Human Breast Cancer Cells Research Articles

Bioactivity of biogenic silver/silver chloride nanoparticles from Maranta arundinacea rhizome extract: Antibacterial and antioxidant properties with anticancer potential against Ehrlich ascites carcinoma and human breast cancer cell lines

IntroductionThis study explores the synthesis and characterization of silver/silver chloride nanoparticles (Ag/AgCl-NPs) using Maranta arundinacea rhizome extract and evaluates their bioactivities, including antibacterial, antioxidant, and anticancer potentials. MethodsThe synthesis of Ag/AgCl-NPs was initially confirmed by a color change and a sharp peak at 463 nm in UV–visible spectroscopy. Further characterization was conducted using scanning electron microscopy (SEM), X-ray powder diffraction (XRD), and fourier transform infrared spectroscopy (FTIR). Antibacterial properties were checked against four pathogenic bacteria (Shigella boydii, Escherichia coli, Shigella dysenteriae, and Staphylococcus aureus), and antioxidant activities were assessed using DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) assay. In addition, the anticancer potential was evaluated in vitro using MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) colorimetric assay and in vivo using the mouse models. Finally, toxicity was determined by employing the brine shrimp nauplii lethality assay. ResultsAg/AgCl-NPs most effectively inhibited the growth of Staphylococcus aureus, showing maximum zone of inhibition and 7 μg/mL of minimum inhibitory concentration (MIC), and prevented the biofilm formation by Escherichia coli at 40 μg/mL. They displayed antioxidant activities against DPPH and ABTS with IC50 values of 90.65 and 24.34 μg/mL, respectively. In vitro, they inhibited 61.96 % EAC and 49.63 % MCF-7 cells growth at 32 and 128 μg/mL, respectively. Subsequently, inhibition rates of EAC cells growth in mice were measured as 38.30 %, 57.38 %, and 31.81 % after employing 2.5, 5, and 10 mg/kg/day of Ag/AgCl-NPs, respectively. Moreover, Ag/AgCl-NPs treated mice were found to carry more apoptotic EAC cells with distorted morphology. Treated mice showed decreased tumor weight, increased mean survival time, and a lifespan increase of up to 30 %, with improved hematological parameters. Later, Ag/AgCl-NPs exhibited moderate toxicity with an LC50 value of 208.41 μg/mL in brine shrimp nauplii lethality assay. ConclusionThe promising antibacterial, antioxidant, and anticancer activities along with mild toxicity suggest the potential biomedical uses of Maranta arundinacea rhizome extract-mediated Ag/AgCl-NPs.

Iron and siRNA co-encapsulated ferritin nanocages induce ferroptosis synergistically for cancer therapy

Ferroptosis has received great attention as an iron-dependent programmed cell death for efficient cancer therapy. However, with the accumulation of iron in tumor cells, the antioxidant system is activated by reducing glutathione (GSH) with glutathione peroxidase 4 (GPX4), which critically limits the ferroptosis therapeutic effect. Herein, an iron and GPX4 silencing siRNA (siGPX4) co-encapsulated ferritin nanocage (HFn@Fe/siGPX4) was developed to enhance ferroptosis by disruption of redox homeostasis and inhibition of antioxidant enzyme synergistically. The siGPX4 were loaded into the nanocages by pre-incubated with iron, which could significantly improve the loading efficiency of the gene drugs when compared with the reported gene drug loading strategy by ferritin nanocages. And more iron was overloaded into the ferritin through the diffusion method. When HFn@Fe/siGPX4 was taken up by human breast cancer cell MCF-7 in a TfR1-mediated pathway, the excess iron ions in the drug delivery system could for one thing induce ferroptosis by the production of reactive oxygen species (ROS), for another promote siGPX4 escaping from the lysosome to exert gene silencing effect more effectively. Both the in vitro and in vivo results demonstrated that HFn@Fe/siGPX4 could significantly inhibit tumor growth by synergistical ferroptosis. Thus, the developed HFn@Fe/siGPX4 afforded a combined ferroptosis strategy for ferroptosis-based antitumor as well as a novel and efficient gene drug delivery system.

Isoliquiritigenin Suppresses Breast Tumor Development by Enhancing Host Antitumor Immunity.

