Nanoparticle-Catalyzed Transamination under Tumor Microenvironment Conditions: A Novel Tool to Disrupt the Pool of Amino Acids and GSSG in Cancer Cells.

Catalytic cancer therapy targets cancer cells by exploiting the specific characteristics of the tumor microenvironment (TME). TME-based catalytic strategies rely on the use of molecules already present in the TME. Amino groups seem to be a suitable target, given the abundance of proteins and peptides in biological environments. Here we show that catalytic CuFe2O4 nanoparticles are able to foster transaminations with different amino acids and pyruvate, another key molecule present in the TME. We observed a significant in cellulo decrease in glutamine and alanine levels up to 48 h after treatment. In addition, we found that di- and tripeptides also undergo catalytic transamination, thereby extending the range of the effects to other molecules such as glutathione disulfide (GSSG). Mechanistic calculations for GSSG transamination revealed the formation of an imine between the oxo group of pyruvate and the free -NH2 group of GSSG. Our results highlight transamination as alternative to the existing toolbox of catalytic therapies.

Cellular Alterations Due to Direct and Indirect Interaction of Nanomaterials with Nucleic Acids.

Deoxyribonucleic acid (DNA) represents the main reservoir of genetic information in the cells, which is why it is protected in the nucleus. Entry into the nucleus is, in general, difficult, as the nuclear membrane is a selective barrier to molecules longer than 40 kDa. However, in some cases, the size of certain nanoparticles (NPs) allows their internalization into the nucleus, thus causing a direct effect on the DNA structure. NPs can also induce indirect effects on DNA through reactive oxygen species (ROS) generation. In this context, nanomaterials are emerging as a disruptive tool for the development of novel therapies in a broad range of biomedical fields; although their effect on cell viability is commonly studied, further interactions with DNA or indirect alterations triggered by the internalization of these materials are not always clarified, since the small size of these materials makes them perfectly suitable for interaction with subcellular structures, such as the nucleus. In this context, and using as a reference the predicted interactions presented in a computational model, we describe and discuss the observed direct and indirect effects of the implicated nanomaterials on DNA.

Impact of the COVID-19 pandemic in colorectal cancer diagnosis and presentation / Impacto de la pandemia del COVID-19 en el diagnóstico y la presentación del cáncer colorrectal

Background and objective: The COVID-19 pandemic has been associated with a decrease in the colorectal cancer (CRC) incidence, due to the disruption of screening programmes and a downscaling of endoscopic activity. The endpoint of this study is to evaluate if the pandemic has led to a change in CRC diagnostic rate and presentation in our population. Methods: Multicenter retrospective study of all public hospitals of the Aragon region, attending a population of 1,329,391 inhabitants. We have analyzed all CRC cases detected and endoscopic units workload the year before the pandemic onset (1 March 2019–14 March 2020) and the first year of the COVID-19 pandemic (15 March 2020–28 February 2021). Results: The diagnosis of CRC cases dropped a 38.9% (888 pre-pandemic vs 542 pandemic cases). Also, there were 30.3% less colonoscopies performed (24,860 vs 17,337). During the pandemic, CRC cases were diagnosed in older patients (72.4±12.2 vs 71.2±12.1 years, p=0.021), and had more frequently severe complications at diagnosis (14.6% vs 10.4%, p=0.019). Moreover, most CRC cases were diagnosed in symptomatic patients (81.4%). No significant difference was found in CRC stage at diagnosis, although stage IV was more frequent (20.1% vs 16.1%). Most hospitals reported a lower workload of endoscopic activity. Conclusion: CRC diagnostic rate was lower after the onset of the pandemic. CRC was diagnosed in older patients and was more frequently associated with complications. After the onset of the pandemic, the endoscopic units did not reach the workload performed previously. (AU)

Introducción y objetivo: La pandemia del COVID-19 ha provocado una disminución en la incidencia de cáncer colorrectal (CCR) tras la suspensión de los programas de cribado y la reducción de la actividad endoscópica. El objetivo del estudio es evaluar si la pandemia se ha asociado a un cambio en la incidencia y presentación del CCR en nuestra población. Métodos: Estudio multicéntrico, retrospectivo de todos los hospitales públicos de Aragón, con 1.329.391 habitantes. Analizamos todos los CRC detectados y la carga laboral de las unidades de endoscopia del año prepandémico (1 marzo 2019 – 14 marzo 2020) y el primer año de la pandemia (15 marzo 2020 – 28 febrero 2021). Resultados: El diagnóstico de CRC descendió un 38,9% (888 casos prepandemia vs. 542 en pandemia). Se realizaron un 30,3% menos de colonoscopias (24.860 vs. 17.337). El CRC en pandemia se diagnosticó en pacientes de mayor edad (72,4±12,2 vs 71,2±12,1 años; p=0,021) y presentaron más complicaciones graves en el momento del diagnóstico (14,6 vs. 10,4%; p=0.019). La mayoría de los CRC se diagnosticaron en pacientes sintomáticos (81,4%). No hubo diferencias en el estadio al diagnóstico aunque el estadio iv fue más frecuente (20,1 vs. 16,1%). La mayoría de los hospitales reiniciaron la actividad endoscópica con una menor carga laboral. Conclusión: La tasa diagnóstica de CRC descendió tras el inicio de la pandemia, el CRC fue diagnosticado en pacientes mayores y se asoció con más complicaciones al diagnóstico. Tras el inicio de la pandemia, la mayoría de los hospitales reiniciaron su actividad con una menor carga laboral. (AU)

X-ray Photoelectron Spectroscopy (XPS) Analysis of Nitrogen Environment in Small Extracellular Vesicle Membranes: A Potential Novel Technique with Application for Cancer Screening.

Small extracellular vesicle (EV) membranes display characteristic protein-lipidic composition features that are related to their cell of origin, providing valuable clues regarding their parental cell composition and real-time state. This could be especially interesting in the case of cancer cell-derived EVs, as their membranes could serve as valuable tools in liquid biopsy applications and to detect changes in the tumor malignancy. X-Ray Photoelectron Spectroscopy (XPS) is a powerful surface analysis technique able to detect every chemical element present, being also sensitive to their chemical environment. Here we explore the use of XPS as a fast technique to characterize EV membrane composition, with possible application in cancer research. Notably, we have focused on the nitrogen environment as an indicator of the relative abundance of pyridine-type bonding, primary, secondary and tertiary amines. Specifically, we have analyzed how tumoral and healthy cells have different nitrogen chemical environments that can indicate the presence or absence of malignancy. In addition, a collection of human serum samples from cancer patients and healthy donors was also analyzed. The differential XPS analysis of EVs collected from patients confirmed that the patterns of amine evolution could be related to markers of cancer disease, opening the possibility of their use as a non-invasive blood biomarker.

Nanoparticle (NP) Loading by Direct Incubation with Extracellular Vesicles-Secretor Cells: NP Encapsulation and Exosome Characterization.

Exosomes are small vesicles released by all types of cells, and they have been postulated as a promising natural way to carry information amongst cells. Exosomes might serve as mediators for intercellular communication through the delivery of their endogenous cargo to neighbor or distant cells. Recently, this ability to transfer their cargo has open a new therapeutic approach and exosomes have been investigated as vectors for the delivery of the loaded cargo, for instance nanoparticles (NPs).Currently, several methods to load exosomes with NPs have been described; however, the maintenance of the membrane integrity on the vesicle has to be taken into consideration, in order to choose one or another methodology. Here we describe the NP encapsulation through the incubation of the cells with the NPs and the subsequential methods to determine their cargo and to discard detrimental alterations on the loaded exosomes.

Cellular Alterations in Carbohydrate and Lipid Metabolism Due to Interactions with Nanomaterials.

Nanoparticles (NPs) have unique physicochemical properties that are useful for a broad range of biomedical and industrial applications; nevertheless, increasing concern exists about their biosafety. This review aims to focus on the implications of nanoparticles in cellular metabolism and their outcomes. In particular, some NPs have the ability to modify glucose and lipid metabolism, and this feature is especially interesting to treat diabetes and obesity and to target cancer cells. However, the lack of specificity to reach target cells and the toxicological evaluation of nontargeted cells can potentially induce detrimental side effects, closely related to inflammation and oxidative stress. Therefore, identifying the metabolic alterations caused by NPs, independent of their application, is highly needed. To our knowledge, this increase would lead to the improvement and safer use with a reduced toxicity, increasing the number of available NPs for diagnosis and treatment of human diseases.

