MCF-7 Tumor Cell Lines Research Articles

Synthesis, Absolute Configuration, Biological Profile and Antiproliferative Activity of New 3,5-Disubstituted Hydantoins.

Hydantoins, a class of five-membered heterocyclic compounds, exhibit diverse biological activities. The aim of this study was to synthesize and characterize a series of novel 3,5-disubstituted hydantoins and to investigate their antiproliferative activity against human cancer cell lines. The new hydantoin derivatives 5a-i were prepared as racemic mixtures of syn- and anti-isomers via a base-assisted intramolecular amidolysis of C-3 functionalized β-lactams. The enantiomers of syn-5a and anti-hydantoins 5b were separated by preparative high-performance liquid chromatography (HPLC) using n-hexane/2-propanol (90/10, v/v) as the mobile phase. The absolute configuration of the four allyl hydantoin enantiomers 5a was assigned based on a comparison of the experimental electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra with those calculated using density functional theory (DFT). The antiproliferative activity evaluated in vitro against three different human cancer cell lines: HepG2 (liver hepatocellular carcinoma), A2780 (ovarian carcinoma), and MCF7 (breast adenocarcinoma), and on the non-tumor cell line HFF1 (normal human foreskin fibroblasts) using the MTT cell proliferation assay. In silico drug-like properties and ADMET profiles were estimated using the ADMET Predictor ver. 9.5 and the online server admetSAR. Eighteen new 3,5-disubstituted hydantoins were synthesized and characterized. The compound anti-5c showed potent cytotoxic activity against the human tumor cell line MCF7 (IC50 = 4.5 µmol/L) and the non-tumor cell line HFF1 (IC50 = 12.0 µmol/L). In silico analyzes revealed that the compounds exhibited moderate water solubility and membrane permeability and are likely substrates for CYP3A4 and P-glycoprotein and have a high probability of antiarthritic activity.

Chemical Composition of Anabasis articulata, and Biological Activity of Greenly Synthesized Zinc Oxide Composite Nanoparticles (Zn-NPs): Antioxidant, Anticancer, and Larvicidal Activities

The synthesis of nanoparticles utilizing green techniques is becoming increasingly important due to its low cost, biocompatibility, high productivity, and eco-friendliness. Herein, the current work focused on the biosynthesis, characterization, and biological applications of zinc oxide nanoparticles (ZnO-NPs) from Anabasis articulata, including antioxidant anticancer and larvicidal properties, as well as modifications to the phytochemical ingredients. Hence, the tannin, phenolic, and flavonoid concentrations of the produced nanoparticle samples were lower than those of the original aqueous extract. When compared to the results of ascorbic acid (12.78 mg/mL), the produced extract of A. articulata and its zinc nanoparticles showed remarkable efficacy as antioxidant agents with IC50 values of 27.48 and 69.53 mg/mL, respectively. A normal lung fibroblast cell line (WI-38) and three tumor cells were used to test the compounds’ anticancer properties. With an IC50 of 21.19 µg/mL, the ZnO-NPs of A. articulata showed the greatest cytotoxicity against HePG-2 cell lines. Additionally, A. articulata zinc nanoparticles showed significant cytotoxicity against MCF-7 and PC3 tumor cell lines, with IC50 values of 30.91 and 49.32 µg/mL. The biogenic ZnO-NPs had LC50 and LC90 values of 13.64 and 26.23 mg/L, respectively, and are very effective against Aedes aegypti larval instar (III). Additionally, the percentages of larval mortality increased from 28.61% at 5 ppm to 84.69% at 25 ppm after 24 h post-treatment. The overall results of this study point to the potential of A. articulata as a substitute biological agent for potential therapeutic/leutic uses in the medical domains and for preventing the proliferation of malarial vector insects.

Dehydroabietylamine-substituted trifluorobenzene sulfonamide rhodamine B hybrids as anticancer agents overcoming drug resistance

Attachment of a conjugate assembled from a novel fluorinated carbonic anhydrase inhibitor and rhodamine B onto dehydroabietylamine (DHA) or cyclododecylamine led to first-in-class conjugates of good cytotoxicity; thereby IC50 values (from SRB assays; employed tumor cell lines A2780, A2780Cis, A549, HT29, MCF7, and non-malignant human fibroblasts CCD18Co) between 0.2 and 0.7 μM were found. Both conjugates showed similar cytotoxic activity but the dehydroabietylamine derived conjugate outperformed its cyclododecyl analog in terms of tumor cell/non-tumor cell selectivity. Both conjugates accumulate intracellular, and the DHA conjugate was able to overcome drug resistance which is effective independent of the expression status of carbonic anhydrase IX.

