Breast Adenocarcinoma Cell Line MCF-7 Research Articles

Unveiling the therapeutic potential of anthocyanin/cisplatin-loaded chitosan nanoparticles against breast and liver cancers

Abstract Background Liver and breast cancers are among the leading causes of cancer-related deaths worldwide, prompting researchers to seek natural anticancer agents and reduce chemotherapy side effects. Red beetroot (Beta vulgaris Linnaeus), rich in polyphenols and powerful antioxidants, has shown potential in cancer prevention. This study aimed to evaluate the impact of red beetroot-derived anthocyanin (Ant), Ant-loaded chitosan nanoparticles (Ant NPs), cisplatin (Cis), Cis-loaded chitosan (Cis NPs), and Cis + Ant-loaded chitosan NPs on human hepatoma HepG2 and breast adenocarcinoma MCF7 cell lines. Methods NPs preparation was evaluated by zeta potential, FTIR, and SEM. The cytotoxic, apoptotic, antioxidant, anti-inflammatory, anti-metastatic, and anti-angiogenic effects were assessed by MTT assay, qPCR, AO/EB staining, and flow cytometry. Results Treatment with Ant, Ant NPs, Cis, Cis NPs, and Cis + Ant NPs caused cytotoxicity in HepG2 and MCF7 with best effect in Cis-treated cells. The anticancer effects were attributed to mitochondrial-dependent apoptosis (with high Bax and low Bcl2 expression), chromatin disintegration, and cell cycle arrest in G2/M and S phases. All treatments inhibited migration by downregulating the migration-related gene MMP9 and upregulating the anti-migratory gene TIMP1 and decreased the angiogenesis-related gene VEGF and the inflammatory gene TNFα with best results in Cis NPs-treated cells. Interestingly, Ant, Ant NPs, and Cis + Ant NPs increased the antioxidant status (high GSH and upregulated expression of Nrf2 and OH-1) and decreased drug resistance-related MAPK1 and MDR1 genes compared to Cis and Cis NPs-treated cells. Conclusions Anthocyanin and cisplatin-loaded chitosan nanoparticles effectively combat breast and liver cancers by inducing cancer cell apoptosis, enhancing antioxidant defenses, and reducing inflammation. They also inhibit tumor spread and blood vessel formation through downregulation of MMP9 and VEGF, highlighting their therapeutic potential.

Antiproliferative potential of Plinia edulis leaves

Plinia edulis, popularly known as ‘cambucá’, is used in Brazilian folk medicine to treat several conditions. The aim of this study was to evaluate the mutagenicity and cytotoxicity of cambucá leaf extract, as well as its antioxidant activity. The ‘Ames’ test and Orac assay were used to evaluate the mutagenicity and antioxidant activity, respectively, and the phytochemical analysis of the ethanol leaf extract (LE) was performed by chromatographic and spectrometric methods. The in vitro cytotoxicity was tested on CHO (Chinese Hamster Ovary) cells and on nine human tumor cell lines. Phytochemical analysis of LE has revealed the presence of triterpenes and flavonoids. The LE did not show mutagenicity at the concentration of 20 mg plate-1 and exhibited high antioxidant activity, with Orac value of 3948 µmol TE g-1. The LE and its fractions (ethyl acetate; methanol) showed antiproliferative activity against cancer cell lines and proliferative activity in normal cells. The ethyl acetate fraction was the most active extract, presenting antiproliferative activity against strains of breast cancer, prostate cancer, colon cancer, lung cancer, melanoma and leukemia (GI50 0.01-8.57 µg mL-1), being more effective than the reference drug (doxorubicin) against human breast adenocarcinoma MCF-7 cell line. These results suggest P. edulis as a potential adjuvant in cancer therapy and chemoprevention.

