Lymphocytes from B-acute lymphoblastic leukemia patients present differential regulation of the adenosinergic axis depending on risk stratification.

PURPOSE: Although risk-stratified chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a significant number of cases. The identification of new therapeutic targets as well as prognostic and diagnostic biomarkers can improve B-ALL patients' clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients' lymphocyte subpopulations. METHODS: Peripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of [Formula: see text], and [Formula: see text] in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC). RESULTS: Comparing B-ALL patients to health donors, we observed an increase of CD4 + and CD8 + T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+ frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1ß, and ADO concentrations, together with an increase in AMP in B-ALL patients' plasma. CONCLUSION: As immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of different cellular subsets. We observed a pro-tumor immunity expression profile in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL.

EGFRvIII peptide nanocapsules and bevacizumab nanocapsules: a nose-to-brain multitarget approach against glioblastoma.

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.

Development of bozepinib-loaded nanocapsules for nose-to-brain delivery: preclinical evaluation in glioblastoma.

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.

New pharmacological findings linked to biphenyl DHPMs, kinesin Eg5 ligands: anticancer and antioxidant effects.

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.

1,2,3-Triazole tethered 2-mercaptobenzimidazole derivatives: design, synthesis and molecular assessment toward C6 glioma cell line.

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.

Genotoxicity and cytotoxicity of oxindole alkaloids from Uncaria tomentosa (cat's claw): Chemotype relevance.

ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria tomentosa (Willdenow ex Roemer & Schultes) DC. (Rubiaceae) or cat's claw is a climber vine from the South American rainforest used in folk medicine for cancer treatment. Its antitumor activity has been mostly ascribed to pentacyclic oxindole alkaloids (POA) from stem bark and leaves while the activity of tetracyclic oxindole alkaloids (TOA) remains unknown. In recent times, the occurrence of three chemotypes based on its oxindole alkaloid profile was noticed in U. tomentosa, namely, chemotype I (POA cis D/E ring junction); chemotype II (POA trans D/E ring junction) or chemotype III (TOA). Consequently, the relationship between the chemotype and cytotoxic and genotoxic activities deserves attention. AIM OF THE STUDY: To evaluate the influence of cat's claw chemotypes on genotoxicity and cytotoxicity against non malignant and malignant human cell line models. MATERIAL AND METHODS: Four authentic stem bark cat's claw samples (SI-SIV) and two leaf samples (LII and LIII) were analyzed by HPLC-PDA, properly extracted and fractioned by ion-exchange to obtain oxindole alkaloid purified fractions (OAPFs). The freeze-dried fractions were assayed for genotoxicity and cytotoxicity against human leukocytes (non malignant cell line) by the micronuclei frequency method and the alkaline comet DNA assay, and the trypan blue method, respectively. Moreover, the cytotoxicity of each OAPF was evaluated against a human bladder cancer cell line (T24) and human glioblastoma cell line (U-251-MG) by MTT method (malignant cell lines). Additionally, the isomerization of oxindole alkaloids throughout the course of cell incubation was monitored by HPLC-PDA. RESULTS: Based on HPLC-PDA analyses, sample SI was characterized as chemotype I, while samples SII and LII were characterized as chemotype II, and samples SIII, SIV and LIII as chemotype III. The chemotypes showed comparable cytotoxic activity toward malignant cell lines (T24 and U-251-MG) unlike human leukocytes (non malignant cell line), where this activity was clearly distinct. Chemotype II (POA trans D/E ring junction) showed a higher selectivity index (SI) against malignant cells (SI=1.11-3.04) than chemotype I (SI=0.10-0.19) and III (SI=0.21-0.57). No important genotoxic potential was found by micronuclei frequency and alkaline comet DNA assays. Despite the isomerization of oxindole alkaloids during the cell incubation, the chemotype of the cat's claw samples remained unchanged. CONCLUSION: Cat's claw chemotypes showed different selectivity against human malignant cells, so that the correct identification of each chemotype seems to be important to better understand its antitumor potential.

Insights into Ecto-5'-Nucleotidase as a New Target for Cancer Therapy: A Medicinal Chemistry Study.

Ecto-5'-nucleotidase (ecto-5'-NT, 5'-NT, eN, CD73) is a membrane ecto-enzyme that is primarily responsible for the extracellular production of adenosine from AMP. Ecto-5'-NT is over expressed in various types of cancer cells, leading to elevated concentrations of adenosine in the tumor microenvironment. Adenosine has also been found to be important in cancer pathogenesis, showing strong immunosuppressive effects over antitumor T cells and macrophages and promoting neovascularization and cell adherence. These actions support tumor growth and development. It has been suggested that the inhibition of ecto-5'-NT results in lower extracellular concentrations of adenosine within the tumor microenvironment, which would directly affect cancer cells and render malignant cells more susceptible to host defense systems. Such mechanisms are proposed to represent promising new targets for cancer therapy. The aim of this review is to explore the biochemical and structural features of ecto-5'-NT, including a brief analysis of its active site by molecular modeling, as a means of evaluating whether the inhibition of this enzyme does indeed represent a feasible strategy for treating cancer. Known inhibitors and possible prototypes that could be used to target ecto-5'-NT during cancer therapy are also discussed.

