Glioblastoma stem cell metabolism and immunity.

Despite enormous efforts being invested in the development of novel therapies for brain malignancies, there remains a dire need for effective treatments, particularly for pediatric glioblastomas. Their poor prognosis has been attributed to the fact that conventional therapies target tumoral cells, but not glioblastoma stem cells (GSCs). GSCs are characterized by self-renewal, tumorigenicity, poor differentiation, and resistance to therapy. These characteristics represent the fundamental tools needed to recapitulate the tumor and result in a relapse. The mechanisms by which GSCs alter metabolic cues and escape elimination by immune cells are discussed in this article, along with potential strategies to harness effector immune cells against GSCs. As cellular immunotherapy is making significant advances in a variety of cancers, leveraging this underexplored reservoir may result in significant improvements in the treatment options for brain malignancies.

RNA Microarray-Based Comparison of Innate Immune Phenotypes between Human THP-1 Macrophages Stimulated with Two BCG Strains.

Currently, the only available vaccine against tuberculosis is Mycobacterium bovis Bacille Calmette-Guérin (BCG). Pulmonary tuberculosis protection provided by the vaccine varies depending on the strain, the patient's age and the evaluated population. Although the adaptive immune responses induced by different BCG strains have been widely studied, little conclusive data is available regarding innate immune responses, especially in macrophages. Here, we aimed to characterize the innate immune responses of human THP-1-derived macrophages at the transcriptional level following a challenge with either the BCG Mexico (M.BCG) or Phipps (P.BCG) strains. After a brief in vitro characterization of the bacterial strains and the innate immune responses, including nitric oxide production and cytokine profiles, we analyzed the mRNA expression patterns and performed pathway enrichment analysis using RNA microarrays. Our results showed that multiple biological processes were enriched, especially those associated with innate inflammatory and antimicrobial responses, including tumor necrosis factor (TNF)-α, type I interferon (IFN-I) and IFN-γ. However, four DEGs were identified in macrophages infected with M.BCG compared to P. BCG. These findings indicated the proinflammatory stimulation of macrophages induced by both BCG strains, at the cytokine level and in terms of gene expression, suggesting a differential expression pattern of innate immune transcripts depending on the mycobacterial strain.

Association of BCAT2 and BCKDH polymorphisms with clinical, anthropometric and biochemical parameters in young adults.

BACKGROUND AND AIM: Circulating amino acids are modified by sex, body mass index (BMI) and insulin resistance (IR). However, whether the presence of genetic variants in branched-chain amino acid (BCAA) catabolic enzymes modifies circulating amino acids is still unknown. Thus, we determined the frequency of two genetic variants, one in the branched-chain aminotransferase 2 (BCAT2) gene (rs11548193), and one in the branched-chain ketoacid dehydrogenase (BCKDH) gene (rs45500792), and elucidated their impact on circulating amino acid levels together with clinical, anthropometric and biochemical parameters. METHODS AND RESULTS: We performed a cross-sectional comparative study in which we recruited 1612 young adults (749 women and 863 men) aged 19.7 ± 2.1 years and with a BMI of 24.9 ± 4.7 kg/m2. Participants underwent clinical evaluation and provided blood samples for DNA extraction and biochemical analysis. The single nucleotide polymorphisms (SNPs) were determined by allelic discrimination using real-time polymerase chain reaction (PCR). The frequencies of the less common alleles were 15.2 % for BCAT2 and 9.83 % for BCKDH. The subjects with either the BCAT2 or BCKDH SNPs displayed no differences in the evaluated parameters compared with subjects homozygotes for the most common allele at each SNP. However, subjects with both SNPs had higher body weight, BMI, blood pressure, glucose, and circulating levels of aspartate, isoleucine, methionine, and proline than the subjects homozygotes for the most common allele (P < 0.05, One-way ANOVA). CONCLUSION: Our findings suggest that the joint presence of both the BCAT2 rs11548193 and BCKDH rs45500792 SNPs induces metabolic alterations that are not observed in subjects without either SNP.