Antiapoptotic and Prometastatic Roles of Cytokine FAM3B in Triple-Negative Breast Cancer

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. FAM3B, a secreted protein, has been extensively studied in various types of tumors. However, its function in breast cancer remains poorly understood. MethodsWe analyzed FAM3B expression data from breast cancer patients available at TCGA database and overall survival was analyzed by using the Kaplan-Meier plotter. MDA-MB-231 TNBC tumor cell line and hormone-responsive MCF-7 cell lines were transfected to overexpress FAM3B. We assessed cell death, tumorigenicity, and invasiveness in vitro through MTT analysis, flow cytometry assays, anchorage-independent tumor growth, and wound healing assays, respectively. We performed in vivo evaluation by tumor xenograft in nude mice. ResultsIn silico analysis revealed that FAM3B expression was lower in all breast tumors. However, TNBC patients with high FAM3B expression had a poor prognosis. FAM3B overexpression protected MDA-MB-231 cells from cell death, with increased expression of Bcl-2 and Bcl-xL, and reduced caspase-3 activity. MDA-MB-231 cells overexpressing FAM3B also exhibited increased tumorigenicity and migration rates in vitro, displaying increased tumor growth and reduced survival rates in xenotransplanted nude mice. This phenotype is accompanied by the upregulation of EMT-related genes Slug, Snail, TGFBR2, vimentin, N-cadherin, MMP-2, MMP-9, and MMP-14. However, these effects were not observed in the MCF-7 cells overexpressing FAM3B. ConclusionFAM3B overexpression contributes to tumor growth, promotion of metastasis, and, consequently, leads to a poor prognosis in the most aggressive forms of breast cancer. Future clinical research is necessary to validate FAM3B as both a diagnostic and a therapeutic strategy for TNBC.

ADAM12-Generated Basigin Ectodomain Binds β1 Integrin and Enhances the Expression of Cancer-Related Extracellular Matrix Proteins.

Desmoplasia is a common feature of aggressive cancers, driven by a complex interplay of protein production and degradation. Basigin is a type 1 integral membrane receptor secreted in exosomes or released by ectodomain shedding from the cell surface. Given that soluble basigin is increased in the circulation of patients with a poor cancer prognosis, we explored the putative role of the ADAM12-generated basigin ectodomain in cancer progression. We show that recombinant basigin ectodomain binds β1 integrin and stimulates gelatin degradation and the migration of cancer cells in a matrix metalloproteinase (MMP)- and β1-integrin-dependent manner. Subsequent in vitro and in vivo experiments demonstrated the altered expression of extracellular matrix proteins, including fibronectin and collagen type 5. Thus, we found increased deposits of collagen type 5 in the stroma of nude mice tumors of the human tumor cell line MCF7 expressing ADAM12-mimicking the desmoplastic response seen in human cancer. Our findings indicate a feedback loop between ADAM12 expression, basigin shedding, TGFβ signaling, and extracellular matrix (ECM) remodeling, which could be a mechanism by which ADAM12-generated basigin ectodomain contributes to the regulation of desmoplasia, a key feature in human cancer progression.

Antioxidant and Anticancer Potential of Extracellular Polysaccharide from Porphyridium aerugineum (Rhodophyta)

