Invasion Of Human Breast Cancer Cells Research Articles

Epigenetic Effects of Resveratrol on Oncogenic Signaling in Breast Cancer.

The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy.

A Standardized Extract of Petasites hybridus L., Containing the Active Ingredients Petasins, Acts as a Pro-Oxidant and Triggers Apoptosis through Elevating of NF-κB in a Highly Invasive Human Breast Cancer Cell Line.

Common butterbur (Petasites hybridus L.) is a traditional medicinal plant with numerous therapeutic properties among which is its recently uncovered anti-tumor activity. The present study aims to examine the activity of a standardized Bulgarian Petasites hybridus L. root extract, containing the active ingredients petasins, on the human breast cancer cell line MDA-MB-231 and non-cancerous MCF-10A cells. Specifically, we examined cell death, oxidative stress, and nuclear factor kappa-B (NF-κB) signaling. A standardized butterbur powdered extract containing a minimum of 15% petasins was used. A lipophilic extract was obtained from subterranean portion of the plant of Bulgarian populations of Petasites hybridus using liquid-liquid extraction after completely removing pyrrolizidine alkaloids. The induction of apoptosis and necrosis was analyzed by flow cytometry, and oxidative stress biomarkers and NF-κB were measured using enzyme-linked immunosorbent assay (ELISA). Petasites hybridus L. root extract triggered apoptosis in a cancer-specific fashion and induced a moderate oxidative stress characterized by diminished glutathione (GSH) levels and elevated malondialdehyde (MDA) levels in MDA-MB-231 72 h after treatment. NF-κB levels were higher in cancer cells after treatment with IC50 and IC75 doses, this suggested that the NF-κB pathway was activated in response to oxidative stress leading to the induction of apoptosis. MCF-10A cells were affected to a lesser extent by the Petasites hybridus extract, and the adaptive response of their antioxidant defense system halted oxidative stress. Overall, these results indicate that Petasites hybridus L. root extract selectively acts as a pro-oxidant in breast cancer cells and thus represents a potential therapeutic option for cancer treatment with fewer side effects.

Naturally Occurring Rhamnopyranosides as Anticancer Agents: Molecular Docking and ADMET Study

After heart disease, cancer continues to be the second most prevalent cause of death in the USA. Several chemotherapeutic treatments (drugs) are available for cancer that use powerful chemicals to kill the body's rapidly proliferating cells. However, recent research disclosed that many clinically viable anticancer drugs have been developed with the help of chemicals originating from plants. A number of phytochemicals isolated from plants possess rhamnopyranoses and some of them are acyl rhamnopyranoses. Encouragingly, such compounds were reported for their cell proliferation and migration inhibition activities against invasive human triple-negative breast cancer cells. In this study, four naturally occurring rhamnopyranose esters were checked against three cancer-related proteins (PDB IDs: 3TJM, 4OAR, and 5FGK) via molecular docking. Rhamnose compounds 3-6 showed better binding energy compared to the related standard drugs in use in the hospitals. Compound 6 was found highly potential against all the proteins (-8.5 to -11.3 kcal/mol). ADMET studies have also been discussed in this respect. This study indicated that natural rhamnopyranose esters could be used to stop the spreading of cancer cells like other reported sugar fatty acid esters (SFAEs).

MitoQ Inhibits Human Breast Cancer Cell Migration, Invasion and Clonogenicity.