Synthesis, cytotoxicity and antioxidant activity of new conjugates of N-acetyl-d-glucosamine with α-aminophosphonates

A series of the first conjugates of N-acetyl-d-glucosamine with α-aminophosphonates was synthesized using the Kabachnik-Fields reaction, the Pudovik reaction, a copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) and evaluated for the in vitro cytotoxicity against human cancer cell lines M − HeLa, HuTu-80, A549, PANC-1, MCF-7, T98G and normal lung fibroblast cells WI-38. The tested conjugates, with exception of compound 21b, considered as a lead compound, were either inactive against the used cancer cells or showed moderate cytotoxicity in the range of IC50 values 33−80 μM. The lead compound 21b, being non cytotoxic against normal human cells WI-38 (IC50 = 90 μM), demonstrated good activity (IC50 = 17 μM) against breast adenocarcinoma cells (MCF-7) which to be 1.5 times higher than the activity of the used reference anticancer drug tamoxifen (IC50 = 25.0 μM). A flexible receptor molecular docking simulation showed that the cytotoxicity of the synthesized conjugates of N-acetyl-d-glucosamine with α-aminophosphonates against breast adenocarcinoma MCF-7 cell line is due to their ability to inhibit EGFR kinase domain. In addition, it was found that conjugates 22a and 22b demonstrated antioxidant activity that was not typical for α-aminophosphonates.

Phyto-synthesized silver nanoparticles from Sargassum subrepandum: anticancer, antimicrobial, and molluscicidal activities.

In the realm of nanotechnology, the use of algae to produce nanoparticles is an environmentally friendly, sustainable, and economically viable strategy. In the present study, the brown macroalgae Sargassum subrepandum was utilized to effectively produce silver nanoparticles (AgNPs). Through various characterization techniques, the AgNPs' structural integrity was confirmed. AgNPs exhibited significant antimicrobial activity against Pseudomonas aeruginosa and Fusarium equiseti. AgNPs showed cytotoxic effects on the MCF-7 breast adenocarcinoma cell line with an IC50 of 12.5 µg/ml. Treatment with AgNPs resulted in a marked reduction in cell viability, alongside evident apoptotic and necrotic morphological changes in the cancer cells. Through molecular docking studies, a deeper understanding of the interaction between AgNPs and crucial proteins related to cancer has been achieved, AgNPs showed a promising molluscicidal action on Biomphalaria alexandrina snails, a Schistosoma mansoni intermediate host. The half-lethal dose (LC50) of AgNPs was determined to be 0.84 mg/L. The potential consequences of its administration include potential disruptions to the glycolysis profile, as well as potential impacts on the steroidal hormone's estrogen and testosterone and certain kidney function tests. This study highlights the diverse uses of algae-synthesized AgNPs, ranging from healthcare to environmental management, demonstrating their importance in advancing nano-biotechnological solutions.

Fabrication and characterization of a new eco-friendly sulfonamide-chitosan derivative with enhanced antimicrobial and selective cytotoxicity properties

Chitosan (CH) exhibits low antimicrobial activity. This study addresses this issue by modifying the chitosan with a sulfonamide derivative, 3-(4-(N,N-dimethylsulfonyl)phenyl)acrylic acid. The structure of the sulfonamide-chitosan derivative (DMS-CH) was confirmed using Fourier transform infrared spectroscopy and Nuclear magnetic resonance. The results of scanning electron microscopy, thermal gravimetric analysis, and X-ray diffraction indicated that the morphology changed to a porous nature, the thermal stability decreased, and the crystallinity increased in the DMS-CH derivative compared to chitosan, respectively. The degree of substitution was calculated from the elemental analysis data and was found to be moderate (42%). The modified chitosan exhibited enhanced antimicrobial properties at low concentrations, with a minimum inhibitory concentration (MIC) of 50 µg/mL observed for B. subtilis and P. aeruginosa, and a value of 25 µg/mL for S. aureus, E. coli, and C. albicans. In the case of native chitosan, the MIC values doubled or more, with 50 µg/mL recorded for E. coli and C. albicans and 100 μg/mL recorded for B. subtilis, S. aureus, and P. aeruginosa. Furthermore, toxicological examinations conducted on MCF-7 (breast adenocarcinoma) cell lines demonstrated that DMS-CH exhibited greater toxicity (IC50 = 225.47 μg/mL) than pure CH, while still maintaining significant safety limits against normal lung fibroblasts (WI-38). Collectively, these results suggest the potential use of the newly modified chitosan in biomedical applications.