Isoliquiritigen (ISL), a constituent of licorice, has been shown to possess antitumorigenic effects in diverse cancer types. In this study, we observed that ISL suppressed breast tumor development significantly more effectively in immunocompetent mice than in immunocompromised ones. In exploring the cause of such a discrepancy, we detected robust tumor infiltration of CD8[Formula: see text] T lymphocytes in mice treated with ISL, not seen in tumors derived from vehicle-treated mice. Moreover, we found a dramatic reduction in PD-L1 in both experimental breast tumors and cultured breast cancer cells upon ISL treatment. In further experiments, we showed that ISL selectively elevated miR-200c in breast cancer and confirmed that PD-L1 mRNA is the target of miR-200c in both murine and human breast cancer cells. ISL suppression of PD-L1 was functionally linked to miR-200c/ZEB1/2 because (1) ISL diminished ZEB1/2; (2) knockdown of ZEB1/2 led to the disappearance of PD-L1; and (3) miR-200c antagomiR disabled ISL to reduce PD-L1. We found evidence that ISL reduced the level of PD-L1 by simultaneously intercepting the ERK and Src signaling pathways. In agreement with clinical finding that PD-L1 antibodies enhance efficacy of taxane-based therapy, we showed that ISL improved the tumoricidal effects of paclitaxel in an orthopedic murine breast tumor model. This study demonstrates that ISL-led tumor suppression acts through the augmentation of host antitumor immunity.

Honokiol Exhibits Anti-Tumor Effects in Breast Cancer by Modulating the miR-148a-5p-CYP1B1 Axis.

Breast cancer (BC) is the most frequently diagnosed malignancy in female patients. There is a significant lack of therapeutic strategies for BC, particularly triple-negative breast cancer (TNBC). Honokiol (HNK), a lignin extracted from the Magnolia genus plant, has demonstrated numerous pharmacological effects. Therefore, this study aims to investigate the antitumor effect of HNK on BC cells and employ high-throughput sequencing to elucidate its potential mechanism. We found that HNK significantly inhibited proliferation and induced apoptosis on BC cell lines in a dose-dependent manner. Moreover, HNK treatment suppressed migration and colony formation and initiated the intrinsic apoptotic pathway specifically in MDA-MB-231 cells. High-throughput sequencing and bioinformatics analysis revealed that miR-148a-5p expression was significantly up-regulated, whereas CYP1B1 expression was down-regulated following HNK treatment. Importantly, survival analysis based on TCGA database showed high miR-148a-5p expression was correlated with a better prognosis for BC patients. Inhibition of miR-148a-5p by inhibitor not only increased cell viability but also attenuated apoptosis induced by HNK.Finally, a strong synergistic effect between HNK and paclitaxel was observed in vitro. In conclusion, our study validated the antitumor efficacy of HNK against human BC cells and elucidated its underlying mechanism through high-throughput sequencing, thereby providing compelling evidence for further exploration of the potential clinical application of HNK towards the treatment of BC.

Oral delivery of Sunitinib malate using carboxymethyl cellulose/poly(acrylic acid-itaconic acid)/Cloisite 30B nanocomposite hydrogel as a pH-responsive carrier.

This work aimed to fabricate a Cloisite 30B-incorporated carboxymethyl cellulose graft copolymer of acrylic acid and itaconic acid hydrogel (Hyd) via a free radical polymerization method for controlled release of Sunitinib malate anticancer drug. The synthesized samples were characterized by FTIR, XRD, TEM, and SEM-dot mapping analyses. The encapsulation efficiency of Hyd and Hyd/Cloisite 30B (6 wt%) was 81 and 93%, respectively, showing the effectiveness of Cloisite 30B in drug loading. An in vitro drug release study showed that drug release from all samples in a buffer solution with pH 7.4 was higher than in a buffer solution with pH 5.5. During 240min, the cumulative drug release from Hyd/Cloisite 30B (94.97% at pH 7.4) is lower than Hyd (53.71% at pH 7.4). Also, drug-loaded Hyd/Cloisite 30B (6 wt%) demonstrated better antibacterial activity towards S. Aureus bacteria and E. Coli. High anticancer activity of Hyd/Cloisite 30B against MCF-7 human breast cancer cells was shown by the MTT assay, with a MCF-7 cell viability of 23.82 ± 1.23% after 72-hour incubation. Our results suggest that Hyd/Cloisite 30B could be used as a pH-controlled carrier to deliver anticancer Sunitinib malate.

