Inhibition Of Protein Biosynthesis Research Articles

New tricks of an old enemy: isolates of Fusarium graminearum produce a type A trichothecene mycotoxin.

SummaryThe ubiquitous filamentous fungus F usarium graminearum causes the important disease Fusarium head blight on various species of cereals, leading to contamination of grains with mycotoxins. In a survey of F . graminearum (sensu stricto) on wheat in North America several novel strains were isolated, which produced none of the known trichothecene mycotoxins despite causing normal disease symptoms. In rice cultures, a new trichothecene mycotoxin (named NX‐2) was characterized by liquid chromatography‐tandem mass spectrometry. Nuclear magnetic resonance measurements identified NX‐2 as 3α‐acetoxy‐7α,15‐dihydroxy‐12,13‐epoxytrichothec‐9‐ene. Compared with the well‐known 3‐acetyl‐deoxynivalenol (3‐ADON), it lacks the keto group at C‐8 and hence is a type A trichothecene. Wheat ears inoculated with the isolated strains revealed a 10‐fold higher contamination with its deacetylated form, named NX‐3, (up to 540 mg kg−1) compared with NX‐2. The toxicities of the novel mycotoxins were evaluated utilizing two in vitro translation assays and the alga C hlamydomonas reinhardtii. NX‐3 inhibits protein biosynthesis to almost the same extent as the prominent mycotoxin deoxynivalenol, while NX‐2 is far less toxic, similar to 3‐ADON. Genetic analysis revealed a different TRI 1 allele in the N‐isolates, which was verified to be responsible for the difference in hydroxylation at C‐8.

Identification of direct targets of plant transcription factors using the GR fusion technique.

The glucocorticoid receptor-dependent activation of plant transcription factors has proven to be a powerful tool for the identification of their direct target genes. In the absence of the synthetic steroid hormone dexamethasone (dex), transcription factors fused to the hormone-binding domain of the glucocorticoid receptor (TF-GR) are held in an inactive state, due to their cytoplasmic localization. This requires physical interaction with the heat shock protein 90 (HSP90) complex. Hormone binding leads to disruption of the interaction between GR and HSP90 and allows TF-GR fusion proteins to enter the nucleus. Once inside the nucleus, they bind to specific DNA sequences and immediately activate or repress expression of their targets. This system is well suited for the identification of direct target genes of transcription factors in plants, as (A) there is little basal protein activity in the absence of dex, (B) steroid application leads to rapid transcription factor activation, (C) no side effects of dex treatment are observed on the physiology of the plant, and (D) secondary effects of transcription factor activity can be eliminated by simultaneous application of an inhibitor of protein biosynthesis, cycloheximide (cyc). In this chapter, we describe detailed protocols for the preparation of plant material, for dex and cyc treatment, for RNA extraction, and for the PCR-based or genome-wide identification of direct targets of transcription factors fused to GR.

Analysis of Protein Stability by the Cycloheximide Chase Assay.

Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a full cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model.

Insect-derived proline-rich antimicrobial peptides kill bacteria by inhibiting bacterial protein translation at the 70S ribosome.

Proline-rich antimicrobial peptides (PrAMPs) have been investigated and optimized by several research groups and companies as promising lead compounds to treat systemic infections caused by Gram-negative bacteria. PrAMPs, such as apidaecins and oncocins, enter the bacteria and kill them apparently through inhibition of specific targets without a lytic effect on the membranes. Both apidaecins and oncocins were shown to bind with nanomolar dissociation constants to the 70S ribosome. In apidaecins, at least the two C-terminal residues (Arg17 and Leu18) interact strongly with the 70S ribosome, whereas residues Lys3, Tyr6, Leu7, and Arg11 are the major interaction sites in oncocins. Oncocins inhibited protein biosynthesis very efficiently in vitro with half maximal inhibitory concentrations (IC50 values) of 150 to 240 nmol L(-1). The chaperone DnaK is most likely not the main target of PrAMPs but it binds them with lower affinity.

