Human Breast Epithelial Cells Research Articles

Mutant RAS-driven secretome causes skeletal muscle defects in breast cancer.

Cancer-induced skeletal muscle defects differ in severity between individuals with the same cancer type. Cancer subtype-specific genomic aberrations are suggested to mediate these differences, but experimental validation studies are very limited. We utilized three different breast cancer patient-derived xenograft (PDX) models to correlate cancer subtype with skeletal muscle defects. PDXs were derived from brain metastasis of triple negative breast cancer (TNBC), Estrogen Receptor-positive/Progesterone Receptor-positive (ER+/PR+) primary breast cancer from a BRCA2-mutation carrier, and pleural effusion from an ER+/PR- breast cancer. While impaired skeletal muscle function as measured through rotarod performance and reduced levels of circulating and/or skeletal muscle miR-486 were common across all three PDXs, only TNBC-derived PDX activated phospho-p38 in skeletal muscle. To further extend these results, we generated transformed variants of human primary breast epithelial cells from healthy donors using HRASG12V or PIK3CAH1047R mutant oncogenes. Mutations in RAS oncogene or its modulators are found in ~37% of metastatic breast cancers, which is often associated with skeletal muscle defects. Although cells transformed with both oncogenes generated adenocarcinomas in NSG mice, only HRASG12V-derived tumors caused skeletal muscle defects affecting rotarod performance, skeletal muscle contraction force, and miR-486, Pax7, pAKT, and p53 levels in skeletal muscle. Circulating levels of the chemokine CXCL1 were elevated only in animals with tumors containing HRASG12V mutation. Since RAS pathway aberrations are found in 19% of cancers, evaluating skeletal muscle defects in the context of genomic aberrations in cancers, particularly RAS pathway mutations, may accelerate development of therapeutic modalities to overcome cancer-induced systemic effects.

Drug repositioning for immunotherapy in breast cancer using single-cell analysis.

Immunomodulatory peptides, while exhibiting potential antimicrobial, antifungal, and/or antiviral properties, can play a role in stimulating or suppressing the immune system, especially in pathological conditions like breast cancer (BC). Thus, deregulation of these peptides may serve as an immunotherapeutic strategy to enhance the immune response. In this meta-analysis, we utilized single-cell RNA sequencing data and known therapeutic peptides to investigate the deregulation of these peptides in malignant versus normal human breast epithelial cells. We corroborated our findings at the chromatin level using ATAC-seq. Additionally, we assessed the protein levels in various BC cell lines. Moreover, our in-house drug repositioning approach was employed to identify potential drugs that could positively impact the relapse-free survival of BC patients. Considering significantly deregulated therapeutic peptides and their role in BC pathology, our approach aims to downregulate B2M and SLPI, while upregulating PIGR, DEFB1, LTF, CLU, S100A7, and SCGB2A1 in BC epithelial cells through our drug repositioning pipeline. Leveraging the LINCS L1000 database, we propose BRD-A06641369 for B2M downregulation and ST-4070043 and BRD-K97926541 for SLPI downregulation without negatively affecting the MHC complex as a significantly correlated pathway with these two genes. Furthermore, we have compiled a comprehensive list of drugs for the upregulation of other selected immunomodulatory peptides. Employing an immunotherapeutic approach by integrating our drug repositioning pipeline with single-cell analysis, we proposed potential drugs and drug targets to fortify the immune system against BC.

Abstract A089: Role of Vardhamana Pippali Rasayana (VPR) in human breast cancer cells

Abstract Serious concerns include delayed diagnosis and adverse side-effects of conventional treatment leading to residual morbidity in Breast cancer (BC) globally. Traditional Indian medicines/herbs via combination of conventional cancer are gaining increasing acceptance worldwide, playing a pivotal role as proven by their efficacy evaluation studies. Many plants are known to reduce the cell proliferation and tumor development after their treatment. Vardhamana Pippali Rasayana (VPR), an Ayurvedic herbal preparation of pippali (Piper longum) in gradually increasing and tapering dose is used to treat many cancers without any adverse side-effects. However, its anti-cancer role in breast cancer is not known yet. The present study has explored the cytotoxic and apoptotic effects of VPR in triplicates on human breast cancer (MCF7) and normal breast epithelial (MCF10A) cell lines at different concentrations after extracting piperine in aqueous and hydro-alcoholic soxhlet extract (0.25 to 50 μg/μl). The cytotoxic activity was carried out by MTT assay and results showed that a low IC50 was observed at 3.75 μg/μl of VPR against MCF-7 as compared with normal breast epithelial cells. Detection of apoptotic cells using suitable Annexin V was done and there was a time-dependent increase in percentage of annexin V positive cells upon treatment. The above results indicate that VPR may have a strong anti-cancerous potential. But other functional assays are warranted to validate the efficacy of the drug against breast cancer which are under progress in our laboratory. Citation Format: Dr.Richa Tripathi, Shivam Singh, Subhash Gupta, VG Huddar, Tanuja M Nesari, Ashok Sharma. Role of Vardhamana Pippali Rasayana (VPR) in human breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A089.

