S3 Cells Research Articles

Pharmacological Screening of Medicinal Plants for Anticancer, Antiinflammatory and Antidiabetic Activities

We report a detailed pharmacological screening of 11 medicinal plants (36 samples) growing in the Tabuk region of Saudi Arabia for cytotoxicity against HeLa S3 cells, anti-inflammatory activity (Raw cells) and adipogenesis. Three plant samples were found to be cytotoxic against HeLa S3 cells namely Commiphora africana (16.9%), Raetam ratam whole plant (8.2%), Raetam ratam roots (3.8%). Six plant samples were found active as anti-infammatory namely Artemisa seberi (28 – 0%), Commiphora africana (6 – 3%), Raetam ratam (20 – 6%), Citrullus colocynthsis whole plant (33.7%), Citrullus colocynthsis seed (40 – 1%), Ocimum bacillum (45.4%). Three plant samples were found with inhibitory adipogenesis effects namely Citrullus colocynthsis, Raetam ratam and Fagonia indica. We have also isolated several biomolecules that are responsible for all three activities mentioned above, which will be presented.

Polyplex exposure inhibits cell cycle, increases inflammatory response, and can cause protein expression without cell division.

We sought to evaluate the relationship between cell division and protein expression when using commercial poly(ethylenimine) (PEI)-based polyplexes. The membrane dye PKH26 was used to assess cell division, and cyan fluorescent protein (CFP) was used to monitor protein expression. When analyzed at the whole population level, a greater number of cells divided than expressed protein, regardless of the level of protein expression observed, giving apparent consistency with the hypothesis that protein expression requires cells to pass through mitosis in order for the transgene to overcome the nuclear membrane. However, when the polyplex-exposed population was evaluated for the amount of division in the protein-expressing subpopulation, it was observed that substantial amounts of expression had occurred in the absence of division. Indeed, in HeLa S3 cells, this represented the majority of expressing cells. Of interest, the doubling time for both cell lines was slowed by ~2-fold upon exposure to polyplexes. This change was not altered by the origin of the plasmid DNA (pDNA) transgene promoter (cytomegalovirus (CMV) or elongation factor-1 alpha (EF1α)). Gene expression arrays in polyplex-exposed HeLa S3 cells showed upregulation of cell cycle arrest genes and downregulation of genes related to mitosis. Chemokine, interleukin, and toll-like receptor genes were also upregulated, suggesting activation of proinflammatory pathways. In summary, we find evidence that a cell division-independent expression pathway exists, and that polyplex exposure slows cell division and increases inflammatory response.

PARP1 gene expression is downregulated by knockdown of PARG gene

Poly(ADP-ribosyl)ation is a modification of nuclear proteins that regulates DNA replication, repair and transcription. In order to investigate the biological effects of degradation of poly(ADP-ribose), knockdown of the poly(ADP-ribose) glycohydrolase (PARG) gene was performed by introducing a short interfering RNA (siRNA)-pool into HeLa S3 cells. Notably, poly(ADP-ribosyl)ated proteins did not accumulate in the cells. Western blotting, quantitative RT-PCR analysis and a transient transfection assay revealed that poly(ADP-ribose) polymerase 1 (PARP1) gene/protein expression and its promoter activity were reduced in the PARG knockdown cells. These results suggest that the amount of poly(ADP-ribose) in a cell is regulated under the control of PARP1/PARG gene expression balance. Furthermore, in this study, we showed that PARG-siRNA enhanced cell death induced by staurosporine (STS). Thus, we propose a PARG-siRNA utilizing gene-therapy for cancer treatment.

The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components

Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status.