Design, Synthesis and In-Silico Studies of Piperidine-Dihydropyridine Hybrids as Anticancer Agents.

In this study, we designed, synthesized and characterized a novel series of piperidine-dihydropyridine hybrid compounds and characterized them by 1H-NMR, 13C NMR, mass spectrometry (MS), and elemental analysis. Subsequently, we assessed their in vitro anticancer potentials against the human breast adenocarcinoma cell line MCF-7 and the lung cancer cell line A-549. Several of these compounds demonstrated significant activity, with IC50 values ranging from 15.94 μM to 48.04 μM for A-549 and 24.68 μM to 59.12 μM for MCF-7, when compared to the reference drug Cisplatin.Notably, a compound featuring a 3-fluoro substitution in the carboxamide series exhibited robust inhibitory effects, with an IC50 of 15.94±0.201 μM against A-549 cells and an IC50 of 22.12±0.213 μM against MCF-7 cells, respectively. Additionally, a compound containing a cyclobutyl ring displayed potent activity, with an IC50 of 16.56±0.125 μM against A-549 and an IC50 of 24.68±0.217 μM against MCF-7 cells, respectively. Furthermore, molecular docking studies against the Epidermal Growth Factor Receptor (EGFR) (PDB ID: 2J6M) revealed favourable binding scores and interactions, suggesting their potential as promising candidates for further investigation in the context of anticancer drug development.

Synthesis and Characterization of Paclitaxel-Loaded Silver Nanoparticles: Evaluation of Cytotoxic Effects and Antimicrobial Activity.

Carrier system therapies based on combining cancer drugs with nanoparticles have been reported to control tumor growth and significantly reduce the side effects of cancer drugs. We thought that paclitaxel-loaded silver nanoparticles (AgNPs-PTX) were the right carrier to target cancer cells. We also carried out antimicrobial activity experiments as systems formed with nanoparticles have been shown to have antimicrobial activity. In our study, we used easy-to-synthesize and low-cost silver nanoparticles (AgNPs) with biocatalytic and photocatalytic advantages as drug carriers. We investigated the antiproliferative activities of silver nanoparticles synthesized by adding paclitaxel on MCF-7 (breast adenocarcinoma cell line), A549 (lung carcinoma cell line), C6 (brain glioma cell line) cells, and healthy WI-38 (fibroblast normal cell line) cell lines and their antimicrobial activities on 10 different microorganisms. The synthesized AgNPs and AgNPs-PTX were characterized by dynamic light scattering (DLS), scanning transmission electron microscopy, UV-visible spectroscopy, Fourier transform infrared spectroscopy, and X-ray spectroscopy. The nanoparticles were spherical in shape, with AgNPs ranging in size from 2.32 to 5.6 nm and AgNPs-PTXs from 24.36 to 58.77 nm. AgNPs demonstrated well stability of -47.3 mV, and AgNPs-PTX showed good stability of -25.4 mV. The antiproliferative effects of the synthesized nanoparticles were determined by XTT (tetrazolium dye; 2,3-bis-(2-methoxy-4-nitro-5-sulfenyl)-(2H)-tetrazolium-5-carboxanilide), and the proapoptotic effects were determined by annexin V/propidium iodide (PI) staining. The effect of AgNPs-PTX was more effective, and anticancer activity was higher than PTX in all cell lines. When selectivity indices were calculated, AgNPs-PTX was more selective in the A549 cell line (SI value 6.53 μg/mL). AgNPs-PTX was determined to increase apoptosis cells by inducing DNA fragmentation. To determine the antimicrobial activity, the MIC (minimum inhibitory concentration) test was performed using 8 different bacteria and 2 different fungi. Seven of the 10 microorganisms tested exhibited high antimicrobial activity according to the MIC ≤100 μg/mL standard, reaching MIC values below 100 μg/mL and 100 μg/mL for both AgNPs and AgNPs-PTX compared to reference sources. Compared to standard antibiotics, AgNPs-PTX was highly effective against 4 microorganisms.

