The mitochondrial DNA common deletion as a potential biomarker of cancer-associated fibroblasts from skin basal and squamous cell carcinomas.

Cancer-associated fibroblasts (CAFs) are components of the tumor microenvironment and represent appealing therapeutic targets for translational studies. Conventional protein-based biomarkers for CAFs have been reported to be limited in their specificity, rendering difficult the identification of CAFs from normal fibroblasts (NFs) in clinical samples and dampening the development of CAF-targeted therapies to treat cancer. In this study, we propose the mitochondrial RNA and the mitochondrial DNA (mtDNA) common deletion (CD) as novel indicators of CAF identity. We found that cancer-activation correlated with decreased levels of the mtDNA CD, a condition not due to altered mitochondria count or cellular redox state, but potentially linked to the generalized overexpression of mtDNA maintenance genes in CAFs. Decreased mtDNA CD content in CAFs was associated with moderate to strong overexpression of mtDNA-encoded genes and to slightly improved mitochondrial function. We identified similar patterns of upregulation of mtDNA-encoded genes in independent single-cell RNA seq data obtained from squamous cell carcinoma (SCC) patients. By using the identified nucleic acids-based indicators, identification of CAFs from NFs could be improved, leading to potential therapeutic benefits in advancing translational and clinical studies.

Detection of UV-Induced Deletions in Mitochondrial DNA.

Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential genes for mitochondrial function. Over 250 deletion mutations have been reported and the common deletion is the most frequent mtDNA deletion linked to disease. This deletion removes 4977 base pairs of mtDNA. It has previously been shown that exposure to UVA radiation can promote the formation of the common deletion. Furthermore, aberrations in mtDNA replication and repair are associated with formation of the common deletion. However, molecular mechanisms describing the formation of this deletion are poorly characterized. This chapter describes a method to irradiate human skin fibroblasts with physiological doses of UVA and the subsequent detection of the common deletion by quantitative PCR analysis.

A combination of direct reversion and nucleotide excision repair counters the mutagenic effects of DNA carboxymethylation.

Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The occurrence of O6-carboxymethylguanine (O6-CMG) has been linked to meat consumption and hypothesized to contribute to the development of colorectal cancer. However, the cellular fate of O6-CMG is poorly characterized and there is contradictory data in the literature as to how repair pathways may protect cells from O6-CMG mutagenicity. To better address how cells detect and remove O6-CMG, we evaluated the role of two DNA repair pathways in counteracting the accumulation and toxic effects of O6-CMG. We found that cells deficient in either the direct repair protein O6-methylguanine-DNA methyltransferase (MGMT), or key components of the nucleotide excision repair (NER) pathway, accumulate higher levels O6-CMG DNA adducts than wild type cells. Furthermore, repair-deficient cells were more sensitive to carboxymethylating agents and displayed an increased mutation rate. These findings suggest that a combination of direct repair and NER circumvent the effects O6-CMG DNA damage.

Mechanisms of replication and repair in mitochondrial DNA deletion formation.

Deletions in mitochondrial DNA (mtDNA) are associated with diverse human pathologies including cancer, aging and mitochondrial disorders. Large-scale deletions span kilobases in length and the loss of these associated genes contributes to crippled oxidative phosphorylation and overall decline in mitochondrial fitness. There is not a united view for how mtDNA deletions are generated and the molecular mechanisms underlying this process are poorly understood. This review discusses the role of replication and repair in mtDNA deletion formation as well as nucleic acid motifs such as repeats, secondary structures, and DNA damage associated with deletion formation in the mitochondrial genome. We propose that while erroneous replication and repair can separately contribute to deletion formation, crosstalk between these pathways is also involved in generating deletions.

Compartmentalized DNA repair: Rif1 S-acylation links DNA double-strand break repair to the nuclear membrane.

DNA double-strand breaks (DSBs) disrupt the structural integrity of chromosomes. Proper DSB repair pathway choice is critical to avoid the type of gross chromosomal rearrangements that characterize cancer cells. Recent findings reveal S-fatty acylation and membrane anchorage of Rap1-interacting factor 1 (Rif1) as a mechanism providing spatial control over DSB repair pathway choice.

Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane.

Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.

Shepherding DNA ends: Rif1 protects telomeres and chromosome breaks.

