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Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1Mulelu, Andani Errol January 2017 (has links)
The cyanide dihydratase enzyme from Bacillus pumilus C1 (CynDₚᵤₘ) is a member of the nitrilase superfamily and is known to specifically catalyse the conversion of cyanide into formic acid and ammonia. This enzyme is a good candidate for bioremediation of cyanide waste but the high alkaline pH of the cyanide waste water poses a problem in that it inactivates the wild type enzyme and therefore improvement of stability is required in order to synthesize an effective enzyme. Over the pH range of 6–8 the enzyme exists as short 18-subunit spirals which associate to form long, more stable helical fibres at pH 5.4. The reason for this pH dependent transition is not fully understood but it is hypothesized to be due to changes in the charge of histidine residues. The aim of this project is to obtain a high resolution structure of CynDₚᵤₘ, relate this to its function, and investigate the role of the histidines in oligomerisation with aid of the structure. Using Cryo-electron microscopy techniques a three dimensional reconstruction structure of purified CynDₚᵤₘ was obtained at a resolution of ~5Å. By flexibly fitting a CynDₚᵤₘ homology model into this high resolution structure we were able to identify amino acid residues involved in oligomerisation and stability as well as the role of the histidines, with aid from additional mutagenesis studies. Interactions at the C-interfacial region were shown to play the most crucial role in oligomerisation and included the His71-Asp275 and Arg67-Asp275 interactions. Mutations at His128, His184, His241 and His285 were shown to affect the oligomerisation of the enzyme by indirectly disrupting interactions at the interfacial regions. The Q86R+H305K+H308K+H323K mutations were shown to increase the stability of the CynDₚᵤₘ by introducing a stronger arginine-arginine interaction at the D interfacial region and a new strong interaction at the C-terminal region.
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Helical reconstruction of Mycobacterium smegmatis Mycothiol S-conjugate amidase filamentsBurgess, Jeremy Gareth January 2017 (has links)
The metabolic pathway of mycothiol (MSH) is a major cellular defence against oxidative stress, and several antibiotics for mycobacteria, including Mycobacterium tuberculosis. The central enzyme used in the clearance of electrophilic toxins is Mycothiol S-conjugate amidase (Mca). Mca is similar to a biosynthetic enzyme MshB, which has partial overlapping substrate activity and is the closest homologue to Mca with a known structure. The basis for the substrate specificity differences in Mca and MshB is not well understood. Several regions of low sequence similarity between MshB and Mca are contained within an active site pocket, and these may affect the observed substrate preferences. However, these regions cannot be modelled in Mca with confidence, which makes it essential to obtain a structure of Mca experimentally. Mca is also a potential drug target, and a structure of Mca would enhance the rational design of inhibitors against the enzyme. A search for crystalline forms of MsMca (Mycobacterium smegmatis Mca) led to the discovery of regular filaments, which showed helical order. Helical symmetry was estimated using power spectra from single filaments. The number of potential symmetry solutions was reduced using phase information from Fourier transforms of single filaments. Three possible solutions to the helical symmetry were suggested, two of which converged on the same symmetry parameters using Iterative Helical Real-Space Reconstruction. The first solution had a selection rule of l = 18m + n, and the second l = 20m + n. Reconstructions made from the predicted helical symmetries were compared in their power spectra and through rigid-body fitting with an atomic model of MsMca. The first reconstruction, with a final symmetry of Δφ = 20.05o and Δz = 10.27 Å, better matched the predicted helical symmetry than did the second reconstruction. However, rigid-body fitting did not indicate either reconstruction as being superior. Following this, the second reconstruction was improved using a number of additional techniques to those used in the initial reconstruction. These included the use of the fortuitous 3-fold cyclic symmetry, the removal of double-walled filaments, use of a cut-off filter for images with low correlation to projections of the 3D reconstruction, and use of a layer-line filter to reduce the noise in the images. These were used individually, then in a single reconstruction, to improve the and agreement between the predicted helical symmetry and that obtained from the reconstruction. Several of the improved reconstructions were used via rigid-body fitting to assess the favoured handedness of the filament through examination of the major interfaces between subunits. These suggest that the 3-start helix is right-handed. Future work would be to determine the handedness of the filament using alternative techniques, such as metal-shadowing. This work provides a springboard for high resolution cryo-electron microscopy, to determine a high-resolution structure of MsMca, which will enable rational inhibitor design and give the basis for the different substrate specificity in Mca and MshB.