Calreticulin in phagocytosis and cancer: opposite roles in immune response outcomes.

Calreticulin (CRT) is a pleiotropic and highly conserved molecule that is mainly localized in the endoplasmic reticulum. Recently, CRT has gained special interest for its functions outside the endoplasmic reticulum where it has immunomodulatory properties. CRT translocation to the cell membrane serves as an "eat me" signal and promotes efferocytosis of apoptotic cells and cancer cell removal with completely opposite outcomes. Efferocytosis results in a silenced immune response and homeostasis, while removal of dying cancer cells brought about by anthracycline treatment, ionizing-irradiation or photodynamic therapy results in immunogenic cell death with activation of the innate and adaptive immune responses. In addition, CRT impacts phagocyte activation and cytokine production. The effects of CRT on cytokine production depend on its conformation, species specificity, degree of oligomerization and/or glycosylation, as well as its cellular localization and the molecular partners involved. The controversial roles of CRT in cancer progression and the possible role of the CALR gene mutations in myeloproliferative neoplasms are also addressed. The release of CRT and its influence on the different cells involved during efferocytosis and immunogenic cell death points to additional roles of CRT besides merely acting as an "eat me" signal during apoptosis. Understanding the contribution of CRT in physiological and pathological processes could give us some insight into the potential of CRT as a therapeutic target.

[Atypical microbacteriosis: a report of two cases and review of the literature]. / Lipotransferencia complicada con micobacteriosis atípicas. Reporte de dos casos y revisión de la literatura.

Lipotransference is a technique that has evolved within the aesthetic and reconstructive surgery area to change body shape in the individual. However, it has been associated occasionally with infections of varying degrees of morbidity and mortality. We report two cases of patients who underwent abdominal and waist area lipotransference to buttocks, and who developed postoperative infection. Using polymerase chain reaction of DNA extracted from a tissue sample and from a culture, with subsequent sequencing, Mycobacterium chelonae and M. massiliense were identified as causative agents.

Expression and characterization of scFv-6009FV in Pichia pastoris with improved ability to neutralize the neurotoxin Cn2 from Centruroides noxius.

Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % ß-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.

O-GlcNAcylation of FOXK1 orchestrates the E2F pathway and promotes oncogenesis.

Gene transcription is a highly regulated process, and deregulation of transcription factors activity underlies numerous pathologies including cancer. Albeit near four decades of studies have established that the E2F pathway is a core transcriptional network that govern cell division in multi-cellular organisms1,2, the molecular mechanisms that underlie the functions of E2F transcription factors remain incompletely understood. FOXK1 and FOXK2 transcription factors have recently emerged as important regulators of cell metabolism, autophagy and cell differentiation3-6. While both FOXK1 and FOXK2 interact with the histone H2AK119ub deubiquitinase BAP1 and possess many overlapping functions in normal biology, their specific functions as well as deregulation of their transcriptional activity in cancer is less clear and sometimes contradictory7-13. Here, we show that elevated expression of FOXK1, but not FOXK2, in primary normal cells promotes transcription of E2F target genes associated with increased proliferation and delayed entry into cellular senescence. FOXK1 expressing cells are highly prone to cellular transformation revealing important oncogenic properties of FOXK1 in tumor initiation. High expression of FOXK1 in patient tumors is also highly correlated with E2F gene expression. Mechanistically, we demonstrate that FOXK1, but not FOXK2, is specifically modified by O-GlcNAcylation. FOXK1 O-GlcNAcylation is modulated during the cell cycle with the highest levels occurring during the time of E2F pathway activation at G1/S. Moreover, loss of FOXK1 O-GlcNAcylation impairs FOXK1 ability to promote cell proliferation, cellular transformation and tumor growth. Mechanistically, expression of FOXK1 O-GlcNAcylation-defective mutants results in reduced recruitment of BAP1 to gene regulatory regions. This event is associated with a concomitant increase in the levels of histone H2AK119ub and a decrease in the levels of H3K4me1, resulting in a transcriptional repressive chromatin environment. Our results define an essential role of O-GlcNAcylation in modulating the functions of FOXK1 in controlling the cell cycle of normal and cancer cells through orchestration of the E2F pathway.

