Glyphosate and Aminomethylphosphonic Acid (AMPA) Modulate Glutathione S-Transferase in Non-Tumorigenic Prostate Cells.

Glyphosate (GLY) was developed in the early 1970s and has become the most used broad-spectrum herbicide in the world so far. Its main metabolite is aminomethylphosphonic acid (AMPA), and the accumulation of GLY and its derivative compounds raises some concerns regarding possible health outcomes. In this study, we aimed to evaluate the effects of GLY and AMPA on prostate cell lines by evaluating cell viability, proliferation, gene and protein expression, and cellular pathways involved in the response to oxidative stress. Our results indicated that GLY and AMPA reduced the cell viability of tumorigenic and non-tumorigenic prostate cell lines only at higher concentrations (10 mM GLY and 20 mM AMPA). In contrast, both compounds increased the clonogenicity of non-tumorigenic PNT2 cells, mainly at concentrations below the IC50 (5 mM GLY and 10 mM AMPA). Moreover, treatment of non-tumorigenic cells with low concentrations of GLY or AMPA for 48 h increased GSTM3 expression at both mRNA and protein levels. In contrast, the treatments decrease the GST activity and induced an increase in oxidative stress, mainly at lower concentrations. Therefore, both compounds can cause cellular damage even at lower concentrations in non-tumorigenic PNT2 cells, mainly affecting cell proliferation and oxidative stress.

In vitro interaction of polyethylene glycol-block-poly(D,L-lactide) nanocapsule devices with host cardiomyoblasts and Trypanosoma cruzi-infective forms.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is an important public health problem in Latin America. Nanoencapsulation of anti-T. cruzi drugs has significantly improved their efficacy and reduced cardiotoxicity. Thus, we investigated the in vitro interaction of polyethylene glycol-block-poly(D,L-lactide) nanocapsules (PEG-PLA) with trypomastigotes and with intracellular amastigotes of the Y strain in cardiomyoblasts, which are the infective forms of T. cruzi, using fluorescence and confocal microscopy. Fluorescently labeled nanocapsules (NCs) were internalized by non-infected H9c2 cells toward the perinuclear region. The NCs did not induce significant cytotoxicity in the H9c2 cells, even at the highest concentrations and interacted equally with infected and non-infected cells. In infected cardiomyocytes, NCs were distributed in the cytoplasm and located near intracellular amastigote forms. PEG-PLA NCs and trypomastigote form interactions also occurred. Altogether, this study contributes to the development of engineered polymeric nanocarriers as a platform to encapsulate drugs and to improve their uptake by different intra- and extracellular forms of T. cruzi, paving the way to find new therapeutic strategies to fight the causative agent of Chagas disease.

Copper(II) Complex Containing 4-Fluorophenoxyacetic Acid Hydrazide and 1,10-Phenanthroline: A Prostate Cancer Cell-Selective and Low-Toxic Copper(II) Compound.

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men worldwide. The treatment of advanced cases is based on chemotherapy, which lacks specificity and efficacy, due to severe side effects and resistance to the traditional drugs. Copper complexes have shown antitumoral efficacy and low toxicity, being considered a promising class of metal-based drugs for the treatment of malignant neoplasms. Thus, the present study aimed to evaluate the cellular effects of a copper(II) complex with 4-fluorophenoxyacetic acid hydrazide and 1,10-phenanthroline (1) on PCa cell lines, as well as the mutagenic/recombinogenic and anticarcinogenic potential of 1 in Drosophila melanogaster. PNT-2 (non-tumorigenic), LNCaP (hormone-responsive PCa) and PC-3 (androgen-independent PCa) cells were cultured, and cytotoxicity was assessed using the MTT assay. The expression levels of the proliferation markers Ki-67 and Cyclin D1 were analyzed by flow cytometry. Furthermore, the Somatic Mutation and Recombination Test (SMART) and the Epithelial Tumor Test (ETT) were performed. Complex 1 was selective to LNCaP cells, significantly reducing Ki-67 and Cyclin D1 expression levels. Sub-toxic concentrations of complex 1 were defined by the toxicity test in D. melanogaster, and no mutagenic/recombinogenic/carcinogenic effects were observed. Anticarcinogenic potential was observed in D. melanogaster, suggesting modulating activity of the complex 1 against Doxorubicin, a drug used as control by its carcinogenic properties. Therefore, complex 1 is a possible starting point for the development of new antitumor agents for the treatment of PCa.