Simple SummarySolid tumors comprise metabolically hostile areas adjacent to metabolically friendly ones, and their distribution fluctuates in space and time. Selection pressure is maximal in hostile areas where cancer cells develop at least four survival strategies: to adapt themselves, to modify the microenvironment, to hibernate metabolically, and to escape. Escape marks the transition from a localized to an invasive tumor that may ultimately disseminate remotely, forming metastases. Based on the hypothesis that mitochondria are metabolic sensors that control these responses, we previously established that mitochondrial superoxide is a pro-metastatic intracellular signaling agent. Here, we tested MitoQ, a mitochondria-targeted ROS inactivator that already successfully passed Phase I safety clinical trials, as a potential inhibitor of the early steps of the metastatic cascade. Using human breast cancer cells as models in anticipation of preclinical and clinical assays, we report that MitoQ inhibits cancer cell migration, invasion, clonogenicity, sphere formation and spheroid stability.To successfully generate distant metastases, metastatic progenitor cells must simultaneously possess mesenchymal characteristics, resist to anoïkis, migrate and invade directionally, resist to redox and shear stresses in the systemic circulation, and possess stem cell characteristics. These cells primarily originate from metabolically hostile areas of the primary tumor, where oxygen and nutrient deprivation, together with metabolic waste accumulation, exert a strong selection pressure promoting evasion. Here, we followed the hypothesis according to which metastasis as a whole implies the existence of metabolic sensors. Among others, mitochondria are singled out as a major source of superoxide that supports the metastatic phenotype. Molecularly, stressed cancer cells increase mitochondrial superoxide production, which activates the transforming growth factor-β pathway through src directly within mitochondria, ultimately activating focal adhesion kinase Pyk2. The existence of mitochondria-targeted antioxidants constitutes an opportunity to interfere with the metastatic process. Here, using aggressive triple-negative and HER2-positive human breast cancer cell lines as models, we report that MitoQ inhibits all the metastatic traits that we tested in vitro. Compared to other mitochondria-targeted antioxidants, MitoQ already successfully passed Phase I safety clinical trials, which provides an important incentive for future preclinical and clinical evaluations of this drug for the prevention of breast cancer metastasis.

Suppression of SIN3A by miR-183 Promotes Breast Cancer Metastasis.

SIN3A expression is decreased in metastatic breast cancer in part due to miR-183.

The long non-coding RNA ET-20 mediates EMT by impairing desmosomes in breast cancer cells.

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFβ-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

Discovery of triaromatic flexible agents bearing 1,2,3-Triazole with selective and potent anti-breast cancer activity and CDK9 inhibition supported by molecular dynamics

This study reports an efficient and convenient click synthesis of novel series of chromene scaffold linked to 1,2,3 triazole ring and terminal lipophilic fragments. Structures of all newly synthesized compounds were well characterized by IR, 1H NMR, 13C NMR and elemental analyses. In vitro MTT cytotoxic screening was performed using staurosporine as a reference drug against three different types: aggressive and invasive human breast cancer cell line (MDA-MB 231), Michigan cancer foundation-7 (MCF-7) and human colorectal carcinoma cell line (HCT116). These screening data showed considerable anticancer activity for these newly synthesized compounds compared to reference staurosporine drug with high degree of cell line selectivity and excellent activity with micromolar (µM) half maximal inhibitory concentration (IC50) values against tumor cells. In addition, Cyclin-Dependent Kinase1 and 9 (CDK1 and 9) assays were performed for the most active compounds to get more details about their mechanism of action. In order to assess and explain their binding affinities, molecular docking simulation was studied against CDK9 site. The results obtained from molecular docking study and those obtained from cytotoxic screening and enzyme inhibition were correlated. One of the most prominent analogs is 34 that showed high selective cytotoxicity profile against single breast cancer cell line MCF-7 and MDA-MB 231 with IC50 3.4 µM and 1.4 uM respectively. Molecular dynamics (MD) simulation of the CDK9 complex by Desmond revealed stability during 100 ns with RMSD of CDK9 complex converged at 2.1 Å after 20 ns. During MD, the interaction fractions confirmed multiple interactions of 34 with Cys106, a crucial residue for inhibition of CDK9 activity. A novel 34 triazole compound was introduced for drug design works as potent and selective hit for treatment of breast cancers after processing of optimization steps.

Abstract PS17-55: Reactive oxygen species scavengers in triple negative breast cancer

