Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.

BackgroundMost chemotherapeutic drugs for killing cancer cells are highly cytotoxic in normal cells, which limits their clinical applications. Therefore, a continuing challenge is identifying a drug that is hypersensitive to cancer cells but has minimal deleterious effects on healthy cells. The aims of this study were to evaluate the potential of 4β-hydroxywithanolide (4βHWE) for selectively killing cancer cells and to elucidate its related mechanisms.Methodology and Principal FindingsChanges in survival, oxidative stress, DNA damage, and apoptosis signaling were compared between 4βHWE-treated oral cancer (Ca9-22) and normal fibroblast (HGF-1) cells. At 24 h and 48 h, the numbers of Ca9-22 cells were substantially decreased, but the numbers of HGF-1 cells were only slightly decreased. Additionally, the IC50 values for 4βHWE in the Ca9-22 cells were 3.6 and 1.9 µg/ml at 24 and 48 h, respectively. Time-dependent abnormal increases in ROS and dose-responsive mitochondrial depolarization can be exploited by using 4βHWE in chemotherapies for selectively killing cancer cells. Dose-dependent DNA damage measured by comet-nuclear extract assay and flow cytometry-based γ-H2AX/propidium iodide (PI) analysis showed relatively severer damage in the Ca9-22 cells. At both low and high concentrations, 4βHWE preferably perturbed the cell cycle in Ca9-22 cells by increasing the subG1 population and arrest of G1 or G2/M. Selective induction of apoptosis in Ca9-22 cells was further confirmed by Annexin V/PI assay, by preferential expression of phosphorylated ataxia-telangiectasia- and Rad3-related protein (p-ATR), and by cleavage of caspase 9, caspase 3, and poly ADP-ribose polymerase (PARP).Conclusions/SignificanceTogether, the findings of this study, particularly the improved understanding of the selective killing mechanisms of 4βHWE, can be used to improve efficiency in killing oral cancer cells during chemoprevention and therapy.

ABSTRACT:In vivo studies have demonstrated that prenatal or neonatal exposure to polybrominated diphenyl ethers (PBDEs) causes developmental neurotoxicity. However, there is a lack of human data. Our hypothesis was that PBDEs would result in lower infant neurodevelopment scores. This is a post hoc analysis of previous studies. Fourteen PBDEs in 70 breast milk were analyzed using a high-resolution gas chromatograph/high-resolution mass spectrometer. Infant neurodevelopment at the age of 8 -12 mo was determined using the Bayley Scales of Infants and Toddlers Development, third edition (Bayley-III). The median of ⌺ 14 PBDEs (the sum of 14 PBDE congeners) was 2.92 ng/g lipid. The ⌺ 14 PBDE concentrations were not correlated with Bayley-III scores on cognitive, language, motor, social-emotional, or adaptive behavior scales. A significantly inverse association between brominated diphenyl ether (BDE)-209 and the cognitive scale was found after multivariate stepwise linear regression analyses (B ϭ Ϫ0.007, adjusted R ϭ Ϫ0.224, p ϭ 0.032). In contrast, the language scale was positively correlated with BDE-196 (B ϭ 0.096, adjusted R ϭ 0.315, p ϭ 0.002). Our results are consistent with most in vivo studies, suggesting that prenatal or postnatal exposure to BDE-209 potentially delays the neurological development. (Pediatr Res 70: 596-600, 2011) P olybrominated diphenyl ethers (PBDEs), which are only used as brominated flame retardants, are widely found in a variety of commercial and household products including foam furniture padding, plastics, electrical equipment, paints, textiles, construction materials, and vehicles (1). PBDEs are similar in structure to polychlorinated biphenyls; but in contrast to point sources of polychlorinated biphenyl contamination, PBDEs are widespread and released into the environment from more sources (2).A marked period of rapid brain growth and development begins in humans during the third trimester of pregnancy and continues throughout the first 2 y of life. PBDE congeners are considered to be neurotoxicants, although more work needs to be done to determine whether in utero and postnatal exposure to PBDEs has any adverse effects on human neurodevelopment, and the effects of PBDEs on human health remain unclear (3). However, recent studies have shown that human exposure to PBDEs causes a reduction in women's fecundability (4), a prolongation of menstruation periods (5), an increase in serum LH in male infants (6), and disruption of thyroxine (T4), triiodothyronine (T3) and thyroid-stimulating hormones in male adults (7), and a positive association with diabetes prevalence (8).Postnatal infant exposure to PBDEs mainly comes from breast milk and house dust (9); moreover, PBDE levels in infants and children are higher than those in adults (10). Recently, epidemiological studies of in utero exposure to PBDEs have found it to be associated with the physiological and neurological development of infants and children (6,(11)(12)(13)(14)(15)(16). In addition, lower birth weights were correlated with hi...

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Background & Aims Gut dysbiosis plays a role in hepatic encephalopathy (HE), while its relationship at the acute episode of overt HE (AHE), the disease progression and clinical outcomes remains unclear. We aimed to identify AHE-specific microbiome and its association to patients’ outcomes. Methods We profiled fecal microbiome changes for a cohort of 62 patients with cirrhosis and AHE i) before treatment, ii) 2-3 days after medication and iii) 2-3 months after recovery, and three control cohorts i) healthy individuals, patients with ii) compensated or iii) decompensated cirrhosis. Results Comparison of the microbiome shift from compensated, decompensated cirrhosis, AHE to recovery revealed the AHE-specific gut-dysbiosis. The gut microbiome diversity was decreased during AHE, further reduced after medication, and only partially reversed during the recovery. The relative abundance of Bacteroidetes phylum in the microbiome decreased, whereas that of Firmicute , Proteobacteria and Actinobacteria increased in patients during AHE compared with those with compensated cirrhosis. A total of 70 operational taxonomic units (OTUs) were significantly different between AHE and decompensated cirrhosis abundances. Of them, the abundance of Veillonella parvula increased the most during AHE via a metagenomics recovery of the genomes. Moreover, the relative abundances of three ( Alistipes , Bacteroides , Phascolarctobacterium ) and five OTUs ( Clostridium-XI , Bacteroides , Bacteroides , Lactobacillus , Clostridium-sedis ) at AHE were respectively associated with HE recurrence and overall survival during the subsequent one-year follow-up. Conclusions AHE-specific gut OTUs were identified that may be involved in HE development and able to predict clinical outcomes, providing new strategies for the prevention and treatment of HE recurrence in patients with cirrhosis.