Antimetastatic effect of the pharmacological inhibition of serine/arginine-rich protein kinases (SRPK) in murine melanoma.

The Serine/arginine-rich protein kinases (SRPK) are involved in pre-mRNA splicing control through the phosphorylation of the SR protein family of splicing factors. Over the last years, several studies have shown the relevance of SRPK for human cancers and their potential as promising drug targets. In this context, we have previously selected three trifluoromethyl arylamides (named here as SRVIC24, SRVIC30 and SRVIC36) with improved in vitro antileukemia effect and ability of impairing the cellular activity of SRPK. Given the increasing amount of reports on the implication of these kinases in metastatic cancers, in this study, we have evaluated the antimetastatic effect of these compounds and the known SRPK inhibitor (SRPIN340) on a murine model of metastatic melanoma. The compounds were able to impact the melanoma cell metastatic behavior by decreasing migration, invasion, adhesion, and colony formation in in vitro assays. Also, they presented antimetastatic in vivo activity, without apparent signs of systemic toxicity after treatments, as revealed by the histology of organs and analysis of key serum biochemical markers. Moreover, the effect of the treatments on SRPK1 nuclear translocation and SR protein phosphorylation was observed. Finally, molecular docking studies were carried out to gain structural information on the SRPK-compound complexes. Together, these data suggest that SRPK pharmacological inhibition should be considered as an interesting therapeutic strategy against metastatic cancers.

Synthesis, molecular properties prediction and cytotoxic screening of 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones.

In the present investigation, a collection of nineteen 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones was synthesized and screened for their cytotoxic activity against a panel of three leukemia cancer cell lines. The compounds were prepared via ZrOCl2·8H2O catalyzed condensation reactions between phthalaldehydic acid and different acetophenones. The reactions were carried out free of solvent and the isobenzofuran-1(3H)-ones were obtained in good yields (80-92%). The identities of the synthesized compounds were confirmed upon IR and NMR ((1)H and (13)C) spectroscopy as well as high resolution mass spectrometry analyses. Structures of compounds 1, 4 and 16 were also investigated by X-ray analysis. The synthesized compounds were submitted to in vitro bioassays against HL-60, K562 and NALM6 cancer cell lines using MTT cytotoxicity assay. After 48h of treatment, twelve derivatives were able to reduce cell viability and presented IC50 values equal to or below 20µmolL(-1) against at least one of the evaluated lineages. The most active compound corresponded to 3-(3-methylphenyl-2-oxoethyl)isobenzofuran-1(3H)-one (18) (IC50 values obtained for HL-60, K562 and NALM6 were, respectively, 13.5µmolL(-1), 8.83µmolL(-1), and 5.24µmolL(-1)). In addition, compound 18 was capable of triggering apoptosis on NALM6 cells. All isobenzofuranones herein evaluated did not present cytotoxicity on peripheral blood mononuclear cells (PBMC), suggesting selective cytotoxic effect on leukemic cells. A computational study allowed prediction of pharmacokinetics and drug-likeness properties of the synthesized compounds. DFT calculations were performed to obtain the energy values of HOMO, LUMO, and dipole moments of isobenzofuranones.

NMR analysis, cytotoxic activity and theoretical study of a complex between SRPIN340 and p-sulfonic acid calix[6]arene.

Aim: This study aimed to enhance the aqueous dissolution of SRPK inhibitor N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340). Materials & Methods: A complex with p-sulfonic calix[6]arene (Host) and SRPIN340 (Guest) was prepared, studied via 1H nuclear magnetic resonance (NMR) and theoretical calculations and biologically evaluated on cancer cell lines. Results & conclusion: The 1:1 host (H)/guest (G) complex significantly enhanced the aqueous dissolution of SRPIN340, achieving 64.8% water solubility as determined by 1H NMR quantification analysis. The H/G complex reduced cell viability by 75% for HL60, ∼50% for Nalm6 and Jurkat, and ∼30% for B16F10 cells. It exhibited greater cytotoxicity than free SRPIN340 against Jurkat and B16F10 cells. Theoretical studies indicated hydrogen bond stabilization of the complex, suggesting broader applicability of SRPIN340 across diverse biological systems.