Abstract Reactive oxygen species (ROS) are well known to play important roles in cancer. In particular, ROS of mitochondrial origin (mtROS) promote DNA mutations and cell death when produced at high levels, but rather stimulate autophagy and metastatic spread when produced at subcytotoxic levels [1]. Multiple metabolic pathways and oncogenic regulators linked to ROS have been studied in cancer cells, including MAPK/ERK, PI3K/AKT, PTEN, PKD, BREF2, HIF-1, NF-κB and p53 [2]. Abnormalities in these pathways can contribute to tumor development.By producing mtROS, mitochondria control and could even trigger metastatic spread, meaning that the use of specific mitochondria-targeted superoxide inhibitors/scavengers, such as mitoquinol mesylate (MitoQ), could reduce and/or prevent metastatic dissemination. This has been studied and reported with the use of MitoTempo, which prevents metastasis of naturally metastatic MDA-MB-231 human breast cancer cells implanted in mice [1]. Here, we show that MitoQ impairs MDA-MB-231 and SkBr3 human breast cancer cell migration and invasion in vitro, and MDA-MB-231 metastasis to the lungs in mice in vivo. In metastatic take assays, tail vein-injected MDA-MB-231 cells pretreated with MitoQ formed significantly less metastases after 4 weeks compared to vehicle. In spontaneous metastatic assays, mice bearing orthotopic MDA-MB-231 tumors were treated with ± 20 mg/kg MitoQ (per os) for 6 weeks. Primary tumors were surgically removed to allow metastatic outgrowth. In this model, MitoQ decreased primary tumor recurrence as well as the number of lung micro-metastases. Additionally, RNAseq studies in two human breast cancer cell lines identified that MitoQ significantly modulated a common set of metabolic genes in vitro and in vivo. They can be used as biomarkers of response. These results, together with a successful Phase I clinical trial and the fact that MitoQ does not interfere with the cytotoxic effects of chemotherapy, confirm that MitoQ is a potential candidate to be clinically tested in triple negative breast cancer patients.

Catharanthus roseus L. extract downregulates the expression profile of motility-related genes in highly invasive human breast cancer cell line MDA-MB-231

This study aimed to investigate the effect of Catharanthus roseus L. (C. roseus) leaf extract on the migration and invasion of MDA-MB-231 cell line and elucidate the molecular mechanisms of action. Effect of the extract on cell viability was evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazoliumbromide) assay. Anti-migratory and anti-invasive effects were evaluated using scratch and Transwell assays. Effect on the levels and activities of matrix metalloproteinase (MMP)-2 and MMP-9 was determined using ELISA and gelatin zymography. Furthermore, changes in the expression of 84 genes commonly involved in cell motility were assessed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and cell motility RT2 profiler PCR array. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DAVID. Using STRING and Cytoscape software, hub genes were determined. The extract significantly (p < 0.001) inhibited the migration and invasion of MDA-MB-231 cells at non-cytotoxic concentrations. The activities and levels of MMP-2 and MMP-9 were decreased in a dose-dependent manner following C. roseus exposure. At 4 µg/mL, the extract significantly downregulated the expression of 52 genes involved in extracellular matrix degradation, cytoskeleton reorganisation, focal adhesions and invadopodia formation. GO and KEGG pathway analysis revealed that the downregulated genes were significantly enriched in biological processes and pathways closely related to cell motility. Our findings showed that C. roseus inhibited the migration and invasion of MDA-MB-231 cells through altering the expression of various motility-related genes. This study provided data about the potential of C. roseus phytochemicals as promising therapeutic agents against breast cancer metastasis, especially at gene level.

Simvastatin Suppresses Human Breast Cancer Cell Invasion by Decreasing the Expression of Pituitary Tumor-Transforming Gene 1.

Statins, or 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, have been widely used to lower cholesterol and prevent cardiovascular diseases. Recent preclinical and clinical studies have shown that statins exert beneficial effects in the management of breast cancer, while the underlying mechanisms remain to be elucidated. Herein, we sought to investigate the effect of statins on the expression of pituitary tumor-transforming gene 1 (PTTG1), a critical gene involved in human breast cancer invasion and metastasis. Our results showed that PTTG1 is highly expressed in malignant Hs578T and MDA-MB-231 breast cancer cell lines as compared with normal or less malignant breast cancer cells. Furthermore, we found that the expression of PTTG1 was markedly suppressed by lipophilic statins, such as simvastatin, fluvastatin, mevastatin, and lovastatin, but not by hydrophilic pravastatin. In a dose and time dependent manner, simvastatin suppressed PTTG1 expression by decreasing PTTG1 mRNA stability in MDA-MB-231 cells. Both siRNA-mediated knockdown of PTTG1 expression and simvastatin treatment markedly inhibited MDA-MB-231 cell invasion, MMP-2 and MMP-9 activity, and the expression of PTTG1 downstream target genes, while ectopic expression of PTTG1 promoted cancer cell invasion, and partly reversed simvastatin-mediated inhibition of cell invasion. Mechanistically, we found that inhibition of PTTG1 expression by simvastatin was reversed by geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate, suggesting the involvement of geranylgeranyl synthesis in regulating PTTG1 expression. Our results identified statins as novel inhibitors of PTTG1 expression in breast cancer cells and provide mechanistic insights into how simvastatin prevent breast cancer metastasis as observed in recent preclinical and clinical studies.