Abstract A063: CancerTools.org: Research Tools Supporting Breast Cancer Studies

Abstract CancerTools.org is the latest, non-profit cancer-focused biorepository. Driven by a mission to accelerate cancer research discoveries, the platform allows scientists to deposit and access tangible laboratory research materials. Till date, researchers from over 175 academic institutes globally have already deposited their tools, generating a cutting-edge portfolio of over 5,000 reagents, including a range of cell-based models and antibodies. Breast cancer is one of the most frequent malignancies in women worldwide. A comprehensive collection of tools has been curated at CancerTools.org to support this important area of research. Cell-based models include the RMAM007-3 breast cancer organoid which recapitulates in vivo tumour characteristics and is suitable for disease modelling, 3D imaging, and anticancer drug discovery. Cell lines such as human anti-oestrogen resistant lines developed from MCF-7 and T47D cells, demonstrate resistance to hormone-dependent treatments and are useful for studying therapy resistance mechanisms and biomarker development for breast cancer detection. NCI-HPN-F murine cell lines with recurrent chromosomal aneuploidies, have enabled investigation and identification of tumorigenesis related cancer-specific genes and signalling pathways. Further models include gene-edited cell lines to explore BRCA1 hypermethylation pathways; reporter breast cancer cell lines to assess apoptosis, necrosis, and autophagy; and immortalised human breast epithelial cell lines to study differentiation and phenotypic transformation. To provide a physiologically relevant cell culture environment, PlasmaxTM, a defined media that mirrors the concentration of over 50 components to human plasma, is also part of the repertoire. It has been used to culture triple-negative breast cancer cell lines to recapitulate the tumour metabolic environment. Antibodies to study breast cancer biology, including tumour suppressors, angiogenesis and metastasis, form part of the portfolio. Antibodies targeting tumour suppressors include RB1, which is often lost by mutation, deletion, transcriptional silencing and hyper-phosphorylation in malignancies- including breast cancer. Deletion and rearrangement of RB1 is estimated in ~20-25% of breast cancer cell lines. Tools identifying specific RB1 phosphorylation sites, include Anti-Phospho-Rb for Ser608 and Thr356. Additional insights into breast cancer prognosis are offered by Anti-Maspin [16F7] which recognises wildtype and denaturated forms of the protein. Maspin is an inhibitor of serine proteases with tumour suppressor activity in breast cancer. Its nuclear staining associates with favourable outcomes in contrast to cytoplasmic staining. MUC1 is also aberrantly overexpressed in 90% of human breast cancer, thus, representing a promising marker for breast cancer diagnosis and prognosis. Further research support is offered by an extensive range of antibodies for MUC1’s different isoforms. Visit our stand to explore more about depositing and accessing tools and technologies that support breast cancer research. Citation Format: Vera Moiseeva, Sara Omenetti, Abby Henney, Katie Valencia. CancerTools.org: Research Tools Supporting Breast Cancer Studies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A063.

The Garlic Compound, Diallyl Trisulfide, Attenuates Benzo[a]Pyrene-Induced Precancerous Effect through Its Antioxidant Effect, AhR Inhibition, and Increased DNA Repair in Human Breast Epithelial Cells.

