Identification and sequence analysis of two novel cryptic plasmids isolated from the vaginal mucosa of South African women.

The vaginal mucosa is dominated by Gram positive, rod shaped lactobacilli which serve as a natural barrier against infection. In both healthy- and bacterial vaginosis (BV)-infected women Lactobacillus crispatus and Lactobacillus jensenii have been found to be the predominant Lactobacillus species. Many studies have been conducted to assess factors influencing lactobacilli dominance in the vaginal microbiome. In the present study two plasmids, pLc4 and pLc17, isolated from vaginal Lactobacillus strains of both healthy and BV-infected women were characterized. The smaller plasmid, pLc4 (4224 bp), was detected in both L. crispatus and L. jensenii strains, while pLc17 was only detected in L. crispatus. Based on its nucleotide sequence pLc4 appears highly novel, with its replication protein having 44% identity to the replication initiation protein of pSMQ173b_03. Phylogenetic analysis with other Rolling Circle Replication plasmids confirmed that pLc4 shows a low degree of similarity to these plasmids. Plasmid pLc17 (16,663 bp) appears to carry both a RCR replicon and a theta replicon, which is rare in naturally occurring plasmids. pLc4 was maintained at a high copy number of 29, while pLc17 appears to be a medium copy number plasmid maintained at 11 copies per chromosome. While sequence analysis is a valuable tool to study cryptic plasmids, further function-based analysis will be required in order to fully elucidate the role of these plasmids within the vaginal milieu.

Disulfiram with or without metformin inhibits oesophageal squamous cell carcinoma in vivo.

Oesophageal squamous cell carcinoma (OSCC) is highly prevalent in developing countries but there has been little recent progress into efficacious yet affordable treatment strategies. Drug repurposing is one attractive approach for cancer therapy. Disulfiram (DSF), used to treat alcoholism, inhibits cancer growth and we previously found that DSF perturbs protein degradation/turnover pathways in vitro. This was enhanced by combining DSF with the anti-diabetic drug metformin (Met). Here, we investigated DSF with/without Met, against OSCC in vivo. Nude mice injected subcutaneously with the human OSCC cell line WHCO1, were treated with 30 mg/kg or 50 mg/kg DSF three times per week and with/without Met, for 21 days. DSF and DSF/Met-treated animals had significantly smaller tumours compared to untreated, vehicle and positive control cisplatin-treated groups. This effect for DSF was independent of copper, with no significant accumulation of copper in tumours, together with maintained proteasome activity. However, increases in total ubiquitinated proteins, LC3B-II, LAMP1 and p62 in DSF and DSF/Met groups, indicate that autophagy is inhibited. These findings show that DSF and DSF/Met significantly impede OSCC tumour growth in vivo and offer prospective alternative chemotherapy approaches for OSCC.

Metformin-induced alterations in nucleotide metabolism cause 5-fluorouracil resistance but gemcitabine susceptibility in oesophageal squamous cell carcinoma.

5-Fluorouracil (5-FU) is a chemotherapeutic agent used to treat a variety of gastric cancers including oesophageal squamous cell carcinoma (OSCC), for which the 5-year mortality rate exceeds 85%. Our study investigated the effects of metformin, an antidiabetic drug with established anti-cancer activity, in combination with 5-FU as a novel chemotherapy strategy, using the OSCC cell lines, WHCO1 and WHCO5. Our results indicate that metformin treatment induces significant resistance to 5-FU in WHCO1 and WHCO5 cells, by more than five- and sixfolds, respectively, as assessed by MTT assay. We show that this is due to global alterations in nucleotide metabolism, including elevated expression of thymidylate synthase and thymidine kinase 1 (established 5-FU resistance mechanisms), which likely result in an increase in intracellular dTTP pools and a "dilution" of 5-FU anabolites. Metformin treatment also increases deoxycytidine kinase (dCK) expression and, as the chemotherapeutic agent gemcitabine relies on dCK for its efficient activity, we speculated that metformin would enhance the sensitivity of OSCC cells to gemcitabine. Indeed we show that metformin pre-treatment greatly increases gemcitabine toxicity and DNA fragmentation in comparison to gemcitabine alone. Taken together, our findings show that metformin alters nucleotide metabolism in OSCC cells and while responsible for inducing resistance to 5-FU, it conversely increases sensitivity to gemcitabine, thereby highlighting metformin and gemcitabine as a potentially novel combination therapy for OSCC.

