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The risk stratification of B-acute lymphoblastic leukemia (B-ALL) is based on clinical and biological factors. However, B-ALL has significant biological and clinical heterogeneity and 50% of B-ALL patients do not have defined prognostic markers. In this sense, the identification of new prognostic biomarkers is necessary. Considering different cohorts of childhood B-ALL patients, gene (DPP4/CD38/ENTPD1/NT5E) and protein (CD38/CD39/CD73) expressions of ectonucleotidases were analyzed in silico and ex vivo and the association with prognosis was established. In univariate analyses, expression of NT5E was significantly associated with worse progression-free survival (PFS) in bone marrow (BM) samples. In multivariate analyses, Kaplan–Meier analysis, and log-rank test, higher NT5E expression predicted unfavorable PFS in BM samples. Considering minimal residual disease (MRD), higher levels of cellularity were associated with the high NT5E expression at day 8 of induction therapy. In addition, we observed that white blood cells (WBC) of childhood B-ALL patients had more CD38 compared to the same cell population of healthy donors (HD). In fact, MRD > 0.1% patients had higher CD38 protein expression on WBC in comparison to HD. Noteworthy, we observed higher CD38 expression on WBC than blasts in MRD > 0.1% patients. We suggest that NT5E gene and CD38 protein expression, of the ectonucleotidases family, could provide interesting prognostic biomarkers for childhood B-ALL.

Aim: To evaluate the antitumor efficacy of bevacizumab-functionalized nanocapsules in a rat glioblastoma model after the pretreatment with nanocapsules functionalized with a peptide-specific to the epidermal growth factor receptor variant III. Materials & methods: Nanocapsules were prepared, physicochemical characterized and intranasally administered to rats. Parameters such as tumor size, histopathological characteristics and infiltration of CD8+ T lymphocytes were evaluated. Results: The strategy of treatment resulted in a reduction of 87% in the tumor size compared with the control group and a higher infiltration of CD8+ T lymphocytes in tumoral tissue. Conclusion: The block of two different molecular targets using nose-to-brain delivery represents a new and promising approach against glioblastoma.

3-O-Methylquercetin (3OMQ), a natural 3-O-methylflavonoid, was isolated from Achyrocline satureioides and purified using the high-performance counter current chromatography (HPCCC) on a semi-preparative scale. High-purity 3OMQ (98%) was obtained with excellent recovery (81.8% (w/w)) and good yield (190 mg/100 g of plant). Isolated 3OMQ was evaluated against the A375 human amelanotic melanoma cancer cell line and A375-derived with different degrees of aggressiveness (A375-A7, A375-G10, and A375-PCDNA3). The results showed that 3OMQ reduced the cell viability of all strains, demonstrating time- and dose-dependent responses. 3OMQ was used to obtain hydrogels for the topical treatment of melanoma. Thus, 3OMQ was incorporated into hypromellose hydrogels with/without different cyclodextrins (CDs). The 3OMQ formulations showed permeation/retention in all skin layers, namely stratum corneum, epidermis, and dermis. A significant amount of 3OMQ was found in the replication site of the melanoma cells (epidermis and dermis). Altogether, these results demonstrate that 3OMQ can be isolated from Achyrocline satureioides by HPCCC on a semi-preparative scale and exhibit cytotoxic activity against melanoma cells. Its incorporation into an HPMC hydrogel containing HP-β-CD yielded a formulation with excellent technological and biopharmaceutical characteristics for evaluating the topical management of melanoma.

Among several strategies related to cancer therapy targeting the modulation of αβ-tubulin has shown encouraging findings, more specifically when this is achieved by inhibitors located at the colchicine binding site. In this work, we aim to fish new αβ-tubulin modulators through a diverse and rational VS study, and thus, exhibiting the development of two VS pipelines. This allowed us to identify two compounds 5 and 9 that showed IC50 values of 19.69 and 21.97 μM, respectively, towards possible modulation of αβ-tubulin, such as assessed by in vitro assays in C6 glioma and HEPG2 cell lines. We also evaluated possible mechanisms of action of obtained hits towards the colchicine binding site of αβ-tubulin by using docking approaches. In addition, assessment of the stability of the active (5 and 9) and inactive compounds (3 and 13) within the colchicine binding site was carried out by molecular dynamics (MD) simulations, highlighting the solvent effect and revealing the compound 5 as the most stable in the complex. At last, deep analysis of these results provided some valuable insights on the importance of using mixed ligand- and structure-based strategies in VS campaigns, in order to achieve higher chemical diversity and biological effect as well.