Porphyridium aerugineum is a unicellular freshwater red microalga that synthesizes and secretes into the culture medium an extracellular polysaccharide (EPS). In this study, algal growth and polysaccharide production, as well as the antioxidant capacity and antitumor effect of Porphyridium aerugineum EPS (PaEPS), were investigated. Cultivation of the microalgae was carried out in a photobioreactor under controlled conditions. Algal growth and the amount of EPS were monitored daily. The accumulated polysaccharide was extracted and lyophilized. At the end of cultivation, the concentration of microalgal biomass and PaEPS reached 3.3 and 1.2 g L−1, respectively. To examine the antioxidant capacity of PaEPS, FRAP and ABTS assays were performed. The cytotoxic activity of PaEPS was evaluated on the tumor cell lines MCF-7 (breast cancer) and HeLa (cervical adenocarcinoma) and on BJ (a non-tumor human skin fibroblast cell line), using MTT assay. The results obtained indicated that P. aerugineum polysaccharide exhibited a high ABTS radical-scavenging activity reaching up to 55%. The cytotoxic effect was best expressed in MCF-7 cells treated for 72 h with 1000 µg/mL PaEPS, where tumor cell proliferation was inhibited by more than 70%. Importantly, the PaEPS treatments did not significantly affect the viability of BJ cells. These findings promote the biotechnological production of P. aerugineum extracellular polysaccharide and reveal its potential as an anticancer and antioxidant agent for future applications.

Cytotoxic Secondary Metabolites from the Ascomycetous Fungus Pseudogymnoascus roseus

Background: Fungi found in unique and competitive environments are abundant in bioactive compounds. Objective: The objective of this study is to isolate and identify secondary metabolites from fungi of unique ecological niches and evaluate their cytotoxicity. Methods: The compounds were isolated and purified using silica gel column chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high-performance liquid chromatography (HPLC). The structures of the isolated compounds were determined using NMR and MS. The cytotoxic activities of these compounds were tested by the MTS assay. Results: Three diphenyl ethers, dechlorodihydromaldoxin (1), violaceol-I (2) and violaceol-II (3), one quinolinone compound, 2-(2-heptenyl)-3-methyl-4(1H)-quinolinone (4), and one α-pyrone nafuredin (5) were isolated from the fermented extracts of Pseudogymnoascus roseus S161. Compound 1 showed modest cytotoxicity against two human tumor cell lines A549 and MCF-7, with IC50 values of 87.12 and 51.07 µM, respectively. Conclusion: Five compounds were isolated from the fungus P. roseus S161. Compound 1 showed moderate cytotoxicity. This study provided a basis for the development of antitumor drugs.

A novel Pt2+ complex containing 1,4-benzodioxan-6-amine: Stability, DNA binding, and antitumor effects against human breast cancer cells

In this work, a new platinum(II) complex was prepared by the reaction of K2PtCl4 with 1,4-benzodioxan-6-amine (bda) at room temperature in the molar ratio 2:1 (L:M). The complex was characterized by physicochemical (elemental analysis, TGA/DTA, and conductivity measurements) and spectroscopic methods (UV–Vis, FTIR, 1H, 13C and 195Pt NMR). According to the spectroscopic data, two bda ligands coordinate to the platinum ion in a monodentate manner through the nitrogen atom to give the complex cis-[PtCl2(bda)2]. Stability studies were carried out under different conditions and showed that the complex is stable in pure DMF, although it undergoes slow solvolysis in DMSO. The interaction of this complex with calf thymus DNA (CT-DNA) was studied using circular dichroism (CD), electronic absorption and fluorescence spectroscopy. The complex binds to CT-DNA with a Kb value of 1.06 ​× ​103 ​M−1 and according to data from CD and fluorescence spectroscopy, appears to interact with DNA by electrostatic forces and or other no intercalative binding mode. Compared to cisplatin, the complex exhibited good cytotoxic activity and selectivity against the MCF-7 tumor cell line, displaying an IC50 value of 17.33 ​± ​1.1 ​μM, as well as inhibited MCF-7 ​cells migration.

Pachira aquatica: biological activity and chemical composition of leaves, flowers, and seeds

The chemical composition of Pachira aquatica crude extracts flowers, leaves, and seeds was obtained by UHPLC-ESI/qTOF and GC/MS. The antiproliferative activity was evaluated against the human tumour cell lines AGS (gastric), CaCo-2 (colorectal), MCF-7 (breast), and NCI-H460 (lung). The anti-inflammatory and cellular antioxidant activities were also studied. Flavonoids, phenolic acids, coumarins, and saturated fatty acids were identified in the samples. The concentration of extracts responsible for inhibiting 50% of nitric oxide production ranged from (149 to > 400 µg mL−1). Antiproliferative activity against the tumour cell lines was: AGS (GI50 175 to > 400 µg mL−1), Caco-2 (GI50 215 to > 400 µg mL−1), MCF7 (GI50 232 to > 400 µg mL−1) and NCI-H460 (GI50 208 to > 400 µg mL−1). Cellular antioxidant activity remained between 73% to > 2000%. The selectivity index (SI) ranged from 1.00 to 2.78, indicating low antiproliferative activity.