Exposure to B[a]P, the most characterized polycyclic aromatic hydrocarbon, significantly increases breast cancer risk. Our lab has previously reported that diallyl trisulfide (DATS), a garlic organosulfur compound (OSC) with chemopreventive and cell cycle arrest properties, reduces lipid peroxides and DNA damage in normal breast epithelial (MCF-10A) cells. In this study, we evaluated the ability of DATS to block the B[a]P-induced initiation of carcinogenesis in MCF-10A cells by examining changes in proliferation, clonogenic formation, reactive oxygen species (ROS) formation, 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, and protein expression of ARNT/HIF-1β, CYP1A1, and DNA POLβ. The study results indicate that B[a]P increased proliferation, clonogenic formation, ROS formation, and 8-OHdG levels, as well as increasing the protein expression of ARNT/HIF-1β and CYP1A1 compared to the control. Conversely, DATS/B[a]P co-treatment (CoTx) inhibited cell proliferation, clonogenic formation, ROS formation, and 8-OHdG levels compared to B[a]P alone. Treatment with DATS significantly inhibited (p < 0.0001) AhR expression, implicated in the development and progression of breast cancer. The CoTx also attenuated all the above-mentioned B[a]P-induced changes in protein expression. At the same time, it increased DNA POLβ protein expression, which indicates increased DNA repair, thus causing a chemopreventive effect. These results provide evidence for the chemopreventive effects of DATS in breast cancer prevention.

The Anticancer Effects of the Garlic Organosulfide Diallyl Trisulfide through the Attenuation of B[a]P-Induced Oxidative Stress, AhR Expression, and DNA Damage in Human Premalignant Breast Epithelial (MCF-10AT1) Cells.

Benzo[a]pyrene (B[a]P) is the most characterized polycyclic aromatic hydrocarbon associated with breast cancer. Our lab previously reported that the organosulfur compound (OSC), diallyl trisulfide (DATS), chemoprevention mechanism works through the induction of cell cycle arrest and a reduction in oxidative stress and DNA damage in normal breast epithelial cells. We hypothesize that DATS will inhibit B[a]P-induced cancer initiation in premalignant breast epithelial (MCF-10AT1) cells. In this study, we evaluated the ability of DATS to attenuate B[a]P-induced neoplastic transformation in MCF-10AT1 cells by measuring biological endpoints such as proliferation, clonogenicity, reactive oxygen species (ROS) formation, and 8-hydroxy-2-deoxyguanosine (8-OHdG) DNA damage levels, as well as DNA repair and antioxidant proteins. The results indicate that B[a]P induced proliferation, clonogenic formation, ROS formation, and 8-OHdG levels, as well as increasing AhR, ARNT/HIF-1β, and CYP1A1 protein expression compared with the control in MCF-10AT1 cells. B[a]P/DATS's co-treatment (CoTx) inhibited cell proliferation, clonogenic formation, ROS formation, AhR protein expression, and 8-OHdG levels compared with B[a]P alone and attenuated all the above-mentioned B[a]P-induced changes in protein expression, causing a chemopreventive effect. This study demonstrates, for the first time, that DATS prevents premalignant breast cells from undergoing B[a]P-induced neoplastic transformation, thus providing more evidence for its chemopreventive effects in breast cancer.

Interfacial Organization and Forces Arising from Epithelial-Cancerous Monolayer Interactions.

The interfacial interactions between epithelia and cancer cells have profound relevance for tumor development and metastasis. Through monolayer confrontation of MCF10A (nontumorigenic human breast epithelial cells) and MDA-MB-231 (human epithelial breast cancer cells) cells, we investigate the epithelial-cancerous interfacial interactions at the tissue level. We show that the monolayer interaction leads to competitive interfacial morphodynamics and drives an intricate spatial organization of MCF10A cells into multicellular finger-like structures, which further branch into multiple subfinger-like structures. These hierarchical interfacial structures penetrate the cancer monolayer and can spontaneously segregate or even envelop cancer cell clusters, consistent with our theoretical prediction. By tracking the substrate displacements via embedded fluorescent nanobeads and implementing nanomechanical modeling that combines atomic force microscopy and finite element simulations, we computed mechanical force patterns, including traction forces and monolayer stresses, caused by the monolayer interaction. It is found that the heterogeneous mechanical forces accumulated in the monolayers are able to squeeze cancer cells, leading to three-dimensional interfacial bulges or cell extrusion, initiating the p53 apoptosis signaling pathways of cancer cells. We reveal that intercellular E-cadherin and P-cadherin of epithelial cells differentially regulate the interfacial organization including migration speed, directionality, spatial correlation, F-actin alignment, and subcellular protrusions of MCF10A cells; whereas E-cadherin governs interfacial geometry that is relevant to force localization and cancer cell extrusion, P-cadherin maintains interfacial integrity that enables long-range force transmission. Our findings suggest that the collaborative molecular and mechanical behaviors are crucial for preventing epithelial tissues from undergoing tumor invasion.