Abstract 6114: Host and neoplastic cells utilize Axl for breast cancer bone metastasis

Abstract Breast cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related death for women in the United States. Approximately 90% of cancer-related death results from metastasis, and the bone is the most common site of metastasis for breast cancer. Upregulation of signaling pathways can promote the metastatic phenotypes of neoplastic cells, including cell migration, invasion, proliferation and angiogenesis. In addition, when breast cancer cells disseminate to the bone, neoplastic cells upregulate signaling pathways to activate osteoclast-induced bone resorption. A key pathway involved in metastatic spread of cancer cells is the receptor tyrosine kinase Axl. Genetic and pharmacologic Axl inhibition in both human and rodent breast cancer cell lines (MDA-MB-231 and 4T1) impaired tumor cell migration and invasion. Intracardiac inoculation of Axl knockdown (KD) MDA-MB-231 cells significantly decreased the number of bone metastases compared to the scrambled control MDA-MB-231 cells. Another mechanism by which Axl inhibition impaired bone metastasis is through the modulation of osteoclast maturation and activity. Co-culture of Axl KD breast cancer cells with murine osteoclast precursor cells (Raw264.7) significantly reduced the number of tumor cell-induced osteoclast resorption pits on bone slices. In addition, genetic and pharmacologic inhibition of Axl in Raw264.7 cells as well as bone marrow derived osteoclast precursor cells, significantly reduced osteoclast formation and activity. Scrambled control and Axl KD Raw264.7 cells were cultured in serum free medium for 24 hours, and their conditioned media were collected. The conditioned media of Axl KD Raw264.7 cells revealed significantly decreased levels of monocyte chemoattractant protein 1 (MCP-1), which known to promote osteoclastogenesis, compared to the scrambled control Raw264.7 cells. In summary, we have demonstrated that inhibition of Axl decreases breast cancer metastasis to the bone through modulation of the host and neoplastic cell functions. Collectively, these data indicate that Axl signaling may be a promising therapeutic target to impair tumor progression and metastasis. Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cells utilize Axl for breast cancer bone metastasis [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 6114.

Breast Fibroblasts and ECM Components Modulate Breast Cancer Cell Migration through the Secretion of MMPs in a 3D Microfluidic Co-Culture Model

The extracellular matrix (ECM) composition greatly influences cancer progression, leading to differential invasion, migration, and metastatic potential. In breast cancer, ECM components, such as fibroblasts and ECM proteins, have the potential to alter cancer cell migration. However, the lack of in vitro migration models that can vary ECM composition limits our knowledge of how specific ECM components contribute to cancer progression. Here, a microfluidic model was used to study the effect of 3D heterogeneous ECMs (i.e., fibroblasts and different ECM protein compositions) on the migration distance of a highly invasive human breast cancer cell line, MDA-MB-231. Specifically, we show that in the presence of normal breast fibroblasts, a fibronectin-rich matrix induces more cancer cell migration. Analysis of the ECM revealed the presence of ECM tunnels. Likewise, cancer-stromal crosstalk induced an increase in the secretion of metalloproteinases (MMPs) in co-cultures. When MMPs were inhibited, migration distance decreased in all conditions except for the fibronectin-rich matrix in the co-culture with human mammary fibroblasts (HMFs). This model mimics the in vivo invasion microenvironment, allowing the examination of cancer cell migration in a relevant context. In general, this data demonstrates the capability of the model to pinpoint the contribution of different components of the tumor microenvironment (TME).