Impact of the COVID-19 pandemic in colorectal cancer diagnosis and presentation.

BACKGROUND AND OBJECTIVE: The COVID-19 pandemic has been associated with a decrease in the colorectal cancer (CRC) incidence, due to the disruption of screening programmes and a downscaling of endoscopic activity. The endpoint of this study is to evaluate if the pandemic has led to a change in CRC diagnostic rate and presentation in our population. METHODS: Multicenter retrospective study of all public hospitals of the Aragon region, attending a population of 1,329,391 inhabitants. We have analyzed all CRC cases detected and endoscopic units workload the year before the pandemic onset (1 March 2019-14 March 2020) and the first year of the COVID-19 pandemic (15 March 2020-28 February 2021). RESULTS: The diagnosis of CRC cases dropped a 38.9% (888 pre-pandemic vs 542 pandemic cases). Also, there were 30.3% less colonoscopies performed (24,860 vs 17,337). During the pandemic, CRC cases were diagnosed in older patients (72.4±12.2 vs 71.2±12.1 years, p=0.021), and had more frequently severe complications at diagnosis (14.6% vs 10.4%, p=0.019). Moreover, most CRC cases were diagnosed in symptomatic patients (81.4%). No significant difference was found in CRC stage at diagnosis, although stage IV was more frequent (20.1% vs 16.1%). Most hospitals reported a lower workload of endoscopic activity. CONCLUSION: CRC diagnostic rate was lower after the onset of the pandemic. CRC was diagnosed in older patients and was more frequently associated with complications. After the onset of the pandemic, the endoscopic units did not reach the workload performed previously.

Exosomes loaded with ultrasmall Pt nanoparticles: a novel low-toxicity alternative to cisplatin.

BACKGROUND: Platinum nanoparticles have been demonstrated to have excellent anticancer properties. However, because of the lack of specificity they must be delivered to the tumor in amounts sufficient to reach the desired therapeutic objectives. Interestingly, exosomes are considered as excellent natural selective delivery nanotools, but until know their targeting properties have not being combined with the anticancer properties of platinum nanoparticles. RESULTS: In this work we combine the targeting capabilities of exosomes and the antitumoral properties of ultrasmall (< 2 nm) platinum nanoparticles as a novel, low toxicity alternative to the use of cisplatin. A mild methodology based on the room temperature CO-assisted in situ reduction of Pt2+ precursor was employed to preserve the integrity of exosomes, while generating ultrasmall therapeutic PtNPs directly inside the vesicles. The resulting PtNPs-loaded exosomes constitute a novel hybrid bioartificial system that was readily internalized by the target cells inducing antiproliferative response, as shown by flow cytometry and microscopy experiments in vitro. In vivo Pt-Exos showed antitumoral properties similar to that of cisplatin but with a strongly reduced or in some cases no toxic effect, highlighting the advantages of this approach and its potential for translation to the clinic. CONCLUSIONS: In this study, a nanoscale vector based on ultrasmall PtNPs and exosomes has been created exhibiting antitumoral properties comparable or higher to those of the FDA approved cisplatin. The preferential uptake of PtNPs mediated by exosomal transfer between certain cell types has been exploited to create a selective antitumoral novel bioartificial system. We have demonstrated their anticancer properties both in vitro and in vivo comparing the results obtained with the administration of equivalent amounts of cisplatin, and showing a spectacular reduction of toxicity.

Characterization of Kelch domain-containing protein 7B in breast tumours and breast cancer cell lines.