Glioblastoma (GBM) is the most aggressive and lethal among the primary brain tumors, with a low survival rate and resistance to radio and chemotherapy. The P2Y(12) is an adenosine diphosphate (ADP) purinergic chemoreceptor, found mainly in platelets. In cancer cells, its activation has been described to induce proliferation and metastasis. Bearing in mind the need to find new treatments for GBM, this study aimed to investigate the role of the P2Y(12)R in the proliferation and migration of GBM cells, as well as to evaluate the expression of this receptor in patients’ data obtained from the TCGA data bank. Here, we used the P2Y(12)R antagonist, ticagrelor, which belongs to the antiplatelet agent’s class. The different GBM cells (cell line and patient-derived cells) were treated with ticagrelor, with the agonist, ADP, or both, and the effects on cell proliferation, colony formation, ADP hydrolysis, cell cycle and death, migration, and cell adhesion were analyzed. The results showed that ticagrelor decreased the viability and the proliferation of GBM cells. P2Y(12)R antagonism also reduced colony formation and migration potentials, with alterations on the expression of metalloproteinases, and induced autophagy in GBM cells. Changes were observed at the cell cycle level, and only the U251 cell line showed a significant reduction in the ADP hydrolysis profile. TCGA data analysis showed a higher expression of P2Y(12)R in gliomas samples when compared to the other tumors. These data demonstrate the importance of the P2Y(12) receptor in gliomas development and reinforce its potential as a pharmacological target for glioma treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11302-022-09888-w.

Since proline metabolism has been implicated to play an underlying role in apoptotic signaling and cancer, and hyperprolinemic patients present susceptibility to tumors development, this study investigated the effect of proline on cell death, cell cycle, antioxidant enzymes activities, and immunocontent/activity of proteins involved in cell death/survival signaling pathways in C6 glioma cells. C6 cells were incubated with proline (0-5 mM) for 1 h, 24 h, 48 h, 72 h, or 7 days. Proline in high concentrations slightly decreased LDH release, and no cytotoxic effect was seen by Annexin-PI staining. Superoxide dismutase and catalase activities were increased by proline (1 mM) after 72 h, suggesting an increase in reactive species levels. Acetylcholinesterase activity was inhibited by proline at 1, 3, and 5 mM. The cell cycle progression was not altered. Results from Western blot analyses showed that proline at 1 mM after 72 h increased p-NF-ĸB and decreased acetylcholinesterase immunocontent but did not altered AKT, p-AKT, GSK3β, and p-GSK3β. Taken together, the data suggest that high proline levels seems to favor the signaling pathways towards cell proliferation, since acetylcholinesterase, which may act as tumor suppressor, is inhibited by proline. Also, p-NF-κB is increased by proline treatment and its activation is related to tumor cell proliferation and cellular response to oxidants. Proline also induced oxidative stress, but it appears to be insufficient to induce a significant change in cell cycle progression. These data may be related, at least in part, to the increased susceptibility to tumor development in hyperprolinemic individuals.

Immunotherapy as an approach for cancer treatment is clinically promising. CD73, which is the enzyme that produces extracellular adenosine, favors cancer progression and protects the tumor from immune surveillance. While CD73 has recently been demonstrated to be a potential target for glioma treatment, its role in regulating the inflammatory tumor microenvironment has not yet been investigated. Thus, this study explores the immunotherapeutic value of the CD73 blockade in glioblastoma. The immuno-therapeutic value of the CD73 blockade was evaluated in vivo in immunocompetent pre-clinical glioblastoma model. As such, glioblastoma-bearing rats were nasally treated for 15 days with a siRNA CD73-loaded cationic-nanoemulsion (NE-siRNA CD73R). Apoptosis was determined by flow cytometry using Annexin-V staining and cell proliferation was analyzed by Ki67 expression by immunohistochemistry. The frequencies of the CD4+, CD8+, and CD4+CD25highCD39+ (Treg) T lymphocytes; CD11b+CD45high macrophages; CD11b+CD45low-microglia; and CD206+-M2-like phenotypes, along with expression levels of CD39 and CD73 in tumor and tumor-associated immune cells, were determined using flow cytometry, while inflammatory markers associated with tumor progression were evaluated using RT-qPCR. The CD73 blockade by NE-siRNA CD73 was found to induce tumor cell apoptosis. Meanwhile, the population of Tregs, microglia, and macrophages was significantly reduced in the tumor microenvironment, though IL-6, CCL17, and CCL22 increased. The treatment selectively decreased CD73 expression in the GB cells as well as in the tumor-associated-macrophages/microglia. This study indicates that CD73 knockdown using a nanotechnological approach to perform nasal delivery of siRNA-CD73 to CNS can potentially regulate the glioblastoma immune microenvironment and delay tumor growth by inducing apoptosis.

Aim: Glioblastoma multiforme (GBM) is an aggressive cancer with very limited clinical therapies. Herein, we have designed novel mercaptobenzimidazole derivatives (1–7) as multitarget antineoplastic drugs and assessed their antiproliferative profiles on an experimental model for GBM, the C6 glioma line. Results: The target compounds were synthesized in few steps with reasonable yields (33–90%). Compounds 1 (∼18 μM) and 4 (∼20 μM) showed dose-dependent antiproliferative effects on C6 glioma and significantly increased early apoptosis, but only 4 disrupted the cell cycle progression and did not induce autophagy. Docking simulations suggested these compounds as dual kinase and colchicine binding site inhibitors. Conclusion: In spite of the limited selective toxicity, 4 hold the potential to be further optimized for the treatment of GBM.

