Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

BACKGROUND: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). METHODOLOGY/PRINCIPAL FINDINGS: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. CONCLUSIONS: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

High-resolution melting (HRM)-based detection of polymorphisms in the malic enzyme and glucose-6-phosphate isomerase genes for Leishmania infantum genotyping.

BACKGROUND: Leishmaniasis is a zoonotic disease endemic in the Mediterranean region where Leishmania infantum is the causative agent of human and canine infection. Characterization of this parasite at the subspecies level can be useful in epidemiological studies, to evaluate the clinical course of the disease (e.g. resistant strains, visceral and cutaneous forms of leishmaniasis) as well as to identify infection reservoirs. Multilocus enzyme electrophoresis (MLEE), a method currently recognized as the reference method for characterizing and identifying strains of Leishmania, is cumbersome and time-consuming and requires cultured parasites. These disadvantages have led to the development of other methods, such as multilocus microsatellite typing (MLMT) and multilocus sequence typing (MLST), for typing Leishmania parasites; however, these methods have not yet been applied for routine use. In this study, we first used MLST to identify informative polymorphisms on single-copy genes coding for metabolic enzymes, following which we developed two rapid genotyping assays based on high-resolution melting (HRM) analysis to explore these polymorphisms in L. infantum parasites. METHODS: A customized sequencing panel targeting 14 housekeeping genes was designed and MLST analysis was performed on nine L. infantum canine and human strains/isolates. Two quantitative real-time PCR-HRM assays were designed to analyze two informative polymorphisms on malic enzyme (ME) and glucose-6-phosphate isomerase (GPI) genes (390T/G and 1831A/G, respectively). The two assays were applied to 73 clinical samples/isolates from central/southern Italy and Pantelleria island, and the results were confirmed by DNA sequencing in a subset of samples. RESULTS: The MLST analysis, together with sequences available in the Genbank database, enabled the identification of two informative polymorphisms on the genes coding for ME and GPI. The fast screening of these polymorphisms using two HRM-based assays in 73 clinical samples/isolates resulted in the identification of seven genotypes. Overall, genotype 1 (sequence type 390T/1831G) was the most highly represented (45.2%) in the overall sample and correlated with the most common L. infantum zymodemes (MON-1, MON-72). Interestingly, in Pantelleria island, the most prevalent genotype (70.6%) was genotype 6 (sequence type 390T/1831A). CONCLUSIONS: Applying our HRM assays on clinical samples allowed us to identify seven different genotypes without the need for parasite isolation and cultivation. We have demonstrated that these assays could be used as fast, routine and inexpensive tools for epidemiological surveillance of L. infantum or for the identification of new infection reservoirs.

Comparing Heterologous and Homologous COVID-19 Vaccination: A Longitudinal Study of Antibody Decay.

The humoral response after vaccination was evaluated in 1248 individuals who received different COVID-19 vaccine schedules. The study compared subjects primed with adenoviral ChAdOx1-S (ChAd) and boosted with BNT162b2 (BNT) mRNA vaccines (ChAd/BNT) to homologous dosing with BNT/BNT or ChAd/ChAd vaccines. Serum samples were collected at two, four and six months after vaccination, and anti-Spike IgG responses were determined. The heterologous vaccination induced a more robust immune response than the two homologous vaccinations. ChAd/BNT induced a stronger immune response than ChAd/ChAd at all time points, whereas the differences between ChAd/BNT and BNT/BNT decreased over time and were not significant at six months. Furthermore, the kinetic parameters associated with IgG decay were estimated by applying a first-order kinetics equation. ChAd/BNT vaccination was associated with the longest time of anti-S IgG negativization and with a slow decay of the titer over time. Finally, analyzing factors influencing the immune response by ANCOVA analysis, it was found that the vaccine schedule had a significant impact on both the IgG titer and kinetic parameters, and having a Body Mass Index (BMI) above the overweight threshold was associated with an impaired immune response. Overall, the heterologous ChAd/BNT vaccination may offer longer-lasting protection against SARS-CoV-2 than homologous vaccination strategies.

Evaluation of Two-Month Antibody Levels after Heterologous ChAdOx1-S/BNT162b2 Vaccination Compared to Homologous ChAdOx1-S or BNT162b2 Vaccination.

