Nucleotide Excision repair pathway modulating both cancer risk and therapy

Nukleotid Exzisions Reparatur (NER) spielt eine wichtige Rolle bei der Reparatur vieler verschiedener Arten von DNA-Schäden, dazu gehören auch DNA-Addukte, die durch ultraviolette Strahlung und durch Exposition gegenüber schädlichen Substanzen wie Tabakrauch und Alkohol entstehen. Genetische Variationen und somatische Mutationen in NER-Genen können möglicherweise Auswirkungen auf Krebsrisiko und Therapie haben. Der Kenntnisstand hierüber ist jedoch gering. Der erste Teil der Arbeit untersucht die Rolle von genetischer Variation in NER-Genen bei der Modulation des Kehlkopfkrebs-Risikos. Die wichtigsten Risikofaktoren für Kehlkopfkrebs sind Rauchen und hoher Alkoholkonsum. Polymorphismen in NER-Genen könnten sich daher auf die Anfälligkeit für Kehlkopfkrebs auswirken. In einer populationsbezogenen Fall-Kontroll-Studie mit 248 Fällen und 647 Kontrollen wurden die Assoziationen von Kehlkopfkrebs mit 11 Einzel-Nukleotid-Polymorphismen (SNPs) in 7 NER-Genen (XPC, ERCC1, ERCC2, ERCC4, ERCC5, ERCC6 und RAD23B) unter Berücksichtigung von Rauchen und Alkoholkonsum analysiert. Für die Genotypisierung wurden Sequenz-spezifische Hybridisierungssonden verwendet. Die für Alter und Geschlecht, sowie Rauchen, Alkoholkonsum und Bildung adjustierten Daten wurden mittels logistischer Regressionsanalyse ausgewertet. Träger der Pro-Allele in ERCC6 Arg1230Pro zeigten ein vermindertes Risiko für Kehlkopfkrebs (OR=0,53, 95% KI 0,34 - 0,85), welches bei starken Rauchern und Pro-Allel-Trägern mit hohem Alkoholkonsum am stärksten reduziert war. ERCC5 Asp1104His war mit dem Risiko bei starken Rauchern assoziiert (OR=1,70, 95% KI 1,1 - 2,5). Val-Allel-Träger von RAD23B Ala249Val hatten ein erhöhtes Krebsrisiko, wenn sie starke Raucher waren (OR=1,6, 95% KI 1,1 - 2,5) oder viel Alkohol konsumierten (OR=2,0, 95% KI 1,1 - 3,4). Die kombinierte Wirkung von Rauchen und Alkoholkonsum beeinflusste das Risiko bei starker Exposition und zwar für ERCC6 1230Pro (OR = 0,47, 95% KI 0,22 - 0,98) und RAD23B 249Val (OR=2,6, 95% KI 1,3 - 4,9). Bei der Untersuchung von Gen-Gen-Interaktionen erhöhte die Anwesenheit von 3 Risikoallelen im XPC-RAD23B-komplex das Risiko um das 2,1-fache. SNPs in den anderen Genen zeigten keine signifikante Assoziation mit dem Risiko für Kehlkopfkrebs. Wir schließen daraus, dass häufige genetische Variationen in NER-Genen das Kehlkopfkrebs-Risiko erheblich beeinflussen können. Der zweite Teil der Arbeit hat die Frage zum Thema, wie sich NER-Defizite auf den Erfolg einer Krebstherapie auswirken. NER Gene sind in vielen Krebszellen mutiert. Der Einsatz von Substanzen, die diese Zellen gezielt angreifen, könnte die Therapiewirkung bei gleichzeitiger Schonung des Reparatur-kompetenten Normalgewebes verstärken. Zwei NER-defiziente Zelllinien, XP3BE (XPC-defiziente Zellen) und GM10902 (ERCC6-defiziente Zellen) sowie eine normale Zelllinie GM01310 wurden mit 72 Substanzen aus der traditionellen chinesischen Medizin (TCM) behandelt. Ein Screening ergab, dass 13 dieser Substanzen in NER-defizienten Zellen deutlich zytotoxischer wirken als in normalen Zellen. Abhängig von ihrer Wirkung sowie der verfügbaren Menge wurden 6 dieser Substanzen zur weiteren Analyse ausgewählt, um ihre IC50 Werte, Auswirkungen auf den Zellzyklus und ihre Fähigkeit zur Induktion von DNA-Schäden zu bestimmen. Die wirksamste Substanz war Ascaridol, mit einer über 1000-fach höheren Resistenz von normalen gegenüber NER-defizienten Zellen (IC50 für GM10902 0,025 myg / ml, XP3BE 0,03 myg / ml und GM01310 > 30 myg / ml). Diese Substanz hatte starke und unterschiedliche Auswirkungen auf die Zellzyklus Verteilung der drei Zelllinien. Ascaridol verursachte etwa drei mal mehr DNA-Schäden in NER-defizienten Zellen im Vergleich zu normalen Zellen. Die Ergebnisse für Ascaridol wurden in einem zweiten Set von isogenen Zelllinien validiert. XPC-defiziente (XP4PA; IC50 0,013 myg / ml) und mit ERCC6-siRNA-behandelte Zellen (IC50 0,095 myg / ml) waren empfindlicher gegenüber diesem Wirkstoff als XPC-kompetente (XP4PA-SE2; IC50 2,19 myg / ml) und Luciferase-siRNA-behandelte Kontroll-Zellen (IC50 3,0 myg / ml). Nach Ascaridolexposition zeigten XPC-defiziente und ERCC6-siRNA-behandelte Zellen 1,5 bis drei mal mehr DNA-Schäden als Reparatur-kompetente Zellen. Außerdem wurde eine dosisabhängige Erhöhung der intrazellulären Peroxidmenge durch Ascaridol in den Zellen beobachtet. Mit Ascaridol (1 myg / ml) behandelte XPC-defiziente und ERCC6-siRNA-behandelte Zellen zeigten auch einen starken Anstieg in der Menge der oxidierten Basen, der weder in XPC-kompetenten noch in Luciferase-siRNA-behandelten Kontroll-Zellen sichtbar war. Diese Ergebnisse zeigen zum ersten Mal, dass Ascaridol DNA-Schäden über reaktive oxidative Zwischenprodukte verursacht, und dass Ascaridol besonders NER-defiziente Zellen angreift. Dies könnte eine neue therapeutische Option für das selektive Abtöten von Tumorzellen darstellen

