MCF-7 Cultures Research Articles

In vitro biochemical features of cold plasma application in MCF-7 experimental breast cancer cells

Introduction. Low-temperature plasma is currently used in medicine, including cancer therapy. Plasma-activated biological solutions have already been proposed as potential reagents for cancer treatment. However, the biological effects in cells induced by exposure to cold plasma still remain unexplored. Investigation of the molecular mechanisms of the effects of cold plasma on cells is of great clinical importance for its clinical application. the aim of the present study was to evaluate the effect of cold plasma exposure on apoptosis, catalase activity, and malonic dialdehyde content in MCF-7 breast cancer cell cultures compared to 3T3 normal fibroblast cells. Material and Methods. MCF-7 mammary epithelial cells were used as research objects, and NIH/3T3 mouse embryonic fibroblast cells were used as controls. Cell suspensions were treated using low-temperature atmospheric discharge plasma with escaping electrons. Annexin V and propidium iodide were used to quantify cell line apoptosis. The content of malonic dialdehyde was determined by the developing coloration of its solution with 2-thiobarbituric acid at high temperature in acidic medium. The activity of catalase was estimated by the rate of decomposition of hydrogen peroxide for a certain time of incubation of the mixture. Results. It was found that irradiation of MCF-7 cell culture with plasma led to an increase in the content of malondialdehyde, the main product of lipid peroxidation. the increase in this parameter is an indicator of cell membrane damage and oxidative stress induced by irradiation. In addition, under the same irradiation regime, cold plasma showed a stimulating effect on the culture of 3T3 cells, while on the MCF-7 culture, on the contrary, it stimulated the activation of apoptosis and cell death. Conclusion. In the present study, we found that exposure of tumor and normal cells to cold plasma promotes apoptosis activation. Catalase and MDA activity have been shown to be significant markers capable of assessing the intensity of oxidative stress.

Cytotoxic and cytostatic activity of five new imidazotetrazine derivatives on breast cancer cell cultures MDAMB231, BT474, and MCF-7

Introduction: The work presents the results of the study of new imidazotetrazine derivatives to establish the possibility of using them as anticancer agents, including for chemotherapy of metastatic breast cancer. The relevance of the work is due to the wide spread of oncological diseases and high cancer mortality, which dictates the need to constantly obtain cell lines and improve cultivation protocols for testing new antitumor drugs. The goal of this study is to check the potential of five new imidazotetrazine derivatives to become new antitumor drugs, in the scope of studying their cytotoxic and cytostatic activities on breast cancer cell cultures. Materials and Methods: The culturing MCF-7, MDAMB231, BT474, and MCF-10a cells with determining cytotoxic and cytostatic activities of five new azolotetrazine derivatives are base methods used in this study. Results: For the MCF-7 culture, MCS of comparison drug temozolomide was equal to 2.44 and IC50 was 6.81 mM/L; for other cultures CTA indicators were worse. Imidazotetrazine 2 and imidazotetrazine 3 demonstrated CTA indicators lower than those of temozolomide. IC50 was not achieved, and the MCS value varied between 1.34 and 1.74. These two derivatives were classified as the compounds with an extremely low CTA. Imidazotetrazine 1 and iminothiotriazine 5 showed cytotoxic activity higher than that of the comparison drug and we classified these compounds as the ones with a moderate CTA. Finally, we found imidazotetrazine 4 with IC50 of 0.85 mM/L and CTA of 7.34 as a compound with a potentially strong anticancer effect for further investigation. The cytostatic activity of four of the five azoloazine derivatives studied was in a narrow range corresponding to the survival rate from 0.21 to 0.32, depending on the compound and cell culture. Against this background, imidazotetrazine 4 demonstrated a higher CSA, determined by the survival rate from 0.17 to 0.20. Conclusion: As a result of an in vitro study, we found that five new azolotriazine derivatives can be evaluated in the ascending order of these properties, as a combination of CTA+CSA in order imidazotetrazine 2, imidazotetrazine 3 < temozolomide < imidazotetrazine 1, iminothiotriazine 5 < imidazotetrazine 4, although the CSA of all the studied compounds turned out to be high. Thus, 3-Cyclohexyl-4-oxoimidazo[5,1-d]-[1,2,3,5]tetrazine-8-N-piperidinyl-carboxamide (imidazotetrazine 4) is an unconditional leader in the tested series of new azoloazine derivatives and we recommend it for further preclinical trials.