Preliminary investigation on the mechanism of baicalein regulating the effects of Nischarin on invasion and apoptosis of human breast cancer cells MCF-7 through Wnt3α/β-catenin pathway

BackgroundBreast cancer (BC) remains the leading cause of cancer-related mortality in women. Here, we investigate the anti-tumor effects of baicalein on human BC cells (MCF-7 cells) and explore if it regulates the Nischarin protein via Wnt3α/β-catenin signaling pathway. MethodsWe employed Wnt3α and DKK-1 to activate and inhibit the Wnt/β-catenin signaling pathway, respectively. We used CCK-8 cell viability, flow cytometry apoptosis, wound-healing and transwell migration/invasion assays. Further, using western blotting and real-time quantitative PCR (q-PCR) we analyzed expression levels of Nischarin, MMP-9, Wnt/β-catenin pathway (β-catenin, Axin 1), and apoptotic pathway (Bax, Bcl-2) proteins and their mRNAs. ResultsWe found that baicalein inhibits MCF-7 cell viability and promotes apoptosis (evidenced by increased Bax and decreased Bcl-2 expressions) in a concentration-dependent manner. It also inhibits TPA-induced migration and invasion, and downregulates MMP-9 expression. Baicalein reverses the increase in cell viability caused by Wnt3α-induced Wnt/β-catenin pathway activation. Conversely, baicalein counteracts the increase in apoptosis caused by DKK-1 mediated inhibition of the Wnt/β-catenin pathway. Additionally, baicalein upregulates Nischarin expression via modulating the Wnt/β-catenin pathway as indicated by the antagonistic effects of Wnt3α and DKK-1 on this effect of baicalein. ConclusionBaicalein exerts anti-tumor effects on MCF-7 cells through the Wnt3α/β-catenin signaling pathway, and promotes apoptosis and inhibits migration and invasion. The upregulation of Nischarin by baicalein further suggests a potential therapeutic target for BC treatment.

Effects of Pervari Honey from Türkiye on Proliferation, Oxidative Stress, and Apoptosis of Human Breast Cancer Cells

Breast cancer is one of the most common causes of deaths worldwide. Major obstacles to treatment have contributed to the increasing popularity of complementary or alternative therapies. Although recent results support the mechanism that honey induces cell death, the full mechanisms are still unknown. This study investigates the potential use of Pervari honey (PH) as an in vitro therapeutic agent in breast cancer. Firstly, the antioxidant capacity and total phenolic content of PH were tested. In addition, MCF-7 and MDA-MB-231 breast cancer cells treated with PH were examined for cell viability, reactive oxygen species production, oxidative deoxyribonucleic acid (DNA) damage, and apoptosis. Our results show that PH treatment decreased cell viability dose-dependently and increased reactive oxygen species (ROS) levels, oxidative DNA damage, and apoptosis rate. The present study suggests that honey is a promising source to produce pharmaceuticals and nutraceuticals for breast cancer therapy.

Significant Impact of let-7d MicroRNA on Breast Cancer Cell Lines Post-Radiation Treatment

Introduction: Radiotherapy is a common treatment for breast cancer treatment, that induces DNA damage. These DNA damages are addressed through various repair pathways, which regulate the DNA repair systems and confer radio-resistance. Non-coding RNAs are a big proportion of genome transcripts without the potential to encode proteins. Related studies demonstrated that radiation affects the expression of non-coding RNAs. Let-7d, a tumor suppressor in numerous cancers, has some target genes that play a role in the DNA repair system. Materials and Methods: Human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in a Dulbecco's Modified Eagle Medium. The exponentially growing cells were treated with some doses of X-rays. After radiation treatment and cell harvesting, RNA was extracted, and cDNA synthesis was done. The let-7d miRNA expression changes were calculated with real-time quantitative reverse transcription polymerase chain reaction. Results: The results implied that radiation caused increased let-7d expression in breast cancer cell lines after radiation treatment. In addition, the results showed that 24 h after radiation, the expression of let-7d in the radioresistant cell line was higher than the radiosensitive one; 48 h after radiation, the expression of let-7d in the radiosensitive cell line was higher than the other one. Conclusions: The results demonstrated that radiation treatment increased let-7d miRNA expression in both radiosensitive and radio-resistant breast cancer cell lines. Therefore, let-7d might be involved in the radiosensitivity of breast cancer.