Accurate Identification and Analysis of Human mRNA Isoforms Using Deep Long Read Sequencing

Precise identification of RNA-coding regions and transcriptomes of eukaryotes is a significant problem in biology. Currently, eukaryote transcriptomes are analyzed using deep short-read sequencing experiments of complementary DNAs. The resulting short-reads are then aligned against a genome and annotated junctions to infer biological meaning. Here we use long-read complementary DNA datasets for the analysis of a eukaryotic transcriptome and generate two large datasets in the human K562 and HeLa S3 cell lines. Both data sets comprised at least 4 million reads and had median read lengths greater than 500 bp. We show that annotation-independent alignments of these reads provide partial gene structures that are very much in-line with annotated gene structures, 15% of which have not been obtained in a previous de novo analysis of short reads. For long-noncoding RNAs (i.e., lncRNA) genes, however, we find an increased fraction of novel gene structures among our alignments. Other important aspects of transcriptome analysis, such as the description of cell type-specific splicing, can be performed in an accurate, reliable and completely annotation-free manner, making it ideal for the analysis of transcriptomes of newly sequenced genomes. Furthermore, we demonstrate that long read sequence can be assembled into full-length transcripts with considerable success. Our method is applicable to all long read sequencing technologies.

The anti-tumor activity of Mikania micrantha aqueous extract in vitro and in vivo

Aqueous extract obtained from Mikania micrantha (MMAE) is commonly used as traditional medicine in some countries. We hypothesized that MMAE may inhibit tumor cell growth, both in an in vitro and in vivo setting. In in vitro experiments, two kinds of human cancer cell lines, K562 and Hela were used to test the anti-tumor activity. Inhibitory concentrations (IC50) were obtained from the inhibition curves fitted by regression analysis, inhibitory rates (%) were calculated by MTT assay, morphological changes were observed by transmission electron microscope (TEM), cell cycles were analyzed by flow cytometry (FCM), and DNA ladders were determined by agarose gel electrophoresis. The in vivo anti-tumor activity was evaluated by calculating the tumor inhibitory rates, thymus index and spleen index of S180-bearing mice. Paraffin-embedded sections were used to test the pathologic changes. The result displayed that the growth of K562 and Hela were enhanced when treated with MMAE at 20μg/mL after 48h. Other concentrations of MMAE (50, 100, 200, 400μg/mL) inhibited the proliferation of both kinds of cells. The IC50 values of K562 and Hela at 48h were 167.16 and 196.27 μg/mL and at 72h 98.07 and 131.56 μg/mL, respectively. The effects showed time-dose dependence. MMAE led to damages of organelles and induced apoptosis. These results were confirmed by ladder DNA fragmentation profile. MMAE also increased the percentage of cells in G2/M phase and decreased the percentage of cells undergoing G0/G1 and S phase in in vivo tests using S180 cells. MMAE showed antitummor activity in vivo, with its tumor inhibitory rate ranging from 12.1 to 46.9%. MMAE also induced necrosis, as shown by pathological examination of Hematoxilin-Eosin stained tumor sections. Meanwhile, compared with the control group, the changes of thymus index and spleen index in MMAE treated group were not obvious. This study suggests that MMAE may be an effective agent for cancer therapy with low toxicity.

Induction of apoptosis in osteosarcoma s180 cells by polysaccharide from dictyophora indusiata

Polysaccharides have shown great importance in cancer therapy. The current study showed that a polysaccharide from Dictyophora indusiata (PDI) also possessed anti-cancer properties. Methyl thiazolyl tetrazolium assay revealed a dose-dependent reduction of osteosarcoma S180 growth in response to PDI treatment. Apoptosis was observed following treatments with PDI, as reflected by the appearance of the subdiploid fraction and DNA fragmentations. We then investigated effects of PDI on expression of apoptosis-associated genes and the results revealed an increase of expression of bcl-2 and decreases of cdk4 and p53 protein levels. Finally, PDI treatment significantly increased the activation of caspase-3, a key executioner of apoptosis. These findings indicate that PDI may act as a chemopreventive and/or chemotherapeutic agent in osteosarcoma cells by reducing cell viability and inducing apoptosis.

DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX), a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180) cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF) from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml) significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor) translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore). Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

In vivoantioxidant, hypoglycemic, and anti-tumor activities of anthocyanin extracts from purple sweet potato

Anthocyanin from purple sweet potato (PSP) extracted by microwave baking (MB) and acidified electrolyzed water (AEW) exhibited antioxidant activity. After further purification by macroporous AB-8 resin, the color value of PSP anthocyanin (PSPA) reached 30.15 with a total flavonoid concentration of 932.5 mg/g. The purified extracts had more potent antioxidant activities than the crude extracts. After continuously administering the PSP extracts to 12-mo-old mice for 1 mo, the anti-aging index of the experimental group was not significantly different from that of 5-mo-old mice. To a certain degree, PSPA was also effective for controlling plasma glucose levels in male Streptozocin (STZ)-treated diabetic mice. In addition, the extracts inhibited Sarcoma S180 cell growth in ICR mice. Mice consuming the PSP extracts formed significantly fewer and smaller sarcomas than mice consuming the control diets. The highest inhibition rate was 69.03%. These results suggest that anthocyanin extracts from PSP not only exert strong antioxidant effects in vitro, but also had anti-aging, anti-hyperglycemic, and anti-tumor activities.

Effect of Lignin Glycosides Extracted from Pine Cones on the Human SIRT1 Promoter

Sirtuins (SIRT1-7) and telomerase (TERT) are known to be involved in the regulation of cellular senescence/ aging processes. Resveratrol (Rsv), a natural polyphenol, which has been shown to extend the lifespan of diverse range of species. Furthermore, Rsv has been shown to activate SIRT1, the class III NAD+ dependent histone deacetylases (HDACs), directly in mammalian cells. Here, we examined the changes in promoter activities of the human SIRT1 and TERT genes after treatment with Rsv and lignin glycosidases/lignin carbohydrate complexes (LCC) extracted from pine cones of Pinus Karalensis. We constructed Luciferase (Luc) expression vectors, pGL4- SIRT1 and pGL4-TERT, containing 396-bp and 263-bp of the 5’-upstream regions of human SIRT1 and TERT genes, respectively, which were then transfected into HeLa S3 cells. The Luc reporter plasmid assay revealed that treatment with the LCC increases not only SIRT1 but also TERT promoter activities more than that with Rsv. These results suggest that the LCC from pine cones induces expression of both SIRT1 and TERT genes, which is thought to have anti-aging effects.

A Comprehensive Profile of ChIP-Seq-Based STAT1 Target Genes Suggests the Complexity of STAT1-Mediated Gene Regulatory Mechanisms

Interferon-gamma (IFNγ) plays a key role in macrophage activation, T helper and regulatory cell differentiation, defense against intracellular pathogens, tissue remodeling, and tumor surveillance. The diverse biological functions of IFNγ are mediated by direct activation of signal transducer and activator of transcription 1 (STAT1) as well as numerous downstream effector genes. Because a perturbation in STAT1 target gene networks is closely associated with development of autoimmune diseases and cancers, it is important to characterize the global picture of these networks. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) provides a highly efficient method for genome-wide profiling of DNA-binding proteins. We analyzed the STAT1 ChIP-Seq dataset of IFNγ-stimulated HeLa S3 cells derived from the ENCODE project, along with transcriptome analysis on microarray. We identified 1,441 stringent ChIP-Seq peaks of protein-coding genes. They were located in the promoter (21.5%) and more often in intronic regions (72.2%) with an existence of IFNγ-activated site (GAS) elements. Among the 1,441 STAT1 target genes, 212 genes are known IFN-regulated genes (IRGs) and 194 genes (13.5%) are actually upregulated in response to IFNγ by transcriptome analysis. The panel of upregulated genes constituted IFN-signaling molecular networks pivotal for host defense against infections, where interferon-regulatory factor (IRF) and STAT transcription factors serve as a hub on which biologically important molecular connections concentrate. The genes with the peak location in intronic regions showed significantly lower expression levels in response to IFNγ. These results indicate that the binding of STAT1 to GAS is not sufficient to fully activate target genes, suggesting the high complexity of STAT1-mediated gene regulatory mechanisms.

Immunoaffinity purification of protein complexes from Mammalian cells.