Putative second hit rare genetic variants in families with seemingly GBA-associated Parkinson's disease

Rare variants in the beta-glucocerebrosidase gene (GBA1) are common genetic risk factors for alpha synucleinopathy, which often manifests clinically as GBA-associated Parkinson's disease (GBA-PD). Clinically, GBA-PD closely mimics idiopathic PD, but it may present at a younger age and often aggregates in families. Most carriers of GBA variants are, however, asymptomatic. Moreover, symptomatic PD patients without GBA variant have been reported in families with seemingly GBA-PD. These observations obscure the link between GBA variants and PD pathogenesis and point towards a role for unidentified additional genetic and/or environmental risk factors or second hits in GBA-PD. In this study, we explored whether rare genetic variants may be additional risk factors for PD in two families segregating the PD-associated GBA1 variants c.115+1G>A (ClinVar ID: 93445) and p.L444P (ClinVar ID: 4288). Our analysis identified rare genetic variants of the HSP70 co-chaperone DnaJ homolog subfamily B member 6 (DNAJB6) and lysosomal protein prosaposin (PSAP) as additional factors possibly influencing PD risk in the two families. In comparison to the wild-type proteins, variant DNAJB6 and PSAP proteins show altered functions in the context of cellular alpha-synuclein homeostasis when expressed in reporter cells. Furthermore, the segregation pattern of the rare variants in the genes encoding DNAJB6 and PSAP indicated a possible association with PD in the respective families. The occurrence of second hits or additional PD cosegregating rare variants has important implications for genetic counseling in PD families with GBA1 variant carriers and for the selection of PD patients for GBA targeted treatments

Segregation and potential functional impact of a rare stop-gain PABPC4L variant in familial atypical parkinsonism