P66Shc Protein—Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer

This work presents a comprehensive evaluation of the role of p66Shc protein in mitochondrial physiology in MDA-MB-231 breast cancer cells. The use of human breast cancer cell line MDA-MB-231 and its genetically modified clones (obtained with the use of the CRISPR-Cas9 technique), expressing different levels of p66Shc protein, allowed us to demonstrate how the p66Shc protein affects mitochondrial metabolism of human breast cancer cells. Changes in the level of p66Shc (its overexpression, and overexpressing of its Serine 36-mutated version, as well as the knockout of p66Shc) exert different effects in breast cancer cells. Interestingly, knocking out p66Shc caused significant changes observed mostly in mitochondrial bioenergetic parameters. We have shown that an MDA-MB-231 (which is a strong metastatic type of breast cancer) clone lacking p66Shc protein is characterized by a significant shift in the metabolic phenotype in comparison to other MDA-MB-231 clones. Additionally, this clone is significantly more vulnerable to doxorubicin treatment. We have proved that p66Shc adaptor protein in human breast cancer cells may exert a different role than in noncancerous cells (e.g., fibroblasts).

Styphnolobium japonicum Fruit and Germinated Soybean Embryo Complex Extract for Postmenopausal-Symptom Relief.

Hormonal alterations during menopause result in substantial physiological changes. Although hormone replacement therapy (HRT) is widely used as a treatment strategy for these changes, its use remains controversial due to its associated risks. Plant isoflavones are phytoestrogens that are considered a potential alternative therapy for postmenopausal syndrome. We aimed to investigate the efficacy of ethanolic extracts from Styphnolobium japonicum fruit (SJF) and germinated soybean embryo (GSE) in alleviating prominent menopausal symptoms. A cell model (MCF7 human breast cancer cells) was used to investigate estrogen-like activity. A rat ovariectomy model was used to simulate estrogen depletion after menopause and to evaluate the efficacy of the SJF-GSE complex extract at ratios of 1:1, 1:2, and 2:1. Treatment with the SJF-GSE extract elicited estrogen-like effects, raising pS2 and estrogen receptor α expression in MCF7 cells. The extract was found to contain 48-72 mg/g sophoricoside and 8-12 mg/g soyasaponin 1, identified as active compounds. In ovariectomized rats, the extract effectively reduced body weight and fat content, alleviated vasomotor symptoms, improved vaginal mucosal health, and exerted osteoprotective effects by enhancing bone density and structure, reducing bone-resorption markers and positively altering estradiol levels and lipid profiles. The SJF-GSE extract, working synergistically, provides a safe and effective alternative to HRT for managing postmenopausal symptoms and enhancing bone health, without adverse effects. These findings support the inclusion of SJF and GSE in health-functional foods and underscore the importance of further research into plant-based therapies for menopause.

Evaluation of the performance of Fe3O4/MnO2@doxorubicin hybrid nanozymes on multicellular structure and their therapeutic management to limit the growth of human breast cancer cells

Doxorubicin (DOX) is the most common treatment for breast cancer, but its effectiveness is limited by drug resistance and dose variability. Evidence suggests that nanozymes can significantly improve drug penetration and effectiveness in breast cancer treatment, owing to their stable and targeted catalytic properties. However, their varied responses and concentration-dependent toxicities present challenges. After developing pH-sensitive Fe3O4/MnO2@DOX hybrid nanozymes (FMDHN) and evaluating their physicochemical and functional properties, their efficacy was investigated on MCF-7 cells using both two-dimensional and spheroid models. Our findings reveal that FMDHNs, sized 150–270 nm, inhibit MCF-7 cell growth through drug release triggered by acidity and photothermal therapy (PTT). The catalytic efficiency of FMHN in generating O2 and ·OH further enhances cancer cell suppression. Doubling the effective concentrations of FMHN and DOX by transitioning from two-dimensional to spheroid cell structures could adversely affect normal cells, while a synergistic approach combining the DOX and FMHN effectively inhibits MCF-7 cell growth at non-toxic dose. Combining FMDHN with PTT enhances this inhibition, lowering the effective dose to 1.08 μg/mL and effectively managing toxicity. The cytotoxicity mechanism in MCF-7 spheroids shows that PTT with FMDHN significantly elevates pro-inflammatory cytokines, including TNF-α, CASP9, CASP7 and CASP3. Optimizing the concentration of pH-sensitive nanozymes based on their synergistic effects can minimize side effects and maximize their breast cancer treatment potential.Graphical

Investigating the Size Effect of Metal-Organic Frameworks in Drug Delivery and Anticancer Properties.

