MDA-MB231 Cells Research Articles

Anti-tumoral activity of Allium roseum compounds on breast cancer cells MCF7 and MDA-MB231

Anti-tumoral activity of Allium roseum compounds on breast cancer cells MCF7 and MDA-MB231

Coagulation factor VIIa enhances programmed death-ligand 1 expression and its stability in breast cancer cells to promote breast cancer immune evasion

BackgroundImmunotherapy for breast cancer has not gained significant success. Coagulation factor VIIa (FVIIa)-tissue factor (TF) mediated activation of protease-activated receptor 2 (PAR2) is shown to promote metastasis and secretion of the immune-modulatory cytokines but the role of FVIIa in cancer immunology is still not well understood. ObjectivesHere, we aim to investigate whether FVIIa protects breast cancer cells from CD8 T-cell–mediated killing. MethodsPeripheral blood mononuclear cell-derived CD8 T cells were cocultured with vehicle or FVIIa pretreated MDAMB468 cells. The proliferation and activity of CD8 T cells were measured by flow cytometry and ELISA. An allograft model, using wild-type or TF/PAR2-deleted 4T1 cells, was employed to determine the effect of FVIIa on breast cancer immune evasion in vivo. ResultsHere, we demonstrate that TF-FVIIa induces programmed death-ligand 1 (PD-L1) in breast cancer cells by activating PAR2. PAR2 activation triggers large tumor suppressor kinase 1 (LATS1) inactivation leading to loss of yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) phosphorylation and subsequent nuclear localization of YAP/TAZ. YAP/TAZ inhibition reduces PD-L1 expression and increases CD8 T-cell activity. We further demonstrate that, apart from transcriptional induction of PD-L1, PAR2 activation also increases PD-L1 stability by enhancing its glycosylation through N-glycosyltransferases STT3A and STT3B. ConclusionIn a mouse model of breast cancer, tumor cell-specific PAR2 depletion leads to PD-L1 downregulation and increases anti–PD-1 immunotherapy efficacy. In conclusion, we showed that FVIIa-mediated signaling cascade in cancer cells serves as a tumor intrinsic mechanism of immunosuppression to promote cancer immune evasion.

Chorein-dependent microfilament organization in tumor cells

Chorein is variably expressed in different cancer cells. In various non-malignant cell types, this protein regulates cytoskeleton microstructure and signaling. However, the role of chorein in regulating the actin cytoskeleton in tumor cells remains elusive. Here, we investigated chorein expression in various breast tumor cells and the involvement of this protein in microfilament organization. We used laser scanning microscopy to analyze microfilaments architecture, Triton X-100 fractionation to quantify G and Total actin levels, and quantitative RT-PCR to assess chorein gene transcription. We show that in line with previous observations, the less differentiated MCF7 breast cancer cells exhibited the highest relative expression of chorein compared to MDA-MB231 and T47D cell lines. Contrastingly, in less differentiated ZF rhabdomyosarcoma cells expressing high chorein levels, silencing of this protein was followed by clear depolymerization of actin microfilaments as apparent from IF morphological analysis. Quantification of G- and F-actin levels by Triton X-100 fractionation that revealed a significant increase in this ratio fully supported this finding. These results disclose that chorein is highly expressed in less differentiated tumor cells. In addition, silencing of this protein induces significant structural disorganization of the actin network, providing clear evidence that chorein regulates microfilament cytoskeleton architecture in tumor cells of higher malignant potential.

Planophila laetevirens-Mediated Synthesis of Silver Nanoparticles: Optimization, Characterization, and Anticancer and Antibacterial Potentials.