Dexamethasone Upregulates Nox1 Expression in Vascular Smooth Muscle Cells

Background/Aim: It has been demonstrated that dexamethasone-induced hypertension can be prevented by the NADPH oxidase inhibitor apocynin. The effect of dexamethasone on NADPH oxidase, however, is unknown. The present study was conducted to investigate the effect of dexamethasone on the gene expression of Nox1, the major NADPH oxidase isoform in vascular smooth muscle cells. Results: Oral treatment of Wistar-Kyoto rats with dexamethasone (0.03 mg/kg/day) for 12 days led to an upregulation of Nox1 mRNA expression in the aorta. In cultured A7r5 rat aortic smooth muscle cells, dexamethasone increased Nox1 mRNA expression in a concentration- and time-dependent manner. The upregulation of Nox1 mRNA expression was completely prevented by the glucocorticoid receptor antagonist mifepristone. The effect of dexamethasone on Nox1 expression was likely to be indirect as it could be largely blocked by cycloheximide, an inhibitor of protein biosynthesis. Dexamethasone increased Nox1 mRNA stability as well as Nox1 transcription. The dexamethasone-induced Nox1 expression was completely prevented by scriptaid, a pan-inhibitor of histone deacetylases (HDAC), indicating a crucial role for HDAC enzymes. In total, A7r5 cells expressed 8 HDAC isoforms, with HDAC1, 5, 6 and 7 being the most abundant ones. Knockdown of these 4 individual HDAC enzymes did not prevent the effect of dexamethasone on Nox1 expression, although HDAC5 knockdown markedly reduced basal Nox1 expression. Conclusion: Dexamethasone upregulates Nox1 expression in vascular smooth muscle cells. This effect involves the glucocorticoid receptor and HDAC enzymes.

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes.

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization.

Metabolomics deciphers the host resistance mechanisms in wheat cultivar Sumai-3, against trichothecene producing and non-producing isolates of Fusarium graminearum

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, reduces grain yield and contaminates grains with trichothecene mycotoxins. Host resistance to FHB is quantitatively inherited and more than 100 QTLs have been mapped, but the host resistance mechanisms are poorly understood. Non-targeted metabolic profiling was applied to elucidate the host resistance mechanisms to FHB spread through rachis of wheat cultivar Sumai-3 against both trichothecene producing and non-producing isolates of Fusarium graminearum. The accumulation of deoxynivalenol (DON) in Sumai-3 was low, however the resistance to spread was not due to its detoxification into DON-3-O-glucoside (D3G), as the proportion of total DON converted to D3G in the resistant was not significantly different from that in the susceptible cultivar Roblin. Instead, the resistance was considered to be due to the accumulation of resistance related (RR) metabolites belonging to the phenylpropanoid pathway that reduced pathogen advancement through increased host cell wall thickening and also reduced pathogen growth due to antifungal and/or antioxidant properties which, in turn, reduced subsequent trichothecene biosynthesis. The RR phenylpropanoids accumulated in Sumai-3 were mainly the preformed syringyl rich monolignols and their glucosides, which are precursors of lignin biosynthesis, as well as antimicrobial flavonoids. The resistant cultivar Sumai-3 inoculated with trichothecene producing F. graminearum not only accumulated less RR metabolites but also the abundance of many RR metabolites was lesser than in the trichothecene non-producing F. graminearum. This implies repression of host resistance mechanisms by trichothecenes/DON, which is a protein biosynthesis inhibitor. Enhancement of resistance in wheat against FHB can be exploited through stacking of candidate phenylpropanoid pathway genes.

Melleolides induce rapid cell death in human primary monocytes and cancer cells

The melleolides are structurally unique and bioactive natural products of the basidiomycete genus Armillaria. Here, we report on cytotoxic effects of melleolides from Armillaria mellea towards non-transformed human primary monocytes and human cancer cell lines, respectively. In contrast to staurosporine or pretubulysin that are less cytotoxic for monocytes, the cytotoxic potency of the active melleolides in primary monocytes is comparable to that in cancer cells. The onset of the cytotoxic effects of melleolides was rapid (within <1h), as compared to the apoptosis inducer staurosporine, the protein biosynthesis inhibitor cycloheximide, and the DNA transcription inhibitor actinomycin D (>5h, each). Side-by-side comparison with the detergent triton X-100 and staurosporine in microscopic and flow cytometric analysis studies as well as analysis of the viability of mitochondria exclude cell lysis and apoptosis as relevant or primary mechanisms. Our results rather point to necrotic features of cell death mediated by an as yet elusive but rapid mechanism.