Adenocarcinomas exhibit great heterogeneity, with many genetic and epigenetic alterations. The Kelch domain-containing protein 7B (KLHDC7B) has recently been identified as epigenetically modified and upregulated in breast cancer. The potential reversibility of epigenetic states offers exciting possibilities for novel cancer diagnostics and drugs. However, to properly evaluate specific inhibitors, the role of KLHDC7B in the development and progression of breast cancer should be established. With that objective in mind, the present study investigated a series of human breast tumours and correlated their clinicopathology, according to the Elston-Ellis modification of the Scarff-Bloom-Richardson (SBR) grading system, with KLHDC7B mRNA expression, analysed using quantitative PCR (qPCR). The results revealed that KLHDC7B was significantly upregulated in grade 3 tumours, and that KLHDC7B expression varied according to the tumour grade and the individual, being downregulated in well-differentiated and moderately-differentiated tumours (grade 1-2) and upregulated in poorly-differentiated tumours (grade 3). Immunohistochemical staining revealed that ductal tumours and tumours with a higher percentage of Ki67 positive cells showed the highest levels of KLHDC7B. Receptor expression, HER, p53 status, presence of metastasis, and vascular invasion showed no association with KLHDC7B expression. Previous studies have proposed KLHDC7B as an epigenetic marker of breast cancer. We propose that KLHDC7B should be used as a marker for poorly-differentiated tumours only; use of KLHDC7B without considering tumour grade could lead to an inaccurate diagnosis. Finally, we suggest the appropriate breast cancer cell lines to use to determine the functions of KLHDC7B. KLHDC7B expression was tested in the non-tumour cell line MCF-10A and in the breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468, using qPCR and western blotting. The results revealed that all tested cancer cell lines overexpressed KLHDC7B mRNA, but MDA-MB-468 exhibited a much lower level of protein expression relative to mRNA. Although the breast cancer cell lines used may be appropriate for studying KLHDC7B epigenetic status, MDA-MB-468 should be excluded from functional experiments.

The role of clonal communication and heterogeneity in breast cancer.

BACKGROUND: Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones. METHODS: We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays. RESULTS: Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration. CONCLUSIONS: These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression.

Author Correction: Rev1 contributes to proper mitochondrial function via the PARP-NAD+-SIRT1-PGC1α axis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Rev1 contributes to proper mitochondrial function via the PARP-NAD+-SIRT1-PGC1α axis.

Nucleic acids, which constitute the genetic material of all organisms, are continuously exposed to endogenous and exogenous damaging agents, representing a significant challenge to genome stability and genome integrity over the life of a cell or organism. Unrepaired DNA lesions, such as single- and double-stranded DNA breaks (SSBs and DSBs), and single-stranded gaps can block progression of the DNA replication fork, causing replicative stress and/or cell cycle arrest. However, translesion synthesis (TLS) DNA polymerases, such as Rev1, have the ability to bypass some DNA lesions, which can circumvent the process leading to replication fork arrest and minimize replicative stress. Here, we show that Rev1-deficiency in mouse embryo fibroblasts or mouse liver tissue is associated with replicative stress and mitochondrial dysfunction. In addition, Rev1-deficiency is associated with high poly(ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD+, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via the PARP-NAD+-SIRT1-PGC1α axis.

Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions.

Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and proliferating cells cope with accumulating endogenous DNA lesions and how these ultimately affect the physiology of cells and tissues. Here we have addressed these questions by investigating the hematopoietic system of mice deficient for Rev1, a core factor in DNA translesion synthesis (TLS), the postreplicative bypass of damaged nucleotides. Rev1 hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of Xpc, essential for global-genome nucleotide excision repair (ggNER) of helix-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the Rev1Xpc bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of helix-distorting oxidative nucleotide lesions by ggNER and Rev1-dependent TLS, respectively. The error-prone nature of TLS may provide mechanistic understanding of the accumulation of mutations in the hematopoietic system upon aging.

Effects of donor age and proliferative aging on the phenotype stability of rat aortic smooth muscle cells.