In this chapter, we describe a purification scheme designed to isolate multisubunit protein complexes gently and quickly from crude extracts of mammalian cells using immunoaffinity purification of epitope tagged proteins and the multisubunit complexes with which they associate. As an example we describe isolation of the mammalian Mediator complex from HeLa S3 cells.

Identification of regulatory regions of bidirectional genes in cervical cancer

BackgroundBidirectional promoters are shared promoter sequences between divergent gene pair (genes proximal to each other on opposite strands), and can regulate the genes in both directions. In the human genome, > 10% of protein-coding genes are arranged head-to-head on opposite strands, with transcription start sites that are separated by < 1,000 base pairs. Many transcription factor binding sites occur in the bidirectional promoters that influence the expression of 2 opposite genes. Recently, RNA polymerase II (RPol II) ChIP-seq data are used to identify the promoters of coding genes and non-coding RNAs. However, a bidirectional promoter with RPol II ChIP-Seq data has not been found.ResultsIn some bidirectional promoter regions, the RPol II forms a bi-peak shape, which indicates that 2 promoters are located in the bidirectional region. We have developed a computational approach to identify the regulatory regions of all divergent gene pairs using genome-wide RPol II binding patterns derived from ChIP-seq data, based upon the assumption that the distribution of RPol II binding patterns around the bidirectional promoters are accumulated by RPol II binding of 2 promoters. In HeLa S3 cells, 249 promoter pairs and 1094 single promoters were identified, of which 76 promoters cover only positive genes, 86 promoters cover only negative genes, and 932 promoters cover 2 genes. Gene expression levels and STAT1 binding sites for different promoter categories were therefore examined.ConclusionsThe regulatory region of bidirectional promoter identification based upon RPol II binding patterns provides important temporal and spatial measurements regarding the initiation of transcription. From gene expression and transcription factor binding site analysis, the promoters in bidirectional regions may regulate the closest gene, and STAT1 is involved in primary promoter.

Apoptosis-inducing activity of the actin-depolymerizing agent aplyronine A and its side-chain derivatives

Aplyronine A (1) and mycalolide B (2), which are cytotoxic actin-depolymerizing marine macrolides, were revealed to induce apoptosis in human leukemia HL60 cells and human epithelial carcinoma HeLa S3 cells. Based on these results, actin-depolymerizing compounds were expected to exhibit apoptosis-inducing activity in cancer cells. Compounds 3–6, which were synthesized based on the side-chain structure of aplyronine A, were evaluated for their actin-depolymerizing activities in vitro and cytotoxicities against HL60 cells. The growth-inhibitory activities of 3–6 were well correlated with their actin-depolymerizing activities, and derivative 6 was shown to induce the disruption of actin filaments and apoptosis in HL60 cells. These results suggested that actin-depolymerizing agents 1, 2, and 6-induced apoptosis in HL60 cells may have been due to their actin-depolymerizing activity.

Purification, structural elucidation and antitumor activity of a novel mannogalactoglucan from the fruiting bodies of Lentinus edodes

A mannogalactoglucan, named LE-MGG, was isolated from the basidiocarps of Lentinus edodes by hot water-extraction, ethanol precipitation anion exchange chromatography, and further purified by gel-permeation chromatography (GPC). Its structural features were investigated by high performance liquid chromatography (HPLC), high performance gel-permeation chromatography (HPGPC), methylation analysis, periodate oxidation-Smith degradation, and by IR and NMR spectroscopy, including two-dimensional (2D) NMR. HPLC analysis revealed that LE-MGG contained mannose–galactose–glucose in the molar ratio of 10:18:72. GPC and HPGPC showed that LE-MGG was a homogeneous fraction (d=1.34) and its molecular weight was estimated to be 18kDa. Chemical and spectroscopic studies indicated that LE-MGG consists of (1→6)-, (1→4)- and (1→3)-linked β-d-glucopyranosyl residues, (1→6)-linked α-d-galactopyranosyl residues, (1→3,6)- and (1→2,4)-linked α-d-mannopyranosyl residues and terminal residues of β-d-glucopyranosyl. Cytotoxicity assay showed that LE-MGG presented higher antitumor activities against S-180 cell with a dose-dependent manner, and exhibited lower cytotoxicity to carcinoma HCT-116 and HT-29 cells. Our studies showed also that LE-MGG presented antitumor bioactivities on Sarcoma 180 solid tumor cell implanted in Kunming mice. This finding suggests that mannogalactoglucan should be explored as potential antitumor agents and could be potentially applied as a natural antitumor drug.