Stress-induced expression of NICOTINE2-locus genes and their homologs encoding Ethylene Response Factor transcription factors in tobacco

Plants have evolved diverse defense metabolites as adaptations to biotic and abiotic stresses. The defense alkaloid nicotine is produced in Nicotiana tabacum (tobacco) and its biosynthesis is elicited by jasmonates in the roots. At least seven jasmonate-responsive genes that encode transcription factors of the Ethylene Response Factor (ERF) family are clustered at the nicotine-regulatory locus NICOTINE2 (NIC2) in the tobacco genome. A subset of the NIC2-locus ERFs and their homologs, including ERF189 and ERF199, have been shown to be most effective in controlling nicotine biosynthetic pathway genes. Herein reported is that the ERF genes of this group, other than ERF189 and ERF199, were strongly induced by NaCl in tobacco hairy roots, although salt stress had no effect on expression of nicotine biosynthesis genes. Abscisic acid and osmotic stress also increased expression of a subset of these NaCl-inducible ERF genes. Promoter expression analysis in transgenic tobacco hairy roots confirmed that while methyl jasmonate (MJ) activated the promoters of ERF29, ERF210 and ERF199, salt stress up-regulated the promoters of only ERF29 and ERF210, but not ERF199. The protein biosynthesis inhibitor cycloheximide induced expression of the ERFs, and simultaneous addition of MJ and cycloheximide showed synergistic effects. These results indicate that, after several gene duplication events, the NIC2-locus ERFs and possibly their homologs appear to have diverged in their responses to jasmonates and various environmental inputs, including salt stress, and may have evolved to regulate distinct metabolic processes and cellular responses.

Triapine and a More Potent Dimethyl Derivative Induce Endoplasmic Reticulum Stress in Cancer Cells

Triapine (3-AP; 3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a ribonucleotide reductase inhibitor, has been extensively evaluated in clinical trials in the last decade. This study addresses the role of endoplasmic reticulum (ER) stress in the anticancer activity of 3-AP and the derivative N(4),N(4)-dimethyl-triapine (3-AP-Me), differing from 3-AP only by dimethylation of the terminal nitrogen. Treatment of colon cancer cells with 3-AP or 3-AP-Me activated all three ER stress pathways (PERK, IRE1a, ATF6) by phosphorylation of eIF2α and upregulation of gene expression of activating transcription factors ATF4 and ATF6. In particular, 3-AP-Me led to an upregulation of the alternatively spliced mRNA variant XBP1 (16-fold). Moreover, 3-AP and 3-AP-Me activated the cellular stress kinases c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, and inhibition of JNK activity antagonized the cytotoxic effect of both compounds. Subsequent to induction of the unfolded protein response, a significant upregulation of proapoptotic proteins was detected, including the transcription factor CHOP and Bim, an essential factor for ER stress-related apoptosis. In correlation with the higher degree of ER stress after 3-AP-Me treatment, also a more potent depolarization of mitochondrial membranes was found. These data suggest that 3-AP and 3-AP-Me induce apoptosis via ER stress. This was further corroborated by showing that inhibition of protein biosynthesis with cycloheximide prior to 3-AP and 3-AP-Me treatment leads to a significant reduction of the antiproliferative properties of both compounds. Taken together, this study demonstrates that induction of ER stress contributes to the mode of action of 3-AP and that terminal dimethylation leads to an even more pronounced manifestation of this effect.

Shiga toxins expressed by human pathogenic bacteria induce immune responses in host cells

Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxin-mediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), macrophage inflammatory protein-1α/β (MIP-1α/β), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groβ. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.