Atypical parkinsonian disorders (APDs) comprise a group of neurodegenerative diseases with heterogeneous clinical and pathological features. Most APDs are sporadic, but rare familial forms have also been reported. Epidemiological and post-mortem studies associated APDs with oxidative stress and cellular protein aggregates. Identifying molecular mechanisms that translate stress into toxic protein aggregation and neurodegeneration in APDs is an active area of research. Recently, ribonucleic acid (RNA) stress granule (SG) pathways were discussed to be pathogenically relevant in several neurodegenerative disorders including APDs. Using whole genome sequencing, mRNA expression analysis, transfection assays and cell imaging, we investigated the genetic and molecular basis of a familial neurodegenerative atypical parkinsonian disorder. We investigated a family with six living members in two generations exhibiting clinical symptoms consistent with atypical parkinsonism. Two affected family members suffered from parkinsonism that was associated with ataxia. Magnetic resonance imaging (MRI) of these patients showed brainstem and cerebellar atrophy. Whole genome sequencing identified a heterozygous stop-gain variant (c.C811T; p.R271X) in the Poly(A) binding protein, cytoplasmic 4-like (PABPC4L) gene, which co-segregated with the disease in the family. In situ hybridization showed that the murine pabpc4l is expressed in several brain regions and in particular in the cerebellum and brainstem. To determine the functional impact of the stop-gain variant in the PABPC4L gene, we investigated the subcellular localization of PABPC4L in heterologous cells. Wild-type PABPC4L protein localized predominantly to the cell nucleus, in contrast to the truncated protein encoded by the stop-gain variant p.R271X, which was found homogeneously throughout the cell. Interestingly, the wild-type, but not the truncated protein localized to RasGAP SH3 domain Binding Protein (G3BP)-labeled cytoplasmic granules in response to oxidative stress induction. This suggests that the PABPC4L variant alters intracellular distribution and possibly the stress granule associated function of the protein, which may underlie APD in this family. In conclusion, we present genetic and molecular evidence supporting the role of a stop-gain PABPC4L variant in a rare familial APD. Our data shows that the variant results in cellular mislocalization and inability of the protein to associate with stress granules

Natural products as reservoirs of novel therapeutic agents

Since ancient times, natural products from plants, animals, microbial and marine sources have been exploited for treatment of several diseases. The knowledge of our ancestors is the base of modern drug discovery process. However, due to the presence of extensive biodiversity in natural sources, the percentage of secondary metabolites screened for bioactivity is low. This review aims to provide a brief overview of historically significant natural therapeutic agents along with some current potential drug candidates. It will also provide an insight into pros and cons of natural product discovery and how development of recent approaches has answered the challenges associated with it

A comparative evaluation of the antiproliferative activity against HepG2 liver carcinoma cells of plant-derived silver nanoparticles from basil extracts with contrasting anthocyanin contents

International audienceNanotechnology is a well-established and revolutionized field with diverse therapeutic properties. Several methods have been employed using different reducing agents to synthesize silver nanoparticles (AgNPs). Chemical mediated synthetic methods are toxic and resulted in non-desired effects on biological systems. Herein, we, synthesized silver nanoparticles using callus extract of purple basil (BC-AgNPs) and anthocyanin extract deriving from the same plant (i.e., purple basil) (AE-AgNPs), and systematically investigated their antiproliferative potential against HepG2 Liver Carcinoma Cells. The phyto-fabricated AgNPs were characterized by different techniques like UV–visible spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM) and Energy dispersive X-rays (EDX). Morphologically, both types of NPs were found spherical. The average size of BC-AgNPs and AE-AgNPs as revealed through XRD and SEM analyses were calculated as 50.97 ± 0.10 nm and 42.73 ± 1.24 nm, respectively. FT-IR spectral analysis demonstrates the existence of possible phytochemicals required for the capping and reduction of Ag ions. Herein, following solid phase extraction (SPE) coupled to HPLC analysis, we report for the first-time the anthocyanin mediated synthesis of AgNPs and conforming the successful capping of anthocyanin. Small sized AE-AgNPs showed significant cytotoxic effect against human hepatocellular carcinoma (HepG2) cell line as compared to BC-AgNPs. Therefore, the results revealed that the prevalent group of flavonoids present in purple basil is the anthocyanins and AE-AgNPs could be employed as potential anticancer agents in future treatments strategies

Polyelectrolyte Multicomponent Colloidosomes Loaded with Nisin Z for Enhanced Antimicrobial Activity against Foodborne Resistant Pathogens

