Aggressive Human Breast Cancer Cell Research Articles

Development and characterisation of mouse monoclonal antibody against ‘neonatal’ Nav1.5

‘Neonatal’ Nav1.5 (nNav1.5) is a potent tumour metastasis marker found especially in aggressive human breast cancer cells in vitro, in tumour tissues of in vivo metastatic animal models and in patients positive for lymph-node metastasis. Its expression has been recently described in human brain neuroblastoma and astrocytoma. However, a thorough understanding of nNav1.5’s role in cancers has been limited by the lack of specific antibodies against it. Here, a mouse monoclonal antibody, 4H8 mAb-nNav1.5, was obtained and characterised concerning its efficacy in detecting nNav1.5 using indirect ELISA, surface plasmon resonance (SPR), Western blotting and immunofluorescence microscopy. 4H8 mAb-nNav1.5 was selected from a panel of hybridoma clones raised against nNav1.5 specific peptide (15 mers). The antibody exhibited linear association against nNav1.5 specific-linear peptide in indirect ELISA and was supported by SPR. The antibody also demonstrated strong immunoreactivity in immunofluorescence imaging of nNav1.5-abundant cells, human and mouse aggressive breast cancer cells, MDA-MB-231 and 4T1, respectively, which was not observed in nNav1.5-deficient cells, human less aggressive breast cancer cells, MCF-7 and non-cancerous breast epithelial cells, MCF-10A. This study demonstrates the initial description of 4H8 mAb-nNav1.5, which could serve as a beneficial tool to enhance future studies on nNav1.5 expression and function in cancers.

In Vitro anti-metastasis of Perilla frutescens leaf water extract on aggressive human breast cancer cells

Background: Perilla frutescens is a long-established plant that is often used in foods and traditional medicines in Asian countries. The perilla leaf contains a considerable number of bioactive substances, such as phenolics and flavonoids, which have been demonstrated to possess anticancer activity in vitro and in vivo. Objectives: We aimed to study anti-metastatic activity, anti-invasion activity, and anti-migration activity of perilla leaf water extract (PLW) at 90°C for 1-5 min in MDA-MB-231 aggressive human breast cancer cells. Materials and methods:Dry perilla leaves were extracted using hot water for 1-5 min to obtain crude extract and then lyophilized for PLW powder. PLW was evaluated for total phenolic, total flavonoid, and rosmarinic acid (RA) contents by Folin-Ciocalteau reagent, aluminum chloride colorimetric assay, and ultra-high-pressure liquid chromatography, respectively. Antioxidant activity of PLW was determined by DPPH and ABTS assays. MTT assay was performed to evaluate the cytotoxicity of PLW on MDA-MB-231 cells. Effective PLW was further determined its inhibitory effect on human breast cancer cell metastasis by a Boyden chamber-based transmembrane assay, the MMP-9 activity, and the proteolytic type IV collagenase activity. Results: PLW by 5-min infusion showed the highest amount of total phenolic and flavonoid contents, as well as RA. Moreover, by the 5-min infusion, PLW had the highest antioxidant capacity when compared to PLW by infusions for 1-4 min. Following that, cytotoxicity testing revealed that the PLW is not toxic to MDA-MB-231 cells after a 24-hr exposure. The PLW at non-toxic doses (12.5-100 µg/mL) intensely presents an inhibitory effect on cell invasion and migration. The gelatinolytic activity showed that the PLW at concentrations of 12.5-100 µg/mL decreases MMP-9 activity in a dose-related manner. Furthermore, after treatment with the PLW, the proteolytic type IV collagenase activity was reduced considerably in a dose-related manner. Conclusion: Our findings further showed that the PLW samples inhibit proteolytic enzymes involved in basement membrane breakdown, which might explain the anti-invasion and anti-migration properties of breast cancer cells. From the result, the application of perilla leaf might be developed as an herbal tea and used as an anti-metastatic agent for breast cancer prevention and treatment.