We evaluated the post-vaccination humoral response of three real-world cohorts. Vaccinated subjects primed with ChAdOx1-S and boosted with BNT162b2 mRNA vaccine were compared to homologous dosing (BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S). Serum samples were collected two months after vaccination from a total of 1248 subjects. The results showed that the heterologous vaccine schedule induced a significantly higher humoral response followed by homologous BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S vaccines (p < 0.0001). Moreover, analyzing factors (i.e., vaccine schedule, sex, age, BMI, smoking, diabetes, cardiovascular diseases, respiratory tract diseases, COVID-19 diagnosis, vaccine side effects) influencing the IgG anti-S response, we found that only the type of vaccine affected the antibody titer (p < 0.0001). Only mild vaccine reactions resolved within few days (40% of subjects) and no severe side effects for either homologous groups or the heterologous group were reported. Our data support the use of heterologous vaccination as an effective and safe alternative to increase humoral immunity against COVID-19.

In Vitro Reduced Susceptibility to Pentavalent Antimonials of a Leishmania infantum Isolate from a Human Cutaneous Leishmaniasis Case in Central Italy.

Cutaneous leishmaniasis (CL) caused by Leishmania (Leishmania) infantum is endemic in the Mediterranean basin. Here we report an autochthonous case of CL in a patient living in central Italy with an unsatisfactory response to treatment with intralesional Meglumine Antimoniate and in vitro demonstration of reduced susceptibility to SbIII. Parasitological diagnosis was first achieved by histopathology on tissue biopsy and the patient was treated with a local infiltration of Meglumine Antimoniate. Since the clinical response at 12 weeks from the treatment's onset was deemed unsatisfactory, two further skin biopsies were taken for histopathological examination, DNA extraction and parasite isolation. L. (L.) infantum was identified by molecular typing. The low susceptibility to Meglumine Antimoniate was confirmed in vitro: the promastigotes from the patient strain showed significantly lower susceptibility to SbIII (the active trivalent form of antimonial) compared to the reference strain MHOM/TN/80/IPT1. The patient underwent a new treatment course with intravenous liposomal Amphotericin B, reaching complete healing of the lesion. Additional studies are needed to confirm the epidemiological and clinical relevance of reduced susceptibility to SbIII of human L. (L.) infantum isolate in Italy.

Activation of NRF2 and ATF4 Signaling by the Pro-Glutathione Molecule I-152, a Co-Drug of N-Acetyl-Cysteine and Cysteamine.

I-152 combines two pro-glutathione (GSH) molecules, namely N-acetyl-cysteine (NAC) and cysteamine (MEA), to improve their potency. The co-drug efficiently increases/replenishes GSH levels in vitro and in vivo; little is known about its mechanism of action. Here we demonstrate that I-152 not only supplies GSH precursors, but also activates the antioxidant kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (KEAP1/NRF2) pathway. The mechanism involves disulfide bond formation between KEAP1 cysteine residues, NRF2 stabilization and enhanced expression of the γ-glutamil cysteine ligase regulatory subunit. Accordingly, a significant increase in GSH levels, not reproduced by treatment with NAC or MEA alone, was found. Compared to its parent compounds, I-152 delivered NAC more efficiently within cells and displayed increased reactivity to KEAP1 compared to MEA. While at all the concentrations tested, I-152 activated the NRF2 pathway; high doses caused co-activation of activating transcription factor 4 (ATF4) and ATF4-dependent gene expression through a mechanism involving Atf4 transcriptional activation rather than preferential mRNA translation. In this case, GSH levels tended to decrease over time, and a reduction in cell proliferation/survival was observed, highlighting that there is a concentration threshold which determines the transition from advantageous to adverse effects. This body of evidence provides a molecular framework for the pro-GSH activity and dose-dependent effects of I-152 and shows how synergism and cross reactivity between different thiol species could be exploited to develop more potent drugs.

I-152, a supplier of N-acetyl-cysteine and cysteamine, inhibits immunoglobulin secretion and plasma cell maturation in LP-BM5 murine leukemia retrovirus-infected mice by affecting the unfolded protein response.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 µmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

Rapamycin Re-Directs Lysosome Network, Stimulates ER-Remodeling, Involving Membrane CD317 and Affecting Exocytosis, in Campylobacter Jejuni-Lysate-Infected U937 Cells.