Glioblastoma (GBM) is the most lethal among malignant gliomas. The tumor invasiveness and therapy-resistance are important clinical hallmarks. Growing evidence emphasizes the purinergic signaling contributing to tumor growth. Here we exposed a potential role of extracellular ATPase activity as a key regulator of temozolomide cytotoxicity and the migration process in GBM cells. The inhibition of ATP hydrolysis was able to improve the impact of temozolomide, causing arrest mainly in S and G2 phases of the cell cycle, leading M059J and U251 cells to apoptosis. In addition to eradicating GBM cells, ATP hydrolysis exhibited a potential to modulate the invasive phenotype and the expression of proteins involved in cell migration and epithelial-to-mesenchymal-like transition in a 3D culture model. Finally, we suggest the ATPase activity as a key target to decline temozolomide resistance and the migratory phenotype in GBM cells.

Glioblastoma is the most common and lethal malignant brain tumor. Despite simvastatin (SVT) showing potential anticancer properties, its antitumoral effect against glioblastoma appears limited when the conventional oral administration route is selected. As a consequence, nose-to-brain delivery has been proposed as an alternative route to deliver SVT into the brain. This study aimed to prepare chitosan-coated simvastatin-loaded lipid-core nanocapsules (LNC

The purinergic signaling has drawn attention from academia and more recently from pharmaceutical industries as a potential therapeutic route for cancer treatment, since ATP may act as chemotactic agent and possess in vitro antineoplastic activity. On the other way, adenosine, produced in extracellular medium by ecto-5'-NT, acts as immunosuppressor and is related to neoangiogenesis, vasculogenesis and evasion to the immune system. Consequently, inhibitors of ecto-5'-NT may prevent tumor progression, reducing adenosine concentrations, preventing escape from the host's immune system and slowing cancer's growth. This review aims to highlight important biochemical and structural features of ecto-5'NT, highlight its expression profile in normal and cancer cell lines detailing compounds which may act as expression regulators and to review the several classes of ecto-5'NT inhibitors developed in the past 12 years, in order to build a general structure-activity relationship model to guide further compound design.

Aim: To develop and characterize bozepinib-loaded lipid-core nanocapsules (BZP-LNC+) as a potential treatment for glioblastoma (GBM). Methods: Characterization of nanocapsules was performed by diameter, polydispersity index, Zeta potential, pH and encapsulation efficiency. GBM cell viability, cell cycle and Annexin/PI were evaluated after BZP-LNC+ treatment. Synergism between BZP-LNC+ and temozolomide (TMZ) was performed by CompuSyn software and confirmed in vitro and in vivo. Results: BZP-LNC+ showed adequate particle sizes, positive Zeta potential, narrow size distribution and high encapsulation efficiency. BZP-LNC+ reduces GBM growth by inducing apoptosis. BZP-LNC+ and TMZ showed synergistic effect in vitro and reduced the in vivo glioma growth by approximately 81%. Conclusion: The present study provides proof-of-principle insights for the combination of these drugs for GBM treatment.

Glioblastoma (GB) is a histological and genetically heterogeneous brain tumor that is highly proliferative and vascularized. The prognosis is poor with currently available treatment. In this study, we evaluated the cytotoxicity and antiangiogenic activity of doxorubicin-loaded-chitosan-coated-arginylglycylaspartic acid-functionalized-poly(ε-caprolactone)-alpha bisabolol-LNC (AB-DOX-LNC-L-C-RGD). The nanoformulation was prepared by self-assembling followed by interfacial reactions, physicochemically characterized and evaluated in vitro against GB cell lines (U87MG and U138MG) and in vivo using the chicken chorioallantoic membrane assay (CAM). Spherical shape nanocapsules had a hydrodynamic mean diameter of 138 nm, zeta potential of +13.4 mV, doxorubicin encapsulation of 65%, and RGD conjugation of 92%. After 24 h of treatment (U87MG and U138MG), the median inhibition concentrations (IC50) were 520 and 490 nmol L−1 doxorubicin-equivalent concentrations, respectively. The treatment induced antiproliferative activity with S-phase cell-cycle arrest and apoptosis in the GB cells. Furthermore, after 48 h of exposure, evaluation of antiangiogenic activity (CAM) showed that the relative vessel growth following treatment with the nanocapsules was 5.4 times lower than that with the control treatment. The results support the therapeutic potential of the nanoformulation against GB and, thereby, pave the way for future preclinical studies.

The conversion of adenosine to inosine is catalyzed by adenosine deaminase (ADA) (EC 3.5.4.4), which has two isoforms in humans (ADA1 and ADA2) and belongs to the zinc-dependent hydrolase family. ADA modulates lymphocyte function and differentiation, and regulates inflammatory and immune responses. This study investigated ADA activity in lymphocyte-rich peripheral blood mononuclear cells (PBMCs) in the absence of disease. The viability of lymphocyte-rich PBMCs isolated from humans and kept in 0.9% saline solution at 4-8°C was analyzed over 20 h. The incubation time and biochemical properties of the enzyme, such as its Michaelis-Menten constant (Km) and maximum velocity (Vmax), were characterized through the liberation of ammonia from the adenosine substrate. Additionally, the presence of ADA protein on the lymphocyte surface was determined by flow cytometry using an anti-CD26 monoclonal human antibody, and the PBMCs showed long-term viability after 20 h. The ADA enzymatic activity was linear from 15 to 120 min of incubation, from 2.5 to 12.5 µg of protein, and pH 6.0 to 7.4. The Km and Vmax values were 0.103±0.051 mM and 0.025±0.001 nmol NH3·mg-1·s-1, respectively. Zinc and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) inhibited enzymatic activity, and substrate preference was given to adenosine over 2′-deoxyadenosine and guanosine. The present study provides the biochemical characterization of ADA in human lymphocyte-rich PBMCs, and indicates the appropriate conditions for enzyme activity quantification.