Guanine and adenine nucleotidase activities in rat cerebrospinal fluid.

Adenine and guanine nucleotides have been shown to exert multiple roles in central and peripheral nervous systems, and the sequential breakdown of these nucleotides by enzymatic systems is an important step in the modulation of their extracellular effects. The aim of this study was to investigate whether nucleotide hydrolysis also occurs in the cerebrospinal fluid (CSF) of rats. CSF was able to hydrolyze all guanine and adenine nucleotides investigated (2.0 mM): GDPz.Gt;ADP=ATP=GTPz.Gt;AMP=GMP. More detailed studies with the diphosphate nucleotides showed that the hydrolysis of ADP and GDP was linear with incubation time and protein concentration. The apparent K(M) (Henry-Michaelis-Menten constant) and V (maximal velocity) values for ADP and GDP were 164.3+/-54.7 microM and 12.2+/-3.8 nmol P(i)/min per mg protein, and 841.0+/-90.2 microM and 22.8+/-8.0 nmol P(i)/min per mg protein. The sum of ADP, GDP and UDP hydrolysis (2.0 mM) upon individual incubations with CSF was similar to the hydrolysis observed when all three nucleotides were incubated together. This pattern of hydrolysis strongly suggests the involvement of more than one enzyme activity. The higher maximum activity for GDP and UDP compared to ADP is compatible with presence of a soluble NTDPase5.

Alterations in the extracellular catabolism of nucleotides are involved in the antiproliferative effect of quercetin in human bladder cancer T24 cells.

Bladder cancer is the most prevalent tumor in the genitourinary tract and the current treatments are not efficient to prevent recurrence and progression of tumor cases. Studies have revealed evidence of the involvement of the purinergic system in bladder tumorigenesis, particularly ecto-5'-NT/CD73, the enzyme responsible for AMP hydrolysis. Quercetin (3,3',4',5,7-pentahydroxyflavone) is a plant-derived flavonoid that has been shown to exert a broad range of pharmacologic properties, including potential anticancer activity. Here, we investigated the quercetin effect on the E-NTPDases and ecto-5'-nucleotidase/CD73, which catalyzes the introversion of the extracellular purine nucleotides in T24 human bladder cancer cells. The results showed that this flavonoid was able to increase ADP hydrolysis and inhibit the ecto-5'-nucleotidase/CD73 activity, with no effect on protein expression. The treatment with APCP (α,ß-methyleneadenosine-5'-diphosphate), another ecto-5'-NT/CD73 inhibitor, led to a significant reduction in cell proliferation. In addition, we showed that AMP, which can be accumulating by enzyme inhibition, had an antiproliferative effect on T24 cells, which was enhanced when its hydrolysis was inhibited by APCP treatment. Otherwise, adenosine did not cause any significant effect on cell proliferation and the quercetin effects were not altered by the simultaneous presence of adenosine. Taken together, the results suggest that the antiproliferative effect of quercetin on tumor cells may occur, at least in part, via alterations in the extracellular catabolism of nucleotides, that could be by AMP accumulation, or could be due to blocked adenosine receptors by this flavonoid, supporting the potential use of quercetin in bladder cancer treatment.

Differential ectonucleotidase expression in human bladder cancer cell lines.

Bladder cancer is the most prevalent tumor in the genitourinary tract. Nucleotides are important molecules that regulate many pathophysiological functions in the extracellular space. Studies have revealed evidence of a relationship between purinergic signaling and urothelial malignancies. Nucleotide-mediated signaling is controlled by a highly efficient enzymatic cascade, which includes the members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDases), ectonucleotide pyrophosphatase/phosphodiesterase (E-NPPs), ecto-alkaline phosphatases, and ecto-5'-nucleotidase/CD73. In an attempt to identify possible differential expression of ectonucleotidases during bladder cancer progression, a comparative analysis between RT4 (grade 1) and T24 (grade 3) bladder cancer cell lines was performed. In RT4 cells, the hydrolysis of tri- and diphosphate nucleosides was higher than monophosphonucleosides. T24 cells, however, presented the opposite profile, a low level of hydrolysis of tri- and diphosphate nucleosides and a high level of hydrolysis of monophosphates. Phosphodiesterase activity was negligible in both cell lines at physiological pH, indicating that these enzymes are not active under our assay conditions, although they are expressed in both cell lines. The T24 cells expressed NTPDase5 mRNA, while the RT4 cells expressed NTPDase3 and NTPDase5 mRNA. Both cell lines expressed ecto-5'-nucleotidase/CD73 mRNA. The present work describes, for the first time, the differential pattern of ectonucleotidases in the more malignant bladder cancer cells compared with cells derived from an early stage of bladder cancer. Our results open new avenues for research into the physiological roles of this family of enzymes and their possible therapeutic potential in bladder cancer.