Cells have evolved conserved mechanisms to protect DNA ends, such as those at the termini of linear chromosomes, or those at DNA double-strand breaks (DSBs). In eukaryotes, DNA ends at chromosomal termini are packaged into proteinaceous structures called telomeres. Telomeres protect chromosome ends from erosion, inadvertent activation of the cellular DNA damage response (DDR), and telomere fusion. In contrast, cells must respond to damage-induced DNA ends at DSBs by harnessing the DDR to restore chromosome integrity, avoiding genome instability and disease. Intriguingly, Rif1 (Rap1-interacting factor 1) has been implicated in telomere homeostasis as well as DSB repair. The protein was first identified in Saccharomyces cerevisiae as being part of the proteinaceous telosome. In mammals, RIF1 is not associated with intact telomeres, but was found at chromosome breaks, where RIF1 has emerged as a key mediator of pathway choice between the two evolutionary conserved DSB repair pathways of non-homologous end-joining (NHEJ) and homologous recombination (HR). While this functional dichotomy has long been a puzzle, recent findings link yeast Rif1 not only to telomeres, but also to DSB repair, and mechanistic parallels likely exist. In this review, we will provide an overview of the actions of Rif1 at DNA ends and explore how exclusion of end-processing factors might be the underlying principle allowing Rif1 to fulfill diverse biological roles at telomeres and chromosome breaks.

Rif1 maintains telomeres and mediates DNA repair by encasing DNA ends.

In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions.

Oxidative stress controls the choice of alternative last exons via a Brahma-BRCA1-CstF pathway.

Alternative splicing of terminal exons increases transcript and protein diversity. How physiological and pathological stimuli regulate the choice between alternative terminal exons is, however, largely unknown. Here, we show that Brahma (BRM), the ATPase subunit of the hSWI/SNF chromatin-remodeling complex interacts with BRCA1/BARD1, which ubiquitinates the 50 kDa subunit of the 3' end processing factor CstF. This results in the inhibition of transcript cleavage at the proximal poly(A) site and a shift towards inclusion of the distal terminal exon. Upon oxidative stress, BRM is depleted, cleavage inhibition is released, and inclusion of the proximal last exon is favoored. Our findings elucidate a novel regulatory mechanism, distinct from the modulation of transcription elongation by BRM that controls alternative splicing of internal exons.

Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1.

Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome.

Effect of chirality and lipophilicity in the functional activity of evodiamine and its analogues at TRPV1 channels.

BACKGROUND AND PURPOSE: Evodiamine, a racemic quinazolinocarboline alkaloid isolated from the traditional Chinese medicine Evodiae fructus, has been reported to act as an agonist of the transient receptor potential vanilloid type-1 (TRPV1) cation channel both in vitro and in vivo. Evodiamine is structurally different from all known TRPV1 activators, and has significant clinical potential as a thermogenic agent. Nevertheless, the molecular bases for its actions are still poorly understood. EXPERIMENTAL APPROACH: To investigate the structure-activity relationships of evodiamine, the natural racemate was resolved, and a series of 23 synthetic analogues was prepared, using as the end point the intracellular Ca(2+) elevation in HEK-293 cells stably overexpressing either the human or the rat recombinant TRPV1. KEY RESULTS: S-(+) evodiamine was more efficacious and potent than R-(-) evodiamine, and a new potent lead (Evo30) was identified, more potent than the reference TRPV1 agonist, capsaicin. In general, potency and efficacy correlated with the lipophilicity of the analogues. Like other TRPV1 agonists, several synthetic analogues could efficiently desensitize TRPV1 to activation by capsaicin. CONCLUSIONS AND IMPLICATIONS: Evodiamine qualifies as structurally unique lead structure to develop new potent TRPV1 agonists/desensitizers.

Quinazolinecarboline alkaloid evodiamine as scaffold for targeting topoisomerase I and sirtuins.

This paper reports the synthesis of a series of evodiamine derivatives. We assayed the ability to inhibit cell growth on three human tumour cell lines (H460, MCF-7 and HepG2) and we evaluated the capacity to interfere with the catalytic activity of topoisomerase I both by the relaxation assay and the occurrence of the cleavable complex. Moreover, whose effect on sirtuins 1, 2 and 3 was investigated. Finally, molecular docking analyses were performed in an attempt to rationalize the biological results.

Camptothecin-7-yl-methanthiole: semisynthesis and biological evaluation.

The introduction of a methylenthiol group at position 7 of camptothecin was carried out in four steps. This preparation also yielded the corresponding disulfide, which behaves as a prodrug due to its reactivity with glutathione. Assessment of their antiproliferative activities, investigations of their mechanism of action, and molecular modeling analysis indicated that the 7-modified camptothecin derivatives described herein maintain the biological activity and drug-target interactions of the parent compound.

Molecular dynamics and tubulin polymerization kinetics study on 1,14-heterofused taxanes: evidence of stabilization of the tubulin head-to-tail dimer-dimer interaction.