Food grade micro- or nano-carrier systems (NCS) are being developed to improve the controlled release of antimicrobial agents. To augment the stability of liposomal NCS and to overcome the limitations associated with the use of free bacteriocin (nisin) in the food system, multi-component colloidosomes (MCCS) were developed by electrostatic interactions between anionic alginate and cationic chitosan (multilayer) around phospholipids based liposomes (core). Zeta-sizer results revealed the average diameter of 145 ± 2 nm, 596 ± 3 nm, and 643 ± 5 nm for nano-liposome (NL), chitosomes (chitosan coated NL) and MCCS, respectively. Zeta potential values of NCS varied from −4.37 ± 0.16 mV to 33.3 ± 6 mV, thus both chitosomes (CS) and MCCS were positively charged. Microstructure analysis by scanning electron microscope (SEM) revealed relatively higher size of MCCS with smooth and round morphology. TGA and DSC based experiments revealed that MCCS were thermally more stable than uncoated liposomes. Encapsulation efficiency of nisin in MCCS was observed to be 82.9 ± 4.1%, which was significantly higher than NL (56.5 ± 2.5%). FTIR analyses confirmed the cross-linking between sodium alginate and chitosan layer. Both qualitative (growth kinetics) and quantitative (colony forming unit) antimicrobial assays revealed that nisin loaded MCCS have superior potential to control resistant foodborne pathogens including Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, (5.8, 5.4, and 6.1 Log CFUmL−1 reduction, respectively) as compared to free nisin, loaded NL or CS. Controlled release kinetics data fitted with Korsmeyer–Peppas model suggested that nisin release from MCCS followed Fickian diffusion. Cytotoxic studies on human blood cells and HepG2 cell lines revealed hemocompatibility and non-toxicity of MCCS. Thus, due to enhanced controlled release, stability and biocompatibility; these multi-component colloidosomes can be useful for incorporating antimicrobial agents into functional foods, beverages and pharmaceutical products to combat pathogenic and spoilage bacteria

Epigenetic Regulation of Inflammatory Cytokines and Associated Genes in Human Malignancies

Inflammation is a multifaceted defense response of immune system against infection. Chronic inflammation has been implicated as an imminent threat for major human malignancies and is directly linked to various steps involved in tumorigenesis. Inflammatory cytokines, interleukins, interferons, transforming growth factors, chemokines, and adhesion molecules have been associated with chronic inflammation. Numerous cytokines are reported to be aberrantly regulated by different epigenetic mechanisms like DNA methylation and histone modifications in tumor tissues, contributing to pathogenesis of tumor in multiple ways. Some of these cytokines also work as epigenetic regulators of other crucial genes in tumor biology, either directly or indirectly. Such regulations are reported in lung, breast, cervical, gastric, colorectal, pancreatic, prostate, and head and neck cancers. Epigenetics of inflammatory mediators in cancer is currently subject of extensive research. These investigations may help in understanding cancer biology and to develop effective therapeutic strategies. The purpose of this paper is to have a brief view of the aberrant regulation of inflammatory cytokines in human malignancies

Exploiting in vitro potential and characterization of surface modified zinc oxide nanoparticles of Isodon rugosus extract

Little is known about biogenically synthesized Zinc oxide nanoparticles (ZnONPs) from Isodon rugosus. Synthesis of metal oxide NPs from essential oil producing medicinal plants results in less harmful side effects to the human population as compared to chemically synthesized NPs. In this article, we report biogenic synthesis of ZnONPs from in vitro derived plantlets and thidiazuron (TDZ) induced callus culture of Isodon rugosus. Synthesized NPs were characterized using UV-spectra, XRD, FTIR, SEM and EDX. Furthermore, the NPs were evaluated for their potential cytotoxic (against HepG2 cell line) and antimicrobial (against drug resistant Staphylococcus epidermidis, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa) activities. Pure crystalline ZnONPs with hexagonal and triangular shapes were obtained as a result of callus extract (CE) and whole plant extract (WPE), respectively. ZnONPs showed potent cytotoxic and antimicrobial potential. The antimicrobial and cytotoxic activities of ZnONPs were found to be shape and surface bound phytochemicals dependent. CE mediated hexagonal ZnONPs showed superior anti-cancer and antimicrobial activities as compared to WPE mediated triangular shaped ZnONPs. It is concluded that biogenic ZnONPs have incredible potential as theranostic agents and can be adopted as useful drug delivery system in next generation treatment strategies

The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC50 values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of >1000-fold in resistance between normal and NER-deficient cells (IC50 values for cells with deficiency in ERCC6: 0.15 mu M, XPC: 0.18 mu M, and normal cells: >180 mu M). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. (C) 2012 Elsevier Inc. All rights reserved