NNav1.5 expression is associated with glutamate level in breast cancer cells

BackgroundGlutamate and voltage-gated sodium channels, both have been the target of intense investigation for its involvement in carcinogenesis and progression of malignant disease. Breast cancer with increased level of glutamate often metastasize to other organs (especially bone), whilst re-expression of ‘neonatal’ Nav1.5, nNav1.5 in breast cancer is known to promote cell invasion in vitro, metastasis in vivo and positive lymph node metastasis in patients.MethodsIn this study, the role of nNav1.5 in regulating glutamate level in human breast cancer cells was examined using pharmacological approach (VGSCs specific blocker, TTX, glutamate release inhibitor, riluzole and siRNA-nNav1.5). Effect of these agents were evaluated based on endogenous and exogenous glutamate concentration using glutamate fluorometric assay, mRNA expression of nNav1.5 using qPCR and finally, invasion using 3D culture assay.ResultsEndogenous and exogenous glutamate levels were significantly higher in aggressive human breast cancer cells, MDA-MB-231 cells compared to less aggressive human breast cancer cells, MCF-7 and non-cancerous human breast epithelial cells, MCF-10A. Treatment with TTX to MDA-MB-231 cells resulted in significant reduction of endogenous and exogenous glutamate levels corresponded with significant suppression of cell invasion. Subsequently, downregulation of nNav1.5 gene was observed in TTX-treated cells.ConclusionsAn interesting link between nNav1.5 expression and glutamate level in aggressive breast cancer cells was detected and requires further investigation.

MitoQ Inhibits Human Breast Cancer Cell Migration, Invasion and Clonogenicity.

Simple SummarySolid tumors comprise metabolically hostile areas adjacent to metabolically friendly ones, and their distribution fluctuates in space and time. Selection pressure is maximal in hostile areas where cancer cells develop at least four survival strategies: to adapt themselves, to modify the microenvironment, to hibernate metabolically, and to escape. Escape marks the transition from a localized to an invasive tumor that may ultimately disseminate remotely, forming metastases. Based on the hypothesis that mitochondria are metabolic sensors that control these responses, we previously established that mitochondrial superoxide is a pro-metastatic intracellular signaling agent. Here, we tested MitoQ, a mitochondria-targeted ROS inactivator that already successfully passed Phase I safety clinical trials, as a potential inhibitor of the early steps of the metastatic cascade. Using human breast cancer cells as models in anticipation of preclinical and clinical assays, we report that MitoQ inhibits cancer cell migration, invasion, clonogenicity, sphere formation and spheroid stability.To successfully generate distant metastases, metastatic progenitor cells must simultaneously possess mesenchymal characteristics, resist to anoïkis, migrate and invade directionally, resist to redox and shear stresses in the systemic circulation, and possess stem cell characteristics. These cells primarily originate from metabolically hostile areas of the primary tumor, where oxygen and nutrient deprivation, together with metabolic waste accumulation, exert a strong selection pressure promoting evasion. Here, we followed the hypothesis according to which metastasis as a whole implies the existence of metabolic sensors. Among others, mitochondria are singled out as a major source of superoxide that supports the metastatic phenotype. Molecularly, stressed cancer cells increase mitochondrial superoxide production, which activates the transforming growth factor-β pathway through src directly within mitochondria, ultimately activating focal adhesion kinase Pyk2. The existence of mitochondria-targeted antioxidants constitutes an opportunity to interfere with the metastatic process. Here, using aggressive triple-negative and HER2-positive human breast cancer cell lines as models, we report that MitoQ inhibits all the metastatic traits that we tested in vitro. Compared to other mitochondria-targeted antioxidants, MitoQ already successfully passed Phase I safety clinical trials, which provides an important incentive for future preclinical and clinical evaluations of this drug for the prevention of breast cancer metastasis.

