Activity In MCF-7 Cells Research Articles

Investigating the effect of hesperetin on estrogen receptor alpha (ERα) expression, phosphorylation and activity in MCF-7 cells

PurposeThe most common malignancy among women worldwide is breast cancer. The estrogen receptor plays a vital role in this cancer. One of the most well-known mechanisms that affects the activity of this receptor is its phosphorylation by protein kinase pathways. Hesperetin, a flavonoid abundant in citrus species such as lemons, grapefruits, and oranges, is the aglycone form of hesperidin. It has undergone thorough evaluation for its potential anti-cancer properties, particularly in the context of breast cancer. Studies have shown that hesperetin has an effect on intracellular kinase pathways. The aim of this study was to investigate the effect of hesperetin on the expression, phosphorylation and activity of estrogen receptor alpha (ERα) in MCF-7 breast cancer cell line. Study design and methodsMCF-7 cells were cultured in RPMI-1640 phenol red-free medium supplemented with charcoal-stripped FBS and treated with hesperetin. The MTT method was used to evaluate cell survival. The levels of the ERα protein and its phosphorylated form (Ser118) were determined via western blotting. A luciferase reporter vector was used to evaluate ERE activity. ResultsThe results of this study indicated that hesperetin reduced the survival of MCF-7 cells in a dose-dependent manner. The expression and phosphorylation (at Ser118) of the ERα significantly increased and decreased, respectively, in the groups treated with hesperetin. Hesperetin increased the activity of the ERα in the absence of E2, although these differences were not statistically significant. Conversely, in the presence of E2, hesperetin caused a significant decrease in receptor activity. ConclusionBased on the results of this study, it can be concluded that hesperetin has a significant effect on ERα expression, phosphorylation and activity.

Evaluation of antiproliferative effect of doxorubicin loaded zinc selenium quantum dots to MCF-7 cell lines by linagliptin functionalized lignin nanoparticles

In this research study, biocompatible linagliptin functionalized lignin (LL) nanomaterial is synthesized to deliver doxorubicin and zinc selenide quantum dots (ZnSe QDs). Afterward, doxorubicin and ZnSe QDs are loaded in LL to form LLQD-Doxo and analyzed its tumor cell proliferation. Drug carriers are characterized by SEM, FTIR, XRD, DSC, TGA, and zeta potential. In vitro studies are performed at different temperatures, pH, time, and concentration. According to the results, maximum drug encapsulation efficiency is 78.91 %, and rapid drug release is observed at 45 °C and pH 6.5. Linagliptin attached to lignin acts as an inhibitor of xanthine-based non-peptidomimetic DPP-4, an antiproliferative cancer agent. LLQD-Doxo is released from lignin by diffusion at pH 6.5, which is favorable for the tumor microenvironment (TME). LLQD-Doxo exhibits significant therapeutic, anti-inflammatory, antioxidant, cytotoxic, and increased antioxidant activity for MCF-7 cells. The best anti-inflammatory and lipoxygenase inhibitory activity is 48.05 ± 1.05 % and 76.93 ± 2.03 %, respectively. LLQD-Doxo shows antioxidant activity due to lignin polyphenolic compounds in DPPH assay and free radical scavenging activity with IC = 64 μg/mL. Cytotoxicity assay also shows promising results, i.e., 82.32 ± 3.68 % of viable cells after treatment with LLQD-Doxo. The results depict that this drug carrier is suitable for drug delivery at acidic pH and high temperature conditions in tumor cells compared to normal cells.

Uncovering a novel mechanism: Butyrate induces estrogen receptor alpha activation independent of estrogen stimulation in MCF-7 breast cancer cells.

Butyrate is a promising candidate for an antitumoral drug, as it promotes cancer cell apoptosis and reduces hormone receptor activity, while promoting differentiation and proliferation in normal cells. However, the effects of low-dose butyrate on breast cancer cell cultures are unclear. We explored the impact of sub-therapeutic doses of butyrate on estrogen receptor alpha (ERα) transcriptional activity in MCF-7 cells, using RT-qPCR, Western blot, wound-healing assays, and chromatin immunoprecipitation. Our results showed that sub-therapeutic doses of sodium butyrate (0.1 - 0.2 mM) increased the transcription of ESR1, TFF1, and CSTD genes, but did not affect ERα protein levels. Moreover, we observed an increase in cell migration in wound-healing assays. ChIP assays revealed that treatment with 0.1 mM of sodium butyrate resulted in estrogen-independent recruitment of ERα at the pS2 promoter and loss of NCoR. Appropriate therapeutic dosage of butyrate is essential to avoid potential adverse effects on patients' health, especially in the case of estrogen receptor-positive breast tumors. Sub-therapeutic doses of butyrate may induce undesirable cell processes, such as migration due to low-dose butyrate-mediated ERα activation. These findings shed light on the complex effects of butyrate in breast cancer and provide insights for research in the development of antitumoral drugs.

