Regulation of autophagy by polyphenolic compounds as a potential therapeutic strategy for cancer

Autophagy, a lysosomal degradation pathway for cellular constituents and organelles, is an adaptive and essential process required for cellular homeostasis. Although autophagy functions as a survival mechanism in response to cellular stressors such as nutrient or growth factor deprivation, it can also lead to a non-apoptotic form of programmed cell death (PCD) called autophagy-induced cell death or autophagy-associated cell death (type II PCD). Current evidence suggests that cell death through autophagy can be induced as an alternative to apoptosis (type I PCD), with therapeutic purpose in cancer cells that are resistant to apoptosis. Thus, modulating autophagy is of great interest in cancer research and therapy. Natural polyphenolic compounds that are present in our diet, such as rottlerin, genistein, quercetin, curcumin, and resveratrol, can trigger type II PCD via various mechanisms through the canonical (Beclin-1 dependent) and non-canonical (Beclin-1 independent) routes of autophagy. The capacity of these compounds to provide a means of cancer cell death that enhances the effects of standard therapies should be taken into consideration for designing novel therapeutic strategies. This review focuses on the autophagy- and cell death-inducing effects of these polyphenolic compounds in cancer.     

Ribozymes in the age of molecular therapeutics

Ribozymes are RNA molecules capable of sequence-specific cleavage of other RNA molecules. Since the discovery of the first group I intron ribozyme in 1982, new classes of ribozymes, each with their own unique reaction, target site specifications, and potential applications, have been identified. These include hammerhead, hairpin, hepatitis delta, varkud satellite, groups I and II intron, and RNase P ribozymes, as well as the ribosome and spliceosome. Meanwhile, ribozyme engineering has enabled the in vitro selection of synthetic ribozymes with unique properties. This, along with advances in ribozyme delivery methods and expression systems, has led to an explosion in the potential therapeutic applications of ribozymes, whether for anti-cancer or anti-viral therapy, or for gene repair.     

High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor

Previous studies have demonstrated that phenolic compounds, including genistein (4',5,7-trihydroxyisoflavone) and resveratrol (3,4',5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in phi X-174 plasmid DNA. H(2)O(2)/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H(2)O(2)/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H(2)O(2) suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H(2)O(2)/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H(2)O(2) were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Cellular stress responses and cancer: new mechanistic insights on anticancer effect by phytochemicals

During the tumorigenesis, cancer cells are frequently exposed to metabolic stress which is derived from altered cancer cell metabolism as well as unfavorable tumor microenvironment, such as hypoxia and glucose deprivation. Cancer cells need to respond to these stress stimuli properly through inducing cellular stress responses, such as unfolded protein response and autophagy, for cell survival. Therefore, modulation of these stress responses has been investigated as an alternative anticancer strategy, although their therapeutic clinical roles remain to be determined. In this review, we will discuss the cellular stress responses in cancer cells, the alternative anticancer strategy targeting unfolded protein response and/or autophagy, and the role of phytochemicals, which include resveratrol, genistein, curcumin, epigallocatechin-3-gallate and quercetin, in modulating the cellular stress responses.     

Nucleolar introns from Physarum flavicomum contain insertion elements that may explain how mobile group I introns gained their open reading frames.

Comparison of two group I intron sequences in the nucleolar genome of the myxomycete Physarum flavicomum to their homologs in the closely related Physarum polycephalum revealed insertion-like elements. One of the insertion-like elements consists of two repetitive sequence motifs of 11 and 101 bp in five and three copies, respectively. The smaller motif, which flanks the larger, resembles a target duplication and indicates a relationship to transposons or retroelements. The insertion-like elements are found in the peripheral loops of the RNA structure; the positions occupied by the ORFs of mobile nucleolar group I introns. The P. flavicomum introns are 1184 and 637 bp in size, located in the large subunit ribosomal RNA gene, and can be folded into group I intron structures at the RNA level. However, the intron 2s from both P. flavicomum and P. polycephalum contain an unusual core region that lacks the P8 segment. None of the introns are able to self-splice in vitro. Southern analysis of different isolates indicates that the introns are not optional in myxomycetes.     

Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H₂O₂/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H₂O₂/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H₂O₂ suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H₂O₂/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H₂O₂ were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.     

Assaying RNA chaperone activity in vivo in bacteria using a ribozyme folding trap

Here, we report an assay to evaluate the intracellular RNA chaperone activity of a protein of interest in vivo in bacterial cells. The method is based on self-splicing of the group I intron, which is located in the thymidylate synthase (td) gene of phage T4. A previously described td mutant (tdSH1) has significantly impaired splicing due to formation of splicing-incompetent alternative structures. In this procedure, overexpression of RNA chaperones in the presence of the td mutant SH1 is used to evaluate whether the putative RNA chaperone is able to rescue the incorrectly folded group I intron. The ability of the RNA chaperone to assist during folding is measured indirectly by assessing the difference between the splicing efficiencies of the td mutant in the absence and in the presence of the RNA chaperone. This procedure can be completed in 5-6 d, not including the time needed to clone the putative RNA chaperone.     