Inhibition of Protein Synthesis Alters Protein Degradation through Activation of Protein Kinase B (AKT)

The homeostasis of protein metabolism is maintained and regulated by the rates of protein biosynthesis and degradation in living systems. Alterations of protein degradation may regulate protein biosynthesis through a feedback mechanism. Whether a change in protein biosynthesis modulates protein degradation has not been reported. In this study, we found that inhibition of protein biosynthesis induced phosphorylation/activation of AKT and led to phosphorylation of AKT target substrates, including FoxO1, GSK3α/β, p70S6K, AS160, and the E3 ubiquitin ligase MDM2. Phosphorylation of ribosomal protein S6 was also modulated by inhibition of protein biosynthesis. The AKT phosphorylation/activation was mediated mainly through the PI3K pathway because it was blocked by the PI3K inhibitor LY294002. The activated AKT phosphorylated MDM2 at Ser(166) and promoted degradation of the tumor suppressor p53. These findings suggest that inhibition of protein biosynthesis can alter degradation of some proteins through activation of AKT. This study reveals a novel regulation of protein degradation and calls for caution in blocking protein biosynthesis to study the half-life of proteins.

The pyrimidine analog FNC inhibits cell proliferation and viral protein synthesis in HTLV-1-infected cells

Human T‑cell leukemia virus type 1 (HTLV‑1), the first retrovirus to be identified, is the etiological agent of an aggressive clonal malignancy of mature CD4+ Tlymphocytes known as adult T‑cell leukemia (ATL). The prognosis of ATL patients remains poor despite the availability of a number of clinical chemotherapy drugs. In addition, HTLV‑1‑infected and ATL cells possess an intrinsic resistance to anticancer drugs. 2'‑Deoxy‑2'‑β‑fluoro‑4'‑azidocytidine (FNC) is a novel pyrimidine analog that is efficiently phosphorylated by cellular kinases and is a substrate for RNA and DNA polymerases. In the present study, the antiviral potential of FNC was investigated in HTLV‑1‑infected cell lines. Following FNC treatment, the HTLV‑1‑infected cells underwent G1 or S phase cell cycle arrest. FNC was also observed to reduce cell growth of the HTLV‑1‑infected cell lines in a dose‑dependent manner. Notably, FNC was found to efficiently inhibit the expression of the viral proteins, Tax and p19Gag, in a dose‑ and time‑dependent manner. Treatment with FNC and the protein biosynthesis inhibitor, cycloheximide (CHX), accelerated the inhibition of viral protein synthesis in the HTLV‑1‑infected cells. Collectively, these results demonstrated the efficient antiretroviral effect of FNC in HTLV‑1‑infected cells and indicate that FNC may be utilized as a valuable therapy in HTLV‑1‑infected patients and those with ATL.

Solithromycin Inhibition of Protein Synthesis and Ribosome Biogenesis in Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae

The continuing increase in antibiotic-resistant microorganisms is driving the search for new antibiotic targets and improved antimicrobial agents. Ketolides are semisynthetic derivatives of macrolide antibiotics, which are effective against certain resistant organisms. Solithromycin (CEM-101) is a novel fluoroketolide with improved antimicrobial effectiveness. This compound binds to the large 50S subunit of the ribosome and inhibits protein biosynthesis. Like other ketolides, it should impair bacterial ribosomal subunit formation. This mechanism of action was examined in strains of Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae. The mean 50% inhibitory concentrations (IC50s) for solithromycin inhibition of cell viability, protein synthesis, and growth rate were 7.5, 40, and 125 ng/ml for Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae, respectively. The net formation of the 50S subunit was reduced in all three organisms, with IC50s similar to those given above. The rates of 50S subunit formation measured by a pulse-chase labeling procedure were reduced by 75% in cells growing at the IC50 of solithromycin. Turnover of 23S rRNA was stimulated by solithromycin as well. Solithromycin was found to be a particularly effective antimicrobial agent, with IC50s comparable to those of telithromycin and significantly better than those of azithromycin and clarithromycin in these three microorganisms.