Age-related effects of the vascular wall have been associated with several hemodynamic dysfunctions, including medial vascular calcification. Vascular aging has been traditionally addressed using proliferative senescence of vascular smooth muscle cells (VSMC) in vitro, which induces osteoblastic transition and favors calcification in vitro. In this work, we have analyzed the relationship between organismal aging and proliferative senescence by comparing the proliferative aging of VSMC obtained from young, mature, and old rats (2-, 12-, and 24-month cell lines [CL], respectively). VSMC proliferated to more than 100 cumulative population doublings (CPD) without evidence of proliferative senescence, most likely as a consequence of telomerase induction. The apoptosis rate increased with CPD in all three CL, but the oxidation status of the cells was not modified. The magnitude of all gene expression changes caused by CPD was higher than the magnitude of the changes caused by donor age: the expressions of VSMC markers α-actin and SM22α decreased, while the expressions of transcription factors Msx2 and Runx2 and of bone morphogenetic protein-2 increased. Treatment of the cells with 2 mmol/L Pi revealed that the intensity of the effect of CPD on calcium deposition was greater than the effect of donor age. In conclusion, the proliferative lifespan of VSMC magnifies the effect of donor age on the osteoblastic transition of VSMC, therefore suggesting that in vivo vascular aging changes can be less dramatic than what is shown by in vitro aging.

Effect of water fluoridation on the development of medial vascular calcification in uremic rats.

Public water fluoridation is a common policy for improving dental health. Fluoride replaces the hydroxyls of hydroxyapatite, thereby improving the strength of tooth enamel, but this process can also occur in other active calcifications. This paper studies the effects of water fluoridation during the course of vascular calcification in renal disease. The effect of fluoride was studied in vitro and in vivo. Rat aortic smooth muscle cells were calcified with 2mM Pi for 5 days. Fluoride concentrations of 5-10 µM--similar to those found in people who drink fluoridated water--partially prevented calcification, death, and osteogene expression in vitro. The anticalcifying mechanism was independent of cell activity, matrix Gla protein, and fetuin A expressions, and it exhibited an IC50 of 8.7 µM fluoride. In vivo, however, fluoridation of drinking water at 1.5mg/L (concentration recommended by the WHO) and 15 mg/L dramatically increased the incipient aortic calcification observed in rats with experimental chronic kidney disease (CKD, 5/6-nephrectomy), fed a Pi-rich fodder (1.2% Pi). Fluoride further declined the remaining renal function of the CKD animals, an effect that most likely overwhelmed the positive effect of fluoride on calcification in vitro. Ultrastructural analysis revealed that fluoride did not modify the Ca/P atomic ratio, but it was incorporated into the lattice of in vivo deposits. Fluoride also converted the crystallization pattern from plate to rode-like structures. In conclusion, while fluoride prevents calcification in vitro, the WHO's recommended concentrations in drinking water become nephrotoxic to CKD rats, thereby aggravating renal disease and making media vascular calcification significant.

Arsenic increases Pi-mediated vascular calcification and induces premature senescence in vascular smooth muscle cells.

Several mechanisms have been proposed to explain the vascular toxicity of arsenic. Some of them are described in this work, such as stress-induced premature senescence (SIPS), dedifferentiation, and medial vascular calcification, and they all affect vascular smooth muscle cells (VSMC). Rat aortic VSMC were treated with 1-100 µM of either sodium arsenate (As(V)), sodium arsenite (As(III)), monomethylarsonic acid, or dimethylarsinic acid. None of the treatments induced VSMC calcification in the presence of 1mM inorganic phosphate (Pi), but 1 µM As(III) did increase calcification when induced with 2.5mM Pi. A lactate dehydrogenase assay revealed that this increase was explained by a rise in cytotoxicity due to simultaneous incubation with 1 µM As(III) and 2.5mM Pi. This calcification increase was also observed in the aortas of a vascular calcification model: 5/6 nephrectomized rats fed with a high Pi diet and treated with vitamin D(3). Several known mechanisms that might explain arsenic toxicity in our experimental model were discarded: apoptosis, oxidative stress, and inflammasome activation. Nevertheless, both senescence-associated ß-galactosidase activity and p21 expression were increased by As(III), which reveals the induction of SIPS. As(III) also caused dedifferentiation of VSMC, as shown by the reduced expression of the VSMC markers SM22α and calponin. Senescence and gene expression were also observed in the aortas of healthy rats treated with 50 ppm As(V) in drinking water for 1 month. In conclusion, both premature senescence in aortic VSMC with phenotypic dedifferentiation and the increase of Pi-induced calcification are novel mechanisms of arsenic vasculotoxicity.