Irpexols A-C, xanthone derivatives from the endophyte fungus Irpex laceratus A878

Three new xanthone derivatives irpexols A-C (1–3) and five known xanthones including three dimeric ones were successfully isolated from Irpex laceratus A878, an endophytic fungus of the family Irpicaceae from the medicinal plant Pogostemon cablin (Blanco) Bentham (Lamiaceae). The structures of these compounds were elucidated by extensive spectroscopic analyses including ultraviolet-visible spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), and nuclear magnetic resonance (NMR). All of the three new compounds (1–3) share a de-aromatic and highly‑oxygenated xanthone skeleton. In addition, the cytotoxic activity of compounds 1–8 were evaluated against SF-268, MCF-7, HepG2, and A549 tumor cell lines. The results revealed that compound 6 showed moderate cytotoxic activity with the IC50 values ranging from 24.83 to 45.46 μM, while the IC50 values of the positive control adriamycin was ranging from 1.11 to 1.44 μM.

Pararorine A, isoindolinone alkaloid from the endophytic fungus Paramyrothecium roridum and its anti-tumor activity

Pararorine A, a new isoindolinone alkaloid was isolated from Paramyrothecium roridum, an endophytic fungus from the medicinal plant Gynochthodes officinalis (F.C. How) Razafim. & B. Bremer. The structure of this compound was elucidated by extensive spectroscopic (UV, IR, MS, and NMR) analyses. In addition, the antitumor activity of pararorine A was evaluated against SF-268, MCF-7, HepG2, and A549 tumor cell lines. The results revealed that pararorine A exhibited potent antitumor activities with the IC50 values ranging from 1.69 to 8.95 μM. Moreover, the tumor cell inhibitory activity of pararorine A was evidenced by promoting cytochrome C release and cell cycle arrest as well as the induction of apoptosis by the up-regulation of the protein expressions of JNK and Bax through PARP-cleavage and caspase 3-cleavage.

Phytochemical Characterization, Antioxidant, and Anti-Proliferative Activities of Wild and Cultivated Nigella damascena Species Collected in Sicily (Italy).

The use of Nigella damascena seeds in the culinary field or as aerial parts infusions in the pharmaceutical and cosmetic fields is widely reported. The biological activity of this plant, as demonstrated over the years, is closely related to its phytochemical content. This investigation focused on the comparative study of the same plants of N. damascena, one totally wild (WND), while the other two, one with white flowers (CWND) and the other with blue flowers (CBND), were subject to cultivation, irrigation, and manual weeding. Using the potential of 1D and 2D-NMR spectroscopy, coupled with MS/MS spectrometric studies, the three methanolic extracts of N. damascena were investigated. Chemical studies have highlighted the presence of triterpene saponin compounds and various glycosylated flavonoids. Finally, the in vitro antiproliferative and antioxidant activities of the three individual extracts were evaluated. The antiproliferative activity performed on U-937, HL-60, and MCF-7 tumor cell lines highlighted a greater anticancer effect of the CBND and CWND extracts compared to the data obtained using WND. The antioxidant activity, however, performed to quantify ROS generation is comparable among the extracts used.