A molecular toolbox to study progesterone receptor signaling

Progesterone receptor (PR) signaling is required for mammary gland development and homeostasis. A major bottleneck in studying PR signaling is the lack of sensitive assays to measure and visualize PR pathway activity both quantitatively and spatially. Here, we develop new tools to study PR signaling in human breast epithelial cells. First, we generate optimized Progesterone Responsive Element (PRE)-luciferase constructs and demonstrate that these new reporters are a powerful tool to quantify PR signaling activity across a wide range of progesterone concentrations in two luminal breast cancer cell lines, MCF7 and T47D. We also describe a fluorescent lentiviral PRE-GFP reporter as a novel tool to visualize PR signaling at the single-cell level. Our reporter constructs are sensitive to physiological levels of progesterone. Second, we show that low background signaling, and high levels of PR expression are a prerequisite for robustly measuring PR signaling. Increasing PR expression by transient transfection, stable overexpression in MCF7 or clonal selection in T47D, drastically improves both the dynamic range of luciferase reporter assays, and the induction of endogenous PR target genes as measured by qRT-PCR. We find that the PR signaling response differs per cell line, target gene and hormone concentration used. Taken together, our tools allow a more rationally designed approach for measuring PR signaling in breast epithelial cells.

Gene Signature Associated with Nervous System in an Experimental Radiation- and Estrogen-Induced Breast Cancer Model.

Breast cancer is frequently the most diagnosed female cancer in the world. The experimental studies on cancer seldom focus on the relationship between the central nervous system and cancer. Despite extensive research into the treatment of breast cancer, chemotherapy resistance is an important issue limiting the efficacy of treatment. Novel biomarkers to predict prognosis or sensitivity to chemotherapy are urgently needed. This study examined nervous-system-related genes. The profiling of differentially expressed genes indicated that high-LET radiation, such as that emitted by radon progeny, in the presence of estrogen, induced a cascade of events indicative of tumorigenicity in human breast epithelial cells. Bioinformatic tools allowed us to analyze the genes involved in breast cancer and associated with the nervous system. The results indicated that the gene expression of the Ephrin A1 gene (EFNA1), the roundabout guidance receptor 1 (ROBO1), and the kallikrein-related peptidase 6 (KLK6) was greater in T2 and A5 than in the A3 cell line; the LIM domain kinase 2 gene (LIMK2) was greater in T2 than A3 and A5; the kallikrein-related peptidase 7 (KLK7), the neuroligin 4 X-linked gene (NLGN4X), and myelin basic protein (MBP) were greater than A3 only in T2; and the neural precursor cell expressed, developmentally down-regulated 9 gene (NEDD9) was greater in A5 than in the A3 and E cell lines. Concerning the correlation, it was found a positive correlation between ESR1 and EFNA1 in BRCA-LumA patients; with ROBO1 in BRCA-Basal patients, but this correlation was negative with the kallikrein-related peptidase 6 (KLK6) in BRCA-LumA and -LumB, as well as with LIMK2 and ROBO1 in all BRCA. It was also positive with neuroligin 4 X-linked (NLGN4X) in BRCA-Her2 and BRCA-LumB, and with MBP in BRCA-LumA and -LumB, but negative with KLK7 in all BRCA and BRCA-LumA and NEDD9 in BRCA-Her2. The differential gene expression levels between the tumor and adjacent tissue indicated that the ROBO1, KLK6, LIMK2, KLK7, NLGN4X, MBP, and NEDD9 gene expression levels were higher in normal tissues than in tumors; however, EFNA1 was higher in the tumor than the normal ones. EFNA1, LIMK2, ROBO1, KLK6, KLK7, and MBP gene expression had a negative ER status, whereas NEDD9 and NLGN4X were not significant concerning ER status. In conclusion, important markers have been analyzed concerning genes related to the nervous system, opening up a new avenue of studies in breast cancer therapy.