Testosterone protects female embryonic heart H9c2 cells against severe metabolic stress by activating estrogen receptors and up-regulating IES SUR2B

A recent clinical study demonstrated that a testosterone supplementation improves functional capacity in elderly female patients suffering from heart failure. These findings prompted us to consider possible mechanisms of testosterone-induced cardioprotection in females. To address this question we have used a pure female population of rat heart embryonic H9c2 cells. Pre-treatment of cells with testosterone for 24h significantly increased survival of H9c2 cells exposed to 2,4-dinitrophenol (DNP), an inhibitor of oxidative phosphorylation. These cells expressed low level of androgen receptors and the effect of testosterone was not modified by hydroxyflutamide, an antagonist of androgen receptor. In contrast, cyclohexamide, an inhibitor of protein biosynthesis, and tamoxifene, a partial agonist of estrogen receptors, abolished cardioprotection afforded by testosterone. In addition, finasteride, an inhibitor of 5α-reductase, and anastrazole, an inhibitor of α-aromatase, also blocked testosterone-induced cytoprotection. Real time RT-PCR revealed that testosterone did not regulate the expression of nine subunits and accessory proteins of sarcolemmal ATP-sensitive K+ (KATP) channels. On the other hand, testosterone, as well as 17β-estradiol, up-regulated a putative mitochondrial KATP channel subunit, mitochondrial sulfonylurea receptor 2B intraexonics splice variant (IES SUR2B), without affecting expression of IES SUR2A. Tamoxifene inhibited testosterone-induced up-regulation of IES SUR2B without affecting IES SUR2A. In conclusion, this study has shown that testosterone protect female embryonic heart H9c2 cells against severe metabolic stress by its conversion into metabolites that activate estrogen receptors and up-regulate IES SUR2B.

Antimycin A sensitizes cells to TRAIL-induced apoptosis through upregulation of DR5 and downregulation of c-FLIP and Bcl-2

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been the focus as a potential anticancer drug, because it induces apoptosis in a wide variety of cancer cells but not in most normal human cell types. In this study, we showed that combination treatment with sub-toxic doses of antimycin A (AMA), an inhibitor of electron transport, plus TRAIL induced apoptosis in human renal cancer cells, but not in normal tubular kidney cells. Treatment of Caki cells with AMA upregulated the death receptor 5 (DR5) protein and downregulated c-FLIP and Bcl-2 proteins in a dose-dependent manner. AMA-induced decrease of c-FLIPL and c-FLIPs protein levels which were caused by increased protein instability, which was confirmed by the result showing that treatment with a protein biosynthesis inhibitor, CHX, accelerated degradation of c-FLIPL and c-FLIPs proteins caused by AMA treatment. We also found that AMA induced upregulation of DR5 and downregulation of Bcl-2 at the transcriptional level. Pretreatment with N-acetyl-l-cysteine (NAC) partly recovered the expression levels of c-FLIPL and c-FLIPs proteins were downregulated by the AMA treatment, suggesting that AMA appears to be partially dependent on the generation of ROS for downregulation of c-FLIPL and c-FLIPs. Collectively, this study demonstrates that AMA enhances TRAIL-induced apoptosis in human renal cancer cells by upregulation of DR5 as well as downregulation of c-FLIP and Bcl-2. Furthermore, this study shows that AMA markedly increases sensitivity to cisplatin in Caki human renal cancer cells.

The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells. Preventing the basal level ethylene response facilitated cell elongation, and the cells exhibited wall loosening and separation phenotype. Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes, the presence of paramural bodies, and the circular Golgi formation. The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide. The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses. Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking, remodeling, and wall modifications were differentially expressed. FM4-64 staining supported the proteomic changes, which indicated reduced endocytosis activity with alleviation of the ethylene response. The basal level ethylene response has an important role in endomembrane trafficking, biological materials transport and maintenance of the endomembrane organization. It is possible that endomembrane alterations may partly associate with the wall modifications, though the biological significance of the alterations should be addressed in future studies.