Differences in nonoxidative sulfur metabolism between normal human breast MCF-12A and adenocarcinoma MCF-7 cell lines

Recent studies have revealed the role of endogenous hydrogen sulfide (H2S) in the development of breast cancer. The capacity of cells to generate H2S and the activity and expression of the main enzymes (cystathionine beta synthase; CBS, cystathionase γ-lyase; CGL, 3-mercaptopyruvate sulfurtransferase; MPST and thiosulfate sulfurtransferase; TST) involved in H2S metabolism were analyzed using an in vitro model of a non-tumourigenic breast cell line (MCF-12A) and a human breast adenocarcinoma cell line (MCF-7). In both cell lines, MPST, CGL, and TST expression was confirmed at the mRNA (RT-PCR) and the protein (Western Blot) level, while CBS expression was detected only in MCF-7 cells. Elevated levels of GSH, sulfane sulfur and increased CBS and TST activity were presented in the MCF-7 compared to the MCF-12A cells. It appears that cysteine might be mainly a substrate for GSH synthesis in breast adenocarcinoma. Increased capacity of the cells to generate H2S was shown for MCF-12A compared to MCF-7 cell line. Results suggest an important function of CBS in H2S metabolism in breast adenocarcinoma. The presented work may contribute to further research on new therapeutic possibilities for breast cancer - one of the most frequently diagnosed types of cancer among women.

Design, Synthesis of Novel 1,2,3-Triazole Pendent Quinazolinones and Their Cytotoxicity against MCF-7 Cell Line.

A library of 6-(((1-(substitutedphenyl)-1H-1,2,3-triazol-4-yl)methyl) amino)-3-methylquinazolin-4(3H)-one analogues synthesized from Isatin precursor through a series of nitration, reduction, hydrolysis, cyclization and click reaction. The structures of compounds were characterized by spectral data including IR, 1 H-NMR, 13 C NMR and Mass. The novel quinazolinone - 1,2,3-triazoles were screened for their cytotoxicity against the human breast adenocarcinoma cell lines MCF-7 by MTT assay. 4-Isopropyl and 2-bromo substituted analogues executed high activity against MCF-7 cell line with IC50 value of 10.16±0.07 μM and 11.23±0.20 μM compared to the Doxorubicin whose IC50 value is 10.81±0.03 μM. The activity of remaining compounds is good to moderate. Further, the molecular docking studies against the crystal structure of Epidermal Growth Factor Receptor delivered the best binding energies and the interactions such as H-bond and hydrophobic are inevitable. The predicted pharmacokinetic properties results showed that these compounds have more drug likeness properties.

Polysciasoside B and C: new dammarane-type triterpene glycosides from the leaves of Australian rainforest plant Polyscias australiana (F.Muell.) Philipson (Araliaceae)

Phytochemical investigation of the leaves of Polyscias australiana (F.Muell.) Philipson (family Araliaceae) led to the isolation and identification of two new analogues belonging to the rare dammarane-type triterpene glycosides, polysciasosides B (1) and C (2). Also isolated in high yields from this plant was the known saponin, β-hedrin (3). The two new polysciasoside analogues exhibited no anti-inflammatory activity (inhibitory effects on NO inhibition and cell viability in RAW 264.7 macrophages) or cytotoxic activity against AGS gastric adenocarcinoma or the MCF7 breast adenocarcinoma cell lines. In contrast, the known compound β-hedrin exhibited potent anti-inflammatory and cytotoxicity in these biological assays.

Phenolic Compounds from Sour Cherry Pomace: Microencapsulation, in Vitro Digestion, and Cell Growth Activities.