Abstract 3706: Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system

Abstract Introduction: Liquid biopsy-based testing has increasingly been adopted by oncologists for biomarker-driven diagnostics, therapy selection, and patient monitoring. Unlike tissue-based approaches, the target analytes in blood specimens are often rare events that must be discriminated from a background of non-tumor derived material. Droplet digital PCR (ddPCR) is an ideal platform for detection of rare nucleic acid markers in the clinic, because it is highly sensitive and specific, robust, and quantitative. Additionally, the workflow is simple to adopt and execute. The QX600 System is a new ddPCR instrument designed for advanced multiplexing which enables simultaneous detection of up to 6 unique fluorophores in a single well. This capability can decrease the sub-sampling needed for detection of multiple analytes. The objective of our study was to develop multiplexed ddPCR assays for detection of cell-based and cell-free circulating biomarkers from a single tube of whole blood. Methods: Tumor cells were enriched from the buffy coat fraction of whole blood collected in fixative-containing cfDNA BCT based on size and morphology using a microfluidics-based system. Enriched tumor cells were enumerated by immunofluorescence (DAPI+/Pan-CK+/CD45-), and gene expression was measured using the QX600 for a panel of cytokeratins (CK) and PD-L1 in CTC negative, NSCLC donor specimens (n=8). Results were compared to CTC positive, contrived specimens (whole blood spiked with breast tumor cell line MCF7 cells; n=5). For ctDNA analysis of the plasma fraction, a multiplexed ddPCR assay was designed to test for five actionable mutations prevalent in NSCLC, including EGFR del19, L858R, T790M, KRAS G12C, BRAF V600E, and wildtype EGFR. To evaluate performance of the multiplexed assay, concordance evaluation was conducted using cfDNA from 8 mutation-positive NSCLC donors on the QX600 and compared to single assays run on the QX200 system. Results: CK8 and CK19 were differentially expressed in the cells enriched from the CTC negative NSCLC cohort as compared to the contrived positives (p=0.0058, and p=0.0049, respectively). All the NSCLC cfDNA samples were positive for the reference variant, and linear regression analysis of the minor variant frequency for ctDNA biomarkers showed good concordance between QX systems (R2=0.8847). Conclusions: These studies demonstrate proof of concept for single cell and cell-free analysis from a single blood draw into a cfDNA BCT using the QX600 system. Liquid biopsy analysis of CTCs and ctDNA can enable minimally invasive, comprehensive decision making in the diagnostic workup of patients. Future studies will focus on further optimizing the cell enrichment methodology from cfDNA BCT and development of additional multiplexed ddPCR assays for NSCLC-relevant biomarkers on the QX600. Citation Format: Leisa Jackson, Colin Cochran, Claire Gould, Sarah Kreston, Jazmin Bello, Brittany D’Alessio, Janice Riley, Alisa Tubbs, Gary A. Pestano. Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3706.

Evaluation of Nanoparticles Covalently Bound with BODIPY for Their Photodynamic Therapy Applicability.

Photodynamic therapy (PDT) relies on the combined action of a photosensitizer (PS), light at an appropriate wavelength, and oxygen, to produce reactive oxygen species (ROS) that lead to cell death. However, this therapeutic modality presents some limitations, such as the poor water solubility of PSs and their limited selectivity. To overcome these problems, research has exploited nanoparticles (NPs). This project aimed to synthesize a PS, belonging to the BODIPY family, covalently link it to two NPs that differ in their lipophilic character, and then evaluate their photodynamic activity on SKOV3 and MCF7 tumor cell lines. Physicochemical analyses demonstrated that both NPs are suitable for PDT, as they are resistant to photobleaching and have good singlet oxygen (1O2) production. In vitro biological analyses showed that BODIPY has greater photodynamic activity in the free form than its NP-bounded counterpart, probably due to greater cellular uptake. To evaluate the main mechanisms involved in PDT-induced cell death, flow cytometric analyses were performed and showed that free BODIPY mainly induced necrosis, while once bound to NP, it seemed to prefer apoptosis. A scratch wound healing test indicated that all compounds partially inhibited cellular migration of SKOV3 cells.

Assessment of bioactive peptides derived from laminin-111 as prospective breast cancer-targeting agents