[Box: see text].

A new copper(II) complex containing long-chain aliphatic hydrazide and 1,10-phenanthroline upregulates ADP hydrolysis in triple-negative breast cancer cells.

Copper can be opportunely complexed to modulate oncogenic pathways, being a promising strategy for cancer treatment. Herein, three new copper(II) complexes containing long-chain aliphatic hydrazides and 1,10-phenanthroline (1,10-phen), namely, [Cu(octh)(1,10-phen)(H2O)](NO3)21, [Cu(dech)(1,10-phen)(H2O)](NO3)22 and [Cu(dodh)(1,10-phen)(H2O)](NO3)2.H2O 3 (where octh = octanoic hydrazide, dech = decanoic hydrazide, dodh = dodecanoic hydrazide) were successfully prepared and characterized by several physical-chemical methods. Furthermore, X-ray structural analysis of complex 2 indicated that the geometry around the copper(II) ion is distorted square-pyramidal, in which hydrazide and 1,10-phenanthroline act as bidentate ligands. A water molecule in the apical position completes the coordination sphere of the metal ion. All new copper(II) complexes were cytotoxic to breast cancer cell lines (MCF7, MDA-MB-453, MDA-MB-231, and MDA-MB-157) and selective when compared to the non tumor lineage MCF-10A. In particular, complex 2 showed half-maximal inhibitory concentration (IC50) values ranging between 2.7 and 13.4 µM in MDA-MB231 cells after 24 and 48 h of treatment, respectively. Furthermore, this complex proved to be more selective for tumor cell lines when compared to doxorubicin and docetaxel. Complex 2 inhibited the clonogenicity of MDA-MB231 cells, increasing adenosine diphosphate (ADP) hydrolysis and upregulating ecto-nucleoside triphosphate diphosphohydrolase 1 (ENTPD1) transcriptional levels. In this sense, we suggest that the inhibitory effect on cell proliferation may be related to the modulation of adenosine monophosphate (AMP) levels. Thus, a novel copper(II) complex with increased cytotoxic effects and selectivity against breast cancer cells was obtained, contributing to medicinal chemistry efforts toward the development of new chemotherapeutic agents.

Tailoring Potential Natural Compounds for the Treatment of Luminal Breast Cancer.

Breast cancer (BC) is the most diagnosed cancer worldwide, mainly affecting the epithelial cells from the mammary glands. When it expresses the estrogen receptor (ER), the tumor is called luminal BC, which is eligible for endocrine therapy with hormone signaling blockade. Hormone therapy is essential for the survival of patients, but therapeutic resistance has been shown to be worrying, significantly compromising the prognosis. In this context, the need to explore new compounds emerges, especially compounds of plant origin, since they are biologically active and particularly promising. Natural products are being continuously screened for treating cancer due to their chemical diversity, reduced toxicity, lower side effects, and low price. This review summarizes natural compounds for the treatment of luminal BC, emphasizing the activities of these compounds in ER-positive cells. Moreover, their potential as an alternative to endocrine resistance is explored, opening new opportunities for the design of optimized therapies.

Photodynamic therapy with the dual-mode association of IR780 to PEG-PLA nanocapsules and the effects on human breast cancer cells.