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain-Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER-lysosome and ER-mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells.

Metformin prevents cell tumorigenesis through autophagy-related cell death.

Autophagy is a cellular mechanism by which cells degrade intracellular components in lysosomes, maintaining cellular homeostasis. It has been hypothesized that autophagy could have a role in cancer prevention through the elimination of damaged proteins and organelles; this could explain epidemiological evidence showing the chemopreventive properties of the autophagy-inducer metformin. In this study, we analyzed the autophagy-related effect of metformin in both cancer initiation and progression in non-tumorigenic cells. We also analyzed the induction of tumorigenesis in autophagy-deficient cells, and its correlation with the ER stress. Our results showed that metformin induced massive cell death in preneoplastic JB6 Cl 41-5a cells treated with tumor promoter (phorbol) and in NIH/3T3 treated with H2O2. Inhibiting autophagy with wortmannin or ATG7 silencing, the effect of metformin decreased, indicating an autophagy-related cytotoxic activity under stress conditions. We also found an induction of tumorigenesis in ATG7-silenced NIH/3T3 cell clone (3T3-619C3 cells), but not in wild-type and in scrambled transfected cells, and an upregulation of unfolded protein response (UPR) markers in 3T3-619C3 cells treated with H2O2. These findings suggest that autophagic cell death could be considered as a new mechanism by which eliminate damaged cells, representing an attractive strategy to eliminate potential tumorigenic cells.

Glutathione increase by the n-butanoyl glutathione derivative (GSH-C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus.

During aging, glutathione (GSH) content declines and the immune system undergoes a deficiency in the induction of Th1 response. Reduced secretion of Th1 cytokines, which is associated with GSH depletion, could weaken the host defenses against viral infections. We first evaluated the concentration of GSH and cysteine in organs of old mice; then, the effect of the administration of the N-butanoyl GSH derivative (GSH-C4) on the response of aged mice infected with influenza A PR8/H1N1 virus was studied through the determination of GSH concentration in organs, lung viral titer, IgA and IgG1/IgG2a production, and Th1/Th2 cytokine profile. Old mice had lower GSH than young mice in organs. Also the gene expression of endoplasmic reticulum (ER) stress markers involved in GSH metabolism and folding of proteins, that is, Nrf2 and PDI, was reduced. Following infection, GSH content remained low and neither infection nor GSH-C4 treatment affected Nrf2 expression. In contrast, PDI expression was upregulated during infection and appeared counterbalanced by GSH-C4. Moreover, the treatment with GSH-C4 increased GSH content in organs, reduced viral replication and induced a predominant Th1 response. In conclusion, GSH-C4 treatment could be used in the elderly to contrast influenza virus infection by inducing immune response, in particular the Th1 profile.

Exploiting genetic polymorphisms in metabolic enzymes for rapid screening of Leishmania infantum genotypes.

BACKGROUND: Leishmania infantum is the aetiological agent of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Numerous strains and/or zymodemes have been identified and characterized by multilocus enzyme electrophoresis (MLEE). MLEE is considered the reference method for L. infantum parasite typing and it is based upon enzyme electrophoretic mobility analysis from promastigote cultures. However, the MLEE technique is cumbersome, time-consuming and does not detect silent genetic mutations or nucleotide changes that give rise to amino acid changes that do not alter electrophoretic mobility. As a result of these difficulties, many DNA-based typing methods have been developed over the past few years. However, relative to the enzymes utilized in MLEE analysis, we observed a shortage of DNA sequences available in the GenBank database or an absolute lack of sequences belonging to specific zymodemes. The aims of the present study were to (i) implement the number of sequences coding for metabolic enzymes used in MLEE; (ii) identify polymorphisms that characterize L. infantum zymodemes most prevalent in the Mediterranean basin; and (iii) exploit these polymorphisms to develop a rapid screening test that would give results comparable with existing MLEE typing. RESULTS: Partial sequences of seven metabolic enzyme genes (malic enzyme, 6-phosphogluconate dehydrogenase, mitochondrial isocitrate dehydrogenase, glucose-6-phosphate isomerase, glucose-6-phosphate dehydrogenase, phosphoglucomutase and mannose phosphate isomerase) were obtained from 11 L. infantum strains. The comparison of these sequences with those obtained from GenBank allowed for the identification of a few polymorphisms that could distinguish several zymodemes. In particular, the polymorphism 390T>G in the malic enzyme gene has been exploited to develop a high-resolution melt (HRM)-based assay to rapidly differentiate the genotype 390T, associated with zymodemes MON-1, MON-72 and MON-201, evidencing a partial agreement between genotyping results and MLEE. The assay has been successfully applied to L. infantum clinical isolates and clinical samples. CONCLUSIONS: A HRM-based assay for rapid identification of genotypes associated with the most common L. infantum zymodemes in the Mediterranean basin has been developed and its potential application in epidemiological research for L. infantum population screening, without parasite isolation and culturing, has been demonstrated.

