Antiproliferative activities of some selected Nigerian medicinal plants against breast, liver, and cervical cancer cells.

BACKGROUND: Phytochemicals have become a growing source of alternative medicine in developing countries due to the poor prognosis, high cost of conventional pharmaceuticals, and undesirable effects associated with mainstream cancer treatment. OBJECTIVE: This study was aimed at investigating the anticancer effect of some selected Nigerian medicinal plants used in cancer treatment. These include ethanol extracts of Dialium guineense root (DGR), Dialium guineense leaves (DGL), Jateorhiza macrantha leaves (JML), Musanga cecropioides leaves (MCL), Musanga cecropioides stembark (MCSB), Piptadeniastrum africanum stembark (PASB), Piptadeniastrum africanum root (PAR), Pupalia lappacea flower tops (PLF), Raphiostylis beninensis root (RBR), Raphiostylis beninensis leaves (RBL), Ritchiea capparoides leaves (RCL), Ritchiea capparoides stembark (RCSB), and Triplochiton scleroxylon stembark (TSB). METHODS: The cytotoxic activity of the extracts was examined using a brine shrimp lethality assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against three cancer cell lines, including MCF-7, HUH-7, and HeLa. The selectivity of all extracts towards cancer cells was investigated using normal lung fibroblasts (MRC-5). Cell migration and colony-forming assays of active extracts against MCF-7 cells were also performed. Additionally, the total polyphenolic contents of the active extracts were estimated using standard methods. RESULTS: The extract of PASB had the highest cytotoxicity (LC50 = 1.58 µg/mL) on the brine shrimps compared to vincristine sulphate (LC50 = 2.24 µg/mL). In the cell viability assay, all the extracts produced significant (p < 0.05) growth inhibitory effects against all cell lines tested in a dose-dependent manner. All extracts were selective to cancer cells at varying degrees. Worth mentioning are the extracts of MCL, DGR, RBR, and PASB, which exhibited 14-, 7-, 6- and 2-fold selectivity toward MCF-7 cancer cells relative to normal lung fibroblast (MRC-5), respectively. These four extracts also significantly inhibited cell migration and colony formation in MCF-7-treated cells in dose-dependent manners. Considerable amounts of phenolics, flavonoids, and proanthocyanidins were detected in all extracts evaluated. CONCLUSION: These findings advocate the continued development of MCL, DGR, RBR, and PASB as potential chemotherapeutic agents.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Investigating histidinylated highly branched poly(lysine) for siRNA delivery.

The temporary silencing of disease-associated genes utilising short interfering RNA (siRNA) is a potent and selective route for addressing a wide range of life limiting disorders. However, the few clinically approved siRNA therapies rely on lipid based formulations, which although potent, provide limited chemical space to tune the stability, efficacy and tissue selectivity. In this study, we investigated the role of molar mass and histidinylation for poly(lysine) based non-viral vectors, synthesised through a fully aqueous thermal condensation polymerisation. Formulation and in vitro studies revealed that higher molar mass derivatives yielded smaller polyplexes attributed to a greater affinity for siRNA at lower N/P ratios yielding greater transfection efficiency, albeit with some cytotoxicity. Histidinylation had a negligible effect on formulation size, yet imparted a moderate improvement in biocompatibility, but did not provide any meaningful improvement over silencing efficiency compared to non-histidinylated derivatives. This was attributed to a greater degree of cellular internalisation for non-histidinylated analogues, which was enhanced with the higher molar mass material.

Investigating the intracellular effects of hyperbranched polycation-DNA complexes on lung cancer cells using LC-MS-based metabolite profiling.

Cationic polymers have emerged as a promising alternative to viral vectors in gene therapy. They are cheap to scale up, easy to functionalise and are potentially safer than viral vectors, however many are cytotoxic. The large number of polycations, designed to address the toxicity problem, raises a practical need to develop a fast and reliable method for assessing the safety of these materials. In this regard, metabolomics provides a detailed and comprehensive method that can assess the potential toxicity at the cellular and molecular level. Here, we applied metabolomics to investigate the impact of hyperbranched polylysine, hyperbranched polylysine-co-histidine and branched polyethyleneimine polyplexes at sub-toxic concentrations on the metabolic pathways of A459 and H1299 lung carcinoma cell lines. The study revealed that the polyplexes downregulated metabolites associated with glycolysis and the TCA cycle, and induced oxidative stress in both cell lines. The relative changes of the metabolites indicated that the polyplexes of polyethyleneimine and hyperbranched polylysine affected the metabolism much more than the polyplexes of hyperbranched polylysine-co-histidine. This was in line with transfection results, suggesting a correlation between the toxicity and transfection efficiency of these polyplexes. Our work highlights the importance of the metabolomics approach not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanisms underlying any adverse effects, which could help in designing more efficient vectors.

Water Solubility Enhancement of Pyrazolo[3,4-d]pyrimidine Derivatives via Miniaturized Polymer-Drug Microarrays.