Algal-mediated synthesis of nanoparticles (NPs) opens the horizon for green and sustainable synthesis of NPs that can be used in many fields, such as medicine and industry. We extracellularly synthesized silver NPs (Ag-NPs) using the novel microalgae Planophila laetevirens under optimized conditions. The isolate was collected from freshwater/soil, purified, morphologically identified, and genetically identified using light, inverted light, scanning electron microscopy, and 18S rRNA sequencing. The phytochemicals in the algal extract were detected by GC-MS. Aqueous biomass extracts and cell-free media were used to reduce silver nitrate to Ag-NPs. To get small, uniformly shaped, and stable Ag-NPs, various abiotic parameters, including precursor concentration, the ratio between the reductant and precursor, temperature, time of temperature exposure, pH, illumination, and incubation time, were controlled during the synthesis of Ag-NPs. B-P@Ag-NPs and S-P@Ag-NPs (Ag-NPs synthesized using biomass and cell-free medium, respectively) were characterized using UV-vis spectroscopy, transmission electron microscopy, scanning electron microscopy, energy-dispersive X-ray analysis (EDX) and mapping, Fourier transform infrared (FTIR) spectroscopy, and a zeta sizer. S-P@Ag-NPs had a smaller size (10.8 ± 0.3 nm) than B-P@Ag-NPs (19.0 ± 0.6 nm), while their shapes were uniform quasispherical (S-P@Ag-NPs) and spherical to oval (B-P@Ag-NPs). EDX and mapping analyses demonstrated that Ag was the dominant element in the B-P@Ag-NP and S-P@Ag-NP samples, while FTIR revealed the presence of O-H, C-H, N-H, and C-O groups, indicating that polysaccharides and proteins acted as reductants, while polysaccharides/fatty acids acted as stabilizers during the synthesis of NPs. The hydrodynamic diameters of B-P@Ag-NPs and S-P@Ag-NPs were 37.7 and 28.3 nm, respectively, with negative charges on their surfaces, suggesting their colloidal stability. Anticancer activities against colon cancer (Sw620 and HT-29 cells), breast cancer (MDA-MB231 and MCF-7 cells), and normal human fibroblasts (HFs) were screened using the MTT assay. B-P@Ag-NPs and S-P@Ag-NPs had a greater antiproliferative effect against colon cancer than against breast cancer, with biocompatibility against HFs. The biocidal effects of the B-P@Ag-NPs and S-P@Ag-NPs were evaluated against Escherichia coli, Bacillus cereus, and Bacillus subtilis using agar well diffusion and resazurin dye assays. B-P@Ag-NPs and S-P@Ag-NPs caused higher growth inhibition of Gram-negative bacteria than of Gram-positive bacteria. B-P@Ag-NPs and S-P@Ag-NPs synthesized by P. laetevirens are promising antitumor and biocidal agents.

Synergistic antitumor effects of circularly permuted TRAIL with doxorubicin in triple-negative breast cancer.

Circularly permuted TRAIL (CPT), a novel recombinant TRAIL mutant, is a potent antitumor agent. However, its efficacy in triple-negative breast cancer (TNBC) remains unclear. Treatment with CPT alone and in combination with doxorubicin (Dox) is explored for its effects on the proliferation and apoptosis of MDA-MB-231 (MB231) and MDA-MB-436 (MB436) breast cancer cells in vitro and in vivo. Here, we show that CPT combined with Dox exhibits time- and dose-dependent synergy to inhibit cell viability and enhance apoptosis of MB231 and MB436 cells. Combined treatment substantially increases caspase-8, caspase-3, and PARP cleavage in both cell lines and significantly suppresses tumor growth in nude mice bearing MB231 xenografts. Collectively, our findings demonstrate that treatment with CPT in combination with Dox exerts synergistic antitumor effects through activation of the caspase cascade pathway, a mechanism that is partly dependent on the Dox-induced upregulation of death receptor 4 and death receptor 5. Therefore, CPT combined with Dox may be a feasible therapeutic strategy for the management of TNBC.

Studying Luminal, A and B subtypes of the breast cancer under paracrine secretion of fibroblasts