Chalcone-based imidazo[2,1-b]thiazole derivatives: synthesis, crystal structure, potent anticancer activity, and computational studies

In this work, two novel chalcone-based imidazothiazole derivatives ITC-1 and ITC-2 were synthesized and characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry with electrospray ionization, and chemical structure of ITC-1 was confirmed by single-crystal X-ray diffraction. Also, the anticancer activity of ITC-1 and ITC-2 was evaluated. First, antiproliferative activity tests were performed against cancer cells namely, human-derived breast adenocarcinoma (MCF-7), lung carcinoma (A-549), and colorectal adenocarcinoma (HT-29) cell lines, and mouse fibroblast healthy cell line (3T3-L1) by XTT assay. Afterward, mitochondrial membrane disruption (MMP), caspase activity, and apoptosis tests were performed on MCF-7 cells to elucidate the anticancer mechanism of action of the test compounds by flow cytometry analysis. XTT results revealed that both compounds exhibited a very high degree of antiproliferative effects on each tested cancer cell line with very low IC50 values while showing much lower antiproliferation on 3T3-L1 normal cells with much higher IC50 values. Besides, ITC-2 was determined to have a striking cytotoxic power competing with the chemotherapeutic drug carboplatin. Flow cytometry results demonstrated the mitochondrial-mediated apoptotic effects of both compounds through membrane disruption and multi-caspase activation in MCF-7 cells. Finally, molecular docking studies were performed to determine the structural understanding of the test compounds by their interactions on caspase-3 and DNA dodecamer enzymes, respectively. The interactions between the compound and the crystal structure were determined according to parameters such as free binding energies (ΔGBind), Glide score values, and determination of the active binding site. The obtained data suggest that ITC-1 and ITC-2 may be considered remarkable anticancer drug candidates. In addition to molecular docking via in silico approaches, the pharmacokinetic properties of compounds ITC-1 and ITC-2 were calculated using the Schrödinger 2021-2 Qikprop wizard. Communicated by Ramaswamy H. Sarma

Estrogenic activity of Bryonia dioica Jacq. through in silico and in vitro studies on pS2 gene expression in the breast cancer cell line MCF-7

Bryonia dioica is a member of the Cucurbitaceae that produces bioactive secondary metabolites (PSM), especially steroidal cucurbitacins. Tubers accumulate cucurbitacins, tetrahydrocucurbitacins, bryonioside, and polyphenols such as kaempferol and isovitexin. Through in vitro and simulation studies, this study explores the potential estrogenic and antiestrogenic properties of Bryonia dioica. In silico study was performed by molecular docking simulation to predict the binding affinity of BryoniaPSM in Estrogen Receptor Alpha using Molegro Virtual Docker ver 6.0. The in vitro investigation of a methanolic tuber extract used MCF-7 cells, a breast cancer cell line with estrogen receptor (ER), in a medium with or without estradiol. We also analyzed the effect of the extract on the expression of the estrogen-dependent pS2 gene using real-time polymerase chain reaction (RT-PCR). Molecular docking showed that Cucurbitacin-E exhibited the best binding affinity to the estrogen receptor with binding sites at His 524, Gly 521, Arg 394, Glu 353, and Leu 391. Methanolic extract of Bryonia dioica exhibited cytotoxic activity in MCF-7 cells with IC50 = 185 µg/mL (in medium without E2) and IC50 = 125 µg/mL (in medium with E2). In media with and without estrogen, it can stimulate the expression of the pS2 gene at a concentration of 50 µg/mL, a marker for estrogenic activity. The results suggest that Bryonia dioica contains phytoestrogenic PSM with ER agonistic and cytotoxic activities.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Increase Human MCF7 Breast Cancer Cell Proliferation associated with OCT4 Expression and ALDH Activity.

The aim of this study was to investigate the effect of mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) on the human MCF7 breast cancer cell proliferation that have been considered to contain limited CSC population and its association with the expression of OCT4 and ALDH1 stemness markers. EVs were successfully isolated from the conditioned medium of umbilical cord MSCs using size exclusion chromatography. The isolated EV fraction was verified under a transmission electron microscope (TEM). Five and ten percent (v/v) concentration of MSC-EVs were then co-cultured with MCF7 cells. To investigate MSC-EV uptake by MCF7 cells, we performed confocal microscopy analysis. Subsequently, the proliferation of co-cultured MCF7 cells was determined using trypan blue exclusion assay, while their mRNA and protein expression of OCT4 as well as ALDH activity as the marker of stemness properties were analyzed using quantitative reverse transcription polymerase chain reaction, Western Blot, and Aldefluor™ assays, respectively. MSC-EVs were detected as round-shaped, ~100 nm sized particles under TEM. We also demonstrate that MSC-EVs can be internalized by MCF7 cells. Notably, MSC-EVs of 5% concentration increased OCT4 mRNA expression and ALDH1 activity in MCF7 cells. At 10% concentration, MSC-EVs reduced the OCT4 expression and ALDH1 activity. MSC-derived EVs modulate the stemness of MCF7 cells, either OCT4 expression or ALDH1 activity, in a concentration dependent manner along with the increase of cell proliferation.