Discovery of triaromatic flexible agents bearing 1,2,3-Triazole with selective and potent anti-breast cancer activity and CDK9 inhibition supported by molecular dynamics

This study reports an efficient and convenient click synthesis of novel series of chromene scaffold linked to 1,2,3 triazole ring and terminal lipophilic fragments. Structures of all newly synthesized compounds were well characterized by IR, 1H NMR, 13C NMR and elemental analyses. In vitro MTT cytotoxic screening was performed using staurosporine as a reference drug against three different types: aggressive and invasive human breast cancer cell line (MDA-MB 231), Michigan cancer foundation-7 (MCF-7) and human colorectal carcinoma cell line (HCT116). These screening data showed considerable anticancer activity for these newly synthesized compounds compared to reference staurosporine drug with high degree of cell line selectivity and excellent activity with micromolar (µM) half maximal inhibitory concentration (IC50) values against tumor cells. In addition, Cyclin-Dependent Kinase1 and 9 (CDK1 and 9) assays were performed for the most active compounds to get more details about their mechanism of action. In order to assess and explain their binding affinities, molecular docking simulation was studied against CDK9 site. The results obtained from molecular docking study and those obtained from cytotoxic screening and enzyme inhibition were correlated. One of the most prominent analogs is 34 that showed high selective cytotoxicity profile against single breast cancer cell line MCF-7 and MDA-MB 231 with IC50 3.4 µM and 1.4 uM respectively. Molecular dynamics (MD) simulation of the CDK9 complex by Desmond revealed stability during 100 ns with RMSD of CDK9 complex converged at 2.1 Å after 20 ns. During MD, the interaction fractions confirmed multiple interactions of 34 with Cys106, a crucial residue for inhibition of CDK9 activity. A novel 34 triazole compound was introduced for drug design works as potent and selective hit for treatment of breast cancers after processing of optimization steps.

Influence of nNav1.5 on MHC class I expression in breast cancer

Cancer metastasis occurs due to the ability of the tumour to evade immune recognition and response by altering the major histocompatibility complex class I (MHC class I). A comprehensive understanding of the MHC class I alteration in breast cancer metastasis is vital for the rational design and improvement of immunotherapies for advanced cancer. In this study, we associate it with the elevation of 'neonatal' Nav1.5 (nNav1.5) mRNA expression in the aggressive type of human breast cancer cells. Using real-time PCR, lower expression of TAP1 (by - 4.83-fold), which is a part of the MHC class I antigen processing machinery (APM) pathway component, in contrast to the greater expression of nNav1.5 (by 3.76-fold), along with other metastatic markers, MMP1 (by 57.48-fold) and fibronectin (by 2.88-fold) in aggressive human breast cancer cell line, MDA-MB231 were obtained. Subsequent knockdown of nNav1.5 (by 52.6%) in MDA-MB-231 via siRNA-SCN5A resulted in downregulation of another metastatic marker, fibronectin (by 52.9%) but importantly, rescued MHC class I expression (by 347% or 3.47-fold). Furthermore, the siRNA-SCN5A transfected cells failed to form a unified spheroid and incapable of efficiently invading the surrounding invasion matrix. In summary, the influence of nNav1.5 in regulating MHC class I mRNA expression to allow for breast cancer invasion is demonstrated, supporting the potential of nNav1.5 as an immunotherapy target to overcome tumour immune evasion thus preventing metastasis.