The Tumorigenicity of Breast Cancer Cells Is Reduced upon Treatment with Small Extracellular Vesicles Isolated from Heparin Treated Cell Cultures.

As a member of the HPSG family, heparin is often used as a specific probe of their role in cell physiology; indeed, we have previously shown a reduction in the tumorigenicity of breast cancer cells when cultured in its presence. However, a partial reversal of the anti-tumorigenic effect occurred when the treated cells were cultured in fresh medium without heparin, which led us to consider whether a more persistent effect could be achieved by treatment of the cells with small extracellular vesicles (sEV) from heparin-treated cells. The tumorigenicity was analyzed using sEV isolated from the culture medium of heparin-treated MCF-7 and MDA-MB231 breast cancer cells (sEV-HT) or from conditioned medium following the termination of treatment (heparin discontinued, sEV-HD). Tumorigenicity was reduced in cells cultured in the presence of sEV-HT compared to that of cells cultured in the presence of sEV from untreated cells (sEV-Ctrl). sEV-HD were also observed to exert an anti-tumorigenic effect on the expression of pro-tumorigenic and cell cycle regulatory proteins, as well as signaling activities when added to fresh cultures of MCF-7 and MDA-MB231 cells. The anti-tumorigenic activity of the heparin-derived sEV may arise from observed changes in the miRNA content or from heparin, which was observed to be bound to the sEV. sEV may constitute a relatively stable reservoir of circulating heparin, allowing heparin activity to persist in the circulation even after therapy has been discontinued. These findings can be considered as a special additional pharmacological characteristic of heparin clinical therapy.

Xanthohumol hinders invasion and cell cycle progression in cancer cells through targeting MMP2, MMP9, FAK and P53 genes in three-dimensional breast and lung cancer cells culture

BackgroundDespite recent advances in the treatment of lung and breast cancer, the mortality with these two types of cancer is high. Xanthohumol (XN) is known as a bioactive compound that shows an anticancer effect on cancer cells. Here, we intended to investigate the anticancer effects of XN on the breast and lung cancer cell lines, using the three-dimensional (3D) cell culture.MethodsXN was isolated from Humulus lupulus using Preparative-Thin Layer Chromatography (P-TLC) method and its authenticity was documented through Fourier Transform Infrared spectroscopy (FT-IR) and Hydrogen Nuclear Magnetic Resonance (H-NMR) methods. The spheroids of the breast (MCF-7) and lung (A549) cancer cell lines were prepared by the Hanging Drop (HD) method. Subsequently, the IC50s of XN were determined using the MTT assay in 2D and 3D cultures. Apoptosis was evaluated by Annexin V/PI flow cytometry and NFκB1/2, BAX, BCL2, and SURVIVIN expressions. Cell cycle progression was determined by P21, and P53 expressions as well as PI flow cytometry assays. Multidrug resistance was investigated through examining the expression of MDR1 and ABCG2. The invasion was examined by MMP2, MMP9, and FAK expression and F-actin labeling with Phalloidin-iFluor.ResultsWhile the IC50s for the XN treatment were 1.9 µM and 4.74 µM in 2D cultures, these values were 12.37 µM and 31.17 µM in 3D cultures of MCF-7 and A549 cells, respectively. XN induced apoptosis in MCF-7 and A549 cell lines. Furthermore, XN treatment reduced cell cycle progression, multidrug resistance, and invasion at the molecular and/or cellular levels.ConclusionsAccording to our results of XN treatment in 3D conditions, this bioactive compound can be introduced as an adjuvant anti-cancer agent for breast and lung cancer.

High-dose Irradiation Stimulated Breast Tumor Microenvironment to Enhance Tumor Cell Growth and Decrease Tumor Cell Motility.