The objective of this work was the valorisation of sour cherry (Prunus cerasus L.) pomace as a source of biologically active compounds. To formulate microcapsules, polyphenolic compounds were extracted and encapsulated with maltodextrin as wall material, by freeze-drying. An in vitro digestion study was carried out on obtained encapsulates but also on sour cherry pomace extract and sour cherry pomace freeze-dried powder. The results indicated that encapsulation, as well as freeze-drying, provided a good protective effect on bioactive compounds during digestion. Furthermore, the potential antiproliferative and cytotoxic activities of encapsulates and sour cherry pomace extract were evaluated using breast adenocarcinoma MCF7 cell lines, colon adenocarcinoma HT-29 cell lines, and noncancer cell line. Encapsulates and sour cherry pomace extract showed variable anti-proliferative activity towards all cell lines. Obtained results showed that encapsulation of sour cherry pomace could be useful for improving the stability of polyphenolic compounds in the gastrointestinal tract. The results highlight the bioactive potential of sour cherry pomace as a nutraceutical resource and the protective effects of microencapsulation on the digestion of bioactive compounds.

Cytotoxic Activity of Atmospheric Cold Plasma Jet Towards 3D Human Breast Cancer Cell Model

The treatment of solid tumors with a cold atmospheric plasma jet (CAP) is an innovative approach, which began to be actively developed only in the last decade. As a consequence, the studies aimed at revealing the conditions of selectivity of such effects on tumor cells, including in 3D tumor models, are important. It is known that the main cytotoxic effects of CAP are caused by reactive oxygen and nitrogen species, which are formed in the plasma flow and the availability of which for the cells in the classical 2D and 3D cultivation models may be different. We used multicellular spheroids of MCF7-EGFR cells with hyperexpression of epidermal growth factor receptor (EGFR), the parental MCF7 breast adenocarcinoma cell line, and MCF10A non-transformed human breast cells. Irradiation of MCF7-EGFR spheroids led to destruction of multicellular 3D structures into individual cells with activation of death processes. It was shown that cells of CAP-irradiated spheroids underwent phagocytosis by activated macrophages. When comparing direct exposure to CAP and cultivation of MCF7-EGFR spheroids in CAP-irradiated medium (CAP-IM), a higher content of reactive oxygen and nitrogen species in spheroid cells was found when cultured in CAP-IM, which further leads to a greater cytotoxic effect than in direct irradiation. The cytotoxicity of CAP-IM has been shown to be valid longer when such medium is stored at 4 than at −20°С. Thus, it was shown that the treatment of spheroids with CAP-IM was more effective in death induction than direct CAP irradiation.

QUASSINOIDS FROM THE ROOTS OF Brucea javanica AND THEIR POTENTIAL CYTOTOXICITY

A phytochemical study of the roots of Brucea javanica was conducted and four alkaloids and three quassinoids were isolated. The structures of the compounds were verified using physical and spectroscopic data, which were compared to the literature. The cytotoxicity of the compounds was evaluated against KB (oral cavity carcinoma), NCI-H187 (small cell lung cancer), and MCF-7 (breast adenocarcinoma) cell lines. The results showed that the quassinoid, Bruceine A, exhibited very strong cytotoxicity against KB, and high potency against NCI-H187, with both being 581- and 100-fold more active than ellipticine, and 99- and 10-fold more active than doxorubicin, respectively. These results suggest the potential use of bruceine A as an anticancer agent

3-(1,2,3-Triazol-4-yl)-β-Carbolines and 3-(1H-Tetrazol-5-yl)-β-Carbolines: Synthesis and Evaluation as Anticancer Agents.