IR780 is a near-infrared fluorescent dye, which can be applied as a photosensitizer in photodynamic (PDT) and photothermal (PTT) therapies and as a biodistribution tracer in imaging techniques. We investigated the growth and migration inhibition and mechanism of death of breast tumor cells, MCF-7 and MDA-MB-231, exposed to polymeric nanocapsules (NC) comprising IR780 covalently linked to the biodegradable polymer PLA (IR-PLA) and IR780 physically encapsulated (IR780-NC) in vitro. Both types of NC had mean diameters around 120 nm and zeta potentials around -40 mV. IR-PLA-NC was less cytotoxic than IR780 NC to a non-tumorigenic mammary epithelial cell line, MCF-10A, which is an important aspect of selectivity. Free-IR780 was more cytotoxic than IR-PLA-NC for MCF-7 and MDA-MB-231 cells after illumination with a 808 nm laser. IR-PLA NC was effective to inhibit colony formation (50%) and migration (30-40%) for both cancer cell lines. MDA-MB-231 cells were less sensitive to all IR780 formulations compared to MCF-7 cells. Cell uptake was higher with IR-PLA-NC than with IR780-NC and free-IR780 in both cancer cell lines (p < 0.05). NC uptake was higher in MCF-7 than in MDA-MB-231 cells. IR-PLA-NC induced a higher percentage of apoptosis upon illumination in MDA-MB-231 than in MCF-7 cells. The necrosis mechanism of death predominated in treatments with free-IR780 and with encapsulated IR780 NC, suggestive of damages at the plasma membrane. IR780 conjugated with PLA increased the apoptotic pathway and demonstrated potential as a multifunctional theranostic agent for breast cancer treatment with increased cellular uptake, photodynamic activity and more reliable tracking in cell-image studies.

Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.

Lipid-based nanocarriers co-loaded with artemether and triglycerides of docosahexaenoic acid: Effects on human breast cancer cells.

Artemether (ART) was combined with triglyceride of docosahexaenoic acid (DHA) as the lipid-core in nanoemulsions (NE), nanostructured lipid carriers (NLC), and PEG-PLA nanocapsules (NC) formulations, and their effects on human breast cancer cells were evaluated. ART has been extensively used for malaria and has also therapeutic potential against different tumor cells in a repositioning strategy. The concentration-dependent cytotoxicity in vitro was determined in tumor lineages, MDA-MB-231 and MCF-7, and non-tumor MCF-10A cells for free-ART/DHA combination and its formulations. The cells were monitored for viability, effects on cell migration and clonogenicity, cell death mechanism, and qualitative and quantitative cell uptake of nanocarriers. The lipid-nanocarriers showed mean sizes over the range of 110 and 280 nm with monodisperse populations and zeta potential values ranging from -21 to -67 mV. The ART encapsulation efficiencies varied from 57 to 83 %. ART/DHA co-loaded in three different lipid nanocarriers reduced the MDA-MB-231 and MCF-7 viability in a dose-dependent manner with enhanced selectivity toward tumor cell lines. They also reduced clonogenicity and the ability of cells to migrate showing antimetastatic potential in both cell lines and triggered apoptosis in MCF-7 cells. Confocal microscopy and flow cytometry analysis showed that NC, NLC, and NE were rapidly internalized by cells, with higher interaction displayed by NE with MCF-7 cells compared to NC and NLC that was correlated with the strongest NE-fluorescence in cells. Therefore, this study not only demonstrated the value of this new combination of ART/DHA as a new strategy for breast cancer therapy but also showed enhanced cytotoxicity and potential metastatic activity of lipid-based formulations against human breast cancer cells that indicate great potential for pre-clinical and clinical translation.

Combined SRPK and AKT pharmacological inhibition is synergistic in T-cell acute lymphoblastic leukemia cells.

The serine/arginine protein kinases respond to the EGFR-PI3K-AKT signaling module in the context of pre-mRNA alternative splicing regulation. These enzymes (notably SRPK1 and SRPK2) have been found dysregulated in a variety of cancers, which suggests them as promising drug targets in oncology. SRPK2 has been related to leukemia cells proliferation and found preferentially overexpressed in T-cell acute lymphoblastic leukemia (T-ALL). Previously, synergistic combination between vincristine and SRPK inhibitors has been observed in leukemia cells in vitro. Herein we sought to evaluate the in vitro combinatory effects of inhibiting SRPK and multiple other kinase targets from the EGFR pathway in T-ALL, a hematological malignancy with a still poor prognosis. We found that the combined SRPK and AKT pharmacological inhibition is synergistic in Jurkat, CCRF-CEM, and TALL-1 (all T-ALL) but not in HL60, an acute myelogenous leukemia cell lineage. Combined treatments also impaired SR proteins phosphorylation in accordance with an improved suppression of SRPK activity. Furthermore, the synergism of treatments seemed associated with apoptosis triggering, as revealed by flow cytometry analyses. Taken together, these results suggest the therapeutic potential of the combined SRPK and AKT pharmacological inhibition against T-ALL.