A miniaturized assay was optimized to evaluate the enhanced apparent water solubility of pyrazolo[3,4-d]pyrimidine derivatives used extensively as anticancer drug scaffolds. The applied amount of drugs used in the reported strategy ranged from 5 to 10 µg per formulation which were dispensed by an inkjet 2D printer directly into a 96-well plate. The selected polymer/drug formulations with high water solubility demonstrated improved cytotoxicity against a human lung adenocarcinoma cancer cell line (A549) compared to the free drugs. We attribute the enhanced efficacy to the improved apparent-solubility of the drug molecules achieved via this methodology. This novel miniaturized method showed promising results in terms of water solubility improvement of the highly hydrophobic pyrazolo[3,4-d]pyrimidine derivatives, requiring only a few micrograms of each drug per tested polymeric formulation. In addition, the reported experimental evidence may facilitate identification of suitable polymers for combination with drug, leading to investigations on biological properties or mechanisms of action in a single formulation.

Bioreducible cross-linked core polymer micelles enhance in vitro activity of methotrexate in breast cancer cells.

Polymer micelles have emerged as promising carriers for controlled release applications, however, several limitations of micelle-based drug delivery have also been reported. To address these issues, we have synthesized a functional biodegradable and cytocompatible block copolymer based on methoxypoly(ethyleneglycol)-b-poly(ε-caprolactone-co-α-azido-ε-caprolactone) (mPEG-b-poly(εCL-co-αN3εCL)) as a precursor of reduction sensitive core-crosslinked micelles. The synthesized polymer was formulated as micelles using a dialysis method and loaded with the anti-inflammatory and anti-cancer drug methotrexate (MTX). The micellar cores were subsequently crosslinked at their pendent azides by a redox-responsive bis(alkyne). The size distributions and morphology of the polymer micelles were assessed using dynamic light scattering (DLS) and transmission electron microscopy, and drug release assays were performed under simplified (serum free) physiological and reductive conditions. Cellular uptake studies in human breast cancer cells were performed using Oregon-green loaded core-crosslinked micelles. The MTX-loaded core-crosslinked micelles were assessed for their effects on metabolic activity in human breast cancer (MCF-7) cells by evaluating the reduction of the dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The apoptosis inducing potential of MTX-loaded core-crosslinked micelles was analysed using Hoechst/propidium iodide (PI) and annexin-V/PI assays. The data from these experiments indicated that drug release from these cross-linked micelles can be controlled and that the redox-responsive micelles are more effective carriers for MTX than non-crosslinked analogues and the free drug in the cell-lines tested.

Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile.

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized. We identify and characterize the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader-helicase-primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.

PRAME is a golgi-targeted protein that associates with the Elongin BC complex and is upregulated by interferon-gamma and bacterial PAMPs.

Preferentially expressed antigen in melanoma (PRAME) has been described as a cancer-testis antigen and is associated with leukaemias and solid tumours. Here we show that PRAME gene transcription in leukaemic cell lines is rapidly induced by exposure of cells to bacterial PAMPs (pathogen associated molecular patterns) in combination with type 2 interferon (IFNγ). Treatment of HL60 cells with lipopolysaccharide or peptidoglycan in combination with IFNγ resulted in a rapid and transient induction of PRAME transcription, and increased association of PRAME transcripts with polysomes. Moreover, treatment with PAMPs/IFNγ also modulated the subcellular localisation of PRAME proteins in HL60 and U937 cells, resulting in targeting of cytoplasmic PRAME to the Golgi. Affinity purification studies revealed that PRAME associates with Elongin B and Elongin C, components of Cullin E3 ubiquitin ligase complexes. This occurs via direct interaction of PRAME with Elongin C, and PRAME colocalises with Elongins in the Golgi after PAMP/IFNγ treatment. PRAME was also found to co-immunoprecipitate core histones, consistent with its partial localisation to the nucleus, and was found to bind directly to histone H3 in vitro. Thus, PRAME is upregulated by signalling pathways that are activated in response to infection/inflammation, and its product may have dual functions as a histone-binding protein, and in directing ubiquitylation of target proteins for processing in the Golgi.

Translational regulation of gene expression during conditions of cell stress.

A number of stresses, including nutrient stress, temperature shock, DNA damage, and hypoxia, can lead to changes in gene expression patterns caused by a general shutdown and reprogramming of protein synthesis. Each of these stress conditions results in selective recruitment of ribosomes to mRNAs whose protein products are required for responding to stress. This recruitment is regulated by elements within the 5' and 3' untranslated regions of mRNAs, including internal ribosome entry segments, upstream open reading frames, and microRNA target sites. These elements can act singly or in combination and are themselves regulated by trans-acting factors. Translational reprogramming can result in increased life span, and conversely, deregulation of these translation pathways is associated with disease including cancer and diabetes.

eIF4A inhibition allows translational regulation of mRNAs encoding proteins involved in Alzheimer's disease.