Surgery and radiotherapy are two main modalities of breast cancer treatment. However, surgery affects the tumor microenvironment negatively and promotes the growth of possible malignant cells remaining in the tumor bed. The present study aimed to investigate the effects of intraoperative radiotherapy (IORT) on the tumor microenvironment. Therefore, the effect of surgical wound fluid (WF), collected from operated and irradiated patients on the growth and motility of a breast cancer cell line (MCF-7) was assessed. In this experimental study, preoperative blood serum (PS) and secreted WF from 18 patients who underwent breast-conserving surgery (IORT-) and 19 patients who received IORT following surgery (IORT+) were collected. The samples were purified and added to MCF-7 cultures. Two groups of the cells were treated with and without fetal bovine serum (FBS) and used as positive and negative controls. Applying 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch wound healing assays, the growth and motility of MCF-7 cells were measured. Cell growth of the cells receiving WF from IORT+ patients (WF+) was statistically higher than the corresponding values of the cells received PS or WF from IORT- patients (WF-) (P<0.01). Both WF+ and WF- decreased the cells' migration ability compared to PS (P<0.02) and FBS (P<0.002), although WF+ caused a more significant reduction (P<0.02). Wound fluid extracted from breast cancer patients who underwent both surgery and IORT increased the growth of breast tumor cells, but decreased their ability to migrate.

Transcriptomics indicate nuclear division and cell adhesion not recapitulated in MCF7 and MCF10A compared to luminal A breast tumours

Breast cancer (BC) cell lines are useful experimental models to understand cancer biology. Yet, their relevance to modelling cancer remains unclear. To better understand the tumour-modelling efficacy of cell lines, we performed RNA-seq analyses on a combined dataset of 2D and 3D cultures of tumourigenic MCF7 and non-tumourigenic MCF10A. To our knowledge, this was the first RNA-seq dataset comprising of 2D and 3D cultures of MCF7 and MCF10A within the same experiment, which facilitates the elucidation of differences between MCF7 and MCF10A across culture types. We compared the genes and gene sets distinguishing MCF7 from MCF10A against separate RNA-seq analyses of clinical luminal A (LumA) and normal samples from the TCGA-BRCA dataset. Among the 1031 cancer-related genes distinguishing LumA from normal samples, only 5.1% and 15.7% of these genes also distinguished MCF7 from MCF10A in 2D and 3D cultures respectively, suggesting that different genes drive cancer-related differences in cell lines compared to clinical BC. Unlike LumA tumours which showed increased nuclear division-related gene expression compared to normal tissue, nuclear division-related gene expression in MCF7 was similar to MCF10A. Moreover, although LumA tumours had similar cell adhesion-related gene expression compared to normal tissues, MCF7 showed reduced cell adhesion-related gene expression compared to MCF10A. These findings suggest that MCF7 and MCF10A cell lines were limited in their ability to model cancer-related processes in clinical LumA tumours.

Selected Statins as Dual Antiproliferative-Antiinflammatory Compounds

We hypothesized that superlative dual cytotoxicity-antiinflammtion bioefficacies of 9 selected lipophilic statins correlate to their chelation effect of 3,5-dihydroxyheptanoic acid. Lipophilic-acid chelating statins have been screened for in vitro duality of proliferation inhibition and NO-radical scavenging capacities. Their spectrum of selectivity indices for safety in PDL fibroblasts -based 72h incubations was reported. Surprisingly despite its lack on macrophages LPS-triggered inflammation over 5-200 µM and unlike the 8 statins; cerivastatin had growth inhibition IC50 values of 40nM (SW620), 110nM (HT29), 2.9 µM (HCT116), 6µM (SW480), and most notably 38µM (<50 µM, in Caco2). Exclusively cerivastatin exerted antitumorigenesis IC50 values <50 µM in all T47D, MCF7 and PANC1 72h cultures. In statins with greater antiinflammation affinity than indomethacin's; lovastatin had cytotoxicity IC50 values <20 µM in SW620<HT29<ACT116100 µM in Caco2. Atorvastatin was found of viability reduction IC50 value <20 µM in HCT11650µM in T47D, MCF7 and PANC1. Rosuvastatin had antineoplastic IC50 values (<50 µM) in SW620<SW48050 µM in remaining colorectal, breast and pancreatic cancer cell lines. In statins with appreciable antiinflammation but reasonably lower affinity than indomethacin's and cytotoxicity IC50 values >50µM in T47D, MCF7 and PANC1; pravastatin had viability reduction IC50 values <50µM in HT2950 µM were for statins in remaining colorectal cancer cell lines, breast cancer and pancreatic cancer cell lines. Among the rest, cerivastatin warrants further novel scaffold development to maximize efficacy and optimal molecular action mechanisms of chemotherapy/prevention.

