Complementary Omics Strategies to Dissect p53 Signaling Networks Under Nutrient Stress

Signaling trough p53 is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Transcriptome Analysis Reveals the Anti-cancerous Mechanism of Licochalcone A on Human Hepatoma Cell HepG2

Hepatocellular carcinoma is a malignancy with a low survival rate globally, and there is imperative to unearth novel natural phytochemicals as effective therapeutic strategies. Licochalcone A is a chalcone from Glycyrrhiza that displayed various pharmacological efficacy. A globally transcriptome analysis was carried out to reveal the gene expression profiling to explore Licochalcone A's function as an anti-cancer phytochemical on HepG2 cells and investigate its potential mechanisms. Altogether, 6,061 dysregulated genes were detected (3,414 up-regulated and 2,647 down-regulated). SP1 was expected as the transcription factor that regulates the functions of most screened genes. GO and KEGG analysis was conducted, and the MAPK signaling pathway and the FoxO signaling pathway were two critical signal pathways. Protein-protein interaction (PPI) network analysis based on STRING platform to discover the hub genes (MAPK1, ATF4, BDNF, CASP3, etc.) in the MAPK signaling pathway and (AKT3, GADD45A, IL6, CDK2, CDKN1A, etc.) the FoxO signaling pathway. The protein level of essential genes that participated in significant pathways was consistent with the transcriptome data. This study will provide an inclusive understanding of the potential anti-cancer mechanism of Licochalcone A on hepatocellular, signifying Licochalcone A as a promising candidate for cancer therapy.

Experimental Study of Rhein Inhibits the Invasion and Migration of HepG2 Cells by ERK Signaling Pathway

Objective: To investigate the effectiveness of Rhein on the proliferation, invasion and migration of human hepatoma cell line HepG2 and its possible mechanism.Methods: Human hepatoma cell line HepG2 was treated with different concentrations of Rhein (Rhein treatment group) and culture in culture medium alone (control group).The proliferation activity of the cells was determined by methyl Thiazolyl Tetrazolium (MTT) colorimetry.Transwell assay detected the invasion and migration of cells in each group.Cell scratch test was used to detect the migration ability of cells in each group.Excella-phospho-excellar signal-regulated kinase (P-ERK) activity was determined by ELISA after treatment with 50μ mol/L Rhein at different times.Western blot was used to detect ERK protein expression in HepG2 cells treated with 50 μmol/L Rhein.Results: Compared with the control group, the proliferation activity, invasion and migration ability of HepG2 cells in the Rhein treatment group were all decreased (P< 0.05), and the p-ERK relative activity of HepG2 cells treated with Rhein was decreased (P < 0.05).Conclusion: Rhein inhibits the invasion and migration of HCC cells, possibly by inhibiting the ERK pathway

Layered Double Hydroxides as A Drug Delivery Vehicle for S-Allyl-Mercapto-Cysteine (SAMC)

The intercalations of anionic molecules and drugs in layered double hydroxides (LDHs) have been intensively investigated in recent years. Due to their properties, such as versatility in chemical composition, good biocompatibility, high density and protection of loaded drugs, LDHs seem very promising nanosized systems for drug delivery. In this work, we report the intercalation of S-allyl-mercapto-cysteine (SAMC), which is a component of garlic that is well-known for its anti-tumor properties, inside ZnAl-LDH (hereafter LDH) nanostructured crystals. In order to investigate the efficacy of the intercalation and drug delivery of SAMC, the intercalated compounds were characterized using X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). The increase in the interlayer distance of LDH from 8.9 Å, typical of the nitrate phase, to 13.9 Å indicated the intercalation of SAMC, which was also confirmed using FT-IR spectra. Indeed, compared to that of the pristine LDH precursor, the spectrum of LDH-SAMC was richly structured in the fingerprint region below 1300 cm−1, whose peaks corresponded to those of the functional groups in the SAMC molecular anion. The LDH-SAMC empirical formula, obtained from UV-Vis spectrophotometry and thermogravimetric analysis, was [Zn0.67Al0.33(OH)2]SAMC0.15(NO3)0.18·0.6H2O. The morphology of the sample was investigated using SEM: LDH-SAMC exhibited a more irregular size and shape of the flake-like crystals in comparison with the pristine LDH, with a reduction in the average crystallite size from 3 µm to about 2 µm. In vitro drug release studies were performed in a phosphate buffer solution at pH 7.2 and 37 °C and were analyzed using UV-Vis spectrophotometry. The SAMC release from LDH-SAMC was initially characterized by a burst effect in the first four hours, during which, 32% of the SAMC is released. Subsequently, the release percentage increased at a slower rate until 42% after 48 h; then it stabilized at 43% and remained constant for the remaining period of the investigation. The LDH-SAMC complex that was developed in this study showed the improved efficacy of the action of SAMC in reducing the invasive capacity of a human hepatoma cell line.