Alginate microbead-encapsulated silver complexes for selective delivery of broad-spectrum silver-based microbicides.

In sub-Saharan Africa, human immunodeficiency virus (HIV) infections are predominantly acquired via heterosexual contact, and women are at greatest risk of being infected. This region also has the highest rates of sexually transmitted infections (STIs) per capita worldwide; STIs are strongly associated with increased HIV transmission. Therefore, there is an urgent requirement for microbicides that are active against HIV and STIs. Silver compounds exhibit broad antimicrobial activity, making them potentially ideal broad-spectrum microbicides. However, for silver compounds to be effective microbicides, they must be active within seminal fluid and the delivery vehicle used must protect the silver microbicide from vaginal fluid components but selectively release it during intercourse and/or following ejaculation. In this study, silver complexes were synthesised from the ligands saccharin, benzimidazole and 8-hydroxyquinoline and their microbicidal activity was assessed. We show that a silver saccharinate-benzimidazole complex (AgSB) exhibited activity against HIV-1, herpes simplex virus type 2 (HSV-2) and Neisseria gonorrhoeae at concentrations significantly below LD(50) levels for the vaginal mucosal cell line SiHa. Furthermore, we show that alginate microbeads are stable in vaginal fluid simulant but rapidly dissolve in seminal fluid simulant. Finally, we have established that microbead-encapsulated AgSB, dissolved in seminal fluid simulant, is active against the above pathogens, albeit at higher concentrations for HIV-1. This research therefore highlights, for the first time, the potential use of silver complexes encapsulated in alginate microbeads as a novel system for the delivery and selective release of broad-spectrum silver-based microbicides within the vaginal milieu during sexual intercourse/after ejaculation.

Disulfiram/copper-disulfiram Damages Multiple Protein Degradation and Turnover Pathways and Cytotoxicity is Enhanced by Metformin in Oesophageal Squamous Cell Carcinoma Cell Lines.

Disulfiram (DSF), used since the 1950s in the treatment of alcoholism, is reductively activated to diethyldithiocarbamate and both compounds are thiol-reactive and readily complex copper. More recently DSF and copper-DSF (Cu-DSF) have been found to exhibit potent anticancer activity. We have previously shown that the anti-diabetic drug metformin is anti-proliferative and induces an intracellular reducing environment in oesophageal squamous cell carcinoma (OSCC) cell lines. Based on these observations, we investigated the effects of Cu-DSF and DSF, with and without metformin, in this present study. We found that Cu-DSF and DSF caused considerable cytotoxicity across a panel of OSCC cells, and metformin significantly enhanced the effects of DSF. Elevated copper transport contributes to DSF and metformin-DSF-induced cytotoxicity since the cell-impermeable copper chelator, bathocuproinedisulfonic acid, partially reversed the cytotoxic effects of these drugs, and interestingly, metformin-treated OSCC cells contained higher intracellular copper levels. Furthermore, DSF may target cancer cells preferentially due to their high dependence on protein degradation/turnover pathways, and we found that metformin further enhances the role of DSF as a proteasome inhibitor. We hypothesized that the lysosome could be an additional, novel, target of DSF. Indeed, this acid-labile compound decreased lysosomal acidification, and DSF-metformin co-treatment interfered with the progression of autophagy in these cells. In summary, this is the first such report identifying the lysosome as a target of DSF and based on the considerable cytotoxic effects of DSF either alone or in the presence of metformin, in vitro, and we propose these as novel potential chemotherapeutic approaches for OSCC.