New finding What is the central question of this study? How does moderate-intensity aerobic exercise affect the behaviour of purinergic enzymes in sedentary, overweight and physically active subjects? What is the relationship between purinergic and inflammatory responses triggered by exercise? What is the main finding and its importance? Moderate-intensity aerobic exercise modifies the activity of purinergic enzymes and the levels of nucleotides and nucleosides. These results are similar in subjects with different biological characteristics. 5'-Nucleotidase activity and adenosine levels are associated with inflammatory responses. This study suggests that a purinergic pathway is related to the inflammatory responses triggered by exercise. Abstract Purinergic signalling is a mechanism of extracellular communication that modulates events related to exercise, such as inflammation and coagulation. Herein, we evaluated the effects of acute moderate-intensity exercise on the activities of purinergic enzymes and plasma levels of adenine nucleotides in individuals with distinct metabolic characteristics. We analysed the relationship between purinergic parameters, inflammatory responses and cardiometabolic markers. Twenty-four healthy males were assigned to three groups: normal weight sedentary (n = 8), overweight sedentary (n = 8) and normal weight physically active (n = 8). The volunteers performed an acute session of moderate-intensity aerobic exercise on a treadmill at 70% of V O 2 peak ; blood samples were drawn at baseline, immediately post-exercise and at 1 h post-exercise. Immediately post-exercise, all subjects showed increases in ATP, ADP, AMP and p-nitrophenyl thymidine 5'-monophosphate hydrolysis, while AMP hydrolysis remained increased at 1 h after exercise. High-performance liquid chromatography analysis demonstrated lower levels of ATP and ADP at post- and 1 h post-exercise in all groups. Conversely, adenosine and inosine levels increased at post-exercise, but only adenosine remained augmented at 1 h after exercise in all groups. With regard to inflammatory responses, the exercise protocol increased tumour necrosis factor α (TNF-α) and interleukin 8 (IL-8) concentrations in all subjects, but only TNF-α remained elevated at 1 h after exercise. Significant correlations were found between the activity of 5'-nucleotidase, adenosine levels, V O 2 peak , triglyceride, TNF-α and IL-8 levels. Our findings suggest a purinergic signalling pathway that participates, at least partially, in the inflammatory responses triggered by acute moderate-intensity exercise. The response of soluble nucleotidases to acute moderate exercise appears to be similar between subjects of different biological profiles.

Extracellular vesicles (EVs) are released especially by cancer cells. They modulate the tumor microenvironment by interacting with immune cells while carrying immunosuppressive or immunostimulatory molecules. In this review, we will explore some conflicting reports regarding the immunological outcomes of EVs in cancer progression, in which they might initiate an antitumor immune response or an immunosuppressive response. Concerning immunosuppression, the role of tumor-derived EVs’ in the adenosinergic system is underexplored. The enhancement of adenosine (ADO) levels in the tumor microenvironment impairs T-cell function and cytokine release. However, some tumor-derived EVs may deliver immunostimulatory factors, promoting immunogenic activity, even with ADO production. The modulatory role of ADO over the tumor progression represents a piece in an intricate microenvironment with anti and pro tumoral seesaw-like mechanisms.

Glioblastoma (GBM) is the most malignant and deadly brain tumor. GBM cells overexpress the CD73 enzyme, which controls the level of extracellular adenosine, an immunosuppressive molecule. Studies have shown that some nonsteroidal anti-inflammatory drugs (NSAIDs) and methotrexate (MTX) have antiproliferative and modulatory effects on CD73 in vitro and in vivo. However, it remains unclear whether the antiproliferative effects of MTX and NSAIDS in GBM cells are mediated by increases in CD73 expression and adenosine formation. The aim of this study was to evaluate the effect of the NSAIDs, naproxen, piroxicam, meloxicam, ibuprofen, sodium diclofenac, acetylsalicylic acid, nimesulide, and ketoprofen on CD73 expression in GBM and mononuclear cells. In addition, we sought to understand whether the effects of MTX may be mediated by CD73 expression and activity. Cell viability and CD73 expression were evaluated in C6 and mononuclear cells after exposure to NSAIDs. For analysis of the mechanism of action of MTX, GBM cells were treated with APCP (CD73 inhibitor), dipyridamole (inhibitor of adenosine uptake), ABT-702 (adenosine kinase enzyme inhibitor), or caffeine (P1 adenosine receptor antagonist), before treatment with MTX and AMP, in the presence or not of mononuclear cells. In summary, only MTX increased the expression of CD73 in GBM cells decreasing cells viability by mechanisms independent of the adenosinergic system. Further studies are needed to understand the role of MTX in the GBM microenvironment.