Introduction Understanding the role of the tumor microenvironment in specifying molecule markers of breast cancer subtypes could involve tumor microenvironment properties in subtyping and clinical assessment of breast cancer. Therefore, interconversion of luminal states and or their specific markers alteration under the control of TME particularly CAFs deserve to be more investigated. Methods To activate normal human fibroblasts, liquid overlay technique or nemesis was used and α-SMA expression, CAFs marker, in fibroblastic spheroids was measured by blotting. The luminal A, MCF-7, and luminal B, MDA-MB 361, cell lines are treated with normal and spheroidal/activated fibroblast conditioned medium for 48 hours. The morphological changes of luminal A and B cells were evaluated by invert microscopy and analyzed by the shape factor formula. Moreover, sensitivity changes to chemotherapeutic agents, Ki67 expression, and gene expression level including ER-related and proliferative genes were assessed respectively by MTT assay, Immunofluorescence, and real time qRT-PCR methods. Results Activated fibroblast, spheroid form, by LOT/nemosis expressed α-SMA marker two folds more than monolayer cultured fibroblasts. Our study underlined a significant increase in IC50 of both luminal A and B cell lines after being co-cultured with conditioned medium markedly in spheroidal conditioned medium. Morphological changes study by shape factor formula demonstrated more aggressiveness with gaining mesenchymal features both in luminal A and B subtypes by increasing exposure time. Changes in Ki67 expression level were observed after fibroblasts and spheroidal paracrine conditioning. Only this data supports the luminal interconversion by elevated Ki67 expression in luminal A and lowered Ki67 expression in luminal B. Gene expression results revealed that the MCF7 and MDA-MB cells as representatives luminal A and B subtypes in this study did not interchange their own specific gene pattern, however both luminal types in co-culture with condition medium increased the expression of anti-apoptotic Bcl2 gene. Conclusion Under the paracrine influence of fibroblast cells, both luminal A(MCF7) and B (MDA-MB) subtypes of breast cancer gained invasive, anti-apoptotic, and chemoresistance features which are mostly increased by activated fibroblast conditioned medium mimicking CAFs. There was no strong proof for interconversion of luminal A and B, with more similarities, subtypes of breast cancer.

Eco-Friendly Green Synthesis and Characterization of Silver Nanoparticles by Scutellaria multicaulis Leaf Extract and Its Biological Activities.

Scutellaria multicaulis is a medicinal plant indigenous to Iran, Afghanistan, and Pakistan. It has been widely used as a prominent herb in traditional medicine for thousands of years. This plant is reported to contain baicalein, wogonin, and chrysin flavonoids, which are a significant group of chemical ingredients which can cure different diseases, such as breast cancer. S. multicaulis leaf extract was used for the bioreduction of silver nanoparticles (SmL-Ag-NPs), and their phytochemical contents and antioxidant, antibacterial, anti-proliferative, and apoptotic activity were evaluated. Optimal physicochemical properties of SmL-Ag-NPs were obtained by mixing 5% of leaf extract and 2 mM of aqueous AgNO3 solution and confirmed by characterization studies including UV-visible spectrophotometry, Field Emission Scanning Electron Microscope (FE-SEM), Energy Dispersive X-ray (EDX), Dynamic Light Scattering (DLS), zeta potential, Thermogravimetric analysis (TGA), Surface-enhanced Raman spectroscopy (SERS), X-ray crystallography (XRD), and Fourier transform infrared (FTIR) Spectroscopy. SmL-Ag-NPs exhibited a higher content of total phenol and total flavonoid and potential antioxidant activity. SmL-Ag-NPs also demonstrated dose-dependent cytotoxicity against MDA-MB231 cell multiplication with an IC50 value of 37.62 μg/mL through inducing cell apoptosis. Results show that SmL-Ag-NPs is effective at inhibiting the proliferation of MDA-MB231 cells compared to tamoxifen. This demonstrates that SmL-Ag-NPs could be a bio-friendly and safe strategy to develop new cancer therapies with a reduction in the adverse effects of chemotherapy in the near future.

Chemically-Modified Sepharose 6B Beads for Collection of Circulating Tumor Cells.

The isolation and quantitative characterization of circulating tumor cells (CTCs) are of great importance in cancer diagnosis and prognosis. However, isolating and detecting CTCs in whole blood presents a significant challenge due to the low numbers of CTCs (often ranging from one to five) in samples containing billions of erythrocytes. Recently, point-of-care devices that use antibody trapping coupled with remote immunofluorescence analyses have been described to identify the number and type of CTCs in blood. In this study, we propose a novel method for trapping and quantifying CTCs using Sepharose 6B beads of 45-160 μm size that are engineered with capture antibodies. Specifically, we employed CD44 antibody conjugates (bearing a maleimide group) that are specific to the CTCs of breast cancer to thiol-Sepharose beads 6B. These beads, when mixed with MDAMB231 and Jurkat cells and filtered through a 40 μm filter, can capture ~80% of MDAMB231 cells. Furthermore, the antibody-modified Sepharose 6B can be stored at four degrees Celsius for a period exceeding six months.