Preparation, Characterization and In vitro Biological activity of 5-Fluorouracil Loaded onto poly (D, L-lactic-co-glycolic acid) Nanoparticles

Nanoscale devices offer a lot of potential in drug delivery because of their small size. The goal of this work was to increase the oral bioavailability of the anti-cancer hydrophilic drug as 5-fluorouracil (5-FU) by incorporating it into poly (D, L-lactide-co-glycolide) nanoparticles (PLGNPs) using the double emulsion process, 5-FU- PLGNPs nanoparticles were created. Various factors, such as drug, polymer, and stabilizer concentrations, were investigated for assembly in order to arrive at the most effective formulation of 5-FU-PLGNPs. PLGNPs had a drug encapsulation efficiency of 9.75 to 24.8%. The prepared nanoparticles had a spherical shape and an average size of 212.3–285 nm, as shown by TEM. The dispersion of the drug into the prepared PLGNPs was confirmed by XRPD and FTIR. The optimized nanoparticles (F225) had high encapsulation efficiency 24.8 ± 0.21%, low particles size 212.3 ± 48.2 nm with an appropriate PDI value of 0.448, and ZP of − 48.3 ± 2.7 mV. The molecular dispersion of the medication within the system was validated by thermal behavior studies (DSC). In vitro drug release from the best-selected formulations revealed a sustained release of nanoparticles, with slower release reported when lower PVA concentrations were utilized. Three 5-FU-PLGNPs formulations were tested for anticancer efficacy against cell cultures of HCT-116 (human colorectal carcinoma), MCF-7 (human breast carcinoma), and HepG2 (human hepatocellular carcinoma). The created formulations were examined for in vitro cytotoxic activity, revealing that they appeared to be promising effective anticancer formulations when compared to the positive controlled (doxorubicin).

Study of the laser radiation effect in combination with doxorubicin on the survival of MCF7 and MCF7DOX culture cells

The use of lasers in oncology is constantly evolving. In recent years, the mechanisms of the effect of laser radiation, particularly of the infrared spectrum, on tumour cells in combination with various chemotherapeutic agents have been studied.&#x0D; The aim. To study the effect of laser radiation with a wavelength of 660 and 810 nm on the survival of tumour cell cultures MCF7 and MCF7DOX in the presence of doxorubicin.&#x0D; Materials and methods. Tumour cells of MCF7 and MCF7DOX cultures were cultured in DMEM medium (Biowest, France) with 10 % fetal calf serum (FST) (Biowest, France) and 40 μg / ml gentamicin (Sigma, USA). Doxorubicin was added to the cells to a final concentration of 1 μg / ml. Cells were irradiated with a laser (Photonica-Plus, Ukraine) with a wavelength of 660 and 810 nm (irradiation time – 5 min, power density – 50 mV/cm2, irradiation dose 15 J/cm2). The results were recorded using a multiwell spectrophotometer (Labsystems Multiskan PLUS, Finland). Photomicrographs of cells were taken using a Carl Zeiss microscope, Germany.&#x0D; Results. Cell survival assessment and morphological characteristics in micropreparations indicate the antitumor efficacy of the combined effects of laser irradiation and doxorubicin. The most pronounced cytotoxic effect on MCF7-DOX culture cells was caused by doxorubicin exposure for 90 min in combination with infrared laser irradiation (λ=810 nm).&#x0D; Conclusions. Infrared laser light synergises with the toxic effects of doxorubicin and creates favourable conditions for apoptosis of tumour cells, as evidenced by cytomorphological data.

Разработка и влияние добавки из лимфоидной ткани птицы на жизнеспособность клеток в культуре

Introduction. Today, dietary supplements are an integral part of human diet. Some of them are made of hydrolysates of animal origin. Biologically active additives of immunomodulatory action can prevent various diseases. The research objective was to develop a dietary supplement from the bursa of Fabricius obtained from broiler chickens and evaluate its effect on cell viability in culture.&#x0D; Study objects and methods. The study featured biologically active supplement obtained by enzymatic hydrolysis of the bursa of Fabricius, immature stem cells, and adult differentiated cells of human dermal fibroblasts, HeLa and MCF-7 cancer cells, and extract of the bursa of Fabricius.&#x0D; Results and discussion. The research resulted in a new technology of dietary supplement production from the bursa of Fabricius of broiler chickens. It included washing, cutting, homogenization, proteolytic enzyme fermentation, and ultrafiltration. When introduced into the culture of mesenchymal stem cells, the dietary supplement caused a slight decrease in the cell viability at concentrations of 25 and 50%, which indicated a possible cytotoxic effect of the extract on mesenchymal cells. The extract did not affect the viability of human fibroblast culture and caused no cytotoxic effect. In MCF-7 culture, the extract had a dose-dependent cytotoxic effect, which lowered the relative cell viability. &#x0D; Conclusion. The new dietary supplement based on the bursa of Fabricius of broiler chickens had a cytotoxic effect on stem cell cultures. However, it did not affect the cell viability and had no cytotoxic effect on human dermal fibroblasts. The effect depended on the cell culture. In the case of HeLa, the supplement stimulated proliferative activity, and in the case of MCF-7, it had a cytotoxic effect. Therefore, the new dietary supplement demonstrated some prospects as an active ingredient for various biologically active additives and immunomodulatory drugs.