Distinct photo-oxidation-induced cell death pathways lead to selective killing of human breast cancer cells

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

65P The new mouse anti-nNav1.5 monoclonal antibody

Neonatal Nav1.5 (nNav1.5) is a potent tumour metastatic marker found re-expressed in human breast cancer cells with aggressive phenotype and breast tumour tissue positive for lymph node metastasis. Mouse anti-nNav1.5 monoclonal antibody, 4H8 was recently obtained.Neonatal Nav1.5 (nNav1.5) is a potent tumour metastatic marker found re-expressed in human breast cancer cells with aggressive phenotype and breast tumour tissue positive for lymph node metastasis. Mouse anti-nNav1.5 monoclonal antibody, 4H8 was recently obtained. This study aimed to assess the specificity of the 4H8 against nNav1.5. 4H8 was used as primary antibody in Western blot and immunocytochemistry tested on cells and cell lysates, respectively of two human breast cancer cell line; MDA-MB-231+nNav1.5 and MCF-7-nNav1.5, a mouse mammary cancer cell line, 4T1+nNav1.5, a non-cancerous human breast epithelial, MCF-10A-nNav1.5 and tumour tissue lysate from an orthotopic syngeneic mouse model (4T1+nNav1.5/BALB/c). were consistent with nNav1.5 protein being more abundantly expressed in aggressive human breast cancer cell line, MDA-MB-231+nNav1.5 and mouse mammary cancer cell line, 4T1+nNav1.5 and tumour tissue lysate from an orthotopic syngeneic mouse model (4T1+nNav1.5/BALB/c) as compared to MCF-7-nNav1.5 and MCF-10A-nNav1.5. The new antibody described in this work can be a novel tool in studying nNav1.5 expression in breast cancer.

Focal drug administration via heparin-containing cryogel microcarriers reduces cancer growth and metastasis

Developing drug delivery systems that release anticancer drugs in a controlled and sustained manner remains challenging. We hypothesized that highly sulfated heparin-based microcarriers would allow electrostatic drug binding and controlled release. In silico modelling showed that the anticancer drug doxorubicin has affinity for the heparin component of the microcarriers. Experimental results showed that the strong electrostatic interaction was reversible, allowing both doxorubicin loading and a subsequent slow release over 42 days without an initial burst release. The drug-loaded microcarriers were able to reduce cancer cell viability in vitro in both hormone-dependent and highly aggressive triple-negative human breast cancer cells. Focal drug treatment, of an in vivo orthotopic triple-negative breast cancer model significantly decreased tumor burden and reduced cancer metastasis, whereas systemic administration of an equivalent drug dose was ineffective. This study proves that heparin-based microcarriers can be used as drug delivery platforms, for focal delivery and sustained long-term drug release.

Cu(II)-Metallacryptands Self-Assembled to Vesicular Aggregates Capable of Encapsulating and Transporting an Anticancer Drug Inside Cancer Cells.

Crystallographically characterized M2 L4 type cationic Cu(II)-metallacryptands [MC(X)] derived from a series of bis-pyridyl-bis-urea ligands (LX ; X = O, S, C) are self-assembled to single-layered vesicular aggregates in DMSO, DMSO/water, and DMSO/DMEM (biological media). One such vesicle is MC(O)-vesicle that is demonstrated to be able to load and release (pH responsive) an anticancer drug, namely doxorubicin hydrochloride (DOX). DOX-loaded MC(O)-vesicle is also successfully transported within MDA-MB-231 cells-a highly aggressive human breast cancer cell line. Such self-assembling behavior to form vesicular aggregates by metallacryptands (MCs) is hitherto unknown.

Design and Synthesis of ZnII -Coordination Polymers Anchored with NSAIDs: Metallovesicle Formation and Multi-drug Delivery.