Metformin induces an intracellular reductive state that protects oesophageal squamous cell carcinoma cells against cisplatin but not copper-bis(thiosemicarbazones).

BACKGROUND: Oesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a poor survival rate. One of the most commonly used chemotherapeutic drugs, cisplatin, displays varied and often poor efficacy in vivo. Therefore, alternative, cost-effective and more efficacious treatments are required. Metformin has been previously shown to reduce proliferative rates in various carcinoma cell lines. We report for the first time, the effect of metformin on OSCC cell proliferation and show that it antagonises cisplatin-induced but not copper-bis(thiosemicarbazone)-induced cytotoxicity in OSCC cells. METHODS: Cell proliferation and stage of the cell cycle were quantified by trypan blue counts and flow cytometry, respectively. All cytotoxicity measurements were made using the tetrazolium based MTT assay. Metabolic alterations to cells were determined as follows: glycolysis via a lactate dehydrogenase assay, reducing equivalents by MTT reduction and reduced intracellular thiols by monobromobimane-thiol fluorescence, and glutathione depletion using buthionine sulfoximine. Inductively coupled plasma mass spectrometry was used to quantify cisplatin-DNA adduct formation. RESULTS: Metformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle. However, metformin significantly protected OSCC cells against cisplatin toxicity. Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin. We subsequently show that the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, which are trapped in cells under reducing conditions, cause significant OSCC cytotoxicity, both alone and in combination with metformin. CONCLUSIONS: This is the first study showing that metformin can be used to decrease cell proliferation in OSCC cells. However, metformin protects against cisplatin cytotoxicity by inducing a reducing intracellular environment leading to lower cisplatin-DNA adduct formation. As such, we advise that caution be used when administering cisplatin to diabetic patients treated with metformin. Furthermore, we propose a novel combination therapy approach for OSCC that utilises metformin with metformin-compatible cytotoxic agents, such as the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM.

Identification of predominant culturable vaginal Lactobacillus species and associated bacteriophages from women with and without vaginal discharge syndrome in South Africa.

Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri and Lactobacillus vaginalis were identified by 16S rRNA gene sequencing as the predominant culturable vaginal Lactobacillus species in a group of South African women, comprising 24, 22, 10, 10 and 9 %, respectively. A significant effect of vaginal discharge syndrome (VDS) and bacterial vaginosis (BV) on the distribution of predominant Lactobacillus species was observed. Whilst L. crispatus isolates were almost equally distributed between individuals with and without VDS and were not significantly reduced in women with BV versus normal microflora, L. jensenii isolates were significantly reduced in women with VDS (P=0.022) and reduced in women with BV versus normal microflora (P=0.053). Unlike L. crispatus, L. jensenii isolates were also significantly reduced in women with BV-associated VDS versus women without VDS and with normal microflora (P=0.051). In addition, lysogeny was commonly observed for L. crispatus, with 77 % of isolates yielding phage particles with contractile and non-contractile tails. Only 29 % of L. jensenii isolates yielded phage particles, and these were visible as tailless or podo-like particles.

Plasmid transduction using bacteriophage Phi(adh) for expression of CC chemokines by Lactobacillus gasseri ADH.

Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Phiadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Phiadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.

The use of 3-aminophthalimide as a pro-chemiluminescent label in chemiluminescence and fluorescence-based cellular assays.

We present a labeling system for direct chemiluminescence-based cellular bioassays using the stable pro-chemiluminescent, luminol precursor, 3-aminophthalimide (API). API-coupled reporter molecules are detected chemiluminometrically after treatment with hydrazine, which converts the API label to luminol. API derivatives containing a variety of functional groups are readily synthesized, allowing for ease of coupling via the imide nitrogen to a host of reporter molecules. The fluorescent nature of APIs further allows for dual fluorescence and chemiluminescence studies. To highlight the utility of this label, we show that API-labeled insulin can be successfully utilized in cellular binding and transport assays and that an API-coupled mitochondrial probe (API-triphenylphosphonium(+)) can be used to both fluorescently and chemiluminometrically investigate mitochondrial function. We also assess the use of API as a polysaccharide and nucleic acid label, and we show that API-labeled palmitic acid undergoes cellular transport and lipid metabolism.