Hybrids of 4-hydroxy derivatives of goniothalamin and piplartine bearing a diester or a 1,2,3-triazole linker as antiproliferative agents

A library of nine hybrids of 4-hydroxygoniothalamin (2), 4-hydroxypiplartine (4), monastrol (5) and oxo-monastrol (6) was prepared via a modular synthetic route with a diester or a 1,2,3-triazole as linkers. The compounds were assayed against a panel of human cancer cell lines, including MCF-7 (breast adenocarcinoma), HeLa (cervical adenocarcinoma), Caco-2 (colorectal adenocarcinoma) and PC3 (prostate adenocarcinoma), as well as against normal breast (MCF10A) and prostate (PNT2) cells. In general, hybrids with an ester linker containing 4-hydroxypiplartine (4) were more potent than the corresponding hybrids with 4-hydroxygoniothalamin (2). On the other hand, compounds presenting the 1,2,3-triazole linker displayed enhanced cytotoxicity and selectivity when compared to their corresponding hybrids with the diester linker. The 4-hydroxypiplartine-based hybrids 12 and 22 displayed high cytotoxicity (IC50 values below 10 μM) against all cancer cells studied, especially in MCF-7 cells with IC50 values of 1.7 ± 0.1 and 1.6 ± 0.9 μM, respectively. Furthermore, the 4-hydroxygoniothalamin-monastrol hybrid (compound 21) and the 4-hydroxypiplartine-oxo-monastrol hybrid (compound 25), both bearing a 1,2,3-triazole linker, displayed high selectivity and potency towards breast cancer cell line (MCF-7 vs. MCF10 cells, selectivity index = 15.8 and 7.1, respectively), while the 4-hydroxypiplartine -4-hydroxymethylgoniothalamin hybrid with a diester linker (compound 33) showed high selectivity towards melanoma cancer cells (selectivity index = 9.6). Antiproliferative and pro-apoptotic potential of compounds 12 and 22 against MCF-7 cancer cells were further investigated. Cell cycle studies revealed increased G2/M population in MCF-7 cultures as well as reduced G0/G1 population compared to the control groups indicating cell cycle arrest in G2/M phase. In addition, the frequency of positive cells for annexin V was higher in treated samples suggesting that compounds 12 and 22 induce apoptosis in estrogen-positive MCF-7 cells.

In vitro modeling of tumor interclonal interactions using breast cancer cell lines.

To study the peculiarities of ecological relationships of breast cancer (BC) cell lines MCF-7, BT-474and MDA-MD-231under co-culturing conditions. Three BC cell lines: luminal A- MCF-7, luminal B- BT-474and triple-negative- MDA-MD-231were co-cultured pairwise. Immunocytochemistry was used to differentiate the cell lines in the wells. The effect of the cell-free culture medium on the growth rate of the alternate cell line in the pair was also evaluated. It was shown that when BT-474cells were co-cultured with MCF-7and BT-474cells were co-cultured with MDA-MD-231, two types of ecological interactions could be observed: commensalism and amensalism, respectively. While the cells do not interact with each other in contact, the supernatants of single cultures of MCF-7and MDA-MD-231exert the same effect on BT-474as co-cultivation of BT-474with these cells. The paracrine mechanism of intercellular interaction between different human BC cell lines has been demonstrated. The models used in population ecology can be applicable to identify the types of interaction between cell lines.