A series of coordination polymers synthesized from a bis-pyridyl linker, namely 4,4'-azopyridine (L), selected non-steroidal-anti-inflammatory drugs (NSAIDs), namely diclofenac (Dic), ibuprofen (Ibu), flurbiprofen (Flu), mefenamic acid (Mefe), and naproxen (Nap), and Zn(NO3 )2 were characterized by single crystal X-ray diffraction. One of the coordination polymers, namely CP3 derived from Flu, was able to form metallovesicles in DMSO, DMSO/H2 O and DMSO/DMEM (biological media) as revealed by TEM, AFM and DLS. Metallovesicle formation by CP3 was further supported by loading a fluorescent dye, namely calcein, as well as an anti-cancer drug, doxorubicin hydrochloride (DOX), as revealed by UV-vis and emission spectra, and fluorescence microscopy. DOX-loaded metallovesicles of CP3 (DOX@CP3-vesicle) could be delivered in vitro to a highly aggressive human breast cancer cell line, namely MDA-MB-231, as revealed by MTT and cell migration assays, and also cell imaging performed under laser scanning confocal microscope (LSCM). Thus, a proof of concept for developing a multi-drug delivery system derived from a metallovesicle for delivering an anti-cancer drug to cancer cells is demonstrated for the first time.

PTPN13 induces cell junction stabilization and inhibits mammary tumor invasiveness.

Clinical data suggest that the protein tyrosine phosphatase PTPN13 exerts an anti-oncogenic effect. Its exact role in tumorigenesis remains, however, unclear due to its negative impact on FAS receptor-induced apoptosis.Methods: We crossed transgenic mice deleted for PTPN13 phosphatase activity with mice that overexpress human HER2 to assess the exact role of PTPN13 in tumor development and aggressiveness. To determine the molecular mechanism underlying the PTPN13 tumor suppressor activity we developed isogenic clones of the aggressive human breast cancer cell line MDA-MB-231 overexpressing either wild type or a catalytically-inactive mutant PTPN13 and subjected these to phosphoproteomic and gene ontology analyses.We investigated the PTPN13 consequences on cell aggressiveness using wound healing and Boyden chamber assays, on intercellular adhesion using videomicroscopy, cell aggregation assay and immunofluorescence.Results: The development, growth and invasiveness of breast tumors were strongly increased by deletion of the PTPN13 phosphatase activity in transgenic mice. We observed that PTPN13 phosphatase activity is required to inhibit cell motility and invasion in the MDA-MB-231 cell line overexpressing PTPN13. In vivo, the negative PTPN13 effect on tumor invasiveness was associated with a mesenchymal-to-epithelial transition phenotype in athymic mice xenografted with PTPN13-overexpressing MDA-MB-231 cells, as well as in HER2-overexpressing mice with wild type PTPN13, compared to HER2-overexpressing mice that lack PTPN13 phosphatase activity. Phosphoproteomic and gene ontology analyses indicated a role of PTPN13 in the regulation of intercellular junction-related proteins. Finally, protein localization studies in MDA-MB-231 cells and HER2-overexpressing mice tumors confirmed that PTPN13 stabilizes intercellular adhesion and promotes desmosome formation.Conclusions: These data provide the first evidence for the negative role of PTPN13 in breast tumor invasiveness and highlight its involvement in cell junction stabilization.

Therapeutic potential of xanthones from Swertia chirata in breast cancer cells.

Background & objectives:Medicinal plants like Swertia chirata are rich sources of different xanthones. This study was aimed to assess the cytotoxic potential of four most abundant xanthones present in S. chirata both in vivo and in vitro in Ehrlich ascites carcinoma (EAC), a mouse transplantable breast carcinoma cell line and two human breast carcinoma cell lines (MCF-7 and MDA-MB-231).Methods:Four xanthones derived from S. chirata namely 1-hydroxy-3,7,8-trimethoxyxanthone (XA), 1,8-dihydroxy-3,5-dimethoxyxanthone (XB), 1-hydroxy-3,5,8-trimethoxyxanthone (XC) and 1,5,8-trihydroxy-3-methoxyxanthone (XD) were used for determination of sub-lethal dose on the cell lines EAC, MCF-7, MDA-MB-231 and verified toxicity of sub-lethal dose on normal murine fibroblast cells. Cytotoxicity was measured in vivo and survivability of mice was plotted accordingly. Therapeutic efficacy of XD was evaluated both in vivo and in vitro by determination of lipid peroxidation (LPO), reactive oxygen species (ROS) generation and by quantitating the enzyme status (GSH, catalase, superoxide dismutase) in treated and untreated samples. DNA damage was evaluated using comet and DNA fragmentation assays. Furthermore, apoptotic effect was analyzed by flow cytometry and validated by TUNEL assay and Western blotting.Results:Among all the xanthones tested XD showed IC50 at the lowest dose, and normal cells were unaffected at this dose. Survivability of mice increased significantly when treated with XD compared to other xanthones and cisplatin. Significantly increased ROS and LPO were found in cancer cells as a result of XD treatment which was unaltered in normal cell line. XD induced DNA damage and apoptosis in cancer cell lines.Interpretation & conclusions:Our experimental data indicate that XD may potentially act as a chemotherapeutic agent by enhancing ROS in breast cancer cells thereby leading to apoptosis.