Background: Dihydropyrimidin-2-thiones (DHPMs) are a class of heterocyclic compound which have been intensively investigated mainly due to their anticancer activity as kinesin Eg5 inhibitors. Materials & methods: A library of N1 aryl substituted DHPMs were tested against glioma and bladder cancer cell lines. Quantitative structure–activity relationship (QSAR) investigation was performed in order to identify key elements of DHPMs linked with their antiproliferative effect. The toxicity of most active compounds was investigated using Caenorhabditis elegans as the model. Results & conclusion: DHPMs 9, 13 and 17 have been identified as having improved activity against glioma and bladder cell lines as compared with monastrol. Flow cytometry investigations showed that the new compounds induce cell cycle arrest in phase G2/M and cell death by apoptosis. In addition, compound 13 was able to modulate the reactive oxygen species production in vivo in C. elegans. The biphenyl dihydropyrimidinthiones provided a safety profile in C. elegans.

Bevacizumab (BCZ) is a recombinant monoclonal antibody that inhibits the biological activity of the vascular endothelial growth factor, which has an important role in angiogenesis for tumoral growth and progression. In this way, our objective was to develop chitosan-coated lipid-core nanocapsules functionalized with BCZ by an organometallic complex using gold-III. The formulation was produced and characterized in relation to physicochemical characteristics. Furthermore, the antitumoral and antiangiogenic activities were evaluated against C6 glioma cell line and chicken embryo chorioallantoic membrane (CAM), respectively. Final formulation showed nanometric size, narrow polydispersity, positive zeta potential and gold clusters size lower than 2 nm. BCZ in aqueous solution (0.01–0.10 μmol L−1) did not show cytotoxic activity in vitro against C6 glioma cell line; although, MLNC-Au-BCZ showed cytotoxicity with a median inhibition concentration of 30 nmol L−1 of BCZ. Moreover, MLNC-Au-BCZ demonstrated cellular internalization dependent on incubation time and BCZ concentration. BCZ solution did not induce significant apoptosis as compared to MLNC-Au-BCZ within 24 h of treatment. CAM assay evidenced potent antiangiogenic activity for MLNC-Au-BCZ, representing a decrease of 5.6 times in BCZ dose comparing to BCZ solution. MLNC-Au-BCZ is a promising product for the treatment of solid tumors.

Aim: To characterize a method to isolate glioma-derived extracellular vesicles (GEVs) and understand their role in immune system modulation and glioma progression. Materials & methods: GEVs were isolated by differential centrifugation from C6 cell supernatant and characterized by size and expression of CD9, HSP70, CD39 and CD73. The glioma model was performed by injecting C6 glioma cells into the right striatum of Wistar rats in the following groups: controls (C6 cells alone), coinjection (C6 cells + GEVs) and GEVs by intranasal administration followed by immune cells, tumor size and cells proliferation analyses. Results: GEVs presented uniform size (175 nm), expressed CD9, HSP70, CD39, CD73 and produced adenosine. In vivo, we observed a reduction in tumor size, in cell proliferation (Ki-67) and in a regulatory cell marker (FoxP3). Conclusion: GEVs, administered before or at tumor challenge, have antiproliferative properties and reduce regulatory cells in the glioma microenvironment.

Additional file 6: Fig. S6 Correlation matrix to all measurements assessed. ρ coef. Represent the pearson correlation coefficients. Red and green boxes correspond to all negative or positive significant correlations, respectively. Abbreviatures: ‘% Annex+/PI- cells’ - % of annexin+/Propidium Iodide negative cells; ‘% CRT+ cells’ - % of calreticulin-positive cells; ‘% AO+ cells’ - % of acridine orange-positive cells; ‘ATP’ - levels of extracellular ATP; ‘HMGB1’ - levels of extracellular HMGB1; ‘CRT Marg.’ - Margination of Calreticulin; ‘%LR, %N, %SR and %Irreg’ - percentage of ‘Large and Regular’, ‘Normal’, ‘Small and Regular’ and ‘Irregular’ nuclei from NMA analysis; ‘Nuc Area’ - nuclear area.

Additional file 4: Fig. S4 Calreticulin (CRT) exposure and its correlation with cell area. (A) Representative histograms of cell count and CRT exposure in the cell surface, as obtained by flow cytometry. *p

Additional file 7: Fig. S7 Influence of HMGB1 levels in the prognosis of NSCLC. Kaplan–Meier survival analysis according to HMGB1 levels.

Additional file 3: Fig. S3 Nuclear and LC3 co-staining. Representative images from each treatment are shown. Double arrowheads: nuclei classified as small and regular in the NMA; Arrows – nuclei classified as large in the NMA; Single arrowhead: nuclei classified as normal in NMA.

Additional file 5: Fig. S5 Influence of CRT levels in the prognosis and staging of NSCLC. (A) Kaplan-Meier survival curve to patients expressing high or low levels of CRT in the TCGA cohort. (B) Dead/live ratio of patients for grade i-iv tumors according to CRT levels. (C to F) Influence of CRT levels on the outcome (i.e. dead or alive) of patients with stage i to iv NSCLC.