Similarities and divergences in the metabolism of immune cells in cancer and helminthic infections.

Energetic and nutritional requirements play a crucial role in shaping the immune cells that infiltrate tumor and parasite infection sites. The dynamic interaction between immune cells and the microenvironment, whether in the context of tumor or helminth infection, is essential for understanding the mechanisms of immunological polarization and developing strategies to manipulate them in order to promote a functional and efficient immune response that could aid in the treatment of these conditions. In this review, we present an overview of the immune response triggered during tumorigenesis and establishment of helminth infections, highlighting the transition to chronicity in both cases. We discuss the energetic demands of immune cells under normal conditions and in the presence of tumors and helminths. Additionally, we compare the metabolic changes that occur in the tumor microenvironment and the infection site, emphasizing the alterations that are induced to redirect the immune response, thereby promoting the survival of cancer cells or helminths. This emerging discipline provides valuable insights into disease pathogenesis. We also provide examples of novel strategies to enhance immune activity by targeting metabolic pathways that shape immune phenotypes, with the aim of achieving positive outcomes in cancer and helminth infections.

Identification of peptides presented through the MHC-II of dendritic cells stimulated with Mycobacterium avium.

Mycobacterium avium (M. avium) represents a species of concern, because of its ability to modulate the host's innate immune response, and therefore influence trajectory of adaptative immunity. Since eradicative response against mycobacteria, and M. tuberculosis/M. avium, relies on peptides actively presented on a Major Histocompatibility complex-II (MHC-II) context, we assessed paradoxical stimulation of Dendritic Cell resulting on immature immunophenotype characterized by membrane minor increase of MHC-II and CD40 despite of high expression of the pro-inflammatory tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in supernatants. Identification of M. avium leucine rich peptides forming short α-helices shutting down Type 1T helper (Th1), contribute to the understanding of immune evasion of an increasingly prevalent pathogen, and may provide a basis for future immunotherapy to infectious and non-infectious disease.

Dual contribution of the mTOR pathway and of the metabolism of amino acids in prostate cancer.

BACKGROUND: Prostate cancer is the leading cause of cancer in men, and its incidence increases with age. Among other risk factors, pre-existing metabolic diseases have been recently linked with prostate cancer, and our current knowledge recognizes prostate cancer as a condition with important metabolic anomalies as well. In malignancies, metabolic disorders are commonly associated with aberrations in mTOR, which is the master regulator of protein synthesis and energetic homeostasis. Although there are reports demonstrating the high dependency of prostate cancer cells for lipid derivatives and even for carbohydrates, the understanding regarding amino acids, and the relationship with the mTOR pathway ultimately resulting in metabolic aberrations, is still scarce. CONCLUSIONS AND PERSPECTIVES: In this review, we briefly provide evidence supporting prostate cancer as a metabolic disease, and discuss what is known about mTOR signaling and prostate cancer. Next, we emphasized on the amino acids glutamine, leucine, serine, glycine, sarcosine, proline and arginine, commonly related to prostate cancer, to explore the alterations in their regulatory pathways and to link them with the associated metabolic reprogramming events seen in prostate cancer. Finally, we display potential therapeutic strategies for targeting mTOR and the referred amino acids, as experimental approaches to selectively attack prostate cancer cells.

The Resilience of Pseudomonas aeruginosa to Antibiotics and the Designing of Antimicrobial Peptides to Overcome Microbial Resistance.