Here, we show particle size-dependent therapeutic efficacy with a Zn-based metal-organic framework (MOF). The size of MOFs was tuned in specific ranges (∼100, 200, and 300 nm) built upon the manipulation of synthetic conditions. X-ray photoelectron spectroscopy, infrared, PXRD, and dynamic light scattering and scanning electron microscopy analyses were used to identify the synthesized structures. The various analyses revealed minimal changes in the molecular properties of these structures regardless of their size, confirming our hypothesis regarding the preservation of the identity of MOF nanoparticles despite size variation. The synthesized carriers undergo structure relative destruction in response to a weak acidic tumor microenvironment, and this relative degradation allows the release of the Nimesulide drug into the environment. Interestingly, anticancer studies resulting in SKBR3 (Human breast cancer cell) cells indicate that the different sizes resulted in various inhibition capacities against cancer cells. This work shows the importance of optimizing the geometry of the drug carrier, such as size and shape, to achieve the highest cellular uptake and therapeutic performance. Besides, theoretical studies were carried out using B3LYP/6-31G (d,p) and density functional theory methods to more consider the drug adsorption mechanism.

Quantitative Determination of the Cytotoxic Compounds in Different Organs of Arctium minus (Hill) Bernh. by LC-HRESIMS Using Respond Survey Methodology.

Arctium minus (Hill) Bernh., commonly known as "Burdock", is a species within the Arctium genus of the Asteraceae family. Determining the optimum extraction conditions to obtain a concentrated extract with targeted active ingredients guides the most efficient use of natural products. Herein, ultrasound-assisted extraction (UAE) was optimized by using response surface methodology (RSM) to extract bioactive compounds from different organs of A. minus. Furthermore, phytochemical composition of extracts of the A. minus was investigated by using liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS), antioxidant potential by using 2,2-diphenyl-1-picrylhydrazyl (DPPH), diazanium;3-ethyl-2-[(3-ethyl-6-sulfonato-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonate (ABTS), CUPRAC, and metal chelation assays, and cytotoxic activities by using human breast cancer cell line (MDA-MB-231) and hepatocellular carcinoma cancer cell line (HepG2), and compared against conventional methods; Soxhlet and maceration. The RSM was employed to investigate the influence of ultrasound power, extraction time, and extraction temperature on the antioxidant potential assessed by the DPPH free radical scavenging assay. In UAE of A. minus leaves, flowers, and branches, the conditions resulting in the minimum IC50 values: 20 °C for 6 min at 50 W for leaves, 20 °C for 3 min at 100 W for flowers, and 20 °C for 3 min at 100 W for branches. Chlorogenic acid was identified as the major phenolic compound in the extracts obtained by UAE, with concentrations of 24,666.96 μg/g in leaves, 1054.92 μg/g in flowers, and 3,501.24 μg/g in branches. Flowers of A. minus had significantly higher levels of arctiin and arctigenin than those of leaves and branches. Extracts from leaves and flowers were more effective against MDA-MB-231 and HepG2 cancer cell lines than arctiin and arctigenin.

Potent drug candidature of copper-coordinated metallodrug: Experimental study, spectroscopic insights, anticancer activity, apoptosis analysis, cell cycle, theoretical calculations, and molecular docking studies