Evaluation of mouse polyclonal antibody against nNav1.5 to recognize nNav1.5 protein in human breast cancer cells using fluorescence immunostaining

Abstract Background Neonatal splice variant of Nav1.5 (nNav1.5) is a known tumor-associated antigen (TAA), re-expressed in adult aggressive breast cancer/tumor biopsies positive for lymph node metastasis. Accurate assessment of nNav1.5 protein in breast cancer cells/tissue is important in validating its potential value as a prognostic or predictive marker. Mouse polyclonal antibody (pAb) against nNav1.5 has been obtained. Herein, the ability of the pAb to selectively recognize nNav1.5 protein was evaluated on a panel of cell lines using fluorescence immunostaining. Methods Mouse mammary cell line, 4T1, human breast cancer cell lines, MDA-MB-231, and MCF-7 as well as human non-cancerous epithelial cell line, MCF-10A were acquired (ATCC) and maintained. Accordingly, the cells were cultured in its respective complete media supplemented with 5% fetal bovine serum (FBS), L-glutamine (4mM) and penicillin-streptomycin (1%) at 37 °C in a 5% CO2, relatively humidified atmosphere. For fluorescence immunostaining, the cells were seeded overnight on a cover-slip in 24-well culture plate, fixed, permeabilized and stained with primary pAb followed by incubation with secondary antibody conjugated with AlexaFluor-555. Fluorescence microscopy was conducted using Leica Inverted DMi8 Fluorescence and captured using DFC 365 FX Camera. Results Red fluorescence (secondary AlexaFlour-555) signals were prominent in aggressive human breast cancer cells, MDA-MB-231 and aggressive mouse mammary cancer cells, 4T1; both cell lines are known as positive for nNav1.5 expression. On the contrary, in less aggressive human breast cancer cells, MCF-7 and non-cancerous human breast epithelial cells, MCF-10A which lacks nNav1.5 expression, red fluorescence signals were not detected similar to the no-primary and no-primary/secondary controls. Conclusions The obtained mouse pAb against nNav1.5 could serve as a useful tool in validating nNav1.5 protein expression in breast cancer cells. Legal entity responsible for the study The authors. Funding Ministry of Energy, Science, Technology, Environment and Climate Change, Malaysia (Science Fund CIPPM/6113610). Disclosure All authors have declared no conflicts of interest.

Self-Assembly of Spherical Organic Molecules to Form Hollow Vesicular Structures in Water for Encapsulation of an Anticancer Drug and Its Release.

Developing hierarchical supramolecular structures is important for better understanding of various biological functions and possibly generating new materials for biomedical applications. Herein, we report the first examples of functional vesicles derived from cationic spherical organic molecules (C1 -C3 ) which were readily synthesized by reacting a C3 -symmetric tris-benzimmidazole derivative (possessing a 1,3,5-ethyl substituted aromatic core) with 1,3,5-substituted tris-bromomethyl benzene derivatives. Vesicle formation by C1 -C3 was probed by high-resolution microscopy (TEM and AFM), dynamic light scattering (DLS) and fluorescence microscopic imaging of calcein-loaded vesicles. One of the vesicles [Vesicle(C3 )] displayed the ability to load the anticancer drug doxorubicin (DOX). The drug was subsequently released from DOX@Vesicle(C3 ) in a stimuli-responsive manner in presence of the well-known vesicle destroyer Triton X-100, as revealed by in vitro cell migration assay carried out on a highly aggressive human breast cancer cell line (MDA-MB-231).