Gallic acid: a polyphenolic compound potentiates the therapeutic efficacy of cisplatin in human breast cancer cells.

Gallic acid (GA) is a natural polyhydroxyphenolic compound with antioxidant, antimutagenic, anti-inflammatory, and antineoplastic activities. Cisplatin (CPT) is a platinum-based chemotherapeutic drug, and it is the treatment of choice for breast, ovarian, testicular, head, and neck cancers. However, the use of anticancer drugs has undesirable effects on patients due to associated toxicities. Thus, it is necessary to search for alternatives that reduce unintended side effects and enhance anticancer potential. The use of natural compounds with the conventional chemotherapeutic drug is a new aspect of cancer therapy. In the present study, we evaluated the ability of GA in the modulation of anticancer effects of CPT in human breast adenocarcinoma cells (MCF-7) by performing MTT, apoptosis, clonogenic cell survival, and micronucleus assays. GA and CPT showed significant cytotoxic activities in MCF-7 cells in a dose-dependent manner. In combination therapy (GA 2.5, 5.0, and 10μg/mL+CPT10 μg/mL), GA synergistically reduced the MCF-7 cell viability in contrast to the individual therapies. Cancer cells death by GA is through the induction of apoptosis as observed in the acridine orange and ethidium bromide dual staining method. The frequency of micronuclei (MN) was decreased significantly (P<0.001) in combinational therapy, possibly reducing the risk of chemotherapy-induced MN. Moreover, GA in mono or combinational therapy did not induce any cytotoxic effects in normal breast epithelial cells (MCF-10A). GA did not show any significant difference in colony inhibition compared to CPT. This outcome shows its differential effects in normal and cancerous cells. Hence, the combination GA with chemotherapeutic drugs could represent a promising alternative therapy in cancer treatment with minimal side effects.

The effects of Hericium erinaceus extracts on cell viability and telomerase activity in MCF-7 cells

Abstract Objectives This study aims to determine the effects of Hericium erinaceus extracts on cell viability and the effects of H. erinaceus water extract on the telomerase activity of MCF-7 cells. H. erinaceus is an edible mushroom widely used in traditional Chinese medicine. Although its various therapeutic properties, the literature has not yet submitted evidence about H. erinaceus for its effects on the telomerase activity of MCF-7 cells. Methods MCF-7 cells were treated with ethanol, ethanol-water, ether, ethyl acetate, methanol-water, and water extracts to determine the effects on cell viability using the WST8 method. The TeloTAGGG Telomerase PCR ELISA kit was used to assess telomerase activity. Results The water extract was determined to be the most efficient extract to decrease cell viability. The water extract’s half-maximal inhibitory concentration was 250 μg/mL at 72 h. It is found that H. erinaceus has no statistically significant effect compared to positive control on reducing telomerase activity. We found a statistically significant difference in telomerase activity % between H. erinaceus water extracts and negative control (p&lt;0.05). Conclusions Consequently, these differences in telomerase activity are a significant association rather than inferring action. It is considered that water extract shows its cell viability inhibition effects through different mechanisms.

Asiaticoside Increases Caspase-9 Activity in MCF-7 Cells and Inhibits TNF-α and IL-6 Expression in Nude Mouse Xenografts via the NF-κB Pathway.

Background: We hypothesized that the antitumor effects of asiaticoside on breast cancer are driven by its ability to decrease the expression of tumor inflammation-promoting genes and increase apoptotic signaling. In this study, we aimed to better understand the mechanisms of action of asiaticoside as a chemical modulator or as a chemopreventive agent in breast cancer. Methods: MCF-7 cells were cultured and treated with 0, 20, 40, and 80 μM asiaticoside for 48 h. Fluorometric caspase-9, apoptosis, and gene expression analyses were conducted. For the xenograft experiments, we divided nude mice into the following 5 groups (10 animals per group): group I, control mice; group II, untreated tumor-bearing nude mice; group III, tumor-bearing nude mice treated with asiaticoside at weeks 1-2 and 4-7 and injected with MCF-7 cells at week 3; group IV, tumor-bearing nude mice injected with MCF-7 cells at week 3 and treated with asiaticoside beginning at week 6; and group V, nude mice treated with asiaticoside, as a drug control. After treatment, weight measurements were performed weekly. Tumor growth was determined and analyzed using histology and DNA and RNA isolation. Results: In MCF-7 cells, we found that asiaticoside increased caspase-9 activity. In the xenograft experiment, we found that TNF-α and IL-6 expression decreased (p < 0.001) via the NF-κB pathway. Conclusion: Overall, our data suggest that asiaticoside produces promising effects on tumor growth, progression, and tumor-associated inflammation in MCF-7 cells as well as a nude mouse MCF-7 tumor xenograft model.

