Characterization of chikusetsusaponin IV and V induced apoptosis in HepG2 cancer cells.

BACKGROUND: Chikusetsusaponin IV and V (CsIV and CsV), two typical oleanolic acid saponins, are mainly derived from the rhizome of Panax japonicus C.A. Mey. To reveal the anti-cancer effect of CsIV and CsV on liver cancer cells, human hepatic cancer cell lines (HepG2) were exposed to these saponins, and various physiological responses of HepG2 were investigated. METHODS AND RESULTS: HepG2 cells were treated with CsIV, CsV and 5-fluorouracil (5-FU). Cell proliferation was measured by CCK-8 assay. The cell cycle arrest, cell apoptosis and intracellular Ca2+ levels were respectively identified by flow cytometry. The mitochondrial membrane potential was detected by fluorescence microscopy. And, the levels of apoptosis-related proteins were analyzed by western blotting. Both CsIV and CsV were demonstrated to inhibit cell viability, and induce cell cycle arrest and apoptosis of HepG2 in a dose-dependent manner. They also enhanced the intracellular Ca2+ level and decreased the mitochondrial membrane potential in HepG2 cells. Furthermore, p53 and p21 were found up-regulated in HepG2 cells treated by CsIV and CsV. The apoptotic proteins, bax, cytochrome c, cleaved caspase-3/-9, were all found activated in HepG2 cells after CsIV and CsV treatment. The anti-apoptotic protein, bcl-2, was significantly down-regulated in all treated HepG2 cells. CONCLUSION: Our data demonstrated that CsIV and CsV exerted significant cytotoxic effects on HepG2 cells without affecting normal liver cells. And, these chikusetsusaponins, especially for CsIV, showed a potent effect on promoting cell apoptosis in HepG2 cells, which was associated with the activation of p53-mediated apoptosis pathway.

Development of a novel DNA delivery system based on rice bran polysaccharide-Fe(III) complexes.

Metal complexes, as a type of potential non-virus gene carriers, have gained much attention due to their properties of high charge density and unique three-dimensional structure. This study investigated the potential of polyethyleneimine (PEI)-modified rice bran polysaccharide-Fe(III) complex (PEI-PI) as a safe gene delivery system and explored the effect of Fe(III) on the efficiency of gene transfection mediated by PEI modified rice bran polysaccharide (PEI-P) and PEI-PI. Gel retardation assay was used to study the DNA binding and protection capability, MTT assay was performed to evaluate the biocompatibility, and PEI-PI complex-mediated EGFP gene transfection was studied in vitro. Results showed the PEI-PI could induce DNA condensation and protect DNA from degradation by DNase I at a low weight ratio (vector/DNA) of 2. At the same weight ratio, PEI exhibited the strongest DNA binding capability but PEI-PI exhibited the highest gene transfection efficiency among all carrier systems. Compared with the PEI-P + Fe(III) system, PEI-PI not only had a more significant capability to condense DNA but also presented higher gene transfection efficiency. Moreover, PEI-PI exhibited no obvious cytotoxicity to cells. This work provides a strategy for the design and development of gene vectors based on PI complexes.

TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery.

Polysaccharides are considered to be promising candidates for non-viral gene delivery because of their molecular diversity, which can be modified to fine-tune their physicochemical properties. In this work, transcriptional activator protein (TAT) functionalized PEI grafted polysaccharide polymer (PRBP) was prepared by using rice bran polysaccharide as the starting material, and characterized by various methods. The potential of TAT functionalized PRBP (PRBP-TAT) as gene vector was studied in vitro, including DNA loading capacity, DNA protection ability and biocompatibility. The cell uptake and transfection efficiency of the PRBP-TAT/pDNA polyplexes were studied. The results showed that PRBP-TAT could completely condense DNA at N/P 2. The PRBP-TAT/pDNA polyplexes could protect DNA from degrading by DNase and were efficiently internalized by cells. Biocompatibility result showed that PRBP-TAT had no significant cytotoxicity and effect on cell proliferation. At low N/P ratios of 1-3.5, PRBP-TAT showed higher transfection efficiency than PEI30k and PEI30k-grafted rice bran polysaccharide. PRBP-TAT and PEI showed the highest transfection efficiency of 42.8% and 28.1% when pDNA is 2 µg and N/P ratio is 1.5, respectively, while PRBP showed the highest transfection efficiency of 37.3% at N/P 2.5. These results indicate that PTA is a promising candidate vector for safe and efficient gene delivery.