A maturase that specifically stabilizes and activates its cognate group I intron at high temperatures

Folding of large structured RNAs into their functional tertiary structures at high temperatures is challenging. Here we show that I-TnaI protein, a small LAGLIDADG homing endonuclease encoded by a group I intron from a hyperthermophilic bacterium, acts as a maturase that is essential for the catalytic activity of this intron at high temperatures and physiological cationic conditions. I-TnaI specifically binds to and induces tertiary packing of the P4-P6 domain of the intron; this RNA-protein complex might serve as a thermostable platform for active folding of the entire intron. Interestingly, the binding affinity of I-TnaI to its cognate intron RNA largely increases with temperature; over 30-fold stronger binding at higher temperatures relative to 37 °C correlates with a switch from an entropy-driven (37 °C) to an enthalpy-driven (55-60 °C) interaction mode. This binding mode may represent a novel strategy how an RNA binding protein can promote the function of its target RNA specifically at high temperatures.     

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.     

Predominance of spliceosomal complex formation over polyadenylation site selection in TDP-43 autoregulation

TDP-43 is a nuclear protein involved in many aspects of RNA metabolism. To ensure cellular viability, its expression levels within cells must be tightly regulated. We have previously demonstrated that TDP-43 autoregulation occurs through the activation of a normally silent intron in its 3′-UTR sequence that results in the use of alternative polyadenylation sites. In this work, we analyse which is the dominant event in autoregulation: the recognition of the splice sites of 3′-UTR intron 7 or the intrinsic quality of the alternative polyadenylation sites. A panel of minigene constructs was tested for autoregulation functionality, protein production and subcellular messenger RNA localization. Our data clearly indicate that constitutive spliceosome complex formation across intron 7 does not lead to high protein production but, on the contrary, to lower TDP-43 messenger RNA and protein levels. This is due to altered nucleocytoplasmic distribution of the RNA that is mostly retained in the nucleus and degraded. This study provides a novel in-depth characterization of how RNA binding proteins can autoregulate their own levels within cells, an essential regulatory process in maintaining cellular viability.     

CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains

Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.     

Two distinct binding modes of a protein cofactor with its target RNA

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions.     

Effects of endocrine disruptors on genes associated with 17beta-estradiol metabolism and excretion

In order to provide a global analysis of the effects of endocrine disruptors on the hormone cellular bioavailability, we combined 17beta-estradiol (E2) cellular flow studies with real-time PCR and Western blot expression measurements of genes involved in the hormone metabolism and excretion. Three endocrine disruptors commonly found in food were chosen for this study, which was conducted in the estrogen receptor (ER) negative hepatoblastoma HepG2 cell line: bisphenol A (BPA), genistein (GEN) and resveratrol (RES). We showed that 24h after a single dose treatment with genistein, resveratrol or bisphenol A, the expression of ATP-binding cassette transporters (the multidrug resistance or MDR, and the multidrug resistance associated proteins or MRP) uridine diphosphate-glucuronosyltransferases (UGT) and/or sulfotransferases (ST) involved in 17beta-estradiol elimination process were significantly modulated and that 17beta-estradiol cellular flow was modified. Resveratrol induced MDR1 and MRP3 expressions, bisphenol A induced MRP2 and MRP3 expressions, and both enhanced 17beta-estradiol efflux. Genistein, on the other hand, inhibited ST1E1 and UGT1A1 expressions, and led to 17beta-estradiol cellular retention. Thus, we demonstrate that bisphenol A, genistein and resveratrol modulate 17beta-estradiol cellular bioavailability in HepG2 and that these modulations most probably involve regulations of 17beta-estradiol phase II and III metabolism proteins. Up to now, the estrogenicity of environmental estrogenic pollutants has been based on the property of these compounds to bind to ERs. Our results obtained with ER negative cells provide strong evidence for the existence of ER-independent pathways leading to endocrine disruption.     

Binding enhancement by tertiary interactions and suicide inhibition of a Candida albicans group I intron by phosphoramidate and 2'-O-methyl hexanucleotides

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.     

Binding analysis of antioxidant polyphenols with PAMAM nanoparticles

Dietary polyphenols are abundant micronutrients in our diet and paly major role in prevention of degenerative diseases. The binding efficacy of antioxidant polyphenols resveratrol, genistein, and curcumin with PAMAM-G3 and PAMAM-G4 nanoparticles was investigated in aqueous solution at physiological conditions, using multiple spectroscopic methods, TEM images, and docking studies. The polyphenol bindings are via hydrophilic, hydrophobic, and H-bonding contacts with resveratrol forming more stable conjugates. As PAMAM size increased the loading efficacy and the stability of polyphenol-polymer conjugates were increased. Polyphenol binding induced major alterations of dendrimer morphology. PAMAM nanoparticles are capable of delivery of polyphenols in vitro.     

NAR Breakthrough Article: Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.