GPX4 inhibition synergistically boosts mitochondria targeting nanoartemisinin-induced apoptosis/ferroptosis combination cancer therapy.

Artemisinin, originally used for its antimalarial activity, has received much attention in recent years for cancer therapy. The anticancer mechanisms of artemisinin are complicated and debatable. Challenges in the delivery of artemisinin also persist because the anticancer effect of artemisinin alone is often not satisfactory when used with traditional nanocarriers. We herein report the mitochondrial delivery of artemisinin with extremely high anticancer capacity. The action mode of artemisinin in the mitochondria of cancer cells includes heme-participating and oxygen-independent conversion of artemisinin into a carbon-centered radical, which is partly converted into ROS in the presence of molecular oxygen. We reveal that artemisinin alone in the mitochondria can induce strong cancer cell apoptosis. In addition, due to the weak inhibition of GPX4 activity by artemisinin, weak ferroptosis is also observed. We further discover that GPX4 activity in MCF-7 cells is greatly inhibited by RSL3 to synergistically enhance the anticancer capacity of artemisinin via enhancing ferroptosis. The synergistic anticancer activity of artemisinin and RSL3 in the mitochondria not only improves cancer cell-killing ability, but also inhibits the re-proliferation of residual cancer cells. This study provides a new insight into developing highly efficient and practical artemisinin nanomedicines for cancer therapy.

Synthesis, crystal structure, and anticancer activity of four metallo‐supramolecular assemblies from dinuclear silver(I)‐N‐heterocyclic carbene

Four Ag(I)-N-heterocyclic carbene (Ag-NHC) supramolecular assemblies, viz. Ag2(L1)2(PF6)2 (L1 = 2, 5-bis[1-benzenyl-2-ylidene-imidazolium-3-yl)]-pyridine) (5), Ag2(L2)2(PF6)2 (L2 = 1, 3-bis[1-benzenyl-2-ylidene-benzimidazolium-3-yl)]-benzene) (6), Ag2(L3)2(PF6)2 (L3 = 1, 4-bis[1-(quinolin-8-yl)-2-ylidene-imidazolium-3-yl)]-benzene) (9), and [Ag(L4)PF6]n (L4 = 1, 4-bis[1-benzenyl-1, 2, 4-triazolium-4-yl-5-ylidene)]-benzene) (12), were synthesized from silver oxide and the corresponding NHC ligand precursors (3, 4, 8, and 11). They were characterized by 1H NMR, 13C NMR, ESI–MS, and single-crystal X-ray diffraction (XRD) analysis. X-ray crystallography revealed that the Ag-NHC supramolecular assemblies 5, 6, and 9 are a box or square dinuclear Ag-NHC [2+2] metallo-macrocyclic supramolecular assemblies, whereas 12 functioned as a one-dimensional (1D) linear Ag-NHC polymer with the link of carbene CNHC–Ag–CNHC bonds. The structural assignments for 5 were further supported by density functional theory (DFT) calculations. The emission band of 12 has a slight blue shift as compared to the precursors NHC compound 11, owing to the metal–ligand charge transition in 1D linear Ag-NHC polymer. The in vitro cytotoxic activity observed in 5, 6, 9, and 12 against breast cancer MCF-7 cells indicates that the Ag-NHC supramolecular assembly 9 displayed the best anticancer activity in MCF-7 cells at a concentration of 5 × 10−6 mol/L. All four Ag-NHC supramolecular assemblies had significant inhibitory effects on breast cancer cell growth at a concentration of 5 × 10−5 mol/L.

Self-Nanoemulsifying Drug Delivery System of 2-Methoxyestradiol Exhibits Enhanced Anti-Proliferative and Pro-Apoptotic Activities in MCF-7 Breast Cancer Cells.