The effects on tubulin dynamics of paclitaxel, ortataxel and two recently developed taxol derivatives bearing a five-membered heterocyclic ring fused at the 1,14 position were analysed by means of molecular dynamic simulations and the MM-PBSA approach. Tubulin polymerization kinetics and microtubule morphology assays were also conducted, providing support to computational results. In particular, it has been shown that the two recently developed 1,14-heterofused taxanes IDN5839 and IDN5798 are able to speed up the in vitro tubulin assembly by promoting the nucleation phase and to affect the microtubule network in cells earlier than paclitaxel.

The quest for new mild and selective modifications of natural structures: laccase-catalysed oxidation of ergot alkaloids leads to unexpected stereoselective C-4 hydroxylation.

Laccase-catalysed oxidation of ergot alkaloids in the absence of chemical mediators allowed the unexpected isolation of the mono-hydroxylated derivatives of compounds 2-7. Structure determination by NMR techniques clearly indicated that hydroxylation took place at the C-4 benzylic position. Quite notably, the proposed protocol allowed, for the first time, functionalisation at the C-4 position of the ergoline skeleton. Depending on the absence or on the presence of a C-10 α-methoxy substituent, hydroxylation was either stereoselective (furnishing C-4α OH derivatives) or gave rise to a C-4α/C-4ß OH mixture in a 2:1 ratio, respectively.

Synthesis and biological evaluation of new camptothecin derivatives obtained by modification of position 20.

The preparation and biological evaluation of a novel series of dimeric camptothecin derivatives are described. All the new compounds showed a significant ability to inhibit human tumor cell growth with IC(50) values ranging from 0.03 to 12.2 µM. The interference with the activity of the nuclear enzymes topoisomerases has been demonstrated, highlighting the poison effect of one of the obtained byproducts toward topoisomerase I. A moderate antiangiogenic activity has been demonstrated for one of the obtained compounds. Moreover, the effects of four new compounds on caspases activity and ROS generation have been studied on transgenic mouse cell.

Structure-activity relationship study of 16 a-thiocamptothecins: an integrated in vitro and in silico approach.

The instability of the hydroxylactone E ring represents a critical drawback of camptothecins, because the lactone ring is recognized to be essential for stabilization of topoisomerase I-mediated DNA cleavage. In an attempt to investigate the effect of the thiopyridone pharmacophofore on the molecular and pharmacological features of the drug, we prepared a series of novel 16 a-thiocamptothecin analogues. Due to the sulfur atom, a destabilization of the hydrogen bond between the hydroxy group in position 17 of the opened E ring and the carbonyl of the pyridone moiety is predicted, thus shifting the equilibrium toward the closed lactone form and increasing the lipophilic properties of the compounds. This feature was associated with superior antiproliferative potency, with reduced interaction with the human serum albumin and with substantial increase of the persistence of the topoisomerase I-DNA cleavable complex. These effects were prominent for thio-SN38, the most active compound of the series. The favorable interactions at the molecular and cellular level of the reported thiocamptothecins confer promising features, and these compounds warrant preclinical development.

A highly diastereoselective synthesis of α-hydroxy-ß-amino acid derivatives via a Lewis acid catalyzed three-component condensation reaction.

A very efficient three-component synthesis of a series of syn α-hydroxy-ß-amino esters, obtained in high diastereoselection and yield, was realized starting from an aldehyde, benzylamine, and the ketene silyl acetals derived from Ley's lactones. The synthetic protocol was optimized and the above compounds were obtained without the isolation of intermediates. The origin of the observed diastereoselection was investigated through a computational model of the key reaction step.

Design, synthesis and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel proapoptotic agents.

The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of biological properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities; moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone, was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antiproliferative activity, in some cases higher to that of the reference compound resveratrol. The most promising compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disubstitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain excellent antitumor compounds endowed with a apoptosis-inducing capability.

Synthesis and biological evaluation of novel thiocolchicine-podophyllotoxin conjugates.

The synthesis and biological evaluation of 9 dimeric compounds obtained by condensation of thiocolchicine and/or podophyllotoxin with 6 different dicarboxylic acids is described. In particular, tubulin assembly assay and immunofluorescence analysis results are reported. The biological data highlighted three compounds as being more active than the others, having a marked ability to inhibit the polymerization of tubulin in vitro and causing significant disruption to the microtubule network in vivo. The spacer unit was found to have a significant effect on biological activity, reinforcing the importance of the design of conjugate compounds to create new biologically active molecules in which the spacer could be useful to improve the solubility and to modulate the efficacy of well known anticancer drugs.