Phytochemical profiling, in vitro analysis for anti-inflammatory, immunomodulatory activities, structural elucidation and in silico evaluation of potential selective COX-2 and TNF-α inhibitor from Hydrilla verticillata (L.f.) Royle

Hydrilla verticillata (L.f.) Royle is a perennial aquatic plant, which exhibits nutritional as well as therapeutic properties. The present study has been carried out to evaluate anti-inflammatory and immunomodulatory activities along with in silico evaluation of potential selective COX-2 and TNF-α inhibitors from methanolic extract of H. verticillata (L.f.) Royle. The potential therapeutic compounds have been identified by high-resolution GC-MS analysis. Its capacity to inhibit inflammatory responses using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells has been explored. The anti-inflammatory properties of the plant extract were investigated by inhibiting inducible nitric oxide (NO) synthase and reduced NO generation driven by LPS on stimulated RAW 264.7 macrophage cells. Further investigation for the underlying molecular mechanism of the anti-inflammatory activity of plant extract has been carried out by molecular docking and molecular dynamics simulation approaches with COX-2 and TNF-α inhibitors ability against the most potent phytocompound phytol from the plant extract. To evaluate whether the extract causes any toxicity, the cytotoxicity test has been carried out with the Human embryonic kidney cell line (Hek-293), Mouse fibroblast (L929), human mesenchyme stem cells (hMSCs) and human breast epithelial cell line (MCF-10a). Ultimately, our findings suggest that the plant extract have great potential to reduce inflammation without causing any toxicity to normal cell. Communicated by Ramaswamy H. Sarma

Adipocyte-derived kynurenine stimulates malignant transformation of mammary epithelial cells through the aryl hydrocarbon receptor

Anti-hormone therapies are not efficacious for reducing the incidence of triple negative breast cancer (TNBC), which lacks both estrogen and progesterone receptors. While the etiology of this aggressive breast cancer subtype is unclear, visceral obesity is a strong risk factor for both pre- and post-menopausal cases. The mechanism by which excessive deposition of visceral adipose tissue (VAT) promotes the malignant transformation of hormone receptor-negative mammary epithelial cells is currently unknown. We developed a novel in vitro system of malignant transformation in which non-tumorigenic human breast epithelial cells (MCF-10A) grow in soft agar when cultured with factors released from VAT. These cells, which acquire the capacity for 3D growth, show elevated aryl hydrocarbon receptor (AhR) protein and AhR target genes, suggesting that AhR activity may drive malignant transformation by VAT. AhR is a ligand-dependent transcription factor that generates biological responses to exogenous carcinogens and to the endogenous tryptophan pathway metabolite, kynurenine. The serum kynurenine to tryptophan ratio has been shown to be elevated in patients with obesity. Herein, we demonstrate that AhR inhibitors or knockdown of AhR in MCF-10A cells prevents VAT-induced malignant transformation. Specifically, VAT-induced transformation is inhibited by Kyn-101, an inhibitor for the endogenous ligand binding site of AhR. Mass spectrometry analysis demonstrates that adipocytes metabolize tryptophan and release kynurenine, which is taken up by MCF-10A cells and activates the AhR to induce CYP1B1 and promote malignant transformation. This novel hormone receptor-independent mechanism of malignant transformation suggests targeting AhR for TNBC prevention in the context of visceral adiposity.

Theoretical, in Vitro Antiproliferative, and in Silico Molecular Docking and Pharmacokinetics Studies of Heteroleptic Nickel(II) and Copper(II) Complexes of Thiosemicarbazone-Based Ligands and Pefloxacin.

Twelve new heteroleptic nickel(II) and copper(II) complexes of the type [M(L1-6 )(Pfx)2 ] (1-12), where L1-6 =2-benzylidenehydrazinecarbothioamide (L1 ), 2-benzylidene-N-methylhydrazinecarbothioamide (L2 ), 2-benzylidene-N-phenylhydrazinecarbothioamide (L3 ), 2-(4-methylbenzylidene)hydrazinecarbothioamide (L4 ), 2-(4-methylbenzylidene)-N-methylhydrazinecarbothioamide (L5 ) and 2-(4-methylbenzylidene)-N-phenylhydrazinecarbothioamide (L6 ), Pfx=pefloxacin and M=Ni(II) or Cu(II) have been synthesised, and their structures were confirmed by different spectral techniques. The spectral data and density functional theory (DFT) calculations supported the bonding of pefloxacin drug molecule via one of the carboxylate oxygen atoms and the pyridone oxygen atom, and the thiosemicarbazone ligand via the imine nitrogen and the thione sulfur atoms with the metal(II) ion, forming distorted octahedral geometry. In vitro antiproliferative activity of the synthesized complexes was evaluated against three human breast cancer (T47D, estrogen negative (MDA-MB-231) and estrogen positive (MCF-7)) as well as non-tumorigenic human breast epithelial (MCF-10a) cell lines, which showed the higher activity for the copper(II) complexes. The interaction of the synthesized complexes with an oncogenic protein H-ras (121 p) was explored by in silico molecular docking studies. Further, in silico pharmacokinetics and ADMET parameters were also analysed to predict the drug-likeness as well as non-toxic and non-carcinogenic behavior, and safe oral administration of the complexes.

Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects.

Aberrantly up-regulated activity of the type II transmembrane protease Matriptase-1 has been associated with the development and progression of a range of epithelial-derived carcinomas, and a variety of signaling pathways can mediate Matriptase-dependent tumorigenic events. During mammalian carcinogenesis, gain of Matriptase activity often results from imbalanced ratios between Matriptase and its cognate transmembrane inhibitor Hai1. Similarly, in zebrafish, unrestrained Matriptase activity due to loss of hai1a results in epidermal pre-neoplasms already during embryogenesis. Here, based on our former findings of a similar tumor-suppressive role for the Na+/K+-pump beta subunit ATP1b1a, we identify epithelial polarity defects and systemic hypotonic stress as another mode of aberrant Matriptase activation in the embryonic zebrafish epidermis in vivo. In this case, however, a different oncogenic pathway is activated which contains PI3K, AKT and NFkB, rather than EGFR and PLD (as in hai1a mutants). Strikingly, epidermal pre-neoplasm is only induced when epithelial polarity defects in keratinocytes (leading to disturbed Matriptase subcellular localization) occur in combination with systemic hypotonic stress (leading to increased proteolytic activity of Matriptase). A similar combinatorial effect of hypotonicity and loss of epithelial polarity was also obtained for the activity levels of Matriptase-1 in human MCF-10A epithelial breast cells. Together, this is in line with the multi-factor concept of carcinogenesis, with the notion that such factors can even branch off from one and the same initiator (here ATP1a1b) and can converge again at the level of one and the same mediator (here Matriptase). In sum, our data point to tonicity and epithelial cell polarity as evolutionarily conserved regulators of Matriptase activity that upon de-regulation can constitute an alternative mode of Matriptase-dependent carcinogenesis in vivo.

A TGF-β-responsive enhancer regulates SRC expression and epithelial-mesenchymal transition-associated cell migration.

The non-receptor tyrosine kinase, SRC, is overexpressed and/or hyperactivated in various human cancers and facilitates cancer progression by promoting invasion and metastasis. However, the mechanisms underlying SRC upregulation are poorly understood. In this study, we demonstrated that transforming growth factor-β (TGF-β) induces SRC expression at the transcriptional level by activating an intragenic the SRC enhancer. In the human breast epithelial cell line MCF10A, TGF-β1 stimulation upregulated the SRC1A promoter, resulting in increased SRC mRNA and protein levels. Chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that SMAD complex is recruited to three enhancer regions ∼15 kb upstream and downstream of the SRC promoter, and one of them is capable of activating the SRC promoter in response to TGF-β. JUN, a member of the activator protein (AP)-1 family, localises to the enhancer and regulates TGF-β-induced SRC expression. Furthermore, TGF-β-induced SRC upregulation plays a crucial role in epithelial-mesenchymal transition (EMT)-associated cell migration by activating the SRC/focal adhesion kinase (FAK) circuit. Overall, these results suggest that TGF-β-induced SRC upregulation promotes cancer cell invasion and metastasis in a subset of human malignancies.

Preparation and Characterization of Nano-Sized Co(II), Cu(II), Mn(II) and Ni(II) Coordination PAA/Alginate Biopolymers and Study of Their Biological and Anticancer Performance