Reducing PMA-induced COX-2 expression using a herbal formulation in MCF-7 breast cancer cells

Cyclooxygenase-2 (COX-2) is the inducible form of a group of enzymes that catalyze the conversion of arachidonic acid to prostaglandins. This isoform is often overexpressed in breast cancer cells. COX-2 overexpression correlates with an aggressive phenotype in breast cancer, including higher histological grade, larger tumor size, estrogen receptor (ER) negativity, and human epidermal growth factor receptor 2 (HER-2)/neu positivity. Another study, via molecular, animal, and human investigations, supported that COX-2 expression increases as cancer develops, getting progressively worse during the metastatic phase through the release of vascular endothelial growth factor (VEGF). Traditional medicine or herbal compounds have been gaining increasing popularity for alternative treatment for cancer alone and in conjunction with treatments. Green tea and turmeric are famous for their numerous beneficial effects on the body, including anti-oxidative, chemo-preventive, chemo-protective, anti-inflammatory properties. COX-2 expression was induced using phorbol-12-myristate-13-acetate (PMA). This study tested the effect of the herbal formulation HF1 (mainly composed of (-)-epigallocatechin-3-gallate (EGCG) and curcumin) on the COX-2 mRNA levels in the breast cancer cell line MCF-7. We hypothesized that PMA-induced overexpression of COX-2 in MCF7 cells would be eliminated after exposure to HF1. PMA increased COX-2 levels by 3-fold as compared to the untreated MCF-7 culture (no PMA, no HF1). However, when cells were treated with HF1 alongside PMA, we observed a 60% decrease in COX-2 levels.

Abstract LB-180: A human breast cancer 3D spheroid platform for microscopy based high throughput screening

Abstract Breast cancer (BC) tumors originate in epithelial cells and rank second as a cause of cancer-related death in women (in 2020, ~270,000 women will be diagnosed, and 42,000 women will die from this disease in the USA). While important progress has been made on new treatments there is an urgent need to discover new therapeutics. We aim to discover therapeutics that elicits differentiation of BC tumor cells towards a phenotype that ceases proliferation and/or becomes more sensitive to therapeutic drugs. Also, since current high-throughput breast cancer drug discovery screens use conventional 2D cell cultures that do not fully represent the complex microenvironments of tumors in patients and often fail to predict clinical efficacy we are using 3D spheroids to better represent patient tumors. To identify potential therapeutic agents, we will quantify effects of test compounds on epigenetic markers (histone methylation/acetylation) within the nuclei of the tumor cells utilizing the Microscopic Imaging of Epigenetic Landscape (MIEL) assay developed by Alexey Terskikh's laboratory at SBP. The changes in epigenetics occur more rapidly than phenotypic changes resulting from differentiation which enables more rapid screening to identify potential therapeutic agents. Here we show that treating 2D cell cultures of the breast cancer cell line MCF7 with Tamoxifen, DPPE or E2 elicit changes in the epigenetic landscape. These epigenetic changes concur with increase in lipid droplet formation (a feature that is linked to milk-producing breast epithelium), and demonstrates that MIEL can be used as a readout of differentiation status. We are developing protocols to image breast cancer 3D spheroids on Vala Sciences' IC200 Structured Illumination Microscopy (IC200-SIM™ workstation). The SIM™ uses a multi-million array of micro-mirrors to achieve confocal resolution with rapid image acquisition, suitable for high throughput screening of spheroids cultured in the 96- or 384-well format. We envision that when developed, the MIEL-Breast Cancer Differentiation (MIEL-BCD) assay will be used to screen large chemical libraries (e.g., libraries with 100,000 different chemical entities) to identify compounds that promote differentiation of breast cancer cells to non-tumorigenic cells. Citation Format: Christine Gjerdrum Rines, Kara Gordon, Ranor Basa, Alyson Smith, Robert Oshima, Chen Farhy, Alexey Terskikh, Patrick McDonough, Jeffrey Price. A human breast cancer 3D spheroid platform for microscopy based high throughput screening [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-180.

The effect of cutting style on the biosynthesis of phenolics and cellular antioxidant capacity in wounded broccoli