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Modulating the expression and activity of the nuclear import protein, Karyopherin β1, in cancer cellsCarden, Sarah January 2017 (has links)
Cancer is primarily a disease of disordered gene expression; the dysregulation of thousands of different genes has been associated with the progression of many types of cancer. Previous research from our laboratory aimed at identifying genes differentially expressed in cervical cancer compared to normal patient tissue, found Karyopherin β1 (Kpnβ1), the primary nuclear import protein, to be significantly overexpressed in cervical cancer tissue. Further studies showed that inhibition of Kpnβ1 expression by siRNA resulted in cancer cell death, while non-cancer cells were only minimally affected. These results suggest that Kpnβ1 has potential as an anti-cancer therapeutic target, thus warranting further research into the association between Kpnβ1 expression and cancer progression. In this study, we investigated the biological effects associated with Kpnβ1 overexpression in order to further elucidate the relationship between Kpnβ1 and the cancer phenotype. Our data revealed that Kpnβ1 overexpression, above what was already detected in cancer cells, resulted in reduced proliferation and an associated delay in cell cycle progression. Additionally, overexpression of Kpnβ1 caused changes in the morphology and adhesion properties of cells. Co-expression of Ran, an important nuclear transport factor, binding partner and regulator of Kpnβ1, resulted in a further reduction in proliferation (greater than that of overexpression of either Kpnβ1 or Ran alone), suggesting that cells are particularly unable to handle an imbalance in the levels of Kpnβ1 and Ran. Previous work from our laboratory using a newly identified small molecule, Inhibitor of Nuclear Import-43 (INI-43), showed that INI-43 significantly reduced the proliferation of cancer cells of different tissue origins and interfered with the nuclear import function of Kpnβ1. In order to investigate the specificity of INI-43 for Kpnβ1 in this study, we performed experiments to determine whether overexpression of Kpnβ1 could rescue cancer cells from the negative effects associated with INI-43 treatment. Results revealed that stable overexpression of Kpnβ1 was able to rescue cell viability, as well as the inhibitory effects that INI-43 had on the nuclear import of Kpnβ1 cargoes NFκB p65 and NFAT. Kpnβ1 overexpression was also able to rescue cells from an INI-43 induced G2/M cell cycle block. In addition, treatment of cells with INI-43 enhanced the degradation of Kpnβ1, indicating that INI-43 is likely acting by targeting Kpnβ1. In order to determine the effects of Kpnβ1 dysregulation in HeLa cells, live cell time-lapse videomicroscopy was used to study cells as the progressed through the cell cycle. Inducible expression of Kpnβ1-EGFP, as well as the treatment of cells with the small molecule inhibitor INI-43, were used as mechanisms of dysregulation. Results revealed that modulation of Kpnβ1 by either overexpression or inhibition caused a disruption in mitotic progression, with the appearance of distinct mitotic abnormalities. The treatment of cells expressing Kpnβ1-EGFP with INI-43 resulted in a significant reduction in (or rescue of) the negative effects associated with either condition alone. Taken together, this data suggests that a precise balance of Kpnβ1 expression is required for the correct functioning of cancer cells; when the balance is perturbed in either direction (i.e. with overexpression of Kpnβ1 or INI-43-mediated inhibition of Kpnβ1) negative effects associated with a variety of biological processes are observed. In addition, results from rescue experiments conducted using Kpnβ1 overexpression in combination with INI-43 treatment suggest that that INI-43 is acting, at least in part, by targeting Kpnβ1.