Abstract 2815: Sylvestrol inhibits protein synthesis by downregulating the AKT/mTOR pathway

Abstract Silvestrol, a cyclopenta[b]benzofuran lignan present in the fruits and twigs of the tropical tree Aglaia foveolata Pannell (Meliaceae), is toxic to human cancer cell lines in culture with a potency similar to those of FDA approved anticancer chemotherapeutic agents. We report here that silvestrol has a mean GI50 of approximately 3 nM in the 60 cell-line panel of human cancer cell lines maintained at the National Cancer Institute. Furthermore, silvestrol is at least as effective as FDA-approved anticancer agents in the treatment of human cancer cells in vivo when tested in the hollow fiber assay or the inhibition of human tumor xenograft tumors propagated in immunedeficient mice. Others as well as our group have reported that silvestrol inhibits protein biosynthesis specifically without affecting the cellular production of either RNA or DNA. Pelletier and colleagues have shown that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. In the present study, we test the hypothesis that disruption of protein synthesis by silvestrol can impact cell signaling pathways upstream of translation. Human colon cancer cells designated HT-29 were exposed to silvestrol at concentrations as high as 100 nM for as long as 48 h prior to harvest and evaluation by immunoblot. Protein signaling molecules known to regulate the protein synthesis machinery were evaluated for their abundance and state of phosphorylation as a function of silvestrol exposure. We observed down-regulation of the p110α catalytic subunit of PI3K, which phosphorylates PIP2 to PIP3. PIP3 then recruits PDK1 and AKT to the plasma membrane, where PDK1 can phosphorylate AKT. A decrease in the amount of PDK1 protein and a significant decrease in phosphorylation of AKT at ser 473 occurred. Moreover, a decrease in both the abundance and phosphorylation of mTOR was evident, which is a key component of mTORC2. Consistent with these findings, phosphorylation of AKT at thr 308 was significantly reduced. Finally, silvestrol exposure resulted in a decrease in phosphorylation of the S6 ribosomal protein serine 235/236. These data indicate that silvestrol treatment can disrupt key signaling pathways leading to protein synthesis production in human cancer cells. This work was supported by NIH grants U01 CA52956 and P01 CA125066. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2815. doi:1538-7445.AM2012-2815

The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMAIII)-induced cytotoxicity

The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMAIII) and compared with that of trivalent arsenite (iAsIII) and monomethylarsonous acid (MMAIII). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMAIII, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAsIII or MMAIII. The generation of reactive oxygen species (ROS) after DMAIII exposure was found to take place specifically in the endoplasmic reticulum (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMAIII. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMAIII-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAsIII and MMAIII. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMAIII-induced cytotoxicity.

Fosfomycin Enhances the Active Transport of Tobramycin in Pseudomonas aeruginosa

Elevated levels of mucins present in bronchiectatic airways predispose patients to bacterial infections and reduce the effectiveness of antibiotic therapies by directly inactivating antibiotics. Consequently, new antibiotics that are not inhibited by mucins are needed to treat chronic respiratory infections caused by Pseudomonas aeruginosa and Staphylococcus aureus. In these studies, we demonstrate that fosfomycin synergistically enhances the activity of tobramycin in the presence of mucin. The bactericidal killing of a novel 4:1 (wt/wt) combination of fosfomycin-tobramycin (FTI) is superior (>9 log(10) CFU/ml) relative to its individual components fosfomycin and tobramycin. Additionally, FTI has a mutation frequency resulting in an antibiotic resistance >3 log(10) lower than for fosfomycin and 4 log(10) lower than for tobramycin for P. aeruginosa. Mechanistic studies revealed that chemical adducts are not formed, suggesting that the beneficial effects of the combination are not due to molecular modification of the components. FTI displayed time-kill kinetics similar to tobramycin and killed in a concentration-dependent fashion. The bactericidal effect resulted from inhibition of protein biosynthesis rather than cell wall biosynthesis. Studies using radiolabeled antibiotics demonstrated that tobramycin uptake was energy dependent and that fosfomycin enhanced the uptake of tobramycin in P. aeruginosa in a dose-dependent manner. Lastly, mutants resistant to fosfomycin and tobramycin were auxotrophic for specific carbohydrates and amino acids, suggesting that the resistance arises from mutations in specific active transport mechanisms. Overall, these data demonstrate that fosfomycin enhances the uptake of tobramycin, resulting in increased inhibition of protein synthesis and ultimately bacterial killing.