Imine-Linked Covalent Organic Frameworks: A Biocompatible and pH-Dependent Carrier for In Vitro Sustained Release of Doxorubicin.

Among the novel drug delivery systems (DDSs), covalent organic frameworks (COFs) show promising features in pharmaceutical science. In this paper, an imine-linked COF with hexagonal topology was synthesized using the autoclave condition. Then, the prepared COF (APB-COF) was used as a pH-dependent carrier for in vitro release of doxorubicin (DOX). The intrinsic properties of APB-COF caused reaching an excellent drug encapsulation efficiency. DOX@APB-COF shows an exemplary pH-dependent release in two different pHs. DOX release at pH = 7.4 was 32%, which increased to 54% by changing the pH to the cancer cell pH (pH = 5.4). Moreover, the cytotoxicity of APB-COF and DOX@APB-COF was studied using the standard MTT test against MCF10 (normal breast cell line) and MDAmb231 cells (breast cancer cell line), respectively. It was observed that the APB-COF does not affect cell proliferation, whereas the DOX@APB-COF only limits cancer cell proliferation. Using APB-COF as the drug carrier can pave the way for using COFs in innovative DDSs.

Au-TiO2 Coated Photonic Crystal Fiber Based SPR Refractometric Sensor for Detection of Cancerous Cells.

A surface plasmon resonance (SPR) refractometric sensor based on gold (Au) and titanium dioxide (TiO2) coated Photonic Crystal Fiber (PCF) is presented for the quick detection of various types of cancerous cells. The cancerous cells and their corresponding normal cells are both considered to be liquid cells each with their unique refractive index (RI). Normally these cells are found in liquid form in the suitable media (food) required to live the cancerous/normal cell lines. Also in our detection case, liquid samples are easy to pump into the sensing channel of the proposed PCF by employing either pressure or capillary forces.The proposed PCF sensor works on the SPR principle, with the Au coating serving as the plasmonic material. This sensor is investigated using the COMSOL Multiphysics software computational tool that is based on the full-vector finite element method (FEM). A TiO2 coating has been applied to enhance adhesion between the Au layer and the PCF surface. Above the Au coating, cancerous cells samples are filled into the PCF. When the core mode of the PCF is coupled with the surface plasmon polariton (SPP) mode under the specific resonance circumstances, SPR will occur on the interface of the gold-sample cell, and in the core mode, the loss peak is observed at the resonance wavelength. Cancerous cells samples have a distinct loss peak than normal cells samples therefore the cancerous cells can be diagnosed by measuring the shift in resonance wavelength corresponding to the loss peak of cancerous and their normal cells samples. The proposed sensor may identify various cancerous cells such as MDAMB-231, MCF-7, PC12, HeLa, and Jurkat for the diagnosis of breast cancer type-1, breast cancer type-2, adrenal glands, cervical, and blood cancer respectively. The computed wavelengths sensitivities of the proposed PCF are 9428.57nm/RIU, 10714.28nm/RIU, 7571.43nm/RIU, 5500nm/RIU, and 6000nm/RIU for the MDAMB-231, MCF-7, PC12, HeLa, and Jurkat cancerous cells, respectively. However, for various cancerous cells, the maximum amplitude sensitivity varies from -1387 RIU-1 to -1599 RIU-1. Moreover, the sensor resolution ranges between 0.93 ×10-5 RIU and 1.82 ×10-5 RIU with a 0.024 maximum detection limit. Because of its improved sensing capability, the presented SPR refractometric sensor is appropriate for the early detection of cancerous cells.

Insights into the mechanisms governing P01 scorpion toxin effect against U87 glioblastoma cells oncogenesis.