Abstract P5-05-01: Catalogued phospho-sensing peptides identifying active, oncogenic kinase signatures

Abstract Background: Phospho-signaling networks represent one of the most clinically and therapeutically important frontiers of biology and medicine. Yet, measuring the phospho-catalytic activity of kinases, and monitoring the functionality of the entire kinome network at once remains largely unexplored. In particular, a highly convenient tool would be the advent of a microarray-like chip to comprehensively map phosphorylation networks to identify kinase signatures underlying cancer types and guide therapeutic interventions. In order to create such a kinase-sensing platform, we developed a library of peptide probes capable of reporting on the catalytic activity of kinases. The well-studied EGFR and SRC kinase network measurements serve as proof of concept. Methods: We developed a human kinase protein and peptide repertoire from thousands of publications and &amp;gt;30 public databases computationally curated, filtered and merged, suitable to act as peptide beacons to evaluate enzymes' activity. Our database catalogues 916 kinase enzymes, 1913 substrate proteins, 6173 distinctive kinase-substrate active nodes, and compiles 2702 unique biological kinase peptide targets that all represent biologically relevant, latent nano-sensors usable to comprehensively track kinase signaling networks. Here, a representative set of 33 out of 417 peptides predicted to be specifically targeted by EGFR and SRC tyrosine kinase families and 17 control peptides, is tested for their potential kinase-activity reporting ability using a common luminescence ATP-consumption assay. We further validated the peptide screen in biological extracts using an isogenic culture model of basal like breast cancer (HMT-3522 S1 and T4-2). Results: The differential phosphorylation activity of recombinant, active kinase enzymes (EGFR, ERBB2, SRC, HCK, FYN, FRK, LYN, LCK) was successfully detected by ATP-consumption measurement using the 50 peptides in time course experiments (0.25–4 hours) with evidence of selectively. Subsets of these data were confirmed by SRC kinase inhibitor treatments and dilution assays (0.1–5ng/ul kinase enzymes). Increased presence of activated SRC kinase in biological extracts from T4-2 breast cancer cells compared to nonmalignant S1 cells was shown by significantly elevated phosphorylation of 10 tested peptides (maximum elevation was shown with a SRC -specific reporting peptide and increased from 4 to 73% ATP consumption in S1 versus T4-2 cells, respectively). Conclusion: These data demonstrate that the tested peptides can monitor kinases' activity. The peptide database can serve as a resource to provide sensitive and specific phospho-sensing probes, used to directly and specifically evaluate the activity of kinases, and can potentially support the identification of the entire, active, oncogenic kinome. Using this resource, we were able to identify peptides with evidence of specificity in in vitro and ex culture assays. Our efforts now concentrate on expanding this approach into a functional kinomic-screening platform in multiplex array format. Ultimately, we hope to translate such device into the clinic, and map phosphorylation signatures that cause breast malignancies and to guide tailor-made therapies. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-05-01.

