-3-8.This paper mainly evaluates the anti-tumor activity,liver targeting and glutathione responsiveness of these fourβ-elemene derivatives.First,the anti-tumor activity of LFY-105 compound in vivo and in vitro was evaluated.MTT results showed that the IC₅₀of Hep G2 cells treated with LFY-105 for 24 h and 48 h was 7.487±0.283μM and 6.107±0.318μM,respectively.Compared with the IC₅₀ value of 337μM for Hep G2 cells treated withβ-elemene for 48 h,the efficacy of the drug was increased by 55.19 times.The clone formation rate of the 8μM LFY-105 group was 0.83%,which significantly inhibited clone formation.The results of cell scratch and Transwell cell experiments showed that LFY-105 can inhibit Hep G2 cell migration.Hoechst 33342 staining and Annexin V＿FITC-PI staining experiments showed that LFY-105 can induce Hep G2 cell apoptosis.Caspase 3 enzyme activity test results showed that LFY-105 can promote early cell apoptosis by inducing the increase of Caspase 3 protein activity in Hep G2 cells.The results of flow cytometry cell cycle detection experiments showed that LFY-105 can block the cell cycle in G0/G1 phase,thereby hindering DNA replication.The results of anti-tumor experiments in H22 tumor-bearing mice showed that there were no significant differences in organ index and body weight between the experimental group and the control group,suggesting that LFY-105 and LFY-3-8 have no significant side effects on mice.The positive control group（5-FU）,the LFY-105group,and the LFY-3-8 group have tumor inhibition rates of 25.86%,32.79%,and 51.63%,respectively,which were significantly different from the normal saline group（P<0.01）.The tumor inhibition rate of the LFY-3-8 group was significantly different from that of the positive control group（5-FU）（P<0.05）.Secondly,the target evaluation of LFY-1-5,LFY-2-9,LFY-3-8 in vivo and in vitro.The test results of compound uptake by different cell lines showed that A549,Hela,and SGC-7901 had lower compound uptake than liver cancer cells（Hep G2）,with significant differences（P<0.01）,the compound has the targeting ability of liver cancer cells,and the targeting ability was:LFY-1-5（monogalactosyl）<LFY-2-9（double galactosyl）<LFY-3-8（three-half galactosyl）.After pre-incubation with galactose（ASGP-R receptor inhibitor）,the uptake of the three compounds by Hep G2 cells was significantly reduced,indicating that the uptake of the compounds by Hep G2 cells was through the asialoglycoprotein receptor（ASGP-R）mediated endocytosis.The results of in vivo targeting experiments in mice showed that liver tissue had the strongest ability to uptake compounds,which was significantly different from other tissues（P<0.01）.Finally,the results of glutathione（GSH）response cleavage experiments showed that the disulfide bonds of LFY-1-5,LFY-2-9,and LFY-3-8 were ruptured under the action of 250 equ GSH to release the pharmacophore.When the compound reaches the liver cancer tissue（high concentration of GSH）,it can be quickly broken,thereby inhibiting the tumor.In the absence of GSH（p H 6.5or p H 7.4）,the disulfide bonds of LFY-1-5,LFY-2-9,and LFY-3-8 are stable without breaking,indicating that these three compounds are in normal tissues（low concentration GSH）can not release the pharmacophore,and has little toxic and side effects on normal tissues.LFY-105 inhibits Hep G2 cell proliferation and tumor growth in vivo by inducing early apoptosis in vitro.LFY-1-5,LFY-2-9,and LFY-3-8 target Hep G2 cells in vitro by ASGP-R,target liver tissues in vivo,and release drugs in response to glutathione break.The research have great application value and provides new drugs for the treatment of liver cancer.