Breast cancer remains a pressing public health issue primarily affecting women. Recent research has spotlighted bioactive peptides derived from laminin-111, implicated in breast tumor development. Remarkably, the sequences IKVAV, YIGSR, and KAFDITYVRLKF from the α1, β1, and γ1 chains, respectively, have garnered significant attention. This study aims to assess the potential of these radiolabeled peptides as targeting agents for breast cancer. The three peptides were synthesized using the Fmoc strategy, purified via reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized through mass spectrometry. Iodine-131 (131I) radiolabeling was performed using the chloramine T method, exhibiting high radiochemical yield and stability for [131I]I-YIKVAV and [131I]I-YIGSR. Conversely, [131I]I-KAFDITYVRLKF demonstrated low radiochemical yield and stability and was excluded from the biological studies. The lipophilicity of the compounds ranged from − 2.12 to − 1.10. Serum protein binding assay for [131I]I-YIKVAV and [131I]I-YIGSR reached ≅ 48% and ≅ 25%, respectively. Affinity for breast cancer cells was evaluated using MDA-MB-231 and MCF-7 tumor cell lines, indicating the affinity of the radiopeptides with these tumor cells. Ex vivo biodistribution profiles of the radiopeptides were assessed in the MDA-MB-231 breast tumor animal model, revealing tumor tissue accumulation, supported by a high tumor-to-contralateral muscle ratio and autoradiography. These results signify the effective penetration of YIKVAV and YIGSR into tumor tissue. Therefore, the synthesized α1 and β1 peptide fragments exhibit favorable characteristics as potential breast cancer-targeting agents, promising future exploration as radiopharmaceuticals for breast cancer.

Styryl carbamate backbones for the discovery of TME-disrupting agents

Twenty-one styryl carbamates have been designed, synthetized and characterized by their physical properties. Afterwards, their potential activity as tumor microenvironment disruptors has been evaluated. First, antiproliferative studies on tumor cell lines HT-29, A-549 and MCF-7 and on non-tumor cell line HEK-293 were carried out. Then, the antiproliferative activity of some selected compounds was evaluated on co-cultures of A-549 and immune cells, Jurkat T or monocytes THP-1. To determine their potential as oncoinflammation regulators, the secretion levels of IL-6, TNF-α and SAA-1 were, also, determined from these co-culture assays. Then, the effect on the expression levels of anticancer targets PD-L1 in both A-549 and monocytes THP-1 were established and, finally, the expression of CD80 and CD11b in THP-1. Compounds 9–13 bearing halophenyl carbamate units exhibited promising results as TME disruptors and inflammation regulators.

SYNTHESIS AND EVALUATION OF THE CYTOTOXICITY OF FURANOLABDANOIDS CONTAINING A 3-AMINOPROPANOYL SUBSTITUENT IN THE FURAN RING

15-Acetyl- and 16-acetylfuranolabdanoids were synthesised by the acetylation of the methyl ester of lambertianic acid with acetic anhydride in the presence of Mg(ClO4)2 and used as starting compounds for the preparation of 15- or 16-(3-aminopropanoyl)methyllambertianates. The proposed approach involved silylation of 15-acetyl- or 16-acetylmethyllambertianate and the Mannich reaction of the resulting enol silyl ester with N,N-disubstituted methyleneiminium salts. The synthesized compounds possessed cytotoxicity on tumour cell lines CCRF CEM, MCF7 and PC-3 (MTT test). The compounds containing a 3-morpholinopropanoyl or 3-pyrrolidinopropanoyl substituent at the C-16 position of methyl lambertianate selectively inhibited the growth of human T-cell leukemia cells (GI50 was 5.8-6.1 μM) and exhibited cytotoxicity an order of magnitude higher than the parent compound lambertianic acid.

Identification of novel sulfathiazole-triazolo-chalcone hybrids as VEGFR-2/EGFR dual inhibitors with antiangiogenic activity and apoptotic induction.

Certain sulfathiazole-triazolo chalcone hybrids were identified as anticancer agents with dual vascular endothelial growth factor receptor-2 (VEGFR-2)/epidermal growth factor receptor (EGFR) kinase inhibitory effect. All of the compounds were evaluated for their cytotoxic activity against the MCF-7 and HepG-2 tumor cell lines. Compounds 11g, 11h, and 11j exhibited the most potent antiproliferative activity against both cancer cell lines, with good safety toward WI-38 normal cells. Thus, they were further assessed for VEGFR-2 inhibitory activity. They have suppressed VEGFR-2 enzyme at IC50 of 0.316, 0.076, and 0.189 µM, respectively in comparison to sorafenib (IC50 = 0.035 µM). EGFR enzyme inhibition was further screened for the most potent inhibitors, 11h and 11j, where they displayed enhanced potency with IC50 of 0.085 and 0.108 µM, respectively, compared to erlotinib (IC50 = 0.037 µM). Compounds 11h and 11j were additionally investigated for inhibition of comparable kinases, PDGFR-β and B-Raf, where results assessed adequate selectivity of both compounds toward the VEGFR-2 and EGFR kinases. Furthermore, the wound healing assay of compound 11h manifested a percent wound closure of 65.18% in MCF-7 cells compared to doxorubicin (58.51%) and untreated cells (97.77%), proving its antiangiogenic activity. The cell cycle assay of MCF-7 cells treated with 11h demonstrated cell cycle arrest at the S phase. Moreover, compound 11h induced apoptosis with a 44-fold increase compared to that induced in the control MCF-7 cells. Molecular docking results of compounds 11h and 11j established their efficacies, and in silico studies showed convenient safety profiles with drug-likeness properties.