(1) Background: 2-Methoxyestradiol (2ME) is a metabolite of estrogens and possesses promising anti-proliferative and cytotoxic activities. However, it suffers unfavorable pharmacokinetic characteristics such as absorption after oral administration. The aim of this study was to prepare an optimized 2ME self-nanoemulsifying drug delivery system (2ME-SNEDDS) and evaluate its cytotoxicity and pro-apoptotic activities in MCF-7 breast cancer cells. (2) Methods: For optimization of the 2ME-SNEDDS, a three-component system was used in the D-optimal mixture experimental study. MCF-7 cells were incubated with the 2ME-SNEDDS and subjected to an assessment of growth inhibition, cell cycle progression, annexin V staining, caspase-3 concentration, Bax, Bcl-2, and cyclin D1 mRNA expression, and reactive oxygen species (ROS) generation. (3) Results: The optimized formula had a globule size of 94.97 ± 4.35 nm. Zeta potential was found to be −3.4 ± 1.2 mV with a polydispersity index (PDI) of 0.34. In addition, 96.3 ± 4.3% of 2ME was released from the 2ME-SNEDDS within 24 h using the activated analysis bag technique. Moreover, the prepared 2ME-SNEDDS exhibited a significant enhancement of the anti-proliferative activity against MCF-7 cells in comparison to raw 2ME. This was associated with cyclin D1 expression down-regulation and the accumulation of cells in the G0/G1 and G2/M phases. The pro-apoptotic activities of the 2ME-SNEDDS were confirmed by annexin V staining, which indicated enhanced early and late cell death. This accompanied modulation of the mRNA expression of Bax and Bcl-2 in favor of apoptosis. The 2ME-SNEDDS significantly enhanced cleaved caspase-3 concentration in comparison to raw 2ME. In addition, the 2ME-SNEDDS significantly increased the generation of ROS in MCF-7 cells. (4) Conclusions: The 2ME-SNEDDS exhibits enhanced cytotoxicity and pro-apoptotic activity in MCF-7 cells. This is mediated by, at least partially, ROS generation.

A sensing strategy combining T7 promoter-contained DNA probe with CRISPR/Cas13a for detection of bacteria and human methyltransferase

Abnormal DNA methylation is closely related to the occurrence and development of many diseases. The determination of human DNA methyltransferase activity and the screening of its inhibitors are extreme important for the diagnosis and the treatment of methylation-related diseases in clinic. Most of the current detection methods have the disadvantages of sophisticated design, high cost and low detection limit. By combining T7 promoter-contained DNA probe as the substrate for methyltransferase with CRISPR/Cas13a sensing strategy, a novel fluorescent sensing platform is designed to achieve simple, specific, sensitive detection of bacteria DNA methyltransferase (DNA-(N-6-adenine)-methyltransferase, Dam MTase) and also human methyltransferase (DNA (cytosine-5)-methyltransferase 1, Dnmt1). A hairpin DNA probe designed for Dam MTase and a double strand DNA probe for Dnmt1 are both methylated followed by the methylation-dependent site-specific cleavage, which result a T7 promoter-contained product and a T7 promoter-free one to respectively open and close the transcription and subsequent CRISPR/Cas13a target-initiated cleavage of fluorescence-labeled reporter RNA. In virtue of the specificity of methylation-dependent cleavage of probe, the efficient transcription amplification and CRISPR/Cas13a sequence-specific sensing, this strategy exhibited remarkable specificity and sensitivity, with the limit of detection of 3.10 × 10−5 U/mL for Dam MTase. Moreover, Dnmt1 activity in MCF-7 cells was detected and the inhibition of Apt. #9 was evaluated. This strategy for methyltransferase detection is convenient and efficient for inhibitor discovery and early cancer diagnosis.

Isoquinoline alkaloids from Hylomecon japonica and their potential anti-breast cancer activities

Four pairs of undescribed enantiomeric isoquinoline alkaloids (6S/R-(N,N-diethylacetamido)yl-dihydrochelerythrine, 6R/S-acetonyl-9-hydroxy-dihydrochelerythrine, 6S/R-acroleinyl-dihydrochelerythrine, 6S/R-acetatemethyl-dihydrochelerythrine), five undescribed isoquinoline alkaloids (6,10-dimethoxydihydrochelerythrine, 6-ethoxy-ethaniminyl-dihydrochelandine, 9-hydroxy-dihydrochelerythrine, 9-methoxy-10-hydroxy-norchelerythrine, chelidoniumine A), together with 13 known isoquinoline alkaloids were isolated from an extract of the roots and rhizomes of Hylomecon japonica. The structures of the undescribed compounds were identified by NMR, HRESIMS, UV, IR, and their absolute configurations were defined via electronic circular dichroism data and optical rotation. All of the isolated compounds were tested for their anti-breast cancer activities in MCF-7 cells. Among them, the undescribed alkaloids 6S/R-acroleinyl-dihydrochelerythrine, 6,10-dimethoxydihydrochelerythrine, 6-ethoxy-ethaniminyl-dihydrochelandine, 9-methoxy-10-hydroxy-norchelerythrine and other known alkaloids 6-methoxydihydrosanguinarine, 6-acetaldehyde-dihyrochelerythrine, dihydrosanguinaline and 10-methoxy boconoline had good inhibitory effects on MCF-7 cells of breast cancer with an IC50 lower than 20 μM.

Guanidine-Curcumin Complex-Loaded Amine-Functionalised Hollow Mesoporous Silica Nanoparticles for Breast Cancer Therapy.