Pseudomonas aeruginosa (P. aeruginosa) is a bacterium of medical concern known for its potential to persist in diverse environments due to its metabolic capacity. Its survival ability is linked to its relatively large genome of 5.5-7 Mbp, from which several genes are employed in overcoming conventional antibiotic treatments and promoting resistance. The worldwide prevalence of antibiotic-resistant clones of P. aeruginosa necessitates novel approaches to researching their multiple resistance mechanisms, such as the use of antimicrobial peptides (AMPs). In this review, we briefly discuss the epidemiology of the resistant strains of P. aeruginosa and then describe their resistance mechanisms. Next, we explain the biology of AMPs, enlist the present database platforms that describe AMPs, and discuss their usefulness and limitations in treating P. aeruginosa strains. Finally, we present 13 AMPs with theoretical action against P. aeruginosa, all of which we evaluated in silico in this work. Our results suggest that the AMPs we evaluated have a carpet-like mode of action with a membranolytic function in Gram-positive and Gramnegative bacteria, with a clear potential of synthesis for in vitro evaluation.

EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an important effector of the mTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A). METHODS: We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). RESULTS: eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition. CONCLUSIONS: We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Mouse Model for Efficient Simultaneous Targeting of Glycolysis, Glutaminolysis, and De Novo Synthesis of Fatty Acids in Colon Cancer.

Colon cancer is a highly anabolic entity with upregulation of glycolysis, glutaminolysis, and de novo synthesis of fatty acids, which also induces a hypercatabolic state in the patient. The blockade of either cancer anabolism or host catabolism has been previously proven to be a successful anticancer experimental treatment. However, it is still unclear whether the simultaneous blockade of both metabolic counterparts can limit malignant survival and the energetic consequences of such an approach. In this chapter, by using the CT26.WT murine colon adenocarcinoma cell line as a model of study, we provide a method to simultaneously perform a pharmacological blockade of tumor anabolism and host catabolism, as a feasible therapeutic approach to treat cancer, and to limit its energetic supply.

Drug Repurposing: Considerations to Surpass While Re-directing Old Compounds for New Treatments.

Drug repurposing has increased in recent years as an attractive option for treating a number of diseases. Compared to those brought forward via traditional chemical development, drugs intended for repurposing can enter the market faster and with lower investment from pharmaceutical companies. However, a common trend is to focus on diseases that yield higher returns to the industry, such as cancer and common metabolic and inflammatory conditions, resulting in orphan illnesses and neglected tropical diseases having fewer repurposing options for affected patients. In addition, certain legal concerns, including limited patent coverage for the repurposed drugs and pharmacological challenges in performing clinical trials, reduce the likelihood of success. In this review, we discuss the most important concerns that affect the pathway of drug repurposing, with special emphasis on the economic revenues, government-industry associations, and legal considerations that together impact the pharmaceutical industry's decision-making on which compounds may be eligible for repurposing.

Perspectives on Drug Repurposing.

Complex common diseases are a significant burden for our societies and demand not only preventive measures but also more effective, safer, and more affordable treatments. The whole process of the current model of drug discovery and development implies a high investment by the pharmaceutical industry, which ultimately impact in high drug prices. In this sense, drug repurposing would help meet the needs of patients to access useful and novel treatments. Unlike the traditional approach, drug repurposing enters both the preclinical evaluation and clinical trials of the compound of interest faster, budgeting research and development costs, and limiting potential biosafety risks. The participation of government, society, and private investors is needed to secure the funds for experimental design and clinical development of repurposing candidates to have affordable, effective, and safe repurposed drugs. Moreover, extensive advertising of repurposing as a concept in the health community, could reduce prescribing bias when enough clinical evidence exists, which will support the employment of cheaper and more accessible repurposed compounds for common conditions.

Pharmacological inhibition of tumor anabolism and host catabolism as a cancer therapy.

The malignant energetic demands are satisfied through glycolysis, glutaminolysis and de novo synthesis of fatty acids, while the host curses with a state of catabolism and systemic inflammation. The concurrent inhibition of both, tumor anabolism and host catabolism, and their effect upon tumor growth and whole animal metabolism, have not been evaluated. We aimed to evaluate in colon cancer cells a combination of six agents directed to block the tumor anabolism (orlistat + lonidamine + DON) and the host catabolism (growth hormone + insulin + indomethacin). Treatment reduced cellular viability, clonogenic capacity and cell cycle progression. These effects were associated with decreased glycolysis and oxidative phosphorylation, leading to a quiescent energetic phenotype, and with an aberrant transcriptomic landscape showing dysregulation in multiple metabolic pathways. The in vivo evaluation revealed a significant tumor volume inhibition, without damage to normal tissues. The six-drug combination preserved lean tissue and decreased fat loss, while the energy expenditure got decreased. Finally, a reduction in gene expression associated with thermogenesis was observed. Our findings demonstrate that the simultaneous use of this six-drug combination has anticancer effects by inducing a quiescent energetic phenotype of cultured cancer cells. Besides, the treatment is well-tolerated in mice and reduces whole animal energetic expenditure and fat loss.