Nucleus-located PDK1 regulates growth, invasion and migration of breast cancer cells

AimsIt is well known that pyruvate dehydrogenase kinase 1 (PDK1) is highly expressed in breast cancer (BC) tissues and promotes tumor growth, but the underlying mechanisms of this process are unclear. Here, we investigated the effects of nuclear PDK1 on growth, migration and invasion in human BC cells. Main methodsThe sub-cellular localization of PDK1 in BC cells was performed with subcellular fractionation followed by Western blot and immunofluorescence. The localization of PDK1 in breast normal tissue and breast duct carcinoma was detected by Immunohistochemistry. Then the protein-protein interaction between PDK1 and Importin β was verified by co-immunoprecipitation assay. Finally, the effects of nuclear PDK1 on cell proliferation, apoptosis, migration and invasion of BC cells were assessed. Key findingsIn addition to its well-known sub-cellular localization, PDK1 was present in the nucleus of BC cells, and EGF treatment increased nucleus distribution of PDK1. Moreover, the level of nuclear PDK1 accumulation facilitated the growth of BC cells. We also found that the entry of PDK1 into nucleus mainly relied on the nuclear localization signal (NLS), and NLS mutation inhibited the entry of PDK1 into nucleus; as a result, the migration and invasion abilities of BC cells were impaired, and the number of apoptotic cells was significantly increased. SignificanceOur findings provided a new supplement to the sub-cellular localization of PDK1 in BC cells and uncovered the function of nuclear PDK1 in facilitating BC cells growth, migration and invasion.

Screening of a Novel Upregulated lncRNA, A2M-AS1, That Promotes Invasion and Migration and Signifies Poor Prognosis in Breast Cancer.

Understanding of prognostic factors and therapeutic targets for breast cancer is imperative for guidance of patient care. We studied 1203 tumour samples from the Gene Expression Omnibus (GEO) to evaluate potential genes related to breast cancer. R software was used to analyse differentially expressed long noncoding RNAs (lncRNAs) in the RNA microarray expression profiles GSE45827 and GSE65216 and to identify a series of differentially expressed lncRNAs associated with human breast cancer. Of these lncRNAs, A2M-AS1, a lncRNA that has not been previously reported, was significantly upregulated in human breast cancer tissues compared with adjacent nontumour tissues. Importantly, A2M-AS1 upregulation was significantly associated with ER-negative, HER2-positive, and basal-like breast cancer and with poor recurrence-free survival and metastasis-free survival in breast cancer patients. After validating these results in 96 collected human breast cancer tissues and 64 paired adjacent noncancerous tissues, we further investigated the roles of A2M-AS1 in human ER-negative and basal-like breast cancer cells. The results revealed that A2M-AS1 significantly promotes human breast cancer cell proliferation, invasion, and migration. Additionally, bioinformatics analysis of genes coexpressed with A2M-AS1 in the context of human breast cancer combined with qRT-PCR and Western blot assays revealed that A2M-AS1 exerts regulatory effects on downstream factors in the cell adhesion molecule pathway, including CD2 and SELL. These results imply that A2M-AS1 might be a promising candidate prognostic factor and therapeutic target for breast cancer.

Different Mechanical Responses to Substrate Stiffness between Cancer Cells and Normal Cells

Metastatic cancer cells persistently interact with tumor microenvironment in every steps of their migrating processes. Understanding the effects of ECM on cancer cells are vital to decipher cancer cell invasion and metastasis. Thousands of molecules in focal adhesions are involved in the force transduction processes. Among them, talin acts as one of the most important participants. Conformational changes of talin caused by force are well researched by using single molecule force microscopies in the last decade. However, correlation between mechanical properties of the cancer cells and forces in focal adhesions in cancer metastasis are not fully understood. Here, we measured the viscoelastic properties of cancer cells on five different substrates and the force on talin in focal adhesions by using custom-made talin tension sensors. In order to find out the working patterns of metastatic cancer cells, we carried out the same measurements in human breast cells (MCF10A), non-invasive human breast cancer cells (MCF-7), and invasive human breast cancer cells (MDA-MB-231). By comparing the results, we found that invasive cancer cells can tune their mechanical properties, both viscoelastic properties and force on talin, accordingly when substrate stiffness are changed. Nonetheless, non-invasive cancer cells and normal cells lack this ability. Furthermore, membrane tether experiments conducted by atomic force microscopy also showed that strength of membrane tethers changed with substrate stiffness in MDA-MB-231 cells, not in MCF10A and MCF7 cells.

