Inhibition of Migration and Invasion of Hepatocarcinoma HepG2 Cells by Dietary Saponins via Targeting HIF-1α.

BACKGROUND: Different saponins from herbs have been used as tonic or functional foods, and for treatment of various diseases including cancers. Although clinical data has supported the function of these saponins, their underlying molecular mechanisms have not been well defined. METHODS: With the simulated hypoxia created by 8 hours of Cu++ exposure and following 24 hour incubation with different concentration of saponins in HepG2 cells for MTT assay, migration and invasion assays, and for RT-PCR, and with each group of cells for immunofluorescence observation by confocal microscopy. Results：ZC-4 had the highest rate of inhibition of cell proliferation by MTT assay, and the highest inhibition of migration rate by in vitro scratch assay, while ZC-3 had the highest inhibition of invasion ratio by transwell assay. Under the same simulated hypoxia, the molecular mechanism of saponin function was conducted by measuring the gene expression of Hypoxia Inducible Factor (HIF)-1α through RT-PCR, in which ZC-3 showed a potent inhibition of gene HIF-1α. For the protein expression by immunofluorescence staining with confocal microscopy, HIF-1α was also inhibited by saponins, with the most potent one being ZC-4 after eight hours' relatively hypoxia incubation. CONCLUSION: Saponins ZC-4 and ZC-3 have the potential to reduce HepG2 cell proliferation, migration and invasion caused by hypoxia through effectively inhibiting the gene and protein expression of HIF-1α directly and as antioxidant indirectly.

MAGEA10 gene expression in non-small cell lung cancer and A549 cells, and the affinity of epitopes with the complex of HLA-A(∗)0201 alleles.

MAGEA10, a cancer/testis antigens expressed in tumors but not in normal tissues with the exception of testis and placenta, represents an attractive target for cancer immunotherapy. However, suppressive cytoenvironment and requirement of specific HLA-alleles presentation frequently led to immunotherapy failure. In this study MAGEA10 was scarcely expressed in cancer patients, but enhanced by viili polysaccharides, which indicates a possibility of increasing epitopes presentation. Furthermore the correlation of gene expression with methylation, indicated by R(2) value for MAGEA10 that was 3 times higher than the value for other MAGE genes tested, provides an explanation of why MAGEA10 was highly inhibited, this is also seen by Kaplan-Meier analysis because MAGEA10 did not change the patients' lifespan. By using Molecular-Docking method, 3 MAGEA10 peptides were found binding to the groove position of HLA-A(∗)0210 as same as MAGEA4 peptide co-crystallized with HLA-A(∗)0210, which indicates that they could be promising for HLA-A(∗)0201 presentation in immunotherapy.

NaV1.5 sodium channel window currents contribute to spontaneous firing in olfactory sensory neurons.

Olfactory sensory neurons (OSNs) fire spontaneously as well as in response to odor; both forms of firing are physiologically important. We studied voltage-gated Na(+) channels in OSNs to assess their role in spontaneous activity. Whole cell patch-clamp recordings from OSNs demonstrated both tetrodotoxin-sensitive and tetrodotoxin-resistant components of Na(+) current. RT-PCR showed mRNAs for five of the nine different Na(+) channel α-subunits in olfactory tissue; only one was tetrodotoxin resistant, the so-called cardiac subtype NaV1.5. Immunohistochemical analysis indicated that NaV1.5 is present in the apical knob of OSN dendrites but not in the axon. The NaV1.5 channels in OSNs exhibited two important features: 1) a half-inactivation potential near -100 mV, well below the resting potential, and 2) a window current centered near the resting potential. The negative half-inactivation potential renders most NaV1.5 channels in OSNs inactivated at the resting potential, while the window current indicates that the minor fraction of noninactivated NaV1.5 channels have a small probability of opening spontaneously at the resting potential. When the tetrodotoxin-sensitive Na(+) channels were blocked by nanomolar tetrodotoxin at the resting potential, spontaneous firing was suppressed as expected. Furthermore, selectively blocking NaV1.5 channels with Zn(2+) in the absence of tetrodotoxin also suppressed spontaneous firing, indicating that NaV1.5 channels are required for spontaneous activity despite resting inactivation. We propose that window currents produced by noninactivated NaV1.5 channels are one source of the generator potentials that trigger spontaneous firing, while the upstroke and propagation of action potentials in OSNs are borne by the tetrodotoxin-sensitive Na(+) channel subtypes.

Virtual and in vitro bioassay screening of phytochemical inhibitors from flavonoids and isoflavones against xanthine oxidase and cyclooxygenase-2 for gout treatment.

Synthetic drugs such as allopurinol and benzbromarone are commonly used to treat the complex pathogenesis of gout, a metabolic disease that results from an inflammation of the joints caused by precipitation of uric acid. We seek to discover novel phytochemicals that could treat gout, by targeting the xanthine oxidase and cyclooxygenase-2 enzymes. In this study, we report the screening of nine compounds of flavonoids from the ZINC and PubChem databases (containing 2092 flavonoids) using the IGEMDOCK software tool against the xanthine oxidase and cyclooxygenase-2 3D protein structures. Each compound was also evaluated by an in vitro bioassay testing the inhibition of xanthine oxidase and cyclooxygenase-2. Myricetin and luteolin were found to be the potential dual inhibitors of xanthine oxidase and cyclooxygenase-2 as demonstrated by IC(50): 62.7 and 3.29 µg/mL (xanthine oxidase)/70.8 and 16.38 µg/mL (cyclooxygenase-2), respectively. In addition, structure-activity relationships and other important factors of the flavonoids binding to the active site of xanthine oxidase and cyclooxygenase-2 were discussed, which is expected for further rational drug design.

Metagenes associated with survival in non-small cell lung cancer.

NSCLC (non-small cell lung cancer) comprises about 80% of all lung cancer cases worldwide. Surgery is most effective treatment for patients with early-stage disease. However, 30%-55% of these patients develop recurrence within 5 years. Therefore, markers that can be used to accurately classify early-stage NSCLC patients into different prognostic groups may be helpful in selecting patients who should receive specific therapies.A previously published dataset was used to evaluate gene expression profiles of different NSCLC subtypes. A moderated two-sample t-test was used to identify differentially expressed genes between all tumor samples and cancer-free control tissue, between SCC samples and AC/BC samples and between stage I tumor samples and all other tumor samples. Gene expression microarray measurements were validated using qRT-PCR.Bayesian regression analysis and Kaplan-Meier survival analysis were performed to determine metagenes associated with survival. We identified 599 genes which were down-regulated and 402 genes which were up-regulated in NSCLC compared to the normal lung tissue and 112 genes which were up-regulated and 101 genes which were down-regulated in AC/BC compared to the SCC. Further, for stage Ib patients the metagenes potentially associated with survival were identified.Genes that expressed differently between normal lung tissue and cancer showed enrichment in gene ontology terms which were associated with mitosis and proliferation. Bayesian regression and Kaplan-Meier analysis showed that gene-expression patterns and metagene profiles can be applied to predict the probability of different survival outcomes in NSCLC patients.

The application of regular expression-based pattern matching to profiling the developmental factors that contribute to the development of the inner ear.

The biomedical literature has always played a critical role in the development of hypotheses to test, experimental design, and the analysis of study results. Yet, the ever-expanding body of biomedical literature is starting to present new challenges, in which locating pertinent literature from among the millions of published research articles is often a challenging task. A regular expression-based pattern matching method has been developed to profile the various gene and protein factors that may play a role in various tissues contained within an organism. This methodology has been demonstrated through the profiling of the various factors that are involved in the development of the inner ear, and is shown to be both effective and accurate.

Molecular modelling insights into DFNA15 mediated enhancement of POU4F3 stability.

The POU4F3 transcription factor is expressed in the cochlear and vestibular hair cells of the inner ear and its targeted deletion results in a loss of inner ear hair cells. The DFNA15 truncation mutation has been demonstrated to result in a loss of transcriptional activity, but an increase in the stability of the protein. Molecular modelling is utilised to propose a mechanism of stability enhancement, via an interaction between the truncated POU(HD) domain and the POU(S) domain of the transcription factor.

Deafness mutation mining using regular expression based pattern matching.

BACKGROUND: While keyword based queries of databases such as Pubmed are frequently of great utility, the ability to use regular expressions in place of a keyword can often improve the results output by such databases. Regular expressions can allow for the identification of element types that cannot be readily specified by a single keyword and can allow for different words with similar character sequences to be distinguished. RESULTS: A Perl based utility was developed to allow the use of regular expressions in Pubmed searches, thereby improving the accuracy of the searches. CONCLUSION: This utility was then utilized to create a comprehensive listing of all DFN deafness mutations discussed in Pubmed records containing the keywords "human ear".

Oral ethanol potentiates the loss of outer hair cells in cisplatin-exposed rats.

OBJECTIVE: The aim of this study was to examine the effect of oral ethanol on cisplatin ototoxicity. STUDY DESIGN AND SETTING: Twenty-seven-week-old, female Fisher 344 rats were divided into 4 experimental groups. The animals were administered per os (PO) saline (group 1), PO ethanol (group 2), PO saline with intraperitoneal (IP) cisplatin (group 3), or PO ethanol with IP cisplatin (group 4). After 3 days, scanning electron microscopy and counts of outer auditory hair cells were performed. RESULTS: A 2-fold increase in outer hair cell loss was obtained in the basal cochlear turn of rats receiving concomitant cisplatin and ethanol compared with animals receiving cisplatin and saline. No hair cell loss was observed in the middle cochlear turn of any experimental group. CONCLUSION: Our findings support potentiation of ototoxicity when cisplatin is combined with oral ethanol. SIGNIFICANCE: Contraindications for alcohol use in cancer patients receiving cisplatin are implicated.