Background Chemotherapeutics can stimulate immune antitumor response by inducing immunogenic cell death (ICD), which is activated by Damage-Associated Molecular Patterns (DAMPs) like the exposure of calreticulin (CRT) on the cell surface, the release of ATP and the secretion of High Mobility Group Box 1 (HMGB1). Methods Here, we investigated the levels of ICD-associated DAMPs induced by chemotherapeutics commonly used in the clinical practice of non-small cell lung cancer (NSCLC) and the association of these DAMPs with apoptosis and autophagy. A549 human lung adenocarcinoma cells were treated with clinically relevant doses of cisplatin, carboplatin, etoposide, paclitaxel and gemcitabine. We assessed ICD-associated DAMPs, cell viability, apoptosis and autophagy in an integrated way. Results Cisplatin and its combination with etoposide induced the highest levels of apoptosis, while etoposide was the less pro-apoptotic treatment. Cisplatin also induced the highest levels of ICD-associated DAMPs, which was not incremented by co-treatments. Etoposide induced the lower levels of ICD and the highest levels of autophagy, suggesting that the cytoprotective role of autophagy is dominant in relation to its pro-ICD role. High levels of CRT were associated with better prognosis in TCGA databank. In an integrative analysis we found a strong positive correlation between DAMPs and apoptosis, and a negative correlation between cell number and ICD-associated DAMPs as well as between autophagy and apoptosis markers. We also purpose a mathematical integration of ICD-associated DAMPs in an index (IndImunnog) that may represent with greater biological relevance this process. Cisplatin-treated cells showed the highest IndImmunog, while etoposide was the less immunogenic and the more pro-autophagic treatment. Conclusions Cisplatin alone induced the highest levels of ICD-associated DAMPs, so that its combination with immunotherapy may be a promising therapeutic strategy in NSCLC.

Additional file 2: Fig. S2 Morphological changes and caspase activation in A549 cells treated with chemotherapeutics. (A) Analysis of cell phenotype assessed by flow cytometry. Population 1 indicates normal cell morphology. Reduced cell size (low FSC) with increased intracellular granularity (high SSC) is suggestive of early apoptosis (population 3); high intracellular granularity (high SSC) suggests cytoplasmic vacuolization, as observed after autophagy activation (population 2); and cell debrie suggests late apoptosis, which is characterized by cell fragmentation (population 4). A scheme showing these populations is shown on the right. (B) Cell area measured through images from immunocytochemistry. (C) Representative images of nuclear staining with DAPI (magnification: 200x). (D) Representative plots of active caspase 3 versus FSC. Red area delimits the population of cells with small size (i.e. cell shrinkage, a typical change of early apoptotic cells) and active caspase 3. Numbers represent the percentage of cells inside the red area ± SEM. Cisplatin 80 μM was used as a positive control (C+).

Additional file 1: Fig. S1 Dose-response curves in A549 cells. Cells were treated with the chemotherapeutics as indicated, for 48 h. After this, cell number was determined through flow cytometry.

Achyrocline satureioides, popularly known as "marcela", is a medicinal plant found in South America. This plant is rich in flavonoids, which have been reported to exert numerous biological activities. The aim of this study was to purify, identify and evaluate the mechanisms underlining anticancer activity of A. satureioides flavonoids in glioma cell lines (U87, U251 and C6) as well as their comparative toxicity in normal brain cells (primary astrocytes, neurons and organotypic hippocampal cultures). The main flavonoids present in A. satureioides are luteolin, quercetin, 3-O-methyl-quercetin and achyrobichalcone, the later a very unique metabolite present in this plant. Isolated flavonoids as well as A. satureioides extracts reduced proliferation and clonogenic survival, and induced apoptosis of glioma cell lines. In addition, A. satureioides flavonoids potentiated the cytotoxic effect and apoptosis induction by the glioma chemotherapeutic temozolomide (TMZ). Importantly, A. satureioides flavonoids were less cytotoxic to astrocytes, neuron:astrocytes co-cultures and hippocampal cultures if compared to gliomas. Investigation of 10 cancer-related pathways showed a reduced activation of MYC and the Map kinases ERK and JNK by A. satureioides flavonoid-enriched extract, an effect not observed when individual flavonoids were evaluated. Altogether, the herein presented results show that A. satureioides extract possesses a combination of flavonoids, some unique for this plant, which have synergistic anticancer activity and potential for further studies in vivo.

Glioblastoma is the worst and most common primary brain tumor. Here, we demonstrated the role of CD73, an enzyme responsible for adenosine (ADO) production, in glioblastoma progression. ADO increased glioma cell viability via A1 receptor sensitization. CD73 downregulation decreased glioma cell migration and invasion by reducing metalloproteinase-2 and vimentin expression and reduced cell proliferation by 40%, which was related to necrosis and sub-G1 phase blockage of cell cycle. Those effects also involved the stimulation of Akt/NF-kB pathways. Additionally, CD73 knockdown or enzyme inhibition potentiated temozolomide cytotoxic effect on glioma cells by decreasing the IC50 value and sensitizing cells to a non-cytotoxic drug concentration. CD73 inhibition also decreased in vivo rat glioblastoma progression. Delivery of siRNA-CD73 or APCP reduced tumor size by 45 and 40%, respectively, when compared with control. This effect was followed by a parallel 95% reduction of ADO levels in cerebrospinal fluid, indicating the role of extracellular ADO in in vivo glioma growth. Treatment did not induce systemic damage or mortality. Altogether, we conclude that CD73 is an interesting target for glioblastoma treatment and its inhibition may provide new opportunities to improve the treatment of brain tumors. Graphical Abstract ᅟ.