Herein, the synthesis and anticancer activity evaluation of a series of novel β-carbolines is reported. The reactivity of nitrosoalkenes towards indole was explored for the synthesis of novel tryptophan analogs where the carboxylic acid was replaced by a triazole moiety. This tryptamine was used in the synthesis of 3-(1,2,3-triazol-4-yl)-β-carbolines via Pictet-Spengler condensation followed by an oxidative step. A library of compounds, including the novel 3-(1,2,3-triazol-4-yl)-β-carbolines as well as methyl β-carboline-3-carboxylate and 3-tetrazolyl-β-carboline derivatives, was evaluated for their antiproliferative activity against colorectal cancer cell lines. The 3-(1H-tetrazol-5-yl)-β-carbolines stood out as the most active compounds, with values of half-maximal inhibitory concentration (IC50) ranging from 3.3 µM to 9.6 µM against colorectal adenocarcinoma HCT116 and HT29 cell lines. The results also revealed a mechanism of action independent of the p53 pathway. Further studies with the 3-tetrazolyl-β-carboline derivative, which showed high selectivity for cancer cells, revealed IC50 values below 8 μM against pancreatic adenocarcinoma PANC-1, melanoma A375, hepatocarcinoma HEPG2, and breast adenocarcinoma MCF-7 cell lines. Collectively, this work discloses the 3-tetrazolyl-β-carboline derivative as a promising anticancer agent worthy of being further explored in future works.

Cytotoxic Potential of Alternaria tenuissima AUMC14342 Mycoendophyte Extract: A Study Combined with LC-MS/MS Metabolic Profiling and Molecular Docking Simulation.

Breast, cervical, and ovarian cancers are among the most serious cancers and the main causes of mortality in females worldwide, necessitating urgent efforts to find newer sources of safe anticancer drugs. The present study aimed to evaluate the anticancer potency of mycoendophytic Alternaria tenuissima AUMC14342 ethyl acetate extract on HeLa (cervical cancer), SKOV-3 (ovarian cancer), and MCF-7 (breast adenocarcinoma) cell lines. The extract showed potent effect on MCF-7 cells with an IC50 value of 55.53 μg/mL. Cell cycle distribution analysis of treated MCF-7 cells revealed a cell cycle arrest at the S phase with a significant increase in the cell population (25.53%). When compared to control cells, no significant signs of necrotic or apoptotic cell death were observed. LC-MS/MS analysis of Alternaria tenuissima extract afforded the identification of 20 secondary metabolites, including 7-dehydrobrefeldin A, which exhibited the highest interaction score (−8.0156 kcal/mol) in molecular docking analysis against human aromatase. Regarding ADME pharmacokinetics and drug-likeness properties, 7-dehydrobrefeldin A, 4’-epialtenuene, and atransfusarin had good GIT absorption and water solubility without any violation of drug-likeness rules. These findings support the anticancer activity of bioactive metabolites derived from endophytic fungi and provide drug scaffolds and substitute sources for the future development of safe chemotherapy.

New Jatrophane-Type Diterpenoids from Euphorbia kansui as Potential MDR Reversal Agents.

A serial jatrophane-type diterpenoids, comprised with three undescribed compounds kanesulones C-E (1-3) and four known ones (4-7), were obtained from the roots of Euphorbia kansui. The structures of compounds 1-3 were elucidated by detailed interpretation of their spectroscopic data, especially 2D-NMR and HR-ESI-MS, the absolute configuration of 1 was revealed by single crystal X-ray diffraction. These isolates were assayed for their multidrug resistance reversing activities on human breast adenocarcinoma cell line MCF-7/ADR. Compound 1 possessed potential as low toxic MDR modulator that could promote the efficacy of anticancer drug adriamycin ca. 85-fold at 5 μM, as 12 times stronger than the positive drug verapamil.