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The impact of EPHA2 polymorphism on KSHV infectivity and KS prevalence among HIV/AIDS patients in South AfricaBlumenthal, Melissa Jayne Walcott January 2017 (has links)
Kaposi's Sarcoma (KS) is the most common Acquired Immune Deficiency Syndrome (AIDS)-related malignancy globally and is of particular significance in sub-Saharan Africa where, due to the Human Immunodeficiency Virus (HIV) epidemic, KS is the cause of significant morbidity and mortality. The oncogenic Kaposi's Sarcoma-associated herpes virus (KSHV) is the etiological agent of KS. Although KSHV seroprevalence in sub-Saharan Africa is high, not all AIDS patients develop KS, suggesting that host genetic factors contribute to susceptibility. The infection mechanism of KSHV in endothelial cells has recently been elucidated and highlights Eph Receptor A2 (EPHA2) as a specific host receptor for virus entry. Furthermore, EPHA2 has been implicated in oncogenesis and is upregulated in a number of cancers including KS. We therefore hypothesised that mutations in the KSHV host receptor's coding region could result in an altered EPHA2 that could affect susceptibility to KSHV infection and/or KS development among HIV/AIDS patients. To test our hypothesis, we studied three groups of HIV positive South African patients, namely patients with KS and patients without KS who were KSHV positive or KSHV negative. KS status was determined clinically and KSHV seroconversion was assessed using a combination of ELISAs to KSHV lytic antigen K8.1 and latency-associated nuclear antigen in patient plasma samples. All patients with KS were found to be KSHV seropositive as expected, while 45.45% of HIV positive patients without KS were found to be KSHV seropositive. From patient blood cells, we extracted genomic DNA and subsequently PCR amplified and sequenced the coding region of EPHA2, before comparing these sequences to the NCBI reference by multiple alignment. A number of variants were identified throughout the EPHA2 coding region and assessed statistically for association with KSHV susceptibility and/or KS prevalence. A novel heterozygous transition (c.2727C>T), which is predicted to result in the substitution of Cysteine for Arginine at amino acid position 858 in the functionally important tyrosine kinase domain, was identified as statistically associated with KSHV susceptibility as well as KS prevalence. Three additional missense variants (c.2254T>C, c.2257A>C and c.2688G>C) occurring in the tyrosine kinase domain and one occurring in the sterile-α-motif (c.2990G>T), a putative protein interaction domain, were found to be statistically associated with KS prevalence. This is the first study to investigate polymorphism in EPHA2 in HIV/AIDS patients in relation to susceptibility to KSHV infection and/or KS prevalence. The identification of variants in the KSHV entry receptor, EPHA2, opens new doors for the development of biomarkers involved in prognosis and treatment of KSHV-associated pathologies.
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A comparison of the effects of xenobiotics on hepatic haem metabolismZiman, Melanie Ruth January 1983 (has links)
Hepatic microsomal cytochrome P-450 has previously been postulated to be an important factor in determining the rates of hepatic haem biosynthesis and biodegradation. The basis for this proposal is that the haem moiety of cytochrome P-450 appears to be in equilibrium between binding to apocytochrome P-450 and existing in some form in the central hepatic pool of haem concerned with the regulation of the haem metabolic pathways. Consequently, any change in the levels of hepatic cytochrome P-450 would be anticipated to affect the pathways of hepatic haem biosynthesis and biodegradation. At the onset of this project, relatively few chemical agents were known to destroy cytochrome P-450 (either by degradation of the haem moiety of, or dissociation of the haem moiety from hepatic microsomal cytochrome P-450) and to affect hepatic haem biosynthesis and/or haem biodegradation (e.g. AIA, Cs₂ and various metals). We thus attempted to further establish the relationship between the ability of compounds to affect hepatic cytochrome P-450 and to affect hepatic haem metabolism in vivo, using the three anaesthetic agents, fluroxene, halothane and trichloroethylene. During the preparation of this thesis, several other chemicals have been found which destroy cytochrome P-450 and affect hepatic haem metabolism (e.g. norethisterone, morphine). In addition to the above, it has been attempted to clarify the roles of the degradation of different forms of cytochrome P-450 and of the different mechanisms of destruction of cytochrome P-450 in the control of hepatic haem metabolism. The three anaesthetic agents, fluroxene, halothane and trichloroethylene were chosen for study since they destroy cytochrome P-450 by apparently different mechanisms. Both fluroxene and trichloroethylene specifically degrade the haem moiety of different forms of cytochrome P-450, but fluroxene converts the haem moiety of cytochrome P-450 to an N-substituted porphyrin, while TCE apparently degrades the haem into uncoloured products. In contrast, halothane appears to degrade the haem of cytochrome P-450 to uncoloured products as well as to facilitate the dissociation of haem from intact cytochrome P-450.
