Browsing by Author "Ngarande, Ellen"

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    Open Access
    Overexpression of Kpnβ1 and Kpnα2 Importin Proteins in Cancer Derives from Deregulated E2F Activity
    (Public Library of Science, 2011) van der Watt, Pauline J; Ngarande, Ellen; Leaner, Virna D
    The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin β1 (Kpnβ1) and Karyopherin α2 (Kpnα2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnβ1 and Kpnα2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpnβ1 and Kpnα2 levels in cancer cells. Kpnβ1 (−2013 to +100) and Kpnα2 (−1900 to +69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the −637 to −271 Kpnβ1 and −180 to −24 Kpnα2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpnβ1 and Kpnα2 promoters in vivo . Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnβ1 and Kpnα2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpnβ1 and Kpnα2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnβ1 and Kpnα2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activities.
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    Sustained hydrogel-based delivery of RNA interference nanocomplexes for gene knockdown
    (2019) Ngarande, Ellen; Davies, Neil; Bezuidenhout Deon
    Scaffold based delivery of RNA interference (RNAi) molecules such as free small interfering RNA (siRNA) and microRNA has recently begun to be employed towards treatment of diseases such as cancer, bone regeneration, muscular dystrophy and cardiovascular disease. Effective translation from bench side to clinical use of RNAi has been limited in part because upon systemic delivery the RNAi molecules are degraded by RNases and flushed by excretory organs causing an inefficient duration of gene silencing effect at target tissues. These challenges can potentially be minimised by delivering RNAi molecules via non-viral nanoparticle carriers encapsulated in biocompatible, biodegradable and injectable scaffolds such as hydrogels. Various scaffolds have been shown to aid in sustained localised delivery of RNAi molecules and improve gene silencing. This research focused on optimising and establishing such an RNAi hydrogel-siRNA-nanoparticle (hydrogel-nanocomplex) system for targeted and sustained gene knockdown both in vitro and in vivo using dendrimer and lipid based nanoparticles in combination with synthetic polyethylene glycol (PEG) and natural fibrin hydrogel scaffolds. Four siRNA nanocarriers were investigated for siRNA delivery, that is, fourth generation dendrimer nanoparticles poly(amidoamine) (D) and its modified version (MD) with PEG and a lipid 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) molecule, commercial lipid based Lipofectamine® RNAiMax and Invivofectamine® 3.0 nanoparticles. D and MD achieved better RNase protection compared to lipid nanocomplexes though Invivofectamine® 3.0 nanocomplexes protected a small percentage of siRNA over 10 days. The MD nanoparticle displayed improved siRNA release and transfection efficacy compared to D but efficacy of the dendrimers was lower than the lipid particles. Four hydrogels that have not been investigated for RNAi were assessed for sustainability. Namely, hydrolytically and proteolytically degradable PEG-acrylate (PEGAC), proteolytically degradable PEG - vinyl sulfone (PEG-VS) hydrogels, unmodified fibrin and PEGylated fibrin hydrogel. The nanocomplex release rate in vitro from the various hydrogels showed minimal release from PEGylated hydrogels, burst release from unmodified fibrin and sustained release from PEGylated fibrin. Invivofectamine® 3.0 nanocomplexes retained efficacy optimally after release from PEGylated fibrin hence this hydrogel was utilised for downstream analysis. For in vivo sustained delivery to be effective, determination of hydrogel persistence in vivo was required. After injection in the mouse tibialis anterior (TA) muscle PEG-AC and PEGylated fibrin gels degraded within 2 days. The efficacy of the various nanocomplexes was assayed in a 3D assay that more closely resembled delivery in soft tissue. PEGylated fibrin containing nanocomplexes with cell death siRNA sequences was polymerised around a preformed PEGylated fibrin cell containing droplet. Invivofectamine® 3.0 nanocomplex consistently achieved the highest gene knockdown effect with no evidence of cytotoxicity whilst Lipofectamine® RNAiMax was ineffective. MD showed signs of cytotoxicity when delivered in a sustained fashion. Thus Invivofectamine® 3.0 nanocomplexes in PEGylated fibrin hydrogel were found to be the optimal gel-nanocomplex system to proceed to in vivo assessment. BALB/c GFP transgenic injected in their TA muscle with Invivofectamine® 3.0 nanocomplexes made with siRNA targeting GFP or myostatin (siGFP/siMSTN) in the presence or absence of PEGylated fibrin gel were analysed 7 days post treatment for siRNA retention and GFP and Mstn gene knockdown. Increased retention of siRNA after encapsulation in PEGylated fibrin was observed at 7 days. A non-significant reduction in GFP protein was seen for limbs injected with siGFP- fibrin after 7 days. A substantial and significant reduction in Mstn mRNA levels was elicited by delivery of siMstn–fibrin. Furthermore, only siMstn-fibrin resulted in significant increase in muscle mass. In this study, dendrimer based nanoparticles were found to effectively protect siRNA against RNases however lipid based nanocomplexes were the most efficacious at gene knockdown. The combination of Invivofectamine® 3.0 and PEGylated fibrin was shown to be the most effective in 3D assays and as an injectable controlled release scaffold into soft tissue suggesting that this approach has therapeutic potential.
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    The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells
    (2019-03-20) Kaschula, Catherine H; Tuveri, Rosanna; Ngarande, Ellen; Dzobo, Kevin; Barnett, Christopher; Kusza, Daniel A; Graham, Lisa M; Katz, Arieh A; Rafudeen, Mohamed S; Parker, M Iqba; Hunter, Roger; Schäfer, Georgia
    Background Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S-thiolating reactive cysteines in target proteins. Methods Using our fluorescently labelled ajoene analogue called dansyl-ajoene, ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to ajoene was performed using Schrödinger and pKa calculations by Epik software. Cytotoxicity of ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. Results The dominant protein tagged by dansyl-ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that ajoene and dansyl-ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by ajoene. Conclusions The garlic-derived phytochemical ajoene targets and covalently modifies vimentin in cancer cells by S-thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of ajoene in cancer cells.