The biological properties of the thiocarbohydrazone, pyrazole, and copper ions are well-known, thus copper complexes incorporated with pyrazole-thiocarbohydrazone hybrid ligands have potential medical applications. In this study, copper complex [Cu(PMT)2(Cl)2] derived from pyrazole-thiocarbohydrazone hybrid chelator (PMT) was successfully synthesized. With the help of several fundamental techniques, including elemental and thermal studies, molar conductivity, and magnetic moment measurements, the structure and coordination behavior of the PMT chelator and its Cu(PMT) nanocomplex were examined. In addition, records of ESR, FT-IR, and UV–Vis spectroscopy were obtained. The powder X-ray diffraction data has identified significant information about the crystalline forms of the Cu(PMT) chelate. The findings demonstrated that producing octahedral, mono-species of chelate and chelation occurs through the N, and S donor sites of neutral chelator. The cytotoxicity assessment of the PMT and its Cu(PMT) chelate was further evaluated against human ovary cancer cells (SKOV3), human breast cancer cells (MCF-7), and human colon cancer cells (HCT116) by using SRB assay. The Cu(PMT) chelate displayed promising results regarding standard doxorubicin. Furthermore, the apoptosis analysis of the Cu(PMT) chelate strongly induced the late-apoptotic cell population against the tested cancer cells. Also, the cell-cycle distribution of the Cu(PMT) complex arrests cell proliferation at the G1, S, and G2 phases. PMT and its Cu(PMT) were also studied using computational simulations and the DFT approach. Different chemical quantum parameters were determined from the optimized structure. The interaction between copper chelate and the CDK8 kinase active site was examined using docking studies.

Abstract C089: Characterization of the breast tumor immune microenvironment in women of African descent using single-nucleus RNA- and ATAC-sequencing

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor (TF) activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus (sn) ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=33), Kenyan (n=25), and European American (EA, n=24) women in relation to genetic ancestry, risk factor exposures, clinical characteristics, and 5-year survival. To achieve this, we successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue through an optimized combination of enzymatic digestion and automated tissue homogenization. We performed snMultiome sequencing of 82 tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Harmony), our dataset includes a total of 296,557 nuclei. Cancerous (163,419 nuclei) and non-cancerous (133,138 nuclei) cells were distinguished based on DNA copy number (CopyKat). Within the microenvironment, 11 major immune, epithelial, and stromal cell types were successfully annotated, exhibiting distinct patterns by population group (e.g. AA tumors showed markedly increased abundance of myeloid and T-cells, while Kenyan tumors showed increased abundance of pericytes and fibroblasts, relative to EA). A large number of enriched TFs within each cell type varied significantly by population group, suggesting distinct chromatin accessibility patterns related to genetic ancestry. CD45+, EPCAM- cells were further extracted, clustered, and manually annotated for subpopulations based on known marker genes. We detected 26 distinct immune subpopulations across our samples, including 2 inflammatory macrophage, 6 immunosuppressive macrophage, 1 fibronectin+/thrombospondin+ monocyte, 6 T-cell, 1 natural killer cell, 2 B-cell, 1 mast cell, and 4 dendritic cell subpopulations. Differential gene expression analyses of each immune cell subpopulation, adjusted for age, BMI, and tumor subtype, revealed ancestry-specific gene expression patterns, which could provide novel insights into functional differences that may impact tumor immune response. Current efforts focus on in-depth molecular characterization of ancestry-related differences in the tumor immune environment and distinct signatures present in lethal disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany D. Jenkins, Tiffany H. Dorsey, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Characterization of the breast tumor immune microenvironment in women of African descent using single-nucleus RNA- and ATAC-sequencing [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C089.

DDX5-Targeting Fragile X Mental Retardation Protein Regulates the Wnt/β-catenin Signaling Pathway to Promote Epithelial Mesenchymal Transition in Breast Cancer