Simple SummaryThe combination of guanidine carbonate and curcumin-loaded hollow mesoporous silica nanoparticles (GuC-HMSNAP) can be used as a therapeutic and to induce MCF-7 cell death. Due to its biological safety and high drug loading capacity, HMSNAP is becoming an increasingly important nanocarrier for cancer research. The features of drug-loaded nanocarriers are significantly altered particle size, pore size, surface area, and pore volume, confirming that a significant amount of drugs can be loaded in the nanocarriers while simultaneously allowing the maximum amount of drugs to be released from the nanocarriers. Here, guanidine-mediated apoptosis is analysed through western blotting; the results suggest that drug complexes result in downregulation of phosphorylation in Ser471 of Akt, Ser259 of c-Raf, and Ser241 of PDK1, upregulation of phosphorylation in GSK-3β Ser9, cleaved caspases, and cleaved PARP, which then partially induces intrinsic cell death in MCF-7. As a whole, our results demonstrate that GuC-HMNSAP is an efficient nanocarrier for effectively inducing cancer cell death.The current study focuses on developing a tumour-targeted functionalised nanocarrier that wraps hollow mesoporous silica nanoparticles. The guanidine carbonate and curcumin are immobilised on the surface of 3-aminopropyl-triethoxy silane (APTES)-decorated hollow mesoporous silica nanoparticles (HMSNP), as confirmed through XPS and NMR analysis. XPS analysis demonstrates that the shape of the hysteresis loops is modified and that pore volume and pore diameter are consequently decreased compared to control. Guanidine (85%) and guanidine–curcumin complex (90%) were successfully encapsulated in HMSNAP and showed a 90% effective and sustained release at pH 7.4 for up to 72 h. Acridine orange/ethidium bromide dual staining determined that GuC-HMNSAP induced more late apoptosis and necrosis at 48 and 72 h compared with Gu-HMNSAP-treated cells. Molecular investigation of guanidine-mediated apoptosis was analysed using western blotting. It was found that cleaved caspases, c-PARP, and GSK-3β (Ser9) had increased activity in MCF-7 cells. GuC-HMSNAP increased the activity of phosphorylation of oncogenic proteins such as Akt (Ser473), c-Raf (Ser249), PDK1 (Ser241), PTEN (Ser380), and GSK-3β (Ser9), thus inducing cell death in MCF-7 cells. Altogether, our findings confirm that GuC-HMNSAP induces cell death by precisely associating with tumour-suppressing proteins, which may lead to new therapeutic approaches for breast cancer therapy.

Selenium nanoparticles coupling with Astragalus Polysaccharides exert their cytotoxicities in MCF-7 cells by inhibiting autophagy and promoting apoptosis

BackgroundAstragalus Polysaccharides (APS) had been reported to exhibit antitumor activities. Given that nanoparticles possessed unique advantages in cancer treatment, APS was used as the modifier to prepare gold, silver and selenium nanoparticles (APS-Au, APS-Ag and APS-Se NPs) in the present study. MethodsThe three nanoparticles were synthesized via a green approach and characterized by DLS, TEM, XRD, FT-IR and UV-Vis. The inhibitory effects of these nanoparticles on various tumor cells proliferation were examined by MTT assay in vitro. Reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the expression of apoptosis and autophagy-related proteins were also detected. ResultsAmong these, APS-Se NPs displayed the most potent antitumor activities against MCF-7 cells in vitro. Flow cytometric analysis suggested that after cells were exposed to elevated concentrations of APS-Se NPs (10, 20 and 40 μmol/L), the rate of apoptosis was increasing (16.63 ± 0.89, 38.60 ± 3.46 and 44.38 ± 2.62%, respectively). Further analysis by immunofluorescence revealed an increase in intracellular ROS and a loss of MMP. This was accompanied by increased LC3-I to LC3-II conversion. Also, western blot analysis demonstrated that the ratios of Bax/Bcl-2 and cleaved caspase9/caspase 9 rose, and LC3-II and p62 protein levels increased. The addition of chloroquine, an inhibitor of autophagy, further enhanced protein expression of p62 and LC3-II. ConclusionAPS-Se NPs exerted their cytotoxic activity in MCF-7 cells by blocking autophagy and facilitating mitochondrial pathway-mediated apoptosis.

Morphological changes of MCF7 cells incubated with quercetin

The antiproliferative properties of quercetin along with its possible mode of cell death have been established. Nevertheless, it was found interesting to observe the morphological changes of MCF7 cells over the time of incubation with quercetin. In this study, the cytotoxic property of quercetin towards MCF7 was evaluated by MTT assay. No effect on the cellular metabolic activity of MCF7 cells after treatment with quercetin at 24 hour was found. But, 48 hours and 72 hours treatment of quercetin exhibited cytotoxic effect with IC50 values of 43.18 μg/mL and 18.49 μg/mL respectively. Live cell imaging was carried out to monitor the morphological changes of the cells. Apoptosis was found absent at 24 hours, however, long-time exposure allowed quercetin to promote apoptosis. Acquired results from live cell imaging presented an insight to the morphological changes due to the respond of MCF7 cells towards quercetin.