A multi-component synthesis of biphenyl dihydropyrimidin-2-thiones from 1-phenylthiourea, aldehydes and ketones or di-ketones has been demonstrated. The reaction proceeded well for aldehydes with electron donor or acceptor substituents under mild conditions.

In order to search for new approaches to treat glioma, intranasal administration has been proposed as an alternative route to deliver drugs into the brain. Among the drug alternatives, kaempferol (KPF) has been reported to induce glioma cell death. This study aimed to prepare nanoemulsions containing KPF with and without chitosan to investigate their potential for brain delivery following intranasal administration, and to evaluate their antitumor activity against glioma cells. KPF-loaded nanoemulsion (KPF-NE) and KPF-loaded mucoadhesive nanoemulsion (KPF-MNE) were prepared by high-pressure homogenization technique and were characterized for their globule size, zeta potential, drug content, pH, viscosity, mucoadhesive strength and morphology. KPF from KPF-MNE showed significantly higher permeation across the mucosa in ex vivo diffusion studies. Histopathological examination suggests both nanoemulsions to be safe for the nasal mucosa and able to preserve KPF antioxidant capability. KPF-MNE enhanced significantly the amount of drug into rat's brain following intranasal administration (5- and 4.5-fold higher than free drug and KPF-NE, respectively). In addition, KPF-MNE reduced C6 glioma cell viability through induction of apoptosis to a greater extent than either free KPF or KPF-NE. The mucoadhesive nanoemulsion developed for intranasal administration may be a promising system for delivery to the brain, and KPF-MNE is a candidate for further antiglioma trials.

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults and the current treatments only have a modest effect on patient survival. Recent studies show that bozepinib (BZP), a purine derivative, has potential applications in cancer treatment. The aim of this study was to evaluate the effect of BZP against GBM cells, specially concerning the purinergic system. Thus, GBM cells (C6 and U138 cell lines) were treated with BZP and cell viability, cell cycle, and annexin/PI assays, and active caspase-3 measurements were carried out. Besides, the effect of BZP over the purinergic system was also evaluated in silico and in vitro. Finally, we evaluate the action of BZP against important markers related to cancer progression, such as Akt, NF-κB, and CD133. We demonstrate here that BZP reduces GBM cell viability (IC50 = 5.7 ± 0.3 µM and 12.7 ± 1.5 µM, in C6 and U138 cells, respectively), inducing cell death through caspase-dependent apoptosis, autophagosome formation, activation of NF-κB, without any change in cell cycle progression or on the Akt pathway. Also, BZP modulates the purinergic system, inducing an increase in CD39 enzyme expression and activity, while inhibiting CD73 activity and adenosine formation, without altering CD73 enzyme expression. Curiously, one cycle of treatment resulted in enrichment of GBM cells expressing NF-κB and CD133+, suggesting resistant cells selection. However, after another treatment round, the resistant cells were eliminated. Altogether, BZP presented in vitro anti-glioma activity, encouraging further in vivo studies in order to better understand its mechanism of action.

The pathophysiology of bipolar disorder remains incompletely elucidated. The purinergic receptor, P2X7 (P2X7R), plays a central role in neuroinflammation, the establishment, and maintenance of microglial activation and neuronal damage/death, all characteristics of bipolar disorder pathology. The present study aims to explore the participation of the P2X7R in a preclinical pharmacological model of mania. We analyzed the modulatory effects of the P2X7R antagonist, brilliant blue, on behavior, monoamines, gene expression, serum purine levels, and cell typing in a pharmacological model of mania induced by D-amphetamine (AMPH) in mice. Our results corroborate an association between the P2X7 receptor and the preclinical animal model of mania, as demonstrated by the decreased responsiveness to AMPH in animals with pharmacologically blocked P2X7R. This study further suggests a possible dopaminergic mechanism for the action of P2X7 receptor antagonism. Additionally, we observed increased peripheral levels of adenosine, a neuroprotective molecule, and increased central expression of Entpd3 and Entpd1 leading to the hydrolysis of ATP, a danger signal, possibly as an attempt to compensate for the damage induced by AMPH. Lastly, P2X7R antagonism in the AMPH model was found to potentially modulate astrogliosis. Our results support the hypothesis that P2X7R plays a vital role in the pathophysiology of mania, possibly by modulating the dopaminergic pathway and astrogliosis, as reflected in the behavioral changes observed. Taken together, this study suggests that a purinergic system imbalance is associated with the AMPH-induced preclinical animal model of mania. P2X7R may represent a promising molecular therapeutic target for bipolar disorder.