The emerging concept of small conductance Ca2+-activated potassium channels (SKCa) as pharmacological target for cancer treatment has significantly increased in recent years. In this study, we isolated the P01 toxin from Androctonus australis (Aa) scorpion venom and investigated its effect on biological properties of glioblastoma U87, breast MDA-MB231 and colon adenocarcinoma LS174 cancer cell lines. Our results showed that P01 was active only on U87 glioblastoma cells. It inhibited their proliferation, adhesion and migration with IC50 values in the micromolar range. We have also shown that P01 reduced the amplitude of the currents recorded in HEK293 cells expressing SK2 channels with an IC50 value of 3pM, while it had no effect on those expressing SK3 channels. The investigation of the SKCa channels expression pattern showed that SK2 transcripts were expressed differently in the three cancer cell lines. Particularly, we highlighted the presence of SK2 isoforms in U87 cells, which could explain and rely on the specific activity of P01 on this cell line. These experimental data highlighted the usefulness of scorpion peptides to decipher the role of SKCa channels in the tumorigenesis process, and develop potential therapeutic molecules targeting glioblastoma with high selectivity.

Synthesis and investigation of cytotoxic effects of compounds derived from flurbiprofen

New flurbiprofen derivatives containing 1,2,4-triazoline-5-thione (4) and thioethers (5a-r) were synthesized in this study. The structures of synthesized compounds were characterized by spectral methods (FT-IR, 1H NMR, 13C NMR) and 19F NMR (only compound 5l), besides elemental analysis. In addition, molecular binding of these compounds to the human methionine aminopeptidase 2 enzyme was performed using AutoDock 4.2, the software product of the research, computationally. All synthesized compounds were evaluated for cytotoxic effect against MDA-MB231 triple-negative breast cancer cell line by using WST-1 Cell Viability and Proliferation assay. Doxorubicin is in the anthracycline class and is an antineoplastic agent. It is used to provide regression in common neoplastic conditions such as breast carcinoma. Due to the cardiovascular side effects of doxorubicin, a combination study was conducted with the (±)(R,S)-3-{1-[2-fluoro-(1,1′-biphenyl)-4-yl]ethyl}-4-methyl-5-{[2-(trifluoromethyl)benzyl]thio}-4H-1,2,4-triazole (5l) with promising cytotoxic effects. As a result of the combination, it was shown as 7% MDA-MB231 cell viability. Therefore, based on the evaluations, a better cytotoxic effect was achieved with the 5l combination depending on the low dose of doxorubicin.

Danthron, an Anthraquinone Isolated from a Marine Fungus, Is a New Inhibitor of Angiogenesis Exhibiting Interesting Antitumor and Antioxidant Properties.

The role played by a sustained angiogenesis in cancer and other diseases stimulates the interest in the search for new antiangiogenic drugs. In this manuscript, we provide evidence that 1,8- dihydroxy-9,10-anthraquinone (danthron), isolated from the fermentation broth of the marine fungus Chromolaenicola sp. (HL-114-33-R04), is a new inhibitor of angiogenesis. The results obtained with the in vivo CAM assay indicate that danthron is a potent antiangiogenic compound. In vitro studies with human umbilical endothelial cells (HUVEC) reveal that this anthraquinone inhibits certain key functions of activated endothelial cells, including proliferation, proteolytic and invasive capabilities and tube formation. In vitro studies with human breast carcinoma MDA-MB231 and fibrosarcoma HT1080 cell lines suggest a moderate antitumor and antimetastatic activity of this compound. Antioxidant properties of danthron are evidenced by the observation that it reduces the intracellular reactive oxygen species production and increases the amount of intracellular sulfhydryl groups in endothelial and tumor cells. These results support a putative role of danthron as a new antiangiogenic drug with potential application in the treatment and angioprevention of cancer and other angiogenesis-dependent diseases.

Anticancer Potential of Sulfonamide Moieties via In-Vitro and In-Silico Approaches: Comparative Investigations for Future Drug Development.

Several kinds of anticancer drugs are presently commercially accessible, but low efficacy, solubility, and toxicity have reduced the overall therapeutic indices. Thus, the search for promising anticancer drugs continues. The interactions of numerous essential anticancer drugs with DNA are crucial to their biological functions. Here, the anticancer effects of N-ethyl toluene-4-sulphonamide (8a) and 2,5-Dichlorothiophene-3-sulphonamide (8b) on cell lines from breast and cervical cancer were investigated. The study also compared how these substances interacted with the hearing sperm DNA. The most promising anticancer drug was identified as 2,5-Dichlorothiophene-3-sulfonamide (8b), which showed GI50 of 7.2 ± 1.12 µM, 4.62 ± 0.13 µM and 7.13 ± 0.13 µM against HeLa, MDA-MB231 and MCF-7 cells, respectively. Moreover, it also exhibited significant electrostatic and non-electrostatic contributions to the binding free energy. The work utilized computational techniques, such as molecular docking and molecular dynamic (MD) simulations, to demonstrate the strong cytotoxicity of 2,5-Dichlorothiophene-3-sulfamide (8b) in comparison to standard Doxorubicin and cisplatin, respectively. Molecular docking experiments provided additional support for a role for the minor groove in the binding of the 2,5-Dichlorothiophene-3-sulfamide (8b)-DNA complex. The molecular docking studies and MD simulation showed that both compounds revealed comparable inhibitory potential against standard Doxorubicin and cisplatin. This study has the potential to lead to the discovery of new bioactive compounds for use in cancer treatment, including metallic and non-metallic derivatives of 2,5-Dichlorothiophene-3-sulfonamide (8b). It also emphasizes the worth of computational approaches in the development of new drugs and lays the groundwork for future research.

Effective inhibition of breast cancer stem cell properties by quercetin-loaded solid lipid nanoparticles via reduction of Smad2/Smad3 phosphorylation and β-catenin signaling pathway in triple-negative breast cancer

BackgroundThe presence of cancer stem cells (CSCs) is a major cause of resistance to cancer therapy and recurrence. Triple-negative breast cancer (TNBC) is a subtype that responds poorly to therapy, making it a significant global health issue. Quercetin (QC) has been shown to affect CSC viability, but its low bioavailability limits its clinical use. This study aims to increase the effectiveness of QC in inhibiting CSC generation by using solid lipid nanoparticles (SLNs) in MDA-MB231 cells. Materials and methodsAfter treating MCF-7 and MDA-MB231 cells with 18.9 μM and 13.4 μM of QC and QC-SLN for 48 h, respectively, cell viability, migration, sphere formation, protein expression of β-catenin, p-Smad 2 and 3, and gene expression of EMT and CSC markers were evaluated. ResultsThe QC-SLN with particle size of 154 nm, zeta potential of −27.7 mV, and encapsulation efficacy of 99.6% was found to be the most effective. Compared to QC, QC-SLN significantly reduced cell viability, migration, sphere formation, protein expression of β-catenin and p-Smad 2 and 3, and gene expression of CD44, zinc finger E-box binding homeobox 1 (ZEB1), vimentin, while increasing the gene expression of E-cadherin. ConclusionsOur findings demonstrate that SLNs improve the cytotoxic effect of QC in MDA-MB231 cells by increasing its bioavailability and inhibiting epithelial-mesenchymal transition (EMT), thereby effectively inhibiting CSC generation. Therefore, SLNs could be a promising new treatment for TNBC, but more in vivo studies are needed to confirm their efficacy.

Cobalt(II) complexes containing mefenamic acid with imidazole and pyridine based auxiliary ligands: Synthesis, structural investigation and cytotoxic evaluation

The interaction between CoCl2.6H2O and mefenamic acid (2-[(2,3-dimethylphenyl)amino]benzoic acid) in the presence of imidazole and pyridine based ligands resulted in the formation of [Co(mef)2(imi)2]; C-1, [Co(mef)2(nic)2(H2O)2].2H2O; C-2, [Co2(µ-H2O)(µ-mef-O,O')2(mef)2(py)4].CH3CN; C-3, and [Co2(µ-mef-O,O')4(apy)2].2CH3CN; C-4, (mef is monodentate mefenamato, µ-H2O is bridging aqua ligand, µ-mef-O, O' is bridging bidentate mefenamato, imi is imidazole, nic is nicotinamide, py is pyridine, and apy is 2-aminopyridine). The complexes were characterized spectroscopically and by physicochemical properties. Single-crystal X-ray diffraction studies confirmed the structures of the complexes. In C-1, Co(II) is in four-coordinate geometry, while in C-2, Co(II) is in hexa-coordination with two mefenamato carboxylate oxygen atoms, two nitrogen atoms, and two oxygen atoms from two solvent ligands. Complex C-3 consists of two cobalt centers linked by an aqua ligand and bridged by two mefenamato ligands. Furthermore, the octahedral geometry in each Co(II) in C-3 was completed by coordinating two monodentate mefenamato ligands and monodentate pyridine ligands. Similarly, in C-4, a Co-Co bond connects two Co(II) centers in six-coordinate geometry, with four bidentate mefenamato ligands acting as bridges between the two Co(II) and two 2-aminopyridine nitrogen, in monodentate coordination to each Co(II). The lattice solvents in C-2 to C-4 participated in intermolecular hydrogen bonding interactions with the mefenamato ligand. FTIR studies showed the presence of both monodentate and bidentate coordination of the deprotonated carboxylate oxygen atoms of the mefenamato ligand, as well as the coordination of the nitrogen donor ligands in the complexes. TG analysis determined the complexes' thermal stability and decomposition steps and ascertained their compositions, which agreed with the analytical and X-ray crystallography studies. Furthermore, the cytotoxicity of mefenamic acid and the cobalt (II) complexes on lung carcinoma cells (NCIH-460) and breast cells (MDAMB) were assessed using MTT assay. The complexes showed higher cytotoxicity than mefenamic acid against the MDAMB cell line.