Rhytidhylides A and B, Two New Phthalide Derivatives from the Endophytic Fungus Rhytidhysteron sp. BZM-9

Two new phthalide derivatives, rhytidhylides A (1) and B (2), together with ten known compounds (3–12) were isolated from cultures of Rhytidhysteron sp. BZM-9, an endophyte isolated from the leaves of Leptospermum brachyandrum. Their structures were identified by an extensive analysis of NMR, HRESIMS, ECD, and through comparison with data reported in the literature. In addition, the cytotoxic activities against two human hepatoma cell lines (HepG2 and SMMC7721) and antibacterial activities against MRSA and E. coli were evaluated.

Inhibiting Effects of Down-regulating of Fascin 1 on Proliferation and Migration in Hepatoma Cells

Objective: To investigate the inhibiting effects of fascin 1 gene knock-down on the proliferation and migration of hepatoma cells by means of small interfering RNA (siRNA).Methods: SiRNA targeting fascin 1 gene (si-fascin) and non-specific sequence siRNA (si-NC）were constructed and transfected into human hepatoma cell lines (HepG2 and Huh7) to down-regulate the expression of fascin 1. RT-qPCR, Western blotting, and Immunofluorescence technique were used to evaluate the efficiency of si-fascin. The proliferation and migration of cells were detected by MTT method and Transwell experiments, and the protein expression of genes related to proliferation and migration in cells were detected by Western blotting. The apoptosis and pseudopodia formation of cells were observed under scanning electron microscope (SEM).Results: Compared with human normal liver cells (LO2), the expressions of fascin 1 mRNA and protein were significantly higher in HepG2 and Huh7 cells. The expression of fascin 1 was overall inhibited in HepG2 and Huh7 cells transfected by the constructed four si-fascins, among which, fascin_siR3 had the highest inhibitory efficiency, therefore was selected in this study. In HepG2 and Huh7 cells transfected by si-fascin significant knock-down target gene expression, while reducing cell proliferation, migration and the formation of pseudopods, and causes reduced protein expression associated with proliferation and migration. Conclusion: This study further confirmed that fascin 1 gene has the function of promoting hepatoma cells proliferation and migration, suggesting that downregulating the expression of fascin 1 in hepatoma cells may be one of the strategies to intervene in liver cancer.

Structure, Antioxidant Activity and In Vitro Hypoglycemic Activity of a Polysaccharide Purified from Tricholoma matsutake

The structure, antioxidant activity and hypoglycemic activity in vitro of a novel homogeneous polysaccharide from Tricholoma matsutake (Tmp) were investigated. Structural features suggested that Tmp was consisted of arabinose (Ara), mannose (Man), glucose (Glc) and galactose (Gal) with a molar ratio of 1.9:13.6:42.7:28.3, respectively, with a molecular weight of 72.14 kDa. The structural chain of Tmp was confirmed to contain →2,5)-α-l-Arabinofuranose (Araf)-(1→, →3,5)-α-l-Araf-(1→, β-d-Glucopyranose (Glcp)-(1→, α-d-Mannopyranose (Manp)-(1→, α-d-Galacopyranose (Galp)-(1→, →4)-β-d-Galp-(1→, →3)-β-d-Glcp-(1→, →3)-α-d-Manp-(1→, →6)-3-O-Methyl (Me)-α-d-Manp-(1→, →6)-α-d-Galp-(1→, →3,6)-β-d-Glcp-(1→, →6)-α-d-Manp-(1→ residues. Furthermore, Tmp possessed strong antioxidant activity and showed the strong inhibitory effect on α-glucosidase and α-amylase activities. Then, a further evaluation found that there was a dramatic improvement in the glucose consumption, glycogen synthesis and the activities of pyruvate kinase and hexokinase when the insulin-resistant-human hepatoma cell line (IR-HepG2) was treated with Tmp. The above results indicated that Tmp had good hypoglycemic activity and also exhibited great potentials in in terms of dealing with type 2 diabetes mellitus.

Novel interferon-sensitive genes unveiled by correlation-driven gene selection and systems biology

Interferons (IFNs) are key cytokines involved in alerting the immune system to viral infection. After IFN stimulation, cellular transcriptional profile critically changes, leading to the expression of several IFN stimulated genes (ISGs) that exert a wide variety of antiviral activities. Despite many ISGs have been already identified, a comprehensive network of coding and non-coding genes with a central role in IFN-response still needs to be elucidated. We performed a global RNA-Seq transcriptome profile of the HCV permissive human hepatoma cell line Huh7.5 and its parental cell line Huh7, upon IFN treatment, to define a network of genes whose coordinated modulation plays a central role in IFN-response. Our study adds molecular actors, coding and non-coding genes, to the complex molecular network underlying IFN-response and shows how systems biology approaches, such as correlation networks, network’s topology and gene ontology analyses can be leveraged to this aim.