iTRAQ-based proteomic analysis of the molecular mechanisms and downstream effects of fatty acid synthase in osteosarcoma cells.

BACKGROUND: Fatty acid synthase (FASN) is a lipogenic enzyme that participates in tumor progression. We previously showed that FASN is dysregulated in OS malignancy, but the molecular mechanism(s) of these effects remained unclear. METHODS: We examined differentially expressed proteins (DEPs) in FASN-silenced osteosarcoma 143B cells and their parental cells by isobaric tags for relative and absolute quantitation (iTRAQ). Differentially expressed proteins were classified using GO and KEGG analysis. The association between FASN and heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) was confirmed using qPCR, Western blot, and immunohistochemistry. The function of HNRNPA1 in osteosarcoma was determined using CCK-8, colony formation, wound healing, transwell migration, and invasion assays. RESULTS: Among the 4971 identified proteins, 567 DEPs (325 upregulated and 242 downregulated) were identified. The top 10 upregulated proteins comprised HIST1H2AB, INA, INTS5, MTCH2, EIF1, MAPK1IP1L, PXK, RPS27, PM20D2, and ZNF800, while the top 10 downregulated proteins comprised NDRG1, CNTLN, STON2, GDF7, HECTD3, HBB, TPM1, PPP4R4, PTTG1IP, and PLCB3. Bioinformatic analysis indicated that the DEPs were related to cellular processes, metabolic processes, biological regulation, binding, and catalytic activity. HNRNPA1 was dysregulated in FASN-silenced 143B and HOS cells. qPCR, Western blot, and immunohistochemistry showed that FASN expression positively correlates with HNRNPA1 expression. Further studies indicated that HNRNPA1 correlates with OS diagnosis and prognosis. And HNRNPA1 silence inhibits the proliferation, migration, and invasion in OS cells. CONCLUSION: HNRNPA1 acts as targets downstream of FASN and potential biomarker and oncogene in OS.

Preparation of liposomes containing zedoary turmeric oil using freeze-drying of liposomes via TBA/water cosolvent systems and evaluation of the bioavailability of the oil.

The purpose of this study was to enhance the absorption of zedoary turmeric oil (ZTO) in vivo and develop new formulations of a water-insoluble oily drug. This study described a method for preparing ZTO liposomes, which involved freeze-drying (FD) of liposomes with TBA/water cosolvent systems. The TBA/water cosolvent systems were used to investigate a feasible method of liposomes manufacture; the two factors, sugar/lipid mass ratio and TBA content (concentration), of the preparation process were evaluated in this study. The results showed that the addition of TBA content could significantly enhance the sublimation of ice resulting in short FD cycles time, and reduce the entrapment efficiency of liposomes. In addition, the residual TBA solvents levels were determined to be less than 0.37% under all optimum formulations and processing conditions. Several physical properties of liposomes were examined by H-600 transmission electron microscope (TEM) and zetamaster analyser system. The results revealed that the liposomes were smooth and spherical with an average particle size of 457 +/- 7.8 nm and the zeta potential was more than 3.65 Mv. The bioavailability of the liposomes was evaluated in rabbits, compared with the conventional self-emulsifying formulation for oral administration. Compared with the conventional self-emulsifying formulation, the plasma concentration-time profiles with improved sustained-release characteristics were achieved after oral administration of the liposomes with a bioavailability of 257.7% (a good strategy for improving the bioavailability of an oily drug). In conclusion, the present experimental findings clearly demonstrated the usefulness of ZTO liposome vesicles in improving therapeutic efficacy by enhancing oral bioavailability. Our study offered an alternative method for designing sustained-release preparations of oily drugs.

Preparation and characterization of zedoary turmeric oil-loaded insulin-modified sterically stabilized liposomes.

The poor selectivity of anticancer drugs often leads to their multiplicate dose-limiting toxicities in humans, which severely restricts their clinical application. In this study, a novel liposomal formulation of zedoary turmeric oil (ZTO) targeting the insulin receptor (IR) was prepared by covalently conjugating insulin to the terminal of the polyethylene glycol (PEG) chain of sterically stabilized liposomes. In vitro assays indicated that a higher uptake of insulin-modified sterically stabilized liposomes (ISSLs) was observed in SMMC-7721 hepatocarcinoma cells overexpressing insulin receptors. IC(50) values of ISSLs, NTLs (nontargeted liposomes), and ZTO injection (free ZTO) against SMMC-7721, determined by MTT assays, were 157.2, 256.7, and 43.3 microg x ml(-1), respectively. Plasma-clearance profiles of ZTO in the liposomal formulations were then compared with that of ZTO injection. The liposomal formulations showed much longer terminal half-lives (11.24 and 14.73 hours for ISSLs and NTLs, respectively) than that of ZTO injection (1.45 hours). All results above indicated the ISSLs were potentially useful for the treatment of IR (+) tumors and are worthy of further investigation.

Transepithelial transport of Cerulenin across Caco-2 cell monolayers.

The transepithelial transport of Cerulenin across Caco-2 cell monolayers was examined in this study. The permeated amounts of Cerulenin were measured by HPLC method to calculate the permeation rate and the apparent permeability coefficient (P(app)). The transport of Cerulenin was independent on apical pH and exhibited concentration-dependent and nonsatuable even at 10 mM Cerulenin. The permeation rate at 1 mM Cerulenin in the apical-to-basolateral direction was 0.151 ng/min/mg of protein and the P(app) was 3.76 x 10(-6) cm/second. The permeation rate of Cerulenin was affected by neither metabolic inhibitors nor inhibitors for P-glycoprotein, as was the same case in monolayers treated with cytochalasin D. All these data from experiments indicated that transport of Cerulenin across Caco-2 cell monolayers was not mediated by ATP-dependent transport systems nor via paracellular pathway, but via passive diffusion without efflux by P-glycoprotein.

Jadomycin B, an Aurora-B kinase inhibitor discovered through virtual screening.

Aurora kinases have emerged as promising targets for cancer therapy because of their critical role in mitosis. These kinases are well-conserved in all eukaryotes, and IPL1 gene encodes the single Aurora kinase in budding yeast. In a virtual screening attempt, 22 compounds were identified from nearly 15,000 microbial natural products as potential small-molecular inhibitors of human Aurora-B kinase. One compound, Jadomycin B, inhibits the growth of ipl1-321 temperature-sensitive mutant more dramatically than wild-type yeast cells, raising the possibility that this compound is an Aurora kinase inhibitor. Further in vitro biochemical assay using purified recombinant human Aurora-B kinase shows that Jadomycin B inhibits Aurora-B activity in a dose-dependent manner. Our results also indicate that Jadomycin B competes with ATP for the kinase domain, which is consistent with our docking prediction. Like other Aurora kinase inhibitors, Jadomycin B blocks the phosphorylation of histone H3 on Ser10 in vivo. We also present evidence suggesting that Jadomycin B induces apoptosis in tumor cells without obvious effects on cell cycle. All the results indicate that Jadomycin B is a new Aurora-B kinase inhibitor worthy of further investigation.