Four of the crosslinked sodium alginate and polyacrylic acid biopolymers based nanoscale metal natural polysaccharides, [M(AG-PAA)Cl(H2O)3], where M = Co(II), Cu(II), Mn(II) and Ni(II), AG = sodium alginate and PAA = polyacrylic acid, have been synthesized and structurally characterized. Because of their numerous biological and pharmacological activities of polysaccharides, including antimicrobial, immunomodulatory, antitumor, antidiabetic, antiviral, antioxidant, hypoglycemic and anticoagulant activities, polysaccharides are one of the near-promising candidates in the biomedical and pharmaceutical fields. The complexity of the polymeric compounds has been verified by carbon and nitrogen analysis, magnetic and conductance measurements, FT-IR spectra, electronic spectral analysis and thermal analysis (DTA, TG). All the synthesized complexes were non-electrolytes with magnetic moments ranging from 1.74 to 5.94 BM. The polymeric complexes were found to be of octahedral geometry. The developed coordination polymeric was found to be crystalline using X-ray powder diffraction examinations, which is confirmed by the SEM analysis. As a result, the crystallite size of all polymeric nanocrystals was in the range of 14 - 69 nm. The test of four compounds exhibits a broad spectrum of antimicrobial activity against both Gram-positive and Gram-negative bacteria and fungal Candida albicans. Using DPPH as a substrate, studies on radical scavenging tests are carried out. The findings demonstrated the antioxidant activities of each complex. In addition, results showed that the two chosen polymeric complexes had a good ability to kill cancer cells in a dose-dependent way. The copper(II) polymeric complex showed to its superior functionality as evidenced by microbial activity. After 72 h of interaction with the normal human breast epithelial cells (MCF10A), the synthesized polymeric compounds of Cu(II) and Co(II) showed exceptional cytocompatibility with the different applied doses. Compared to poly-AG/PAA/Co(II), poly-AG/PAA/Cu(II) exhibits a greater anticancer potential at various polymeric dosages.

Pharmacokinetic investigation on the mechanism of interaction of anti-breast cancer calycosin with albumin: In vitro

It has been documented that calycosin induces anticancer effects against a wide range of cancer cells, in vitro. However, pharmacokinetic characteristics of calycosin based on its interaction with albumin as a main carrier protein as well as corresponding anticancer mechanisms remain largely unknown. Herein, we inquired into the interaction of calycosin with human serum albumin (HSA) by a wide range collection of spectroscopic and theoretical data. Also, anticancer effects of calycosin on breast cancer cells, MDA-MB 231, were explored by cell viability, lactate dehydrogenase (LDH), caspase-3 activity, and real-time PCR assays. The findings demonstrated that the quenching process resulting from the interaction of calycosin and HSA was largely static in nature and that the calycosin-HSA system spontaneously forms. Cellular and molecular studies revealed that calycosin did not induce a significant cytotoxic effect on the viability of normal human breast epithelial cells, (MCF-10A), even at 200 μM concentration at which it was able to inhibit the proliferation of breast cancer MDA-MB 231 cells up to 50%. Then, it was disclosed that calycosin can disrupt the membrane integrity and induces apoptosis in MDA-MB 231 cells through overexpression of caspase-3 mRNA, down regulation of Bcl-2, and elevation of caspase-3 mRNA and activity mediated by downregulation of mTOR and Akt mRNA. Therefore, it was realized that in breast cancer MDA-MB 231 cells, calycosin is able to suppress cellular proliferation via apoptosis, which is controlled by the Akt/mTOR signaling cascade.

Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells.

Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.

Enhancing the efficacy of radiotherapy with Kalanchoe daigremontiana: A nanotechnological approach

Radiotherapy is widely utilized in the treatment of cancer, but this method is associated with dose-dependent limitation. Herbal compounds contain various secondary metabolites that increase the effectiveness of radiotherapy. However, a significant challenge associated with the use of herbal preparations is their poor biocompatibility. To address this limitation, the active ingredients of the leaves of Kalanchoe daigremontiana were extracted subsequently, phytoniosomes were prepared from the extract. The phytoniosomes were characterized through various analyses, Content analysis was performed with Quadrupole time-of-flight mass spectrometer (QTOF-MS) and encapsulation efficiency was evaluated with High-performance liquid chromatography (HPLC). Then, the cytotoxic effects of the phytoniosomes were evaluated on the Human Breast Epithelial Adenocarcinoma Cell Line (MCF-7), and Human Cervix epithelial Adenocarcinoma Cell Line (HeLa), using the MTT assay over multi-periods. Then radiotherapy studies have been carried out to increase this effect via Linear Accelerator (LINAC). Finally, to further understand the interactions between the phytoniosomes and cells, the niosomes were labelled with Fluorescein isothiocyanate (FITC) and fluorescence images were obtained. The results confirmed that the size of plain niosomes and phytoniosomes were 95.9 ± 0.52 and 126.0 ± 5.14 nm, respectively and localized around cytoplasm. Phytoniosomes combined with radiotherapy on cell lines are more effective than radiotherapy or phytoniosomes alone. The cell viability decreased significantly to 7.102 ± 3.51 for the MCF-7 cell line and to 8.183 ± 4.117 for the HeLa cell line). In conclusion, the combined use of these niosomal preparations with radiotherapy has the potential to be used as a tool for overcoming dose-dependent limitation in radiotherapy.