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The role of the chicken gonadotropin-releasing hormone receptor C-terminal tail in expression and couplingLopes, John January 2000 (has links)
The role of the carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor was determined by testing the activity of a series of chicken gonadotropinreleasing hormone receptors with progressive deletions in their carboxyl terminus. The 55 amino acid carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor was progressively truncated, resulting in cS320STOP, cR330STOP, cS337STOP, cS346STOP, cT35ISTOP, cD356STOP, cS366STOP and cC375STOP truncated mutants, which were all tested in parallel with the wild type chicken gonadotropin-releasing hormone receptor. Truncation of the entire carboxy terminal tail from the chicken gonadotropin-releasing hormone receptor, cS320STOP abolished gonadotropin-releasing hormone binding and gonadotropin-releasing hormone-induced inositol phosphate production. The loss of gonadotropin-releasing hormone binding by the cS320STOP-truncated mutant suggests that this receptor is possibly not expressed on the cell membrane, which might be due to improper receptor folding by cS320STOP. The carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor might therefore be required for proper folding of newly formed chicken gonadotropin-releasing hormone receptors and expression of these receptors on the cell membrane. The cR330STOP mutant had a maximal gonadotropin-releasing hormone binding of ~12%, which is the lowest receptor expression detected. The amino acid region between P³¹⁹ and L³²⁹ might therefore play a role in receptor expression. Progressive increase in the carboxy terminal tail from L³²⁹ resulted in progressive increase in the receptor expression. Maximal gonadotropin-releasing hormone binding levels reached wild type levels at truncation of the cGnRHR at S³⁶⁶. These results indicate that the first 45 amino region, ie. between P³¹⁹ and S³⁶⁶ of the chicken gonadotropin-releasing hormone receptor carboxy terminal tail contains elements that promote receptor expression. Gonadotropin-releasing hormone-induced inositol phosphate production was enhanced for all the truncated receptors except cR330STOP and cS337STOP, though all the truncated receptors had coupling efficiency values larger than the wild type chicken gonadotropin-releasing hormone receptor. This enhanced inositol phosphate production might be due to an increased coupling efficiency between the truncated chicken gonadotropin-releasing hormone receptors and the aq111-type G-protein. However, none of the truncated chicken gonadotropin-releasing hormone receptors have left-shifted EC50 values, indicating that coupling efficiency did not increase. Alternatively, a loss or retardation in receptor desensitization and/ or internalization for the truncated chicken gonadotropin-releasing hormone receptor mutants might be responsible for the enhanced gonadotropin-releasing hormone-induced inositol phosphate production by the truncated chicken gonadotropin-releasing hormone receptors. The chicken gonadotropin-releasing hormone receptor has a highly conserved cysteine residue in position 328 that might be palmitoylated. Replacing this cysteine in the chicken gonadotropin-releasing hormone receptor with an alanine [cC328A] increased receptor expression 2 fold, reduced maximal inositol phosphate production to ~69% and severely impaired coupling efficiency to 30% relative to the wild type levels. This finding indicates that C³²⁸ might be palmitoylated and is required for receptor coupling. In conclusion, the ammo terminal region of the chicken gonadotropin-releasing hormone receptor carboxy terminal tail increases receptor expression, either by affecting the transport of newly synthesized chicken gonadotropin-releasing hormone receptors to the plasma membrane and/or the proper folding of this receptor. The intracellular carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor might play a negative role in G-protein coupling. However, the enhanced inositol phosphate production from the truncated chicken gonadotropin-releasing hormone receptors could be due to reduced internalization and/ or desensitization of the carboxy terminal truncated receptors. Point-mutation of C³²⁸ to A resulted in decreased coupling suggesting that C³²⁸ may be a palmitoylation site and might play a role in coupling or desensitization.