Design, Synthesis, Anticancer Activity and Docking Studies of Thiazole Linked Phenylsulfone Moiety as Cyclin-Dependent Kinase 2 (CDK2) Inhibitors

Inhibition of the cyclin dependent kinase 2 (CDK2) is a well-known cancer treatment approach. Based on the molecular docking simulation analysis, two new series of 2-(1-(4-methoxyphenyl)-2-(phenylsulfonyl)ethylidene)hydrazineylidene)-2,5-dihydrothiazole as CDK-2 inhibitors were designed. The lead compound IV (5-benzoyl-N2-phenyl-1,3-thiazole-2,4-diamine) was modified and optimized prior to this analysis. In contrast to Roniciclib (−8.6 kcal/mol), docking results of CDK2 hits showed high affinity and docking scores ranging from −9.1 to −10.3 kcal/mol. All of the compounds studied were evaluated in vitro for CDK2 inhibitory activity. In comparison to Roscovitine's IC50 of 0.432 µM, compound 11b had a maximum IC50 of 0.416 µM. Additionally, all hits were tested for antiproliferative activities against PC-3 human prostate cancer cells, HEPG-2 human hepatocellular carcinoma, and MCF-7 breast adenocarcinoma cell lines. The arylhydrazine moiety of the tested compounds 11a–e demonstrated high potency against HEPG-2, with 11b (IC50 = 0.11 µM) being more active than doxorubicin (IC50 = 0.12 µM). In addition, compound 11b (IC50 = 0.245 µM) was nearly as effective as doxorubicin (IC50 = 0.246 µM) against MCF-7 cancer cells. 11d showed superior antiproliferative activity against PC-3 cell line (IC50= 0.23 µM) relative to doxorubicin (IC50 = 0.29 µM).

3D Bioprinted Artificial Breast Cancer Tumor Disks: A New Tool for Understanding Intra‐tumor Heterogeneity?

Recently the UWRF Tissue and Cellular Innovation Center and the Century College Biofabrication Program have engaged in a series of new collaborative research projects using bioprinting technology and methods to model and study in‐vitro 3D artificial tumor and stromal tissues. In this presentation, we report on results obtained from efforts to bioprint cancer disks using the human breast adenocarcinoma cell line MCF‐7. This breast cancer cell line is considered to be one of the “gold standard” cultures to study this type tumor. In extensive previous studies in our lab using other 3D modeling methods, we have established a robust library of experience and data with these cells and artificial tissue constructs. In these new studies, bioprinting methods were applied to establish tumor structures which initially contained specific cell numbers per unit volume as well as relatively mono‐cellular distribution patterns. Both of these characteristics had been very difficult to predictably generate and control in earlier studies using precast or molded hydrogels made of chitosan and hyaluronic acid or decellularized natural marine sponge ECM. In this project, two distinct bioinks were tested including one called GelMa, a gelatin/methacrylate hydrogel and another called GelMa‐C which is basically the same, but with nanocellulose added to enhance the structural stability of constructs during printing. While both bioinks generated structures and tissues, the GelMa bioink specifically generated relatively transparent tumor structures that provided a unique window into the cell interactions and eventual early development of these artificial tumor tissues. Using a Cellink “Inkcredible” model printer loaned to the TCIC by Century College for the duration of this project, we printed tumor disks with a diameter of 5 mm and thickness of 2.5 mm and then cultured these in 6 well plates for up to 3 months. These breast cancer disks displayed features and tissue progression similar to that seen with the other methods used in our earlier work, while also providing new and unique insights into the interior artificial tumor tissue development in real‐time. These new observations also demonstrated a unique cell/tissue behavior in the resulting constructs that we hypothesize may directly address the origins of intra‐tumor heterogeneity of various cell subpopulations and their relative contributions to the overall tumor development. Future studies are planned to further evaluate and qualify these observations and to apply this new 3D artificial tissue tool to further elucidate cancer biology as a whole.

ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS

Aim. To determine the role of Ruk/CIN85 in the control of breast adenocarcinoma cells metabolism, we performed systemic analysis of the activity levels/content of key enzymes/components of glycolysis and oxidative phosphorylation using as a model the weakly invasive human breast adenocarcinoma MCF-7 cell line (Mock); and its sublines with stable overexpression (G4 subline) and reverse down-regulation (G4vir subline) of the adaptor protein. Materials and methods. MCF-7 cells were cultured in the complete DMEM medium under standard conditions. Enzymes activity, content of metabolites and protein in cell extracts and the conditioned cell culture medium were estimated by spectrophotometric and fluorometric assays. Results. First of all, biochemical indexes of aerobic glycolysis, activity levels of some key glycolytic enzymes and metabolites were evaluated. A significant increase in the activity of these enzymes, aldolase A (ALDOA) and lactate dehydrogenase A (LDHA), was found in G4 cells compared to Mock by 1.3 and 1.6 times, respectively. In addition, in the conditioned medium of G4 cells, an increase in lactate content by 1.5 times compared with the control was found, which corresponded to a change in LDHA activity. Knockdown of Ruk/CIN85 expression level in G4 subline resulted in a significant decrease of these parameters compared to G4 cells, ALDOA – 4 times, LDHA - 1.4 times, and lactate production - 2.5 times. It should be noted that in G4vir cells, LDHA activity returned to level of control cells, while ALDOA activity and lactate content additionally decreased by 3 times and 1.6 times, respectively. Therefore, the observed changes in the intensity of glycolysis in MCF-7 sublines positively correlate with the expression level of adaptor protein studied. To assess the metabolic status of mitochondria, the level of activity of the Krebs cycle enzyme, NAD-dependent malate dehydrogenase (MDH2), the catalyst of last stage of the cycle, was determined. A 2-fold decrease in MDH2 activity was found in the MCF-7 G4 subline relative to control Mock cells, as well as an increase in this index by 2.4 times in G4vir cells to control values. Unlike glycolysis, we observed the opposite pattern with respect to the intensity of Krebs cycle reactions depending on the expression level of Ruk/CIN85. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.

In Vitro Investigation of the Antioxidant and Cytotoxic Potential of Tabernaemontana ventricosa Hochst. ex A. DC. Leaf, Stem, and Latex Extracts

Tabernaemontana ventricosa (Apocynaceae) a latex-bearing plant is used in traditional medicine for its therapeutic benefits in reducing fever and hypertension and wound healing. Due to limited information on the plant’s pharmacological activities, this study aimed to investigate the antioxidant potential of the leaf, stem, and latex extracts of T. ventricosa, using the Folin-Ciocalteu (total phenolics), aluminum chloride colorimetric (total flavonoids), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. The cytotoxic activity was evaluated in the human HEK293 (embryonic kidney), HeLa (cervical carcinoma), and MCF-7 (breast adenocarcinoma) cell lines using the MTT assay. The latex extracts possessed the highest total phenolic content (115.36 ± 2.89 mg GAE/g), followed by the stem hexane extracts (21.33 ± 0.42 mg GAE/g), the chloroform leaf (7.89 ± 0.87 mg GAE/g), and the chloroform stem (4.69 ± 0.21 mg GAE/g) extracts. The flavonoid content was substantially high ranging from 946.92 ± 6.29 mg QE/g in the stem hexane, 768.96 ± 5.43 mg QE/g in the latex, 693.24 ± 4.12 mg QE/g in the stem chloroform, and 662.20 ± 1.00 mg QE/g in the leaf hexane extracts. The DPPH assays showed the highest percentage of inhibition at 240 µg/mL, for the stem hexane (70.10%), stem methanol (65.24%), and stem chloroform (60.26%) extracts, with their respective IC50 values of 19.26 µg/mL (stem hexane), 6.19 µg/mL (stem methanol), and 22.56 µg/mL (stem chloroform). The FRAP assays displayed minimal inhibition ranging from 4.73% to 14.40%, except for the latex extracts which displayed moderate inhibition at 15 µg/mL (21.82%) and substantial inhibition at 240 µg/mL (98.48%). The HeLa and MCF-7 cell lines were the most sensitive to the extracts, with the hexane, chloroform, and methanol leaf and stem, and latex extracts significantly affecting the percentage cell survival. Overall, the various parts of T. ventricosa exhibited strong antioxidant activity correlating to its cytotoxicity. Further studies should focus on the isolation of specific antioxidant compounds that could be investigated for their anticancer potential.