Synthesis and Characterization of a New Alginate/Carrageenan Crosslinked Biopolymer and Study of the Antibacterial, Antioxidant, and Anticancer Performance of Its Mn(II), Fe(III), Ni(II), and Cu(II) Polymeric Complexes.

Natural polysaccharides are essential to a wide range of fields, including medicine, food, and cosmetics, for their various physiochemical and biological properties. However, they still have adverse effects limiting their further applications. Consequently, possible structural modifications should be carried out on the polysaccharides for their valorization. Recently, polysaccharides complexed with metal ions have been reported to enhance their bioactivities. In this paper, we synthesized a new crosslinked biopolymer based on sodium alginate (AG) and carrageenan (CAR) polysaccharides. The biopolymer was then exploited to form complexes with different metal salts including MnCl2·4H2O, FeCl3·6H2O, NiCl2·6H2O, and CuCl2·2H2O. The four polymeric complexes were characterized by Fourier-transform infrared spectroscopy (FT-IR), elemental analysis, ultraviolet-visible spectroscopy (UV-Vis), magnetic susceptibility, molar conductivity methods, and thermogravimetric analysis. The X-ray crystal structure of the Mn(II) complex is tetrahedral and belongs to the monoclinic crystal system with the space group P121/n1. The Fe(III) complex is octahedral and crystal data fit with the cubic crystal system with the space group Pm-3m. The Ni(II) complex is tetrahedral and crystal data correspond to the cubic crystal arrangement with the space group Pm-3m. The data estimated for the Cu(II) polymeric complex revealed that it is tetrahedral and belongs to the cubic system with the space group Fm-3m. The antibacterial study showed significant activity of all the complexes against both Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and Gram-negative (Escherichia coli and Salmonella typhimurium) pathogenic strains. Similarly, the various complexes revealed an antifungal activity against Candida albicans. The Cu(II) polymeric complex recorded a higher antimicrobial activity with an inhibitory zone reaching 4.5 cm against Staphylococcus aureus bacteria and the best antifungal effect of 4 cm. Furthermore, higher antioxidant values of the four complexes were obtained with DPPH scavenging activity varying from 73 to 94%. The two more biologically effective complexes were then selected for the viability cell assessments and in vitro anticancer assays. The polymeric complexes revealed excellent cytocompatibility with normal human breast epithelial cells (MCF10A) and a high anticancer potential with human breast cancer cells (MCF-7) which increase significantly in a dose-dependent manner.

The activity and surface presence of organic cation/carnitine transporter OCTN2 (SLC22A5) in breast cancer cells depends on AKT kinase

l-carnitine is indispensable for transfer of fatty acids to mitochondria for the process of β-oxidation, a process, whose significance in cancer has drawn attention in recent years. In humans majority of carnitine is delivered by diet and enters the cell due to activity of solute carriers (SLCs), mainly by ubiquitously expressed organic cation/carnitine transporter (OCTN2/SLC22A5). In control and cancer human breast epithelial cell lines the major fraction of OCTN2 is present as a not matured non-glycosylated form. Studies on overexpressed OCTN2 demonstrated an exclusive interaction with SEC24C, as the cargo-recognizing subunit of coatomer II in transporter exit from endoplasmic reticulum. Co-transfection with SEC24C dominant negative mutant completely abolished presence of the mature form of OCTN2, pointing to a possibility of trafficking regulation. SEC24C was previously shown to be phosphorylated by serine/threonine kinase AKT, known to be activated in cancer. Further studies on breast cell lines showed that inhibition of AKT with MK-2206 in control and cancer lines decreased level of OCTN2 mature form. Proximity ligation assay showed that phosphorylation of OCTN2 on threonine was significantly abolished by AKT inhibition with MK-2206. Carnitine transport was positively correlated with the level of OCTN2 phosphorylated by AKT on threonine moiety. The observed regulation of OCTN2 by AKT places this kinase in the center of metabolic control. This points to both proteins, AKT and OCTN2, as druggable targets, in particular in a combination therapy of breast cancer.