Leishmania infantum Induces Mild Unfolded Protein Response in Infected Macrophages.

The Leishmaniases are a group of parasitic diseases caused by protozoa of the Leishmania genus affecting both humans and other vertebrates. Leishmania is an intracellular pathogen able to confer resistance to apoptosis in the early phase of macrophages infection by activation of host PI3K/Akt pathway and inhibition of caspase-3 activation. Intracellular pathogens hijack organelles such as ER to facilitate survival and replication, thus eliciting ER stress and activating/modulating the unfolded protein response (UPR) in the host cell. The UPR is aimed to mitigate ER stress, thereby promoting cell survival. However, prolonged ER stress will activate the apoptotic pathway. The aim of this study was to investigate the ER stress response in Leishmania-infected macrophages to gain insights about the mechanisms underlying the apoptosis resistance in parasitized cells. Macrophages differentiated from human monocytic cell lines (U937 and THP-1) and murine primary macrophages were infected with Leishmania infantum MHOM/TN/80/IPT1 (WHO international reference strain). Several ER stress/autophagy expression markers, as well as cell survival/apoptosis markers (phospho-Akt and cleaved caspase-3) were evaluated by qPCR and/or by western blotting. As ER stress positive control, cells were treated with tunicamycin or dithiothreitol (DTT). The gene expression analyses showed a mild but significant induction of the ER stress/autophagy markers. The western blot analyses revealed that the Leishmania infection induced Akt phosphorylation and significantly inhibited the induction of caspase-3 cleavage, eIF2α phosphorylation and DDIT3/CHOP expression in tunicamycin and DTT treated cells. The mild but significant increase in ER stress expression markers and the delay/attenuation of the effects of ER stress inducers in infected cells support the hypothesis that L. infantum could promote survival of host cells by inducing a mild ER stress response. The host ER stress response could be not only a common pathogenic mechanism among Leishmania species but also a target for development of new drugs.

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics.

UNLABELLED: Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152), an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCE: The first report of an association between Th2 polarization and alteration of the redox state in LP-BM5 infection is presented. Moreover, it provides evidence that LP-BM5 infection causes a decrease in the thiol content of peritoneal macrophages, which can influence IL-12 production. The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection, as it reestablishes the Th1/Th2 balance. Immunotherapy based on the use of pro-GSH molecules would permit LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and a Th1/Th2 imbalance to be more effectively combated.

Pre-implantation genetic diagnosis and screening: now and the future.

Since 1989, the year of the first pre-implantation genetic diagnosis (PGD), many developments occurred both in assisted reproduction techniques and in molecular tools. While PGD is a well-established and documented application, pre-implantation genetic screening (PGS) for the detection of aneuploid embryos is still debated due to the presence of mosaicism in the embryo, but especially to the knowledge of the limits that label an embryo as healthy or as appropriate to the life. The aim of this review is to present the state-of-the-art in the field of PGD and PGS, illustrating its benefits and limitations, along with biopsy techniques and the use of new high-throughput technologies.

Inhibition of Testosterone Aromatization by the Indole-3-carbinol Derivative CTet in CYP19A1-overexpressing MCF-7 Breast Cancer Cells.