Synthesis, X-ray Single-Crystal Analysis, and Anticancer Activity Evaluation of New Alkylsulfanyl-Pyridazino[4,5-b]indole Compounds as Multitarget Inhibitors of EGFR and Its Downstream PI3K-AKT Pathway

The alkylation of 3,5-dihydro-4H-pyridazino[4,5-b]indole-4-thione with benzyl bromide, ethyl chloroacetate, and allyl bromide in the presence of potassium carbonate (K2CO3) yielded new alkylsulfanylpyridazino[4,5-b]indole derivatives (i.e., compounds 4–6). Hydrazinolysis of ester 6 resulted in hydrazide 7. The structure of compound 6 was verified by X-ray single-crystal analysis. Among the synthesized compounds, compound 6 exhibited the most promising cytotoxicity toward MCF-7 cells with an IC50 value of 12 µM. It showed potential inhibition activity toward EGFR, PI3K, and AKT in MCF-7 cells, with 0.26-, 0.49-, and 0.31-fold reductions in concentration compared to an untreated control. Additionally, it showed apoptosis-inducing activity in MCF-7 cells (47.98-fold); overall apoptosis increased to 38.87% compared to 0.81% in the untreated control, which disrupted the cell cycle at pre-G1 and S phases. Moreover, compound 6 exhibited good binding affinities toward the tested proteins (EGFR, PI3K, and AKT) and had binding energies ranging from −15.87 to −24.87 Kcal/mol. It also formed good interactions with essential amino acids inside the binding sites. Hence, compound 6 is recommended as an anti-breast cancer chemotherapeutic due to its effects on the EGFR-PI3K-AKT pathway.

Honeybee Venom Synergistically Enhances the Cytotoxic Effect of CNS Drugs in HT-29 Colon and MCF-7 Breast Cancer Cell Lines

5-fluorouracil (5-FU) and doxorubicin (DOX) are potent anti-tumour agents commonly used for colon and breast cancer therapy, respectively. However, their clinical application is limited by their side effects and the development of drug resistance. Honeybee venom is a complex mixture of substances that has been reported to be effective against different cancer cells. Its active compound is melittin, a positively charged amphipathic peptide that interacts with the phospholipids of the cell membrane, forming pores that enable the internalization of small molecules with cytotoxic activities,. and consequently, causing cell death. Some central nervous system (CNS) drugs have recently demonstrated great anti-cancer potential, both in vitro, in vivo and in clinical trials, being promising candidates for drug repurposing in oncology. The present work evaluated the anti-cancer efficacy of honeybee venom in combination with chemotherapeutic or CNS drugs in HT-29 colon and MCF-7 breast cancer cell lines. The chemical characterization of a Portuguese sample of honeybee venom was done by LC-DAD-ESI/MSn analysis. For single treatments, cells were incubated with increasing concentrations of bee venom. For combination treatments, increasing concentrations of bee venom were first combined with the half-maximal inhibitory concentration (IC50) of 5-FU and DOX, in HT-29 and MCF-7 cells, respectively. Cells were also treated with increasing concentrations of bee venom in combination with the IC50 value of four CNS drugs (fluphenazine, fluoxetine, sertraline and thioridazine). Cytotoxicity was evaluated by MTT and SRB assays. The combination index (CI) value was calculated using CompuSyn software, based on the Chou–Talalay method. Synergy scores of different reference models (HSA, Loewe, ZIP and Bliss) were also calculated using SynergyFinder. The results demonstrate that honeybee venom is active against HT-29 colon and MCF-7 breast cancer cells, having better anti-tumour activity in MCF-7 cells. It was found that bee venom combined with 5-FU and fluphenazine in HT-29 cells resulted in less cytotoxic effects compared to the co-treatment of fluoxetine, sertraline and thioridazine plus bee venom, which resulted in less than 15% of viable cells for the whole range of concentrations. The combination of MCF-7 cells with repurposed drugs plus honeybee venom resulted in better anti-cancer efficacies than with DOX, notably for lower concentrations. A combination of fluoxetine and thioridazine plus honeybee venom resulted in less than 40% of viable cells for all ranges of concentrations. These results support that the combination of honeybee venom with repurposed drugs and chemotherapeutic agents can help improve their anti-cancer activity, especially for lower concentrations, in both cell lines. Overall, the present study corroborates the enormous bioactive potential of honeybee venom for colon and breast cancer treatments, both alone and in combination with chemotherapy or repurposed drugs.