Antitumor effects of ivermectin at clinically feasible concentrations support its clinical development as a repositioned cancer drug.

PURPOSE: Ivermectin is an antiparasitic drug that exhibits antitumor effects in preclinical studies, and as such is currently being repositioned for cancer treatment. However, divergences exist regarding its employed doses in preclinical works. Therefore, the aim of this study was to determine whether the antitumor effects of ivermectin are observable at clinically feasible drug concentrations. METHODS: Twenty-eight malignant cell lines were treated with 5 µM ivermectin. Cell viability, clonogenicity, cell cycle, cell death and pharmacological interaction with common cytotoxic drugs were assessed, as well as the consequences of its use on stem cell-enriched populations. The antitumor in vivo effects of ivermectin were also evaluated. RESULTS: The breast MDA-MB-231, MDA-MB-468, and MCF-7, and the ovarian SKOV-3, were the most sensitive cancer cell lines to ivermectin. Conversely, the prostate cancer cell line DU145 was the most resistant to its use. In the most sensitive cells, ivermectin induced cell cycle arrest at G0-G1 phase, with modulation of proteins associated with cell cycle control. Furthermore, ivermectin was synergistic with docetaxel, cyclophosphamide and tamoxifen. Ivermectin reduced both cell viability and colony formation capacity in the stem cell-enriched population as compared with the parental one. Finally, in tumor-bearing mice ivermectin successfully reduced both tumor size and weight. CONCLUSION: Our results on the antitumor effects of ivermectin support its clinical testing.

Erratum: A combination of inhibitors of glycolysis, glutaminolysis and de novo fatty acid synthesis decrease the expression of chemokines in human colon cancer cells.

[This corrects the article DOI: 10.3892/ol.2019.11008.].

The combination of orlistat, lonidamine and 6-diazo-5-oxo-L-norleucine induces a quiescent energetic phenotype and limits substrate flexibility in colon cancer cells.

Cancer upregulates glycolysis, glutaminolysis and lipogenesis, and induces a catabolic state in patients. The concurrent inhibition of both tumor anabolism and host catabolism, and the energetic consequences of such an approach, have not previously been fully investigated. In the present study, CT26.WT murine colon cancer cells were treated with the combination of anti-anabolic drugs orlistat, lonidamine and 6-diazo-5-oxo-L-norleucine (DON; OLD scheme), which are inhibitors of the de novo synthesis of fatty acids, glycolysis and glutaminolysis, respectively. In addition, the effects of OLD scheme sumplemented with the combination of anti-catabolic compounds, namely growth hormone, insulin and indomethacin (GII scheme), were also evaluated. The effects of the compounds used in combination on CT26.WT cell viability, clonogenicity and energetic metabolism were assessed in vitro. The results demonstrated that the anti-anabolic approach reduced cell viability, clonogenicity and cell cycle progression, and increased apoptosis. These effects were associated with decreased oxidative phosphorylation, glycolysis and fuel flexibility. Furthermore, the anti-catabolic scheme, alone or supplemented with anti-anabolic compounds, did not favor tumor growth. These findings indicated that the simultaneous pharmacological inhibition of tumor anabolism and host catabolism exhibits antitumor effects that should be further evaluated.

In Vitro Employment of Recombinant Taenia solium Calreticulin as a Novel Strategy Against Breast and Ovarian Cancer Stem-like Cells.

BACKGROUND AND AIMS: Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth in vitro. METHODS: We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. RESULTS: rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. CONCLUSION: Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.