Neural network-based prediction of mutation-induced protein stability changes in Staphylococcal nuclease at 20 residue positions.

Protein-based therapeutics are playing an increasingly important role in the treatment of diseases, including diabetes and cancer. The viability of these treatments, however, are highly dependent on the stability of the therapeutic, since stability affects both the shelf life of the therapeutic as well as its active life in the body. Stability engineering can, therefore, be used to increase the effectiveness of protein-based therapeutics. Computational methods of protein stability prediction have been under development for about a decade, but complex molecular interactions make stability prediction difficult and computationally intensive. A rapid computational method of protein stability prediction is developed using feed-forward neural networks and used to predict mutation-induced stability changes in Staphylococcal nuclease. The input to the neural network consisted of sequences of evolutionarily based amino acid similarity scores that were obtained through the comparison of the amino acids in a mutation containing sequence to their positional counterparts in the baseline wild-type amino acid sequence. A training set was created which consisted of similarity score sequences, for which the stabilities of the corresponding amino acid sequences were known, paired with the relative stabilities of the sequences to that of the baseline. Back-propagation of error was used to train the network to output accurate relative stability scores for the sequences in the training set. Neural network-based relative stability predictions for 55 sequences containing mutation combinations not found in the training set had an accuracy of 92.8%.

Nicotine and epibatidine triggered prolonged rise in calcium and TH gene transcription in PC12 cells.

The effect of epibatidine on regulation of [Ca2+]i and tyrosine hydroxylase (TH) transcription was examined. Epibatidine triggers a biphasic rise in [Ca2+]i in PC12 cells similar to that observed with nicotine. There was an immediate transient increase in [Ca2+]i and a subsequent sustained second elevation. In contrast to nicotine, the epibatidine-triggered increase in [Ca2+]i was independent of activation of alpha7 nicotinic acetylcholine receptors, as it was not altered by either methyllycaconitine or alpha-bungarotoxin. The second [Ca2+]i elevation involves calcium release from intracellular stores and is inhibited by dantrolene or xestospongin C. Epibatidine, like nicotine, elevated TH promoter driven reporter transcription, mostly mediated by the cyclic-AMP responsive motifs. Elevation in TH promoter activity requires Ca2+ and cAMP since it is inhibited by 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic Acid Tetra (acetoxymethyl ester) (BAPTA-AM) or 2',5'-dideoxyadenosine (DDA). The results reveal that epibatidine can elevate [Ca2+]i in an alpha7 independent manner and nevertheless induce TH transcription.

Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations.

Multi-conformation continuum electrostatics (MCCE) was used to analyze various structures of the NS3 RNA helicase from the hepatitis C virus in order to determine the ionization state of amino acid side chains and their pK(a)s. In MCCE analyses of HCV helicase structures that lacked ligands, several active site residues were identified to have perturbed pK(a)s in both the nucleic acid binding site and in the distant ATP-binding site, which regulates helicase movement. In all HCV helicase structures, Glu493 was unusually basic and His369 was abnormally acidic. Both these residues are part of the HCV helicase nucleic acid binding site, and their roles were analyzed by examining the pH profiles of site-directed mutants. Data support the accuracy of MCCE predicted pK(a) values, and reveal that Glu493 is critical for low pH enzyme activation. Several key residues, which were previously shown to be involved in helicase-catalyzed ATP hydrolysis, were also identified to have perturbed pK(a)s including Lys210 in the Walker-A motif and the DExD/H-box motif residues Asp290 and His293. When DNA was present in the structure, the calculated pK(a)s shifted for both Lys210 and Asp290, demonstrating how DNA binding might lead to electrostatic changes that stimulate ATP hydrolysis.

Role of Ca2+ in induction of neurotransmitter-related gene expression by butyrate.

We examined the effect of butyrate on neurotransmitter-related gene expression and calcium homeostasis in PC12 cells. Pretreatment with Ca2+ chelators (EGTA or BAPTA-AM) attenuated the butyrate-triggered accumulation of TH and ppEnk mRNA indicating that Ca2+ plays a role in butyrate-induced regulation of neuronal genes. Butyrate alone did not alter intracellular Ca2+ levels as determined by Fura-PE3 fluorescence; however, pretreatment with butyrate (18-24 h) reduced the first Ca2+ peak and prevented the second sustained rise in [Ca2+]i as induced by nicotine or ryanodine. In contrast, butyrate had no effect on Ca2+ transients when added shortly before or during nicotine or ryanodine stimulation. These results suggest that chronic butyrate exposure can modulate cell responses by affecting intracellular Ca2+ signaling.