To explore the effect of cutting style on the biosynthesis of phenolic compounds and cellular antioxidant capacity in wounded broccoli subjected to different cutting styles (heads, florets, 1/2 florets and shredded florets), the mechanism of the accumulation of phenolic compounds was investigated at the transcriptional level, and cellular antioxidant capacity was measured using a breast cancer cell MCF-7 culture model. The results indicated that the relative expression of the genes encoding phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase and 4-coumarin coenzyme A ligase, three enzymes involved in phenylpropanoid metabolism, was upregulated and that contributed to the synthesis of individual phenolic compounds, including catechin, hydroxybenzoic acid, chlorogenic acid, caffeic acid, sinapic acid, catechin gallate, rutin, cinnamic acid and quercetin. This research constructes the phenol synthesis pathway in wounded broccoli. Moreover, the relative expression of critical genes including superoxide dismutase, peroxidase, catalase, glutathione peroxidase and glutathione reductase involved in the metabolism of reactive oxygen species (ROS) increased, resulting in enhanced antioxidant capacity in wounded broccoli. Cell antioxidant capacity (CAA) of heads, florets, 1/2 florets and shredded florets increased by 52.7%, 59.2%, 64.8% and 86.5%, respectively, compared to whole broccoli. The enhancement of CAA was greater as the intensity of wounding increased, indicating that enhancement of antioxidant activity occurred at the cellular level. This research helps provide a reliable and persuasive theoretical basis for nutritional value assessment at the cellular level in wounded broccoli.

Dedifferentiation of MCF-7 Breast Cancer Continuous Cell Line, Development of Breast Cancer Stem Cells (BCSCs) Enriched Culture and Biomarker Analysis

BACKGROUND: Cancer stem cells (CSCs) eradication might serve as a robust approach for cancer eradication. MCF-7 as breast cancer continuous cell line is known to contain breast CSCs (BCSCs) for its capability to maintain its original tumor population. CSCs enriched culture is a fundamental tool for CSCs targeted therapy development. Effective and unsophisticated CSCs dedifferentiation protocol for producing CSCs enriched culture is needed.METHODS: MCF-7 cells were cultured initially in Dulbecco's Modified Eagle Medium (DMEM) low glucose medium then changed to DMEM:F12. Serum starvation was performed during each medium refreshment gradually with fetal bovine serum (FBS) concentration of 10%, 5%, 2.5% until reaching 1% FBS concentration. Stable MCF-7 culture was then adapted to serum free culture system, containing DMEM:F12, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and B27 supplement as dedifferentiation protocol for 18 days. Cluster of differentiation (CD)44 and CD24 double staining immunocytochemistry was performed to evaluate cell stemness.RESULTS: The population of cells expressing BCSCs markers (CD44+/CD24low) in non-adherent single cells subpopulation was significantly increased after the dedifferentiation procedure (70.39%) compared to control groups (0.71%) (p&lt;0.05). In contrast, the expression of BCSCs marker in adherent single cells subpopulation and for both adherent and non-adherent mammosphere the BCSCs markers showed a stable expression.CONCLUSION: BCSCs enrichment of breast cancer cell cultures from MCF-7 breast cancer cell line can be performed. Breast cancer cell plasticity is observed during the dedifferentiation protocol. Development of dedifferentiation inducing protocols can serve as an important foundation for breast cancer therapy development through BCSCs elimination.KEYWORDS: breast neoplasms, cell line, dedifferentiation, immunohistochemistry, neoplastic stem cells

Biological and Spectroscopic Investigations of New Tenoxicam and 1.10-Phenthroline Metal Complexes.

In the present work, tenoxicam (H2Ten) reacted with Mn(II), Co(II), Ni(II), Cu(II) and Zn (II) ions in the presence of 1.10-phenthroline (Phen), forming new mixed ligand metal complexes. The properties of the formed complexes were depicted by elemental analyses, infrared, electronic spectra, proton nuclear magnetic resonance (1H NMR), mass spectrometry, thermogravimetric (TGA) and differential thermogravimetric (DTG) analysis, molar conductance and magnetic moment. IR spectra demonstrated that H2Ten acted as a neutral bidentate ligand, coordinated to the metal ions via the pyridine-N and carbonyl group of the amide moiety, and Phen through the nitrogen atoms. Kinetic thermodynamics parameters activation energy (E*), enthalpy of activation (ΔH*), entropy of activation (ΔS*), Gibbs, free energy (ΔG*) associated to the complexes have been evaluated. Antibacterial screening of the compounds was carried out in vitro against Clavibacter michiganensis, Xanthomonas campestris and Bacillus megaterium. Antifungal activity was performed in vitro against Monilinia fructicola, Penicillium digitatum and Colletotrichum acutatum. The possible phytotoxic effect of the studied compounds was also investigated on Solanum lycopersicum (tomatoes) and Lepidium sativum (garden cress) seeds. The anticancer activity was screened against cell cultures of HCT-116 (human colorectal carcinoma), HepG2 (human hepatocellular carcinoma) and MCF-7 (human breast adenocarcinoma).