Alzheimer's disease (AD) is the main cause of dementia in our increasingly aging population. The debilitating cognitive and behavioral symptoms characteristic of AD make it an extremely distressing illness for patients and carers. Although drugs have been developed to treat AD symptoms and to slow disease progression, there is currently no cure. The incidence of AD is predicted to increase to over one hundred million by 2050, placing a heavy burden on communities and economies, and making the development of effective therapies an urgent priority. Two proteins are thought to have major contributory roles in AD: the microtubule associated protein tau, also known as MAPT; and the amyloid-beta peptide (A-beta), a cleavage product of amyloid precursor protein (APP). Oxidative stress is also implicated in AD pathology from an early stage. By targeting eIF4A, an RNA helicase involved in translation initiation, the synthesis of APP and tau, but not neuroprotective proteins, can be simultaneously and specifically reduced, representing a novel avenue for AD intervention. We also show that protection from oxidative stress is increased upon eIF4A inhibition. We demonstrate that the reduction of these proteins is not due to changes in mRNA levels or increased protein degradation, but is a consequence of translational repression conferred by inhibition of the helicase activity of eIF4A. Inhibition of eIF4A selectively and simultaneously modulates the synthesis of proteins involved in Alzheimer's disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective.

Leucine-rich repeat protein PRAME: expression, potential functions and clinical implications for leukaemia.

PRAME/MAPE/OIP4 is a germinal tissue-specific gene that is also expressed at high levels in haematological malignancies and solid tumours. The physiological functions of PRAME in normal and tumour cells are unknown, although a role in the regulation of retinoic acid signalling has been proposed. Sequence homology and structural predictions suggest that PRAME is related to the leucine-rich repeat (LRR) family of proteins, which have diverse functions. Here we review the current knowledge of the structure/function of PRAME and its relevance in leukaemia.

Dysregulation of protein synthesis and disease.

The regulation of protein synthesis plays as important a role as transcriptional control in the control of gene expression. Once thought solely to act globally, translational control has now been shown to be able to control the expression of most genes specifically. Dysregulation of this process is associated with a range of pathological conditions, notably cancer and several neurological disorders, and can occur in many ways. These include alterations in the expression of canonical initiation factors, mutations in regulatory mRNA sequence elements in 5' and 3' untranslated regions (UTRs), such as upstream open reading frames (uORFs), internal ribosome entry segments (IRESs) and micro-RNA (miR) target sites, and the altered expression of trans-acting protein factors that bind to and regulate these elements. Translational control is increasingly open for study in both fresh and fixed tissue, and this rapidly developing field is yielding useful diagnostic and prognostic tools that will hopefully provide new targets for effective treatments.

The human insulin receptor mRNA contains a functional internal ribosome entry segment.

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5'-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective.

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes.

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5' untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.

Canonical initiation factor requirements of the Myc family of internal ribosome entry segments.

Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5' end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5' untranslated region (5'-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5'-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex.

Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein.

PTB (polypyrimidine-tract-binding protein) is a ubiquitous RNA-binding protein. It was originally identified as a protein with a role in splicing but it is now known to function in a large number of diverse cellular processes including polyadenylation, mRNA stability and translation initiation. Specificity of PTB function is achieved by a combination of changes in the cellular localization of this protein (its ability to shuttle from the nucleus to the cytoplasm is tightly controlled) and its interaction with additional proteins. These differences in location and trans-acting factor requirements account for the fact that PTB acts both as a suppressor of splicing and an activator of translation. In the latter case, the role of PTB in translation has been studied extensively and it appears that this protein is required for an alternative form of translation initiation that is mediated by a large RNA structural element termed an IRES (internal ribosome entry site) that allows the synthesis of picornaviral proteins and cellular proteins that function to control cell growth and cell death. In the present review, we discuss how PTB regulates these disparate processes.

The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the target gene.

MicroRNAs (miRNAs) are noncoding RNAs that base pair imperfectly to homologous regions in target mRNAs and negatively influence the synthesis of the corresponding proteins. Repression is mediated by a number of mechanisms, one of which is the direct inhibition of protein synthesis. Surprisingly, previous studies have suggested that two mutually exclusive mechanisms exist, one acting at the initiation phase of protein synthesis and the other at a postinitiation event. Here, we resolve this apparent dichotomy by demonstrating that the promoter used to transcribe the mRNA influences the type of miRNA-mediated translational repression. Transcripts derived from the SV40 promoter that contain let-7 target sites in their 3' UTRs are repressed at the initiation stage of translation, whereas essentially identical mRNAs derived from the TK promoter are repressed at a postinitiation step. We also show that there is a miR-34 target site within the 3' UTR of c-myc mRNA and that promoter dependency is also true for this endogenous 3' UTR. Overall, these data establish a link between the nuclear history of an mRNA and the mechanism of miRNA-mediated translational regulation in the cytoplasm.