The Effects of Trifolium pratense L. Sprouts' Phenolic Compounds on Cell Growth and Migration of MDA-MB-231, MCF-7 and HUVEC Cells.

Uncontrolled growth and migration and invasion abilities are common for cancer cells in malignant tumors with low therapeutic effectiveness and high mortality and morbidity. Estrogen receptor β (ERβ), as a member of the nuclear receptor superfamily, shows potent tumor suppressive activities in many cancers. Phytoestrogens’ structural resemblance to 17 β-estradiol allows their binding to ERβ isoform predominantly, and therefore, expression of genes connected with elevated proliferation, motility and invasiveness of cancer cells may be downregulated. Among polyphenolic compounds with phytoestrogenic activity, there are isoflavones from Trifolium pratense L. (red clover) sprouts, containing high amounts of formononetin and biochanin A and their glycosides. To determine the source of the most biologically active isoflavones, we obtained four extracts from sprouts before and after their lactic fermentation and/or β-glucosidase treatment. Our previous results of ITC (isothermal titration calorimetry) modelling and a docking simulation showed clover isoflavones’ affinity to ERβ binding, which may downregulate cancer cell proliferation and migration. Thus, the biological activity of T. pratense sprouts’ extracts was checked under in vitro conditions against highly invasive human breast cancer cell line MDA-MB-231 and non-invasive human breast cancer cell line MCF-7 cells. To compare extracts’ activities acquired for cancer cells with those activities against normal cells, as a third model we choose human umbilical vein endothelial cells (HUVEC), which, due to their migration abilities, are involved in blood vessel formation. Extracts obtained from fermented sprouts at IC0 dosages were able to inhibit migration of breast cancer cells through their influence on intracellular ROS generation; membrane stiffening; adhesion; regulation of MMP-9, N-cadherin and E-cadherin at transcriptional level; or VEGF secretion. Simultaneously, isolated phenolics revealed no toxicity against normal HUVEC cells. In the manuscript, we proposed a preliminary mechanism accounting for the in vitro activity of Trifolium pratense L. isoflavones. In this manner, T. pratense sprouts, especially after their lactic fermentation, can be considered a potent source of biological active phytoestrogens and a dietary supplement with anti-cancer and anti-invasion properties.

Effect of miR-200c on migration and proliferation of breast cancer MDA-MB-231 cells and BT-549 cells and the possible mechanism.

This study was designed to investigate the effects of miR-200c on the migration and proliferation of breast cancer MDA-MB-231 cells and BT-549 cells and the possible mechanisms. The effects of miR-200c on the proliferation and migration of highly invasive human breast cancer MDA-MB-231 and BT-549 cells were investigated by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT and wound-healing assay. Effects of miR-200c on the expression of adhesion molecules in cells of 2 cell lines were examined by Western blot. MTT assay showed that miR-200c could inhibit the proliferation of MDA-MB-231 and BT-549 cells. Wound-healing assay results showed that miR-200c could inhibit the migration of MDA-MB-231 and BT-549 cells. Western blot results showed that miR-200c up-regulated the expression of E-cadherin protein and down-regulated the expression of Vimentin protein. The results showed that miR-200c can inhibit the proliferation of highly invasive human breast cancer cells and may inhibit cell migration by up-regulating the expression of E-cadherin protein and down-regulating the expression of Vimentin protein.

Triple-negative breast cancer suppressive activities, antioxidants and pharmacophore model of new acylated rhamnopyranoses from Premna odorata.