Protease Cargo in Circulating Exosomes of Breast Cancer and Ovarian Cancer Patients

Background:As is known, exosomes play an important role in promoting progression of cancers by increasing its invasive potential. The aim of this study was to evaluate the levels of tetraspanine-associated (ADAM-10) and tetraspanine-nonassociated proteases (20S proteasomes) in exosomes from culture medium, plasma exosomes of patients with breast tumors and plasma and ascites of ovarian tumor patients.Methods:MCF-7 and SVO-3 culture mediums and blood samples from healthy females (n = 30, HFs), patients with diffuse dyshormonal dysplasia of the breast (n=28, BBTPs), breast cancer patients (n=32, BCPs), borderline ovarian tumor patients (n=20, BOTPs) and blood and ascites samples ovarian cancer patients (n=35, OCPs) were included in the study. Exosomes from plasma, ascites and culture mediums were isolated and characterized in according to Extracellular Vesicles Society. The expression levels of 20S proteasome and ADAM-10 in exosomes were determined using flow cytometry and western blot analysis, correspondingly.Results:The subpopulation composition of the exosomes from MCF-7 culture medium and from blood plasma of HFs and breast diseases patients is similar, however CD9/CD24 subpopulation significantly increased at cell supernatant. The similar results was obtained for exosomes from SVO-3 medium and blood plasma and ascites of ovary tumor patients, but CD9/CD24 subpopulation significantly decreased at cells and illness samples, however CD63/CD24 exosomes increased significantly from cell supernatant. 20S proteasome level is significantly increased in exosomes from MCF-7 and SVO-3 culture medium, breast tumor patients and OCPs plasma in comparison to HUVEC culture medium and HFs plasma samples. At CD9-positive exosomes from BCPs plasma and MCF-7 was reveal a high expression of ADAM-10 and low expression is from BBDPs plasma and ovarian tumor patients plasma/ascites samples. Exosomes from ascites OCP had high expression of ADAM-10 in the CD24-positive subpopulation.Conclusion:Breast and ovarian cancer development is connected with functioning of immune proteasome forms in plasma and ascites exosomes, while increased ADAM10 expression at CD9-positive exosome was associated with breast cancer and at CD24-positive subpopulation – with ovarian cancer. Obtained data confirm role of exosomal proteases in tumor progression.

Entosis and Cell Cycle in Tumor Cell Culture

Entosis is a type of cell cannibalism during which one tumor cell invades another. The fate of the inner cell can vary. It can leave the entotic vacuole, divide within it, or be subjected to lysosome-mediated degradation. The aim of our work was to determine whether MCF7 (p53+) human breast adenocarcinoma cells and A431 (p53-) human epidermoid carcinoma cells can pass through the cell cycle during entosis. The percentage of entotic cells was 1.01 ± 0.37% in MCF7 culture and 0.42 ± 0.27% in A431 culture. It was shown that inner cells, as well as outer cells, can synthesize DNA (BrdU incorporation) and enter mitosis. Morphometric analysis showed polyploidization of outer cells. This process is most pronounced in the A431 (p53–) cell line. In addition, polyploid cells may be the preferred targets of invasion in this culture. In the MCF-7 cell line, the number of G1-phase entotic cells was higher, probably due to p53-mediated cell cycle arrest or the preferred cell invasion in the G1-phase. Overall, in tumors with active p53 protein expression, the entosis contribution to polyploidy and genetic instability of tumor cells is less than in p53 tumor cells.

Synthesis and Intramolecular Cyclization of a 2,3-seco-Oleanane Triterpenoid with an Ethylketone Fragment

1-Cyano-2,3-seco-18αH-oleanane 3-ethyl-3-ketone was synthesized and underwent base-catalyzed intramolecular oxo-nitrile C(1)–C(3) cyclization to produce the A-pentacyclic 1-cyano-3-ethyl-1(3)-alkene. Nitrile-anionic C(1)–C(31) cyclization of the Br-substituted 3-ethyl-3-ketone occurred with loss of the cyano group and formation of an α,β-unsaturated ketone in a six-membered ring A. The cytotoxic properties of the synthesized compounds against cultures of tumor cell lines HEp-2, HCT 116, MS, RD TE32, A549, MCF-7, and PC3 were studied.