β-1,4-galactosyltransferase Inhibitor Selectively Induces In Vitro Anticancer Effects

Cancer is a life threatening disease that affects the human beings all around the world and is a major global disease that has yet to be overcome.With the breakthrough in molecular biology technology and a deep understanding of the pathogenesis along with tissues,organs,cells to the molecular level,tumor targeted therapy has attracted the most attentions,which was believed to solve the inevitable side-effects of traditional chemotherapy drugs currently used to treat cancer and also overcome the frequent drug resistance and limited treatment effects.The development of novel efficient and safe anticancer drugs and preparations are urgently needed in the clinic.We have previously synthesized a β-1,4-galactosyltransferase inhibitor（GBU）.In this study,the selective anticancer activity of GBU has been studied,and the preparation of GBU via nanotechnology to improve their physicochemical properties has been investigated.Firstly,MTT assays were used to determine the cytotoxicity of GBU against normal cells（HUVEC）and a variety of cancer cell lines（including 786-O,MCF-7,MDA-MB-231,RD,SMMC-7221,HepG2,A549,CT26 and 4T1）.The results showed that GBU exhibited higher cytotoxic against liver cancer cell lines(SMMC-7221,IC₅₀=77.3 ± 2.0 μg/mL;HepG2,IC₅₀= 52.0 ± 1.2 μg/mL)in comparison to other cancer cell lines(A549,IC₅₀=163.7 ± 14.6 μg/mL;NCI-H292,IC₅₀=120.1 ± 3.1 μg/mL;SGC7901,IC₅₀=179.7 ± 2.5 μg/mL;HGC27,IC₅₀=158.7 ± 0.5 μg/mL;SW620,IC₅₀=87.1 ± 1.8 μg/mL;SW480,IC₅₀=98.4 ± 3.5 μg/mL;MCF-7,IC₅₀=143.3 ± 2.2 μg/mL;MDA-MB-231,IC₅₀=119.7 ± 6.2 μg/mL)and HUVEC normal cell lines(IC₅₀= 443.0 ± 6.9 μg/mL),indicating that GBU selectively against tumors with higher specificity to liver cancer cells.The better anti-cancer effect was found to be associated with higher expression levels of β-1,4-galactosyltransferase（β4GalT）in cell lines（HepG2 and SMMC-7221）.Flow cytometry analysis was then applied to analyze the apoptotic characteristics and cell cycle distribution of GBU treated cells.The results showed that GBU treated HepG2 and SMMC-7221 cells exhibited higher levels of apoptosis and induced S-phase cell cycle arrest.Finally,the expression levels of pro-apoptotic and anti-apoptotic factors were determined by Western blot analysis.The results showed that GBU up-regulated the expression of pro-apoptotic protein factor Bax and down-regulated the expression of anti-apoptotic factor Bcl-2（p < 0.05）.GBU activated the apoptotic promoter cleaved Caspase 9（p < 0.05）and eventually led to the activation of effector Caspase 3（p < 0.05）and induced apoptosis in liver cancer cells.In addition,the cyclodextrin drug-loaded nano preparation of GBU-CD was prepared via a thin film hydration method.The particle size,potential,encapsulation rate,drug loading,in vitro release effect of GBU-CD was characterized,and time stability were investigated.Finally,the optimal preparation method that provides appropriate particle size distribution（about 100 nm）,relatively stable potential（about-28 mV）,high encapsulation efficiency（> 95%）,drug loading efficiency（> 9%）and pH-responsive drug release characteristics was established.Subsequently,the cytotoxic effect of cyclodextrin-loaded nano-agent GBU-CD against hepatocellular carcinoma cell cytotoxicity was evaluated.As a result,the cytotoxicity of GBU-CD on HepG2 cells(IC₅₀= 2.4 ± 0.3 μg/mL)and SMMC-7221 cells((IC₅₀= 3.1 ± 0.4 μg/mL)were significantly higher than that of free GBU（p < 0.05）.In summary,GBU showed selective cytotoxic effect against tumor cells and is highly specific to hepatocellular carcinoma cell lines.It can promote the death of liver cancer cells by inducing S-phase cycle arrest and apoptosis in cells.Furthermore,cyclodextrin-based nano preparations can significantly improve the antitumor effect of GBU,provides new insight into the development of highly effective and safe tumor targeted anticancer drugs.