Natural products such as aromatase inhibitors have been the object of growing attention in recent years because of their potential to inhibit aromatase with fewer side effects and the possible translation of their current use as chemotherapeutic agents to future clinical applications in breast cancer chemoprevention. We have previously investigated CTet, a novel anticancer agent obtained from the broccoli-derived compound indole-3- carbinol (I3C), that shows great anticancer potential in both in vitro and in vivo studies. Here we evaluated the potential of CTet as a chemopreventive agent in aromatase expressing MCF-7/AROM-1 breast cancer cells. The testosterone (TE) aromatization in estradiol (E2) was indirectly evaluated in terms of inhibition of TE-induced cell proliferation, ERα phosphorylation/activation and Bcl-2 and IGF-1R ERE-regulated protein accumulation. Our results showed that CTet inhibited TE-driven ERα phosphorylation of both cytosolic and nuclear ERα pools, suggesting an inhibitory effect of TE aromatization in E2. CTet did not inhibit E2-driven nuclear ERα phosphorylation, but partially inhibited E2-driven cytosolic ERα phosphorylation. Moreover, CTet inhibited Bcl-2 and IGF-1R accumulation induced by TE but not that which was induced by E2. A cell-free enzymatic assay showed that CTet did not inhibit aromatase activity directly; however, since CTet treatment induced endoplasmic reticulum stress, the TE aromatization could be affected because the aromatase enzyme is located within the endoplasmic reticulum. Finally, CTet and letrozole synergistically inhibited TE-induced cell proliferation. These results showed the potential of the I3C derivative CTet as a chemopreventive agent that interferes with aromatase activity.

Extracellular embryo genomic DNA and its potential for genotyping applications.

BACKGROUND: Preimplantation genetic diagnosis (PGD) currently relies on biopsy of one or few embryo cells. Our aim was to evaluate the embryo extracellular matrices (spent medium and blastocoele fluid) as source of DNA for embryo genotyping. RESULTS/METHODOLOGY: We first evaluated the amplifiability and the amount of genomic DNA in spent embryo culture media from day 3 (n = 32) and day 5/6 (n = 54). Secondly, we evaluated the possibility to genotype the MTHFR polymorphism C677T from media at day 5/6 (n = 8) and blastocoele fluids (n = 9) by direct sequencing. The C677T polymorphism detection rate was 62.5 and 44.4% in medium and fluid, respectively. CONCLUSION: A noninvasive approach for embryo genotyping was possible, but still with limitations due to low detection rate and possible allele dropout.

The Indole-3-carbinol cyclic tetrameric derivative CTet synergizes with cisplatin and doxorubicin in triple-negative breast cancer cell lines.

BACKGROUND/AIM: The indole-3-carbinol cyclic tetrameric derivative (CTet) inhibits breast cancer cell proliferation by endoplasmic reticulum stress and autophagy-related cell death induction, AKT/PKB (protein kinase B) activity inhibition and p53-independent overexpression of cyclin-dependent kinase inhibitor-1A (p21/CDKN1A). In the present study we evaluated the synergistic activity of CTet in combination with cisplatin and doxorubicin in triple-negative breast cancer cell lines. MATERIALS AND METHODS: Synergisms were evaluated in terms of cell viability, induction of autophagy and overexpression of microtubule-associated protein-1 light chain-3 beta (MAP1LC3B) autophagy-related gene in MDA-MB-231 and BT-20 triple-negative breast cancer cells. RESULTS: We demonstrated that CTet in combination with both cisplatin and doxorubicin synergistically inhibits cell viability and induces autophay. The MAP1LC3B gene was synergistically overexpressed in MDA-MB-231 cells treated with CTet-cisplatin combination. Moreover, the cytotoxic activity of CTet was improved in cells pre-treated with cisplatin and doxorubicin. CONCLUSION: This preliminary in vitro study confirms the potential of CTet as a chemopreventive agent or chemotherapeutic in combination with standard approaches for triple-negative breast cancer.

Antitumoral activity of indole-3-carbinol cyclic tri- and tetrameric derivatives mixture in human breast cancer cells: in vitro and in vivo studies.