Supramolecular Gels Derived from Simple Organic Salts of Flufenamic Acid: Design, Synthesis, Structures, and Plausible Biomedical Application.

Following supramolecular synthon rationale in the context of crystal engineering, a nonsteroidal-anti-inflammatory-drug (NSAID), namely flufenamic acid (FA) and its β-alanine monopeptide derivative (FM), were converted to a series of primary ammonium monocarboxylate (PAM) salts. Majority of the PAM salts (∼90%) showed gelation with various solvents including water and methyl salicylate (important solvents in topical gel formulation). Structure-property correlation studies based on single-crystal X-ray diffraction (SXRD) and powder X-ray diffraction (PXRD) data provided intriguing insights into the structure of the gel network. Furthermore, one of the gelator salts (S7) displayed anticancer activity on a highly aggressive human breast cancer cell line (MDA-MB-231) ,as revealed by MTT, PEG2, and cell migration assays.

Rational Approach Towards Designing Metallogels From a Urea-Functionalized Pyridyl Dicarboxylate: Anti-inflammatory, Anticancer, and Drug Delivery.

A structural rationale was adopted to design a series of metallogels from a newly synthesized urea-functionalized dicarboxylate ligand, namely, 5-[3-(pyridin-3-yl)ureido]isophthalic acid (PUIA), that produces metallogels upon reaction with various metal salts (CuII , ZnII , CoII , CdII , and NiII salts) at room temperature. The gels were characterized by dynamic rheology and transmission electron microscopy (TEM). The existence of a coordination bond in the gel state was probed by FTIR and 1 H NMR spectroscopy in a ZnII metallogel (i.e., MG2). Single crystals isolated from the reaction mixture of PUIA and CoII or CdII salts characterized by X-ray diffraction revealed lattice inclusion of solvent molecules, which was in agreement with the hypothesis based on which the metallogels were designed. MG2 displayed anti-inflammatory response (prostaglandin E2 assay) in the macrophage cell line (RAW 264.7) and anticancer properties (cell migration assay) on a highly aggressive human breast cancer cell line (MDA-MB-231). The MG2 metallogel matrix could also be used to load and release (pH responsive) the anticancer drug doxorubicin. Fluorescence imaging of MDA-MB-231 cells treated with MG2 revealed that it was successfully internalized.

The role of miRNAs 34a, 146a, 320a and 542 in the synergistic anticancer effects of methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC) with doxorubicin in breast cancer cells.

Combination Index (CI) analysis suggested that MBIC and doxorubicin synergistically inhibited up to 97% of cell proliferation in ER+/PR+MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. Moreover, treatment of the breast cancer cells with the combined drugs resulted in lower IC50 values in contrast to the individual drug treatment. Small noncoding microRNAs (miRNA) may function as non-mutational gene regulators at post-transcriptional level of protein synthesis. In the present study, the effect of the combined treatment of MBIC and doxorubicin on the expression level of several miRNAs including miR-34a, miR-146a, miR-320a and miR-542 were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. These miRNAs have the potential to alter the protein level of survivin, the anti-apoptotic protein and reduce the metastatic activity in human breast cancer cell lines by interfering with the nuclear accumulation of NF-κB. Our results demonstrated the several fold changes in expression of miRNAs, which is drug and cell line dependent. This finding demonstrated a functional synergistic network between miR-34a, miR-320a and miR-542 that are negatively involved in post-transcriptional regulation of survivin in MCF-7 cells. While in MDA-MB-231 cells, changes in expression level of miR-146a was correlated with inhibition of the nuclear translocation of NF-κB. The overall result suggested that alteration in protein level and location of survivin and NF-κB by miR-34a, miR-320a, miR-146a and miR-542, remarkably influenced the synergistic enhancement of combined MBIC and doxorubicin in treatment of aggressive and less aggressive human breast cancer cell lines.

