Myricetin suppresses breast cancer metastasis through down‐regulating the activity of matrix metalloproteinase (MMP)‐2/9

Tumour metastasis is the major cause of breast cancer mortality. Myricetin, a natural polyphenol, is found in teas, wines, and berries. The pharmacodynamic action and molecular mechanism of myricetin on breast cancer metastasis remain unknown. Here, we investigated the effect of myricetin on MDA‐Mb‐231Br cell viability, migration, invasion, and 4T1 mouse lung metastasis mouse models. MMP‐2/9 protein expression and ST6GALNAC5 expression were analysed using western blot assays and quantitative real‐time polymerase chain reaction, respectively. Cell migration and invasion were detected by wound‐healing and Boyden transwell assays. The antimetastatic effect in vivo was evaluated by lung metastasis model. Myricetin significantly decreased the activities of MMP‐2/9 and mRNA levels of ST6GALNAC5. In addition, the migration, invasion, and adhesion were effectively inhibited in a concentration‐dependent manner. On the other hand, mice treated with myricetin exhibited smaller tumour nodules compared with the vehicle mice, with only 17.78 ± 15.41% after treatment with 50 mg/kg myricetin. In conclusion, myricetin could significantly block invasion of MDA‐Mb‐231Br cells through suppressing the protein expression of MMP‐2/9 and the expression of ST6GALNAC5, as well as lung metastasis in a mouse model, which suggests that myricetin should be developed as a potential therapeutic candidate for breast cancer.     

Polyphenols from Artemisia annua L Inhibