Heterogeneity beyond tumor heterogeneity—SULF2 involvement in Wnt/β-catenin signaling activation in a heterogeneous side population of liver cancer cells

Introduction:Sulfatase 2(SULF2),an endogenous extracellular sulfatase,can remove 6-O-sulfate groups of glucosamine residues from heparan sulfate(HS)chains to modulate the Wnt/β-catenin signaling pathway,which plays an important role in both liver carcinogenesis and embryogenesis.Side population(SP)cells are widely identified as stem-like cancer cells and are closely related to carcinoma metastasis,recurrence,and poor patient prognosis.However,the roles of SULF2 in SP cells of hepatomas are unclear,and the underlying mechanism is undefined.Objectives:This study aimed to compare the heterogeneity between SP cells and non-side population(NSP)cells derived from three different liver cancer cell lines and to elucidate the involvement of the SULF2-Wnt/β-catenin axis in liver cancer stem cells(CSCs)and its impact on the processes of carcinogenesis and invasiveness.Methods:In this work,three different liver cancer SP cells(HepG2,Huh7,and PRC/PRL/5)were sorted by flow cytometry.We also examined the migration and invasion behaviors of SP and NSP cells.To determine if this high tumorigenic potential of SP cells is correlated to SULF2,qPCR,western blotting,and immunofluorescence analysis were conducted.We also performed nude mouse xenograft experiments for in vivo analysis.Results:The results from the in vitro colony formation assay showed that SP cells exhibited a 2-fold higher colony formation efficiency compared to their NSP counterparts.The SP cells exhibited significantly higher potentials in terms of their migratory capacity and invasive ability compared to NSP cells.We found that higher expression of SULF2 in SP cells was associated with greater capabilities for clonogenicity,migration,and invasion.It was also linked to higher activation of the Wnt/β-catenin signaling pathway via stimulation of key downstream factors,particularlyβ-catenin,c-Myc,and cyclin D1.Further,a positive correlation between the upregulated SULF2 expression and tumorigenesis in the in vivo nude mouse xenograft models was demonstrated,highlighting that the potential underlying mechanism was Wnt/β-catenin signaling pathway activation.Conclusion:Our findings show that variable SULF2 expression was associated with differential activation of the Wnt/β-catenin signaling pathway,which could lead to behavioral differences between SP and NSP cells and also among the SP cells of the three liver cancer cell lines assessed.It was reasonably concluded that the SULF2-Wnt/β-catenin axis could play an important role in the tumorigenicity of liver cancer stem cells.

Preparation of 5-fluorouracil-loaded chitosan nanoparticles and study of the sustained release in vitro and in vivo

The sustained-release properties of the biodegradable nano-drug delivery systems were used to improve the residence time of the chemotherapeutic agent in the body. These drug delivery systems were widely used to deliver chemotherapeutic drugs. The 5-fluorouracil loaded chitosan nanoparticles prepared in this paper have the above advantage. Here, we found that when the mass ratio of 5-fluorouracil and chitosan was 1:1, the maximum drug loading of nanoparticles was 20.13 ± 0.007%, the encapsulation efficiency was 44.28 ± 1.69%, the particle size was 283.9 ± 5.25 nm and the zeta potential was 45.3 ± 3.23 mV. The prepared nanoparticles had both burst-release and sustained-release phases in vitro release studies.In addition, the inhibitory effect of the prepared nanoparticles on gastric cancer SGC-7901 cells was similar to that of 5-fluorouracil injection, and the blank vector had no obvious inhibitory effect on SGC-7901 cells. In the pharmacokinetic study of rats in vivo, we found that AUC(0-t), MRT(0-t) and t1/2 z of nanoparticles were significantly increased in vivo compared with 5-fluorouracil solution, indicating that the prepared nanoparticles can play a role in sustained-release.