Rationally Developed Metallogelators Derived from Pyridyl Derivatives of NSAIDs Displaying Anti-Inflammatory and Anticancer Activities.

Metal-ligand coordination involving hydrogen-bond-functionalized ligands was employed rationally to get an easy access to a series of metallogelators derived from 3-pyridyl derivatives of nonsteroidal anti-inflammatory drugs [e.g., ibuprofen, sulindac, and flurbiprofen designated as 3-pyIBU, 3-pySUL, and 3-pyFLR, respectively] and biogenic metal centers [Zn(II), Cu(II), Mn(II), and Ag(I)]. A total of 13 metallogels (MG1-MG13) were obtained by allowing the ligands and the metal salts to react in dimethyl sulfoxide (DMSO)/water at room temperature. A slightly different solvent system (DMSO/water/MeOH) afforded four crystalline coordination complexes of 3-pyIBU, namely, [{Cu(3-pyIBU)4(DMSO)2}(NO3)2] (CC1), [{Ag(3-pyIBU)2}(BF4)] (CC2), [{Ag(3-pyIBU)2}(ClO4)] (CC3), and [{Cu(3-pyIBU)4(CH3OH)2}(OTf)] (CC4), which were fully characterized by single-crystal X-ray diffraction. However, none of these coordination complexes produced metallogels-the results corroborated well with the rationale, based on which the metallogelators were obtained. Two selected metallogels (MG3 and MG9) could be leached out from the corresponding metallogels to the bulk solvent to the extent of 51 and 59%, respectively after 24 h of incubation at 37 °C, indicating their plausible use in topical application. Moreover, one of the selected metallogelators, i.e., MG9, displayed anti-inflammatory response and was able to inhibit the migration of highly aggressive human breast cancer cells MDA-MB-231, suggesting its plausible use as anticancer agent.

In silico pharmacokinetic and molecular docking studies of N-cinnamoyltetraketide derivatives as inhibitors of cyclooxygenase-2 enzyme

Recent phytochemical analysis of Toussaintia orientalis leaves yielded series of novel bioactive N-cinnamoyltetraketide derivatives namely toussaintines A-G (t_1 - t_8) some portraying cytotoxicity against the triple negative aggressive human breast cancer cell line (MDA-MB-231) among other potencies. Despite having broad bioactivity spectrum, their general drug-likeness profiles and mode of action (simulated or actual) targeting any enzyme remains uninvestigated. In silico pharmacokinetic, drug-likeness descriptors and molecular docking of the compounds t_1-t_8 targeting inhibition of cyclooxygenase-2 (COX-2) enzyme were evaluated. The Lipinski Rule of Five heralded the pharmacokinetic properties of the studied metabolites. The studied compounds were docked with COX-2 following already established protocol. ADMET descriptors fell within the recommended range, except for compound t_3 that was predicted to potentially have positive blood brain barrier (BBB+) penetration. Docking studies indicated N-cinnamoyltetraketide derivatives as potential inhibitors of COX-2 enzyme. Compounds t_3 and t_5 showed lower binding energy of -13 and -12.3 kcal/mol, respectively, being closely comparable to celecoxib (-12.3 kcal/mol) indicating compatibility with the protein receptor. The findings provide baseline information on drug or lead-likeness and potential mode of action of the studied molecules towards inhibition of COX-2 enzyme.Keywords: N-cinnamoyltetraketide derivatives; molecular docking, ADMET, in silico, COX-2.