Abstract P4-06-05: NF-κB Upregulates β1-integrin via Increased Transcriptional Activity in Three-dimensional Culture: a Mechanism by which Malignant Breast Cells Acquire Radioresistance

Abstract The molecular mechanisms giving rise to tumor viability after therapeutic radiation remain elusive. We showed previously that β1-integrin is associated with enhanced cell survival of breast cancer cells after exposure to high dose ionizing radiation (IR). Recently, we discovered that a typical binding site for Nuclear factor-κB (NF-κB), a stress-sensitive heterodimeric transcription factor, was located in human β1-integrin promoter, which indicates that β1-integrin-mediated resistance to radiation may be functionally regulated by NF-κB. The aim of the present study was to investigate the relationship between NF-κB and β1-integrin pathways in radioprotection of malignant T4-2 breast cells in three-dimensional laminin-rich extracellular matrix (3D lrECM). We show that NF-κB is required for radiation-induced β1-integrin transactivation. Inhibition of NF-κB activity significantly reduced clonogenic survival with the inhibition of IR-induced total and phosphorylated (Thr 788/789) β1-integrins, correlated with increased DNA binding activity at the β1- integrin promoter. Treatment of T4-2 colonies with NF-κB activation inhibitor before IR resulted in a reduction of the size of colonies, associated with a decrease in proliferation and increase in apoptosis, indicating an increased susceptibility to IR. Further studies revealed that a direct interaction of β1-integrin with α5-integrin was highly induced, but not with NF-κB p65, in T4-2 cells surviving IR. These interactions were found to be oppositely regulated in radiosensitive S1 cells, suggesting that IR induces the formation of α5β1-integrin complexes leading to increased survival post-IR. Together these results provide the first evidence that NF-κB-induced β1-integrin transactivation is responsible for increased survival post IR in breast cancer. Since β1-integrin is shown to activate NF-κB, our data further suggest a loop-like β1-integrin–NF-κB–β1-integrin pathway in tumor radioresistance. This novel pathway may serve as an efficient drug target to re-sensitize radioresistant tumor cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-05.

Lamins in development, tissue maintenance and stress

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Activation of PLC by an endogenous cytokine (GBP) inDrosophilaS3 cells and its application as a model for studying inositol phosphate signalling through ITPK1

Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117-28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

The spectroscopy analyses of PpIX by ultrasound irradiation and its sonotoxicity in vitro

Protoporphyrin IX (PpIX) has been used as a sensitizer in photodynamic therapy (PDT) as well as in sonodynamic therapy (SDT). The photo-bleaching of PpIX has been well investigated in many experimental systems and some photo-products have also been identified in PDT. But until now, little information has been reported about the sono-damage of PpIX in SDT. So, the present study was to investigate changes of PpIX properties before and after different ultrasound treatment, and the potential interactions between PpIX, ultrasound and the irradiated cells. In cell-free system, the absorption and fluorescence spectra of PpIX in different solutions were measured by ultraviolet spectrometer and fluorescence spectrophotometer, respectively. The terephthalic acid dosimetry was applied to evaluate the efficiency of ultrasound cavitation by monitoring hydroxyl radical (OH) production on the thermolysis of H2O in the ultrasound field. In in vitro study, confocal microscopy was applied to detect the sub-cellular localization of PpIX in S180 cells before and after ultrasound exposure. Flow cytometry was used to detect the reactive oxygen species (ROS) generation during PpIX–SDT. MTT assay was performed to evaluate the cell viability of S180 cells after SDT treatment with or without ROS scavengers. The results show that PpIX displayed different spectral patterns in different solutions. PpIX was decomposed by ultrasound exposure as measured by the decreased absorption and fluorescence peak values in RPMI-1640 medium. In addition, the decomposition of PpIX was found to be simultaneously accompanied by OH production with increasing output power from ultrasound generator. PpIX at 1μg/ml significantly enhanced the ultrasound induced cavitation as measured by OH generation, and which was greatly eliminated by NaN3, histidine, mannitol, EDTA and catalase, but not by SOD. The in vitro study indicates more PpIX entered into S180 cells after ultrasound exposure. And, the extra-cellular PpIX play an important role in the enhanced cell killing of PpIX–SDT. SDT induced obvious ROS generation in S180 cells, which could be mostly inhibited by the general ROS scavenge NAC (N-acetylcysteine). Other scavengers such as NaN3, histidine, mannitol all partially prevented the SDT induced cell viability loss of S180 cells, suggesting OH, 1O2 might be involved during the process.