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Transcriptional regulation of the human alpha 2(I) procollagen geneLeaner, Virna Drucille January 1997 (has links)
The objective of this study was to investigate the cell- and species-specific regulation of the α2(1) pro collagen gene by analysing trans-acting factor interactions within the proximal promoter of the gene and to identify the genes coding for these trans-acting factors. α2(1) procollagen gene expression was examined in a number of diff erentiate<;l cell lines and shown to differ significantly between normal fibroblasts (WI-38, FG₀), transformed fibroblasts (CT-1, SVWI-38), HT1080 fibrosarcoma, HepG2 hepatocellular carcinoma, L77 lymphoblasts and breast cancer epithelial cells (MDA-MB-231, ZR-75-2). These differences were due to changes in transcription of the α2(1) procollagen gene as shown by Northern blot analysis and nuclear runon transcription experiments . Analysis of DNA-protein interactions with the proximal α2(1) procollagen promoter showed the presence of at least two DNA-protein complexes (complexes I and III) in collagen producing cell lines, while cells where collagen synthesis did not occur contained a third DNA-protein complex (complex II). α2(1) procollagen gene expression was therefore shown to be associated with the presence of complexes I and III while repression of the gene was associated with the presence of complexes I and II and the partial or complete absence of complex III. Complex I is a ubiquitous factor which binds the inverted CCAAT box located between -92 and -80 (G/CBE) with an apparent Kd of 2.9nM. Complexes II and III both bind an adjacent DNA sequence between -78 and -67 (the CME) with Kd values of 4.2 and 3.5nM respectively. While the CCAA T boxes in the human and mouse promoters are identical, a 3bp mismatch was detected in the CME. This mismatch abolished the formation of complex II and III on the mouse promoter, even though mouse cells contained complex II proteins. The difference in the CME binding site between rodent and human promoters implied species-specific regulation of the α2(1) procollagen gene. Transfection of human and mouse proximal α2(1) procollagen promoter/CAT constructs into human cells (CT-1) indicated that the human promoter had higher activity than the mouse promoter, whilst the two promoters had equivalent activities in rodent cells. These promoter activities may be accounted for by the differences in trans-acting factor binding to the two promoters. Complex I formation was competed out by the mouse CBF and NF-Y consensus oligonucleotides, while the mouse anti-CBF-B antibody resulted in a supershifted complex I. These results indicate that complex I is a member of the heterologous CCAAT-binding proteins and possibly related to or similar to the mouse CBF. The treatment of nuclear extracts with calf intestinal phosphatase resulted in a loss of complex I formation on the human and CBF binding to the mouse promoters. The Ser/Thr phosphatase, PP2A, specifically inhibited complexes II and ill formation. Nuclear extracts from CT-1 and U937 cell lines treated with the kinase inhibitor, staurosporin, was accompanied by a loss in DNA-protein interaction. This inhibition of DNA-binding activity was not observed using the tyrosine kinase inhibitor, genistein, and the PP2A phosphatase inhibitor, okadaic acid. Staurosporin also had a significant inhibitory effect on α2(1) procollagen promoter activity in CT-1 cells transfected with the human proximal α2(1) procollagen promoter and on steady state collagen mRNA levels. These results indicate that phosphorylation is required for the binding of trans-acting factors to the proximal α2(1) procollagen promoter and in transcriptional regulation of this gene. In support of the suggestion that phosphorylation events play a role in transcriptional regulation of the α2(1) procollagen gene, CT-1 cells treated with the protein kinase C activator, PMA, showed a significant reduction in α2(1) procollagen mRNA levels. A lambda gt11 expression library was screened to obtain cDNA's encoding proteins that bind the CME in the human α2(1) proximal promoter. A cDNA clone of 958 bp with a predicted open reading frame of 116 amino acids (12.5kD) was obtained. No significant DNA or polypeptide sequence homologies existed in the databank, indicating the possibility of a novel trans-acting factor. Binding of this fusion protein was specific for the CME as observed in South Western blotting and gel shift assays using competitor DNA sequences. Northern blot analysis detected a mRNA transcript of approximately 4kb predominantly in cells where α2(1) procollagen expression is repressed.