BRCA-1 is a nuclear protein involved in DNA repair, transcriptional regulation, and cell cycle control. Its involvement in other cellular processes has been described. Here, we aimed to investigate the role of BRCA-1 in macrophages M(LPS), M(IL-4), and tumor cell-induced differentiation. We used siRNAs to knockdown BRCA-1 in RAW 264.7 macrophages exposed to LPS, IL-4, and C6 glioma cells conditioned medium (CMC6), and evaluated macrophage differentiation markers and functional phagocytic activity as well as DNA damage and cell survival in the presence and absence of BRCA-1. LPS and CMC6, but not by IL-4, increased DNA damage in macrophages, and this effect was more pronounced in BRCA-1-depleted cells, including M(IL-4). BRCA-1 depletion impaired expression of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the phagocytic activity of macrophages in response to LPS. In CMC6-induced differentiation, BRCA-1 knockdown inhibited TNF-α and IL-6 expression which was accompanied by upregulation of the anti-inflammatory markers IL-10 and TGF-β and reduced phagocytosis. In contrast, M(IL-4) phenotype was not affected by BRCA-1 status. Molecular docking predicted that the conserved BRCA-1 domain BRCT can interact with the p65 subunit of NF-κB. Immunofluorescence assays showed that BRCA-1 and p65 co-localize in the nucleus of LPS-treated macrophages and reporter gene assay showed that depletion of BRCA-1 decreased LPS and CMC6-induced NF-κB transactivation. IL-4 had no effect upon NF-κB. Taken together, our findings suggest a role of BRCA-1 in macrophage differentiation and phagocytosis induced by LPS and tumor cells secretoma, but not IL-4, in a mechanism associated with inhibition of NF-κB.

Kaempferol (KPF), an important flavonoid, has been reported to exert antioxidant, anti-inflammatory, and anticancer activity. However, this compound has low water solubility and hence poor oral bioavailability. This work aims to prepare a solid dispersion (SD) of KPF using Poloxamer 407 in order to improve the water solubility, dissolution rate, and pharmacokinetic properties KPF. After optimization, SDs were prepared at a 1:5 weight ratio of KPF:carrier using the solvent method (SDSM) and melting method (SDMM). Formulations were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD) analysis, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). The solubility in water of carried-KPF was about 4000-fold greater than that of free KPF. Compared with free KPF or the physical mixture, solid dispersions significantly increased the extent of drug release (approximately 100% within 120 min) and the dissolution rate. Furthermore, after oral administration of SDMM in rats, the area under the curve (AUC) and the peak plasma concentration (Cmax) of KPF from SDMM were twofold greater than those of free KPF (p

Prostate cancer is among the major malignancies that affect men around the world. Adenine nucleotides are important signaling molecules that mediate innumerous biological functions in pathophysiological conditions, including cancer. These molecules are degraded by several ectoenzymes named ectonucleotidases that produce adenosine in the extracellular medium. Some of these ecto-enzymes can be found in soluble in the blood stream. Thus, the present study aimed to evaluate the hydrolysis of adenine nucleotides (ATP, ADP, and AMP) in the plasma blood of patients with prostate cancer. Peripheral blood samples were collected, and questionnaires were filled based on the clinical data of the medical records. The nucleotide hydrolysis was performed by Malachite Green method using ATP, ADP, and AMP as substrates. Plasma from prostate cancer patients presented an elevated hydrolysis of all nucleotides evaluated when compared to healthy individuals. NTPDase inhibitor (ARL67156) and the alkaline phosphatase inhibitor (levamisole) did not alter ATP hydrolysis. However, AMP hydrolysis was reduced by the CD73 inhibitor, APCP, and by levamisole, suggesting the action of a soluble form of CD73 and alkaline phosphatase. On microvesicles, it was observed that there was a low expression and activity of CD39 and almost absent of CD73. The correlation of ATP, ADP, and AMP hydrolysis with clinic pathological data demonstrated that patients who received radiotherapy showed a higher AMP hydrolysis than those who did not, and patients with lower clinical stage (CS-IIA) presented an elevated ATP hydrolysis when compared to those with more advanced clinical stages (CS-IIB and CS-III). Patients of all clinical stages presented an elevated AMPase activity. Therefore, we can suggest that the nucleotide hydrolysis might be attributed to soluble ecto-enzymes present in the plasma, which, in a coordinate manner, produce adenosine in the blood stream, favoring prostate cancer progression.

Lithium is the mainstay of pharmacotherapy for treating bipolar disorder (BD). However, despite its wide use for over 60 years in the clinic, its mechanisms of action are not yet well defined. Elucidating lithium's mechanism of action will not only shed light on the pathophysiology of BD, but also potentially uncover new treatment targets. Previous studies suggest that the purinergic system may be involved in lithium's neuroprotective action; thus, the specific aim of this study is to better understand the neuroprotective action of lithium against ATP-induced cellular effect in both neuronal and microglial cellular lineages. We used PC12 neuronal and N9 microglial cells, evaluating cell death by cell counting and Annexin/PI cytometry assay, P2 × 7R immunocontent and ectonucleotidases activity, together with cytokine and nitrite assessment for microglial activity determination. Our results indicate that cells of different neural origins are responsive to ATP, in the sense of neuronal excitotoxicity and microglial switch into an activated M1-like phenotype respectively. Lithium, in turn, modulates the response in neuronal PC12 cells, preventing ATP-induced cell death. On the other hand, in N9 microglial cells, lithium was unable to prevent ATP-induced activation via P2 × 7R, indicating that lithium protective action against the effects of ATP more likely occurs in neurons rather than in microglia. Further studies are needed to better characterize the involvement of the purinergic system in the mechanism of action of lithium against neuronal death and microglial activation, in order to uncover new therapeutic adjunctive targets, such as antagonism of P2 × 7R, as potential approach for bipolar disorder treatment.