Synthesis and antimetastatic activity evaluation of cinnamic acid derivatives containing 1,2,3-triazolic portions.

It is herein described the preparation and evaluation of antimetastatic activity of twenty-six cinnamic acid derivatives containing 1,2,3-triazolic portions. The compounds were prepared using as the key step the Copper(I)-catalyzed azide (A)-alkyne (A) cycloaddition (C) (CuAAC reaction), also known as click reaction, between alkynylated cinnamic acid derivatives and different benzyl azides. The reactions were carried in CH2Cl2/H2O (1:1 v/v) at room temperature, and the triazole derivatives were obtained in yields ranging from 73%99%. Reaction times varied from 5 to 40 min. The identity of the synthesized compounds was confirmed by IR and NMR (1H and 13C) spectroscopic techniques. They were then submitted to in vitro bioassays to investigate how they act over metastatic behavior of murine melanoma. The most potent compound, namely 3-(1-benzyl-1H-1,2,3-triazol-4-yl)propyl cinnamate (9a), showed significant antimetastatic and antiproliferative activities against B16-F10 cells. In addition, gelatin zymography and molecular docking analyses pointed to the fact that this compound has potential to interact with matrix metalloproteinase 9 (MMP-9) and MMP-2, which are directly involved in melanoma progression. Therefore, these findings suggest that cinnamic acid derivatives containing 1,2,3-triazolic portions may have potential for development of novel candidates for controlling malignant metastatic melanoma.

Synthesis, theoretical studies, and effect on the photosynthetic electron transport of trifluoromethyl arylamides.

BACKGROUND: The photosynthetic apparatus is targeted by various herbicides, including several amides such as diuron and linuron. Considering the need for the discovery of new active ingredients to cope with weed resistance, the synthesis of a series of trifluoromethyl aryl amides is herein described whose inhibitory properties were assessed in vitro on the photosynthetic electron transport chain, and in vivo on the growth of a model cyanobacterial strain. Theoretical studies were also carried out. RESULTS: Starting with 1-fluoro-2-nitro-4-(trifluoromethyl) benzene, the preparation of the amides was achieved via a three-step sequence, namely nucleophilic aromatic substitution, reduction with SnCl2 /HCl, and acylation reactions. The measurement of ferricyanide reduction by functionally intact spinach chloroplasts showed that several derivatives are capable of inhibiting the photosynthetic apparatus. The most active amides presented IC50 values close to 1 µmol L-1 , and showed the presence of a 4-bromophenyl group as a common structural feature. The addition of these brominated amides to the culture medium of a model cyanobacterial strain, Synechococcus elongatus PCC 6301, caused various degrees of growth inhibition. Theoretical studies (molecular modeling and quantitative structure-activity relationship) of all amides and their comparison with some known herbicides confirmed these experimental findings and provided more in-depth information about the possible molecular target of these compounds. CONCLUSION: Trifluoromethyl amides herein described, which were shown to act at the PSII level, may represent a novel scaffold to be exploited aiming at the development of new active ingredients for weed control. © 2017 Society of Chemical Industry.

Trifluoromethyl arylamides with antileukemia effect and intracellular inhibitory activity over serine/arginine-rich protein kinases (SRPKs).

The serine/arginine-rich protein kinases (SRPKs) have frequently been found with altered activity in a number of cancers, suggesting they could serve as potential therapeutic targets in oncology. Here we describe the synthesis of a series of twenty-two trifluoromethyl arylamides based on the known SRPKs inhibitor N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) and the evaluation of their antileukemia effects. Some derivatives presented superior cytotoxic effects against myeloid and lymphoid leukemia cell lines compared to SRPIN340. In particular, compounds 24, 30, and 36 presented IC50 values ranging between 6.0 and 35.7 µM. In addition, these three compounds were able to trigger apoptosis and autophagy, and to exhibit synergistic effects with the chemotherapeutic agent vincristine. Furthermore, compound 30 was more efficient than SRPIN340 in impairing the intracellular phosphorylation status of SR proteins as well as the expression of MAP2K1, MAP2K2, VEGF, and RON oncogenic isoforms. Therefore, novel compounds with increased intracellular effects against SRPK activity were obtained, contributing to medicinal chemistry efforts towards the development of new anticancer agents.

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340).

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.