Abstract P5-06-09: Rivaroxaban targets the procoagulant tumour microenvironment in vitro and thereby inhibits breast cancer progression

Abstract Introduction: Breast cancer patients have a four-fold increased risk of developing a venous thromboembolism and those that do have a significantly increased risk of mortality despite adjusting for cancer stage. Tissue Factor (TF) is expressed by breast cancer-associated fibroblasts as well as breast cancer epithelial cells, and at significantly higher levels than by normal breast fibroblasts. TF signals via PAR-1 and PAR-2 to induce proliferation, invasion, angiogenesis and metastasis. Rivaroxaban is a licensed oral anticoagulant that inhibits the TF-Factor VIIa-Factor Xa complex and could therefore be repurposed to target the procoagulant tumour microenvironment in breast cancer. We hypothesise that a procoagulant microenvironment will induce breast cancer progression in vitro and that these promoting effects will be inhibited by anticoagulants, including Rivaroxaban. Methods: Lentivirally transduced TF over-expressing fibroblasts (TFF) and their control (CF) or conditioned media (TFFCM and CFCM), were cultured with oestrogen receptor positive (MCF-7), triple negative (MDA-MB-231) and HER2 positive (BT474) breast cancer cells, in the presence or absence of Rivaroxaban or anti-TF antibody 10H10. Proliferation (sulforhodamine-B/EdU assay), migration (scratch/transwell assay) and stem cell activity (mammosphere forming efficiency (MFE) assay) were assessed. The underlying mechanism was analysed with western blotting and quantitative PCR. The TFFCM and CFCM were analysed with cytokine arrays, enzyme-linked immunosorbent assays (ELISA) and mass spectrometry whilst the TFF and CF were analysed using RNA sequencing. Results: 3D co-culture of MCF-7s with TFF as compared to CF promoted cancer cell migration (p=0.04) and stem cell activity (MFE: p&amp;lt;0.0001), with these effects abrogated by Rivaroxaban (migration: p= 0.0341, MFE: p=0.0003) and 10H10 (migration: p=0.01, MFE: p=0.0028). TFFCM promoted proliferation, migration and stem cell activity in MCF-7 cells (p&amp;lt;0.05) compared to CFCM, with these effects abrogated by Rivaroxaban (migration, MFE: p&amp;lt;0.05) and 10H10 (proliferation, migration, MFE: p&amp;lt;0.05). The cancer-promoting effects of TFFCM in MCF-7 cells was associated with a decrease in CXCL8 (p&amp;lt;0.0001) and an increase in VEGFA (p&amp;lt;0.05) when compared to CFCM; changes which were reversed by Rivaroxaban (VEGFA: p&amp;lt;0.03) and 10H10 (CXCL8, VEGFA: p&amp;lt;0.05). TFFCM also promoted stem cell activity in both MDA-MB-231 (p&amp;lt;0.006) and BT474 (p&amp;lt;0.002) cell lines compared to CFCM. This increased MFE in BT474 was abrogated by Rivaroxaban (p=0.02) and 10H10 (p&amp;lt;0.005) and associated with an increase in VEGFA (p&amp;lt;0.001) and CXCL8 (p&amp;lt;0.0001). Cytokine array and ELISA analysis of TFFCM versus CFCM indicated increased levels of IL-10 (p&amp;lt;0.006), GRO-α (p&amp;lt;0.0001) and IL1-α (p&amp;lt;0.02). GRO-α and IL1-α production was inhibited by fibroblast treatment with Rivaroxaban (GRO-α, p&amp;lt;0.0001 and IL1-α, p&amp;lt;0.001) and 10H10 (p&amp;lt;0.001). TFFCM versus CFCM had elevated levels of plasminogen activator inhibitor 1, integrin linked kinase, thrombospondin 4 and α -fetoprotein on mass spectrometry. RNA sequencing analysis of the TFF as compared to the CF revealed upregulation of AEBP1 (adipocyte enhancer binding protein 1) (p&amp;lt;0.01), NKX2-5 (homeobox protein NKX-2.5) (p&amp;lt;0.0001), TRIM58 (tripartite motif containing 58) (p&amp;lt;0.0001) and KRT81 (keratin 81) (p&amp;lt;0.0001), all of which were decreased by fibroblast treatment with Rivaroxaban (NKX2-5: p&amp;lt;0.01, KRT81: p&amp;lt;0.0001). Conclusion: A procoagulant microenvironment promotes proliferation, migration and stem cell activity in breast cancer in vitro, with these effects abrogated by Rivaroxaban. Targeting the procoagulant tumour microenvironment is a promising future cancer treatment. Citation Format: Emma L Blower, John Castle, Angelica Santiago-Gomez, Robert Clarke, Cliona C Kirwan. Rivaroxaban targets the procoagulant tumour microenvironment in vitro and thereby inhibits breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-09.