Abstract 2539: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis

Abstract TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting that there are unknown modulatory mechanisms responsible for its lack of TRAIL activity in vivo. We hypothesized that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu, modulating the therapeutic efficacy. To investigate the hypothesis, we performed RNAseq analysis of MDA-MB231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. TRAIL treatment of the TNBC significantly induced expression of a number of cytokines, such as CXCL1, CXCL2, CXCL3, CXCL8, CXCL11, IL6, which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by Death Receptor 5 and Caspase-8 protein, but not Caspase-8 enzymatic activity. Gene Set Enrichment Analysis of the RNAseq indicated that NFKB pathway-mediated TNF-alpha signaling was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL. However, siRNA knockdown experiments indicated that the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs’ (1, 2, 3, 8) and IL6 significantly increased neutrophil chemotaxis in a NFKB2-dependent manner. Moreover, neutrophils pre-incubated with supernatants from TRAIL-treated TNBC significantly inhibited its cytotoxic effect in TNBCs in a NFKB2-dependent pathway. Further RNAseq and RT-PCR of neutrophils incubated with by either TRAIL or supernatant of MDA-MB231 cells treated with TRAIL revealed significant enrichment of inflammatory pathway-related genes as well as increased expression of immune modulating cytokines. These results suggested that TRAIL exerts a pro-inflammatory role towards immune cells in tumor microenvironment. CODEX (CO-Detection by indEXing) analysis of in vivo TNBC xenografts from mice treated with the TRAIL agonist MEDI3039 confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Using TNBC organoids and humanized mice models, the changes in tumor immune environment caused by TRAIL are currently under investigation. Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production that affects neutrophil functions, leading to modulation of the immune response in TNBC. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa A. Ridnour, David A. Wink, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2539.

Abstract 1278: ID3 controlled dynamic Pyk2 localization is important for the mobility of cancer stem cells

Abstract Treatment of brain metastases remains a clinical barrier in breast cancer patients. Secondary breast tumors in the brain are reported to be enriched with breast cancer stem cell (BCSC) markers suggesting that BCSCs infiltrate the brain. Brain infiltration by BCSCs is complex involving detachment from the primary tumor, entering the blood, surviving the microvascular niche, and crossing the blood brain barrier (BBB). It is critical to understand how BCSCs move through the BBB to prevent brain metastases. Nuclear respiratory factor 1 (NRF1) transcription factor, Pyk2 non-receptor tyrosine kinase, and inhibitor of differentiation protein 3 (ID3) have been shown to contribute to the pathogenesis of cancer through inducing CSCs or increasing mobility of these cells. Recently, we showed that NRF1-induced BCSCs crossed the BBB which were guided by ID3 overexpressing endothelial stem cells. In this study, we investigated the NRF1/ID3/Pyk2 axis contributing to BCSCs mobility. NRF1 promoted migration of BCSCs and mesenchymal MDAMB231 cells through brain endothelial cells cultured with astrocytes. ID3 overexpressing endothelial cells promoted adhesion with BCSCs and mobility of BCSCs across the BBB model. In addition, ID3 controlled Pyk2 localization which is important for mobility of stem cells and cancer cells. Endothelial cells were treated with a known risk factors for breast cancer, 17β-estradiol (E2) and environmental pollutant PCB153. Treatment with PCB153 or E2 showed increased growth of BCSCs tumor spheroids and mobility of ESCs. Exosomal ID3 released from endothelial cells also supported the growth of BCSCs and provide the basis for paracrine effects by endothelial stem cells associated with breast tumors. These findings show for the first time a novel role for ID3 and Pyk2 by which endothelial stem cells support breast tumor metastases where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3/Pyk2 modifying agents to prevent the spread of breast cancer to the brain. Citation Format: Jayanta Das, Alok Deoraj, Deodutta Roy, Quentin Felty. ID3 controlled dynamic Pyk2 localization is important for the mobility of cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1278.