To investigate the role of fragile X mental retardation protein (FMRP) in promoting cell migration and epithelial-mesenchymal transition (EMT) in breast cancer (BC) and the potential mechanisms involved. The mRNA and protein expressions of FMRP in MCF-10A, a normal human breast epithelial cell line, and four breast cancer cell lines, including MCF-7, BT474, MDA-MB-231, and HCC1937, were analyzed by RT-PCR and Western blot. The expression of FMRP in BC tissues was measured by immunohistochemistry (IHC). FMRP expression in BC and its relationship with clinical prognosis were analyzed using GEO database. Lentiviral infection and siRNA interference were used to construct FMRP overexpression and interference vectors, respectively, and the human breast cancer cell line MCF-7 was subsequently transfected. A Control group, an interference empty vector group (the NC group), a knockdown vector group (the siFMRP group), an overexpression empty vector group (the Lv-NC group), and an overexpression vector group (the Lv-FMRP group) were set up. The migration and invasion abilities of the cells were assessed by scratch assay and Transwell assay. The expression of EMT markers, including E-cadherin, an epithelial marker, N-cadherin, an mesenchymal markers, vimentin, zinc finger E-box binding homeobox 1 (ZEB1), and snail family zinc finger 2 (Slug), in the cells of each group was determined by Western blot. The interaction between FMRP and DEAD-box RNA helicase-5 (DDX5) protein was analyzed by immunocoprecipitation combined with mass spectrometry (IP-MS). The regulatory effect of FMRP on DDX5 protein expression was assessed using the protein synthesis inhibitor cycloheximide (CHX) and proteasome inhibitor MG132. In addition, transfection with siDDX5 vector was conducted to observe whether DDX5 could reverse the effects of FMRP overexpression on cell migration and EMT. The localization and expression of β-catenin were determined by immunofluorescence staining, and the expression of core markers of Wnt/β-catenin signaling pathway was examined by Western blot. FMRP was highly expressed in BC tissues and cells (P<0.05), and overall survival (OS) and recurrence-free survival (RFS) of the FMRP high expression group were significantly lower than those of the FMRP low expression group (P<0.05). The migration ability of MCF-7 cells was weakened after FMRP knockdown, while overexpression of FMRP promoted cell migration (P<0.05). After FMRP knockdown, the expression of E-cadherin was increased, while the expression levels of N-cadherin, vimentin, ZEB1, and Slug were decreased, which inhibited the occurrence of EMT. In contrast, the overexpression of FMRP promoted the EMT process (P<0.05). FMRP interacted with DDX5 protein and promoted DDX5 protein stability by blocking the ubiquitin-proteasome pathway. DDX5 knockdown reversed the effect of FMRP overexpression to promote cell migration and EMT (P<0.05), effectively inhibited β-catenin nuclear translocation, and decreased β-catenin nuclear distribution. Furthermore, it was found that the expression of p-β-catenin, GSK3β and Axin2 protein was increased and the expression of C-myc protein was decreased after DDX5 downregulation (P<0.05). On the other hand, the expression of these proteins was reversed by combined FMRP overexpression (P<0.05). FMRP targets DDX5 and promotes BC cell migration and EMT via the activation of the Wnt/β-catenin signaling pathway.

Cover Image

On the Cover: The diagram of cellular localization of AMOTL2 in different cell lines MDCK: Madin‐Darby canine kidney cells; HeLa: human cervical cancer cells; MS‐1: MileSven1 pancreatic islet endothelial cells; MTD1A: mouse epithelial cells; MCF‐7: human breast cancer cells by Wang et al.; Article first published: 8 May 2024; https://doi.org/10.1002/iub.2830. image

Organocatalytic[3 + 2]Cycloaddition: Synthesis of Quinazoline Containing Sulfonyl 1,2,3‐Triazoles as Potent EGFR Targeting Anti‐Breast Cancer Agents

ABSTRACTA general strategy was developed for the synthesis of new fully decorated 1,2,3‐triazoles (4a–4m and 5a–5g) containing quinazolines from 1‐(4‐nitrophenyl)‐2‐(quinazolin‐8‐ylsulfonyl) ethan‐1‐one and several azides using Ramachary organocatalytic azide‐ketone cycloaddition method. This reaction is reported for the synthesis of fully substituted sulfonyl‐1,2,3‐triazolyl quinazolins at a temperature of 100°C and the yields of the products produced are satisfactory to excellent. In vitro anticancer activity of all these derivatives demonstrated that six compounds, 4d, 4f, 4i, 4j, 5d, and 5e, were effective against two human breast cancer cell lines, MCF‐7 and MDA‐MB‐231. Compounds 4f, 4j, and 5d had more action against both cell lines than Erlotinib. Later, the findings of inhibitory assays of potent compounds 4d, 4f, 4i, 4j, 5d, and 5e against the tyrosine kinase EGFR revealed that compound 5d proved more potent than the reference erlotinib, while 4f and 4j had comparable efficacy. In silico molecular docking studies were also performed on six strong medicines to identify interactions with the EGFR receptor, and the energy estimations were shown to be comparable with the observed IC50 values. Ultimately, using SWISS/ADME and pkCSM, the in silico pharmacokinetic profile of potent compounds 4d, 4f, 4i, 4j, 5d, and 5e was predicted. All of the compounds precisely followed the principles established by Lipinski, Veber, Egan, and Muegge.