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Lipoprotein receptors in cultured bovine endothelial cellsStrumpfer, A E M January 1983 (has links)
Endothelial cells take up and degrade both low density lipoproteins and low density lipoproteins which have been modified by acetylation (AcLDL). In this study, receptors that may be involved in the uptake of these lipoproteins were characterized. The cells used were aortic endothelial cells obtained from a bovine foetus, with subsequent cloning (A₃Cl₂). A cell culture system which closely resembled the in vivo monolayer was established, by growing the cells on gelatin-coated Petri dishes. Endothelial cell and lipoprotein interactions were examined by incubating the cells with ¹²⁵I-labelled lipoproteins under various conditions. The main findings were the following: The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. The half-maximal rates of degradation, obtained from degradation saturation curves which were linearized using the Scatchard method, were about 20 μg protein/ml for LDL and about 2 μg protein/ml for AcLDL. Analyses of binding data were not accurate due to the large amount of non-saturable material bound. However, the bulk of the lipoproteins was taken up and degraded via the saturable process. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Sparse cultures incubated at low lipoprotein concentrations (10-20 μg protein/ml) had a higher receptor activity for LDL than for AcLDL. In contrast, confluent cultures, catabolized more AcLDL than LDL. In comparing sparse to confluent cell cultures, the rate of ¹²⁵1- labelled LDL degradation decreased about twice, while the degradation rates of ¹²⁵I-labelled AcLDL increased about three times. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Up-regulation was measured by pre-incubation of the cells with lipoprotein-deficient serum medium (LPDS-medium) for 48 h. Using degradation data, the LDL receptor was up-regulated about 4-fold, whereas the AcLDL receptor was not up-regulated under these circumstances. Down-regulation by incubating the cells with 25-hydroxycholesterol for 24 h resulted in a 96 % decrease in the LDL receptor activity and only a 30 % decrease in the AcLDL receptor activity. Furthermore, both LDL and AcLDL could down-regulate the LDL receptor, but neither could down-regulate the AcLDL receptor. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased (by about 50 %). However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form of cholesteryl esters.
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The effect of inhibiting KPNB1-mediated nuclear import on cancer cell biology and inflammatory transcription factor signallingStelma, Tamara January 2018 (has links)
Cancer remains one of the major causes of morbidity and mortality globally. Many novel and innovative approaches have been employed to develop new chemotherapeutic strategies, of which targeted therapies aim to identify a molecular lesion or dysregulated pathway that cancer cells are dependent on. Research in our laboratory and others identified the nuclear import protein, Karyopherin β1 (KPNB1), to be overexpressed in various cancers and that inhibiting its expression blocks the proliferation of cancer cells. However, little is known about the potential role of KPNB1 in other cancer cell phenotypes and inflammatory signalling pathways. The aim of this study was to investigate the anticancer and anti-inflammatory effects of inhibiting nuclear import via KPNB1 and to characterise the in vivo effect of the small molecule inhibitor of nuclear import, INI-43, on tumour formation. Using siRNA and a small molecule inhibitor, INI-43, to inhibit KPNB1 we found that cervical cancer cell migration and invasion was significantly reduced. The reduced motility of cancer cells was associated with a decrease in MMP-2 and -9 expression and an increase in TIMP-1 and -2 expression following INI-43 treatment. This corresponded with a decrease in MMP-9 gelatinase activity in KPNB1-inhibited cervical cancer cells. Extended periods of KPNB1 inhibition lead to decreased proliferation and apoptosis. These changes in cancer cell biology when KPNB1 is inhibited may in part be due to its function as a nuclear transporter of transcription factors associated with cancer cell proliferation, migration and invasion. We therefore investigated the effects of KPNB1 inhibition on the nuclear localisation and transcriptional activity of key transcription factors; NFkB and AP-1, both having been implicated in many of the hallmarks of cancer. Immunofluorescent analysis and nuclear/cytoplasmic fractionation assays showed that KPNB1 inhibition blocked the nuclear localisation of NFkB. Electromobility shift assays confirmed a reduced NFkB binding to an NFkB DNA-binding sequence in the nuclear extract of KPNB1-inhibited cells. Luciferase reporter assays containing NFkB and/or AP-1 consensus binding sites showed reduced transcriptional activity for both transcription factors following KPNB1 inhibition. Associated with these changes in NFkB and AP-1 activity was reduced inflammatory cytokines; IL-6, IL-1β, TNF-α and GM-CSF target gene expression. To further characterise the role of INI-43 as a potential chemotherapeutic, the effects on tumour growth and development were investigated in an ectopic xenograft mouse model. INI-43 treatment significantly reduced tumour growth in mice and associated with the redistribution and reduction in KPNB1 levels. INI-43 treated tumours also showed altered morphological features including; better tissue differentiation and reduced inflammatory stromal infiltration, as well as reduced Ki-67 expression. The expression of extracellular matrix components and the cytoskeletal structure of cancer cells was analysed to further investigate the role of KPNB1 inhibition in tumour development. Inhibition of KPNB1 in cancer cells caused reduced expression of both collagen type IV and MMP-9. The redistribution of B-catenin and F-actin suggested that INI-43 treatment caused a loss of mesenchymal features required for tumour progression. The nuclear transport system has been of particular interest in recent years for the development of targeted anticancer drugs. However, most studies have focused on nuclear export inhibitors with little known on the potential of nuclear import inhibitors as anticancer drugs. This study provides evidence that inhibiting the nuclear import protein, KPNB1, has anti-inflammatory and anticancer effects and shows promise as an anticancer approach requiring further investigation.