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function.

Fat-loaded HepG2 spheroids exhibit enhanced protection from Pro-oxidant and cytokine induced damage.

The mechanisms by which steatosis renders hepatocytes susceptible to damage in non-alcoholic steatohepatitis (NASH) are unclear although fat accumulation is believed to increase hepatocyte susceptibility to inflammatory cytokines and oxidative stress. We therefore investigated the susceptibility of steatotic, hepatocyte-derived cells to TNFalpha and the pro-oxidant, t-butylhydroperoxide (TBH). HepG2 spheroids rendered steatotic by fat-loading with 0.15 mM oleic or palmitic acid for 48 h and treated with TNFalpha or TBH for 18 h exhibited surprisingly lower levels of cytotoxicity, and increased anti-oxidant activity (superoxide dismutase (SOD)) compared with non fat-loaded controls. The protective effect of steatosis was significantly reversed by the inhibition of AMP-activated kinase (AMPK) since spheroids transfected with a kinase-dead AMPKalpha2 subunit, exhibited a significant increase in TBH-induced cytotoxicity when fat-loaded. In conclusion, our findings suggest that fat-loaded hepatocyte-derived cells are surprisingly less susceptible to cytokine and pro-oxidant induced damage via an adaptive mechanism dependent, in part, on AMPK activity.

Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2.

A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver.

Increased expression of uncoupling protein 2 in HepG2 cells attenuates oxidative damage and apoptosis.

INTRODUCTION: Oxidative damage plays a major part in the pathogenesis of liver disease. Uncoupling proteins (UCPs) may be able to limit the generation of reactive oxygen species (ROS) and be cytoprotective. METHODS: We investigated the effect of up-regulation of UCP2 in a hepatoblastoma cell line exposed to menadione or hypoxia/re-oxygenation. RESULTS: Lipid and protein oxidation was increased in HepG2 cells exposed to ROS but this increase was significantly lower in cells over-expressing UCP2 under identical conditions. LDH release increased 2.5-fold in response to hypoxia/re-oxygenation in control HepG2 cells with no significant increase in UCP2 transfected cells. Hypoxia/re-oxygenation resulted in a reduction in liver-specific protein secretion that was attenuated in transfected cells and UCP2 over-expression also resulted in a 66% reduction in apoptosis compared with non-transfected controls. CONCLUSIONS: These data suggest that UCP2 can limit oxidative damage in HepG2 cells in response to oxidative stress resulting in improved cell function and resistance to apoptosis.

Altered mitochondrial function and cholesterol synthesis influences protein synthesis in extended HepG2 spheroid cultures.

Cultures of hepatocytes and HepG2 cells provide useful in vitro models of liver specific function. In this study, we investigated metabolic and biosynthetic function in 3-D HepG2 spheroid cultures, in particular to characterise changes on prolonged culture. We show that HepG2 cells cultured in spheroids demonstrate a reduction in mitochondrial membrane potential and respiration following 10 days of culture. This coincides with a modest reduction in glycolysis but an increase in glucose uptake where increased glycogen synthesis occurs at the expense of the intracellular ATP pool. Lowered biosynthesis coincides with and is linked to mitochondrial functional decline since low glucose-adapted spheroids, which exhibit extended mitochondrial function, have stable biosynthetic activity during extended culture although biosynthetic function is lower. This indicates that glucose is required for biosynthetic output but sustained mitochondrial function is required for the maintenance of biosynthetic function. Furthermore, we show that cholesterol synthesis is markedly increased in spheroids cf. monolayer culture and that inhibition of cholesterol synthesis by lovastatin extends mitochondrial and biosynthetic function. Therefore, increased cholesterol synthesis and/or its derivatives contributes to mitochondrial functional decline in extended HepG2 spheroid cultures.

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling.

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.