Safe and efficient gene delivery based on rice bran polysaccharide.

Polysaccharides are capable of being modified by polycations to adjust their physical and chemical properties, which accordingly are considered as potential candidate materials for safe and efficient gene delivery. Here, we extracted and purified polysaccharides from rice bran, and their physicochemical properties were determined by various methods. Polyethyleneimine (PEI) modified rice bran polysaccharide (PRBP) was prepared by grafting RBP with low molecular weight PEI and the preparation was determined by FTIR. The potential of PRBP as a gene vector was systematically evaluated in vitro. The results show that PRBP can compact DNA and form PRBP/DNA polylexes with a particle size of 50-100 nm. The PRBP/DNA polylexes can protect DNA degradation from DNase I efficiently. Compared with PEI, higher transfection efficiency was achieved by the PRBP. At weight ratio of 3, the highest efficiency of gene transfection mediated by PRBP-2000 was obtained, which was 37.5% and significantly higher than PEI and commercial reagents (calcium phosphate cell transfection kit) and was closed to lipo6000. Furthermore, according to MTT results, the cytotoxicity of PRBP is much lower than that of PEI, especially for PEI2000. We hope these results will provide new strategy for rice bran polysaccharides development and application as biomaterials.

A fluorescent imaging assay of cast in renal disease based on graphene quantum dots and Fe3O4 nanoparticles.

BACKGROUND: Renal disease has become a global public health problem. Cast is a useful disease marker of kidney injury and renal failure. Hence, a cast-targeted fluorescent imaging assay is developed for the laboratory diagnosis of renal disease. METHODS: Firstly, graphene quantum dots (GQDs) were stripped from graphene oxide sheets and amine-modified. Then, anti-human IgG antibody was conjugated with Fe3O4 nanoparticles to identify the cast in urine. Furthermore, the modified GQDs were linked onto the surface of Fe3O4/anti-IgG nanocomposites. Lastly, this Fe3O4/GQD fluorescent probe was added into the sample to detect the cast through fluorescent imaging. RESULTS: Preliminary application of this probe in clinical detection showed that the common types of casts in urine (including RBC, WBC, fatty and granular casts) could be detected by this fluorescent imaging assay. The method has the advantages of fast speed, high sensitivity (lowest detection limit to 2 casts/ml), good selectivity, and wide linear range (2-2000 casts/ml). Regression analysis also showed that there was a good linear relationship (y=0.9495×+10.974, R(2)=0.9879) between the fluorescent counts and the casts in urine. CONCLUSION: This cast-targeted fluorescent imaging assay may be a potential method for the laboratory diagnosis of renal disease.

GLIPR-2 overexpression in HK-2 cells promotes cell EMT and migration through ERK1/2 activation.

The epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells in the adult kidney is one of the key events in renal interstitial fibrosis. Glioma pathogenesis related-2 (GLIPR-2) has been shown to be up-regulated in proximal tubular cells (PTCs) in the fibrotic kidney. However, the biological function of GLIPR-2 remains unknown. In this study, we found that GLIPR-2 expression is elevated in the kidney tissue samples of patients with diabetic nephropathy (DN). Human proximal renal tubular epithelial cells (HK-2 cells) were transfected with pcDNA3.0-GLIPR-2 and selected with G418. To identify the biological function of GLIPR-2, an epithelial-to-mesenchymal transition (EMT) PCR array analysis was performed, and genes that had statistically significantly altered expression levels with more than a two-fold difference compared with the pcDNA3.0-transfected HK-2 cells were considered. Key elements of the EMT process, such as E-cadherin and vimentin, were transcriptionally activated in the pcDNA3.0-GLIPR-2-transfected sublines. In addition, α-SMA gene expression, which is a marker of myofibroblasts, increased in the pcDNA3.0-GLIPR-2-transfected HK-2 cells. The cell migration assay demonstrated that the transfection of HK-2 with GLIPR-2 promoted cell migration following an EMT. Additionally, consistent with the effects of increased EGFR expression levels, we found that the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was highly elevated in the pcDNA3.0-GLIPR-2-transfected group. Our study demonstrates that GLIPR-2 overexpression in HK-2 cells can potentiate EMT-like processes in this cell type through the ERK1/2 signaling pathway. GLIPR-2 may be responsible for the development of renal fibrosis by increasing the accumulation of interstitial fibroblasts.