Studies On The Targeting Antitumor Effects Of 5-FU-galactoside

Background: 5-FU is one of broad-spectrum antitumor drugs which is commonly used in clinical. It kills tumor cells mainly by interfering with DNA synthesis of tumor cells, but it also kills normal cells of the body causing many adverse reactions, such as gastrointestinal tract damage, bone marrow suppression and nervous system damage and so on. Asialoglycoprotein Receptor (ASGPR) is mainly expressed on the surface of liver cells, which can specifically recognize the galactose residues of asialoglycoproteins, and then remove them by inducing receptor-mediated endocytosis. Later research was found that some tumor cells such as HepG2, Caco-2 and HT-29 cells can also express ASGPR on their surfaces. Therefore, researchers began to develop anticancer drugs targeting to asialoglycoprotein receptor hoping that the vehicle with galactose residues can transport the drugs to tumor tissue via blood circulation, and then increase the local concentration of antitumor drugs. Currently most of these drugs are designed by packaging the common antitumor drugs with galactose-based drug delivery system or making the common antitumor drugs physically adsorbed on the galactose-based drug delivery system, so that the drugs can be transported to the tumor tissue and release, naming targeting anti-tumor effect. However, the galactose-based compounds transformed from conventional anticancer drugs that possess targeting anti-tumor effect have hardly been reported. Hence, when galactose was covalently bound to 5-FU, do this kind of compounds get the targeting antitumor effect, causing less adverse reaction?Objective: To investigate the targeting anti-tumor effects of 5-FU-galactoside in vitro and in vivo, so that can offer new experimental evidence to cancer treatment and development of targeting antitumor drugs.Methods:1. The 5-FU-galactoside used in our research is designed and synthesized by members in our laboratory.2. Western blotting was employed to observe the expressions of asialoglycoprotein receptor on the surfaces of HepG2 live cancer cells, Caco-2 colon cancer cells, A549 lung cancer cells; Inhibited effects of 5-FU and 5-FU-galactoside on proliferations of HepG2, Caco-2, A549 tumor cells was observed by MTT assay and IC50 value was calculated; Absorption of 5-FU and 5-FU-galactoside by Caco-2 cells in the 2th , 5th, 10th, 15th, 30th, 60th and 120th minute was detected with High Performance Liquid Chromatography (HPLC).3. A tumor-bearing mice model was established through subcutaneous injection of HepG2 tumor cell suspensions, and tumor growth was observed every day. About three weeks later, the tumor grew to about the size of 0.5×0.5cm, then the mice were randomly divided into 5 groups: model group, 5-FU group and large-dose 5-FU-galactoside, medium-dose 5-FU-galactoside group and small-dose 5-FU-galactoside group. At the 25th day 5-FU and 5-FU-galactoside were administrated via tail vein one time every two days for 2 weeks, and the mice weight, the length and width of tumor was recorded every two days for three weeks. After one week withdrawal of administration, a dose of 0.2 mM/kg body weight 5-FU and 5-FU-galactoside were administrated via tail vein again. And 15 minutes after this administration, the blood of mice were corrected while killing them, and at the same time a small amount of blood was taken for detection of blood cell number with blood cell counter, and the hearts, livers, spleens, lungs, kidneys, brains, intestines and tumors of mice were taken, weighed and kept in ice. Then the remaining blood was centrifuged to obtain blood plasma and all the organs were homogenated, drugs were extracted to make into samples for HPLC detection. Last the 5-FU and 5 - FU galactoside containing in the samples were detected with HPLC to evaluate the tumor and the tissue distribution of 5-FU and 5-FU-galactoside.Results:1. Results of western blotting suggest that HepG2 and Caco-2 cells have a higher expression of asialoglycoprotein receptor, while the A549 cells have almost no expression of asialoglycoprotein receptor.2. 5-FU-galactoside can significantly inhibit the proliferation of HepG2 and Caco-2 cells in a dose-dependent way, and it is more effective than 5-FU. And 5-FU-galactoside can also inhibit the proliferation A549 cells in a dose-dependent way, but there is no significant difference compared with 5-FU. (IC50 of 5-FU, HepG2 cells: 5.95×10-6 M/L, Caco-2 cells: 3.17×10-6 M/L, A549 cells: 6.24×10-6 M/L; IC50 of 5-FU-galactoside, HepG2 cells: 2.75×10-7 M/L, Caco-2 cells: 1.75×10-7 M/L, A549 cells: 6.05×10-6 M/L)3. Results of HPLC show that Caco-2 can uptake 5-FU and 5-FU-galactoside time-dependently in a certain range, but the intake of 5-FU-galactoside is much higher than 5-FU.4. 5-FU-galactoside can significantly inhibit the tumor growth of nude mice in a dose-dependent way, and it is more effective than 5-FU. Compared with model group, the body weight of the nude mice in 5-FU-galactoside group shows no significant difference, but the body weight of the nude mice in 5-FU group shows a decline tendency that is significantly differ from that of model. And 5-FU-galactoside show less impact on the blood of mice than 5-FU indicating smaller toxicity of 5-FU-galactoside.5. 15 minutes after the tail vein administration of 5-FU and 5-FU-galactoside, 5-FU-galactoside can distribute a higher concentration to the tumor tissues of mice than 5-FU indicating the tumor targeting effect of 5-FU-galactoside.Conclusions:1. 5-FU-galactoside can inhibit the tumor cells growth in a dose-dependent way in vitro, and the inhibited effect on tumor cells with higher asialoglycoprotein receptor expression is stronger compared with 5-FU. And tumor cells with higher expression of asialoglycoprotein receptor can uptake higher concentration of 5-FU-galactoside than 5-FU time-dependently in a certain range.2. 5-FU-galactoside can significantly inhibit the tumor growth of nude mice in a dose-dependent way, and it is more efficient and less toxic to the system and bone marrow than 5-FU. 15 minutes after the tail vein administration, 5-FU-galactoside distribute a higher concentration to the tumor tissue of mice than 5-FU.3. Both in vitro and in vivo, 5-FU-galactoside can inhibit tumor growth of mice dose-dependently, with stronger effect and smaller toxicity than 5-FU, and shows a tumor-targeted property.