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Natural product derivative activates autophagy in cancer cellsAndong Koung Edzidzi, Ursula-Claire January 2016 (has links)
Artemisinin, a natural product and its derivatives are potent antimalarial compounds, which have shown anticancer activity. In this study, we further characterized a novel artemisinin derivative namely EXP57EA which was previously designed and synthesized by the Chemistry Department at the University Of Cape Town. We determined the effect of EXP57EA on a panel of cancer cell lines, characterized the mode of cell death and also performed preliminary investigations of the signaling pathways that trigger the mode of cell death. Dihydroartemisinin (DHA), EXP57EA and cisplatin were screened on a selected panel of cancer cell lines: 3 esophageal cancer cell lines WHCO1, WHCO5, KYSE150; one breast cancer cell line MDA-MB-231 and one cervical cancer cell line SiHa. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5- diphenyltetrazolium bromide (MTT) assay, and analysis with GraphPad prism software were used to calculate IC₅₀ values. EXP57EA displayed toxicity in the panel of cancer cell lines studied, and had lower IC₅₀ values (IC₅₀ values were ranging from 15.8 μM to 25.1 μM) than DHA and cisplatin. DHA was only active in two cells lines: WHCO1 (21.3 μM) and WHCO5 (77.3 μM), IC₅₀ values of cisplatin were ranging from 31.2 μM to 108.1 μM. EXP57EA was further investigated to understand the mode of cell death activated in the panel of cancer cell lines. The results showed that EXP57EA did not induce apoptosis in any of the cell lines studied, whereas DHA induced apoptosis, based on the PARP cleavage assay. In contrast, treatment with EXP57EA induced the appearance of vacuoles in treated cells compared to untreated cells, which was suggestive of autophagy. Autophagy was monitored by analyzing the expression level of two autophagy markers, Beclin1 and LC3-II by western blot. It was observed that EXP57EA treatment caused changes in the expression levels of both Beclin1 and LC3-II. We showed that EXP57EA induced elevated levels of autophagy, based on an increase in the flux of autophagy in the treated cells, since the lysosomal inhibitors ammonium chloride (NH₄Cl) and chloroquine substantially blocked LC3-II turnover in WHCO1 (confirmed previous result in our laboratory) and SiHa cancer cell lines. Furthermore, we also showed that treatment with EXP57EA resulted in increased expression of CHOP (by Real-Time PCR), and activated the PERK/eIF2α pathway, since treatment of WHCO1 cells with EXP57EA stimulated phosphorylation of eIF2α, suggesting that ER stress might be involved in mediating EXP57EA-induced cell death. Our results also suggested that EXP57EA activated the JNK pathway since treatment of WHCO1 and WHCO5 cells with EXP57EA stimulated phosphorylation of cjun and resulted in elevated levels of total c-jun. These results suggested the JNK pathway might also be involved in EXP57EA-induced cell death. However, the proposed involvement of the PERK/eIF2α pathway and the JNK pathway in EXP57EA-mediated autophagy is of a preliminary nature, and further work will have to be done to confirm the involvement of these pathways. This study showed that EXP57EA may have potential as an anticancer drug lead.
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