Knock-out of CD73 delays the onset of HR-negative breast cancer by reprogramming lipid metabolism and is associated with increased tumor mutational burden

ObjectiveCD73 (ecto-5′-nucleotidase, NT5E), a cell-surface enzyme converting 5′-AMP to adenosine, is crucial for cancer progression. However, its role in the tumorigenesis process remains mostly obscure. We aimed to demonstrate CD73's role in breast cancer (BC) tumorigenesis through metabolic rewiring of fatty acid metabolism, a process recently indicated to be regulated by BC major prognostic markers, hormone receptors (HR) for estrogen (ER), and progesterone (PR). MethodsA murine model of chemically induced mammary gland tumorigenesis was applied to analyze CD73 knock-out (KO)-induced changes at the transcriptome (RNA-seq), proteome (IHC, WB), and lipidome (GC-EI-MS) levels. CD73 KO-induced changes were correlated with scRNA-seq and bulk RNA-seq data for human breast tissues and BCs from public collections and confirmed at the proteome level with IHC or WB analysis of BC tissue microarrays and cell lines. ResultsCD73 KO delayed the onset of HR/PR-negative mammary tumors in a murine model. This delay correlated with increased expression of genes related to biosynthesis and β-oxidation of fatty acids (FAs) in the CD73 KO group at the initiation stage. STRING analysis based on RNA-seq data indicated an interplay between CD73 KO, up-regulated expression of PR-coding gene, and DEGs involved in FA metabolism, with PPARγ, a main regulator of FA synthesis, as a main connective node. In epithelial cells of mammary glands, PPARγ expression correlated with CD73 at the RNA level. With cancer progression, CD73 KO increased the levels of PUFAn3/6 (polyunsaturated omega 3/6 FAs), known ligands of PPARγ and target for lipid peroxidation, which may lead to oxidative DNA damage. It correlated with the downregulation of genes involved in cellular stress response (Mlh1, Gsta3), PR–or CD73-dependent changes in the intracellular ROS levels and expression or activation of proteins involved in DNA repair or oxidative stress response in mammary tumor or human BC cell lines, increased tumor mutational burden (TMB) and genomic instability markers in CD73 low HR-negative human BCs, and the prolonged onset of tumors in the CD73 KO HR/PR-negative group. ConclusionsCD73 has a significant role in tumorigenesis driving the reprogramming of lipid metabolism through the regulatory loop with PR and PPARγ in epithelial cells of mammary glands. Low CD73 expression/CD73 KO might enhance mutational burden by disrupting this regulatory loop, delaying the onset of HR-negative tumors. Our results support combining therapy targeting the CD73-adenosine axis and tumor lipidome against HR-negative tumors, especially at their earliest developmental stage.

Encapsulation of Inositol Hexakisphosphate with Chitosan via Gelation to Facilitate Cellular Delivery and Programmed Cell Death in Human Breast Cancer Cells.

Inositol hexakisphosphate (InsP6) is the most abundant inositol polyphosphate both in plant and animal cells. Exogenous InsP6 is known to inhibit cell proliferation and induce apoptosis in cancerous cells. However, cellular entry of exogenous InsP6 is hindered due to the presence of highly negative charge on this molecule. Therefore, to enhance the cellular delivery of InsP6 in cancerous cells, InsP6 was encapsulated by chitosan (CS), a natural polysaccharide, via the ionic gelation method. Our hypothesis is that encapsulated InsP6 will enter the cell more efficiently to trigger its apoptotic effects. The incorporation of InsP6 into CS was optimized by varying the ratios of the two and confirmed by InsP6 analysis via polyacrylamide gel electrophoresis (PAGE) and atomic absorption spectrophotometry (AAS). The complex was further characterized by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) for physicochemical changes. The data indicated morphological changes and changes in the spectral properties of the complex upon encapsulation. The encapsulated InsP6 enters human breast cancer MCF-7 cells more efficiently than free InsP6 and triggers apoptosis via a mechanism involving the production of reactive oxygen species (ROS). This work has potential for developing cancer therapeutic applications utilizing natural compounds that are likely to overcome the severe toxic effects associated with synthetic chemotherapeutic drugs.