Abstract A010: Assay development to assess the efficiency and stability of candidate molecules for transcriptional gene silencing of FOXP3, using a human tumor-derived cell line

Abstract Regulatory T cells play an important role to modulate the balance between immunotolerance and immunosurveillance. These cells have the property of inhibiting effector lymphocytes and may antagonize antitumor immunity. Regulatory T cells are originated in the thymus, or even converted from peripheral lymphocytes by factors produced in the tumor microenvironment. Clinical data suggests that regulatory T cell infiltration correlates with poor prognosis in the treatment of solid tumors. The FOXP3 transcription factor is considered a master key to control the phenotype of regulatory T cells. It was previously shown, that the ectopic expression of FOXP3 can induce an immunosuppressive phenotype in lymphocytes. In contrast, it has been observed that mutations in the FOXP3 gene can cause impaired immunosuppressive activity mediated by regulatory T cells, such as the autoimmunity syndrome known as IPEX. In this sense, the FOXP3 transcription factor may be an interesting target, looking for inactivation of the immunosuppressive phenotype of regulatory T cells, to potentiate antitumor response. In this work, we present an assay development to investigate the potential of transcriptional interference RNA candidates to silence FOXP3 expression. In contrast to post-transcriptional gene silencing method, which targets messenger RNA and depends on a constant supply or efficient turn-over of the inhibitory molecule, the transcriptional gene silencing targets the cellular DNA genome, inducing epigenetic changes that may control the target gene transcription. We employed the human MCF-7 tumor cell line, which has endogenous and constitutive expression of FOXP3 as a target model to test transcriptional gene silencing candidates. This cell line is transduced with lentiviral vectors harboring interfering RNA sequences driven to the FOXP3 promoter region. The RNA interference candidates are then evaluated for their ability to induce DNA methylation, by bisulfite method, and transcriptional gene silencing of FOXP3, by qPCR. We performed the screening of candidates for FOXP3 transcriptional silencing, in comparison to a post-transcriptional interference RNA control. The lentiviral transduced cells are easily expanded and allow a temporal analysis of target gene expression. In this model, we observed the possibility of finding transcriptional interference RNA candidates that exhibited high efficiency and stability, that may be used for research purposes, or even for the investigation of new therapeutic possibilities in immuno-oncology. Citation Format: Carolinne T Fogagnolo, Daniela S Mizobuti, Marcio C Bajgelman. Assay development to assess the efficiency and stability of candidate molecules for transcriptional gene silencing of FOXP3, using a human tumor-derived cell line [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A010.

Synthesis and cytotoxic activity of some (+)-salsolidine derivatives

On the basis of typical for secondary amino group reactions a number of derivatives of alkaloid (+)-salsolidine was synthesised. Cytotoxic properties of obtained compounds towards the HEK293, A549, MCF-7 and SH-SY5Y cell lines have been evaluated. As a result of the screening, the hit compound − 2-(chloroacetyl)-6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (11) was identified, that inhibited the metabolic activity of A-549, MCF-7 and SH-SY5Y tumour cell lines with the IC50 values of 3.83 ± 0.78 µM, 5.84 ± 1.62 µM and 2.89 ± 0,92 µM correspondingly. Based on the effect of 11 on the cell cycle progression and the molecular docking data, it was preliminary assumed that the cytotoxic activity of the 11 can be realised through its interaction with the active site of the cyclin-dependent kinase CDK9 (PDB code 3BLR).