Indole-3-carbinol (I3C) and its oligomeric derivatives have been widely studied for their chemopreventive and chemotherapeutic properties. We have previously shown that the I3C cyclic tetrameric derivative CTet inhibits breast cancer cell proliferation in vitro and in xenotrasplanted tumor. Here we report the antitumoral activity of a mixture of tri- and tetrameric cyclic I3C derivatives (CTr/CTet) both in vitro (MCF-7 and MDA-MB-231 breast cancer cell lines) and in vivo in a tumor xenograft model. CTr/CTet mixture avoids the low solubility drawbacks of CTet, thus favouring its solubilization, and reducing purification process, time and costs. CTr/CTet mixture has been shown to inhibit breast cancer cell proliferation (IC50 = 1.3 and 1.6 µg/ml in MCF-7 and MDAMB- 231, respectively) inducing the G0/1 cell cycle phase accumulation. The main molecular events related to CTr/CTet activity are the overexpression of p21, p27 and GADD45A, nuclear translocation of FOXO3a, inhibition of Akt activity and downregulation of estrogen receptor. In vivo, the growth of xenotransplanted tumor has been inhibited and the pro-tumoral low molecular weight cyclin E downregulation has been detected. Our data indicate that CTr/CTet is a potential anticancer combination agent for both hormone-responsive and triple-negative breast tumors.

Induction of endoplasmic reticulum stress response by the indole-3-carbinol cyclic tetrameric derivative CTet in human breast cancer cell lines.

BACKGROUND: Indole-3-carbinol and its metabolic products are considered promising chemopreventive and anticancer agents. Previously we have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In the present study, we further characterize the autophagic response and investigate the mechanism through which CTet regulates these events. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of gene expression microarray data and subsequent confirmation by quantitative real-time PCR, showed that CTet is able to induce up-regulation of key signaling molecules involved in endoplasmic reticulum (ER) stress response (e.g. DDIT3/CHOP, CHAC1, ATF3, HSPA5/BiP/GRP78, CEBPB, ASNS) and autophagy (e.g. MAP1LC3B), in both MCF-7 and MDA-MB-231 cell lines. Moreover, the monitoring of Xbp-1 splicing confirmed the activation of IRE1/Xbp-1 ER stress response branch after CTet treatment. The role of autophagic processes (known to be induced by ER stress) was investigated further through ATG5 gene silencing and pharmacological inhibition of AVOs formation. CTet was shown to induce an autophagy-related cell death. Moreover, CTet-treated cells stained with Hoechst/PI revealed the presence of necrotic processes without evidence of apoptosis. CONCLUSIONS/SIGNIFICANCE: The ER stress response was identified as the main upstream molecular mechanism through which CTet acts in both hormone-responsive and triple-negative breast cancer cells. Because of its important role in cancer development, ER stress is a potential target in cancer therapy. The abiltiy of CTet to induce ER stress response and subsequently activate a death program in tumor cells confirms this molecule as a promising anticancer agent.

Development of a multilevel approach for the evaluation of nanomaterials' toxicity.

AIM: To develop a multilevel approach that includes different toxicity tests and gene-expression studies for toxicity evaluation of engineered nanomaterials developed for biomedical applications. MATERIALS & METHODS: K-562, MCF-7 and U-937 human-derived cell lines were used as models for in vitro toxicity tests. These tests included viability assays (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium [MTS] assay); evaluation of apoptosis/necrosis by propidium iodide staining and DNA laddering assay; evaluation of mitochondrial toxicity (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethyl-benzimidazolcarbocyanine iodide [JC-1] assay); transmission electron microscopy analysis and gene expression analysis by DNA microarray. For in vivo toxicity evaluation, Swiss mice were used for monitoring acute or chronic effects. Two superparamagnetic contrast agents approved for human use (Resovist and Primovist) and two new lanthanide-based luminescent nanoparticles were tested. RESULTS & DISCUSSION: The nanomaterials approved for human use did not show significant toxicities in our assays. Toxicity studies performed on lanthanide-based nanoparticles (EDTA120 and EDTA120D) complexed with the chelating agent EDTA revealed that these nanomaterials induced necrosis in U-937 and K-562 cells while no toxicity was observed in MCF-7 cells. Moreover, no in vivo effects have been observed. The comparative analysis of the nanomaterials and their separated components showed that the toxicity in U-937 and K-562 cells was mainly due to the presence of EDTA. CONCLUSION: The multilevel approach proved to be useful for nanomaterial toxicity characterization. In particular, for the lanthanide-based nanoparticles tested in this work, the EDTA was identified as the main cause of the toxicity in vitro, suggesting a possible applicability of these nanoparticle suspensions for in vivo optical imaging.