Fabrication and Assessment of Orodispersible Tablets Loaded with Cubosomes for the Improved Anticancer Activity of Simvastatin against the MDA-MB-231 Breast Cancer Cell Line.

Various factors limit the use of simvastatin as an anticancer drug. Therefore, this study aimed to analyse simvastatin (SIM)-loaded cubosome efficacy against breast cancer. SIM-loaded cubosomes were prepared using the emulsification method using different glyceryl monooleate, Pluronic F127 (PF-127), and polyvinyl alcohol (PVA) ratios. The best cubosomal formula was subjected to an in vitro cytotoxicity analysis using the human breast cancer cell line, MDA-MB-231 (MDA) (ATCC, HTB-26), and formulated as oral disintegrating tablets through direct compression. PF-127 and PVA positively affected drug loading, and the entrapment efficiency percentage of different SIM-cubosomal formulations ranged from 33.52% to 80.80%. Vesicle size ranged from 181.9 ± 0.50 to 316.6 ± 1.25 nm. PF-127 enhanced in vitro SIM release from cubosome formulations due to its solubilising action on SIM. The in vitro dissolution analysis indicated that SIM exhibited an initial dissolution of 10.4 ± 0.25% within the first 5 min, and 63.5 ± 0.29% of the loaded drug was released after 1 h. Moreover, cubosome formula F3 at 25 and 50 µg/mL doses significantly decreased MDA cell viability compared to the 12.5 µg/mL dose. The untreated SIM suspension and drug-free cubosomes at all doses had no significant influence on MDA cell viability compared to the control.

Anti-cancer Effect and Active Phytochemicals of Houttuynia cordata Thunb. against Human Breast Cancer Cells.

Houttuynia cordata Thunb (HCT) is a medicinal herb used in Southeast Asia. Aim of this work: This study aimed at investigating the cytotoxicity of this plant extract and fractions towards human breast cancer MDA-MB-231 and MCF-7 cells. HCT's phytoactive compounds are determined. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mode of cell death was measured by staining with annexin V-FITC and propidium iodide (PI) employing flow cytometry technique. The oxidative stress was measured by using 2',7'-dihydrodichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE+) fluorescent probes and using a fluorescence microplate reader. HCT phytochemicals were characterized by high performance liquid chromatography (HPLC). The proliferation of MDA-MB-231 and MCF-7 cells was dramatically decreased by the crude extract and individual fraction of HCT. Ethyl acetate was the solvent fraction with the highest toxicity against MCF-7 cells, followed by dichloromethane, crude, and hexane fractions, respectively, whereas in MDA-MB231 cells, dichloromethane, crude, hexane, and ethyl acetate fractions each had the strongest impact, respectively. The methanol fraction had no effect on either cell line up to 200 μg/ml. The extract and fractions were less harmful to the NIH3T3 normal murine fibroblast cell line. The mode of both cell death was apoptosis evidenced by the increase of cell population stained with annexin V-FITC and PI. The fluorescence probes of both DCFH-DA and DHE in MDA-MB-231 cell line were enhanced. Phenolic acids included chlorogenic acid (CA), gallic acid (GA), transcoumaric acid (TCA), vanillic acid (VA), and syringic acid (SA), as well as flavonoids like quercetin and rutin, were identified as the active phytochemicals in the crude and fractions by using HPLC method. MDA-MB-231cells underwent apoptosis via oxidative stress when induced with HCT hexane fraction. Phenolic acids and flavonoids were identified in HCT's extract and fractions.