Phytochemical investigation of Premna odorata Blanco “Lamiaceae” young stems afforded four new acylated rhamnopyranoses 1–4, along with fourteen known compounds 5–19. The structures of the new compounds were confirmed using extensive 1D, 2D NMR, and HRESIMS analysis. The isolated compounds were tested for their cell proliferation and migration inhibition activities against the invasive human triple-negative breast cancer cells MDA-MB-231 and MCF-7, and the normal human breast cell line MCF-10A. In addition, free radical scavenging activities using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) were studied. Compound 1 was the most active as an antiproliferative agent, showing a high to moderate antiproliferative effect with an IC50 value of 4.95 and 17.7 μM against MCF-7 and MDA-MB-231, respectively. The antiproliferative activities of compounds 1–5 against the normal breast cell line MCF-10A were moderate to low with IC50 values of 13.91 to 27.70 μM. On the other hand, compounds 1 and 10 suppressed MDA-MB-231 cell migration in the wound-healing assay at 10 μM concentration. Meanwhile, compounds 1–5 exhibited the highest value of DPPH radical scavenging activities with an IC50 value range of 17.5–20.43 ± 0.5 μg mL−1. The pharmacophore model generated using Molecular Operating Environment (MOE) for compounds 1–5 showed three hydrogen bond acceptors (HBAs), one hydrogen bond donor (HBD), one aromatic ring (Aro), and one hydrophobic (Hyd.) group. The central HBA feature lies at a distance of 4.36 Å and 6.38 Å from the remaining two HBA features. Also, the HBD feature maintains a distance of 2.74 Å from the aromatic feature. Acylated rhamnopyranoses can be considered good scaffolds for developing new anti-breast cancer and antioxidant compounds.

3-Methylindole-substituted zinc phthalocyanines for photodynamic cancer therapy

Novel peripherally and non-peripherally 3-methylindole-substituted zinc phthalocyanine derivatives were synthesized as photosensitizers for photodynamic therapy in cancer treatment. The photophysical, photochemical and photobiological properties of targeted phthalocyanines were also investigated. For this purpose, the fluorescence and singlet oxygen quantum yields, and fluorescence lifetime values of the final compounds were determined in DMF solutions. The phototoxicity and cytotoxicity of the phthalocyanine complexes were tested against the invasive human breast cancer cell line (MDA-MB-231) for determination of their photosensitizing ability in the area of photodynamic therapy. It was revealed that while peripherally 3-methylindole-substituted phthalocyanine was found to be toxic for cells in both dark and light conditions, its non-peripherally substituted phthalocyanine analogue significantly caused cell death following light irradiation. A preliminary assay suggested that the non-peripherally linked phthalocyanine could be a suitable candidate for cancer treatment via photodynamic therapy techniques.

NADPH: Quinone oxidoreductase 1 (NQO1) mediated anti-cancer effects of plumbagin in endocrine resistant MCF7 breast cancer cells

BackgroundPLB is a natural naphthoquinone compound isolated from the roots of Plumbago indica plant. Our previous study reported the inhibitory effect of Plumbagin (PLB) on human endocrine resistant breast cancer cell growth and cell invasion. Hypothesis/PurposeSince PLB is a naphthoquinone compound, it can be reduced by the cytosolic NADPH: quinone oxidoreductase 1 (NQO1) enzyme. NQO1 expression is upregulated in various types of aggressive cancer including breast cancer. This study investigated the impact of NQO1 on anti-cancer effects of PLB in endocrine-resistant breast cancer cells. Study DesignThis study was an in vitro study using ER-positive cell line (MCF7) and endocrine-resistant cell lines (MCF7/LCC2 and MCF7/LCC9 cells). MethodsThe roles of NQO1 in anti-cancer activity of PLB were investigated by using NQO1 knockdown cells, NQO1 inhibitor and NQO1 overexpressed cells. To study the impact of NQO1 on the effects of PLB on cell viability, apoptosis, invasion and generation of ROS, the following assays were used: MTT assays, annexin V-PE/7-ADD staining flow cytometry, matrigel invasion assays and DCFHDA assays. To study the mechanism of how NQO1 mediated PLB effects in tamoxifen response and apoptosis, we assessed the levels of mRNA expression by using qRT-PCR. Results1. In this study, NQO1 was upregulated in endocrine-resistant cells.2. PLB did not change the expression of NQO1 but it was able to increase NQO1 activity.3. The inhibitory effects of PLB on cell proliferation, cell invasion and expression of tamoxifen resistant gene were attenuated in NQO1 knockdown cells or in the presence of NQO1 inhibitor.4. The effects of PLB to induce apoptosis and generate ROS were also decreased when NQO1 activity was inhibited or when the NQO1 expression was reduced.5. The anti-cancer effects of PLB increased when NQO1 was upregulated. ConclusionThe effects of PLB in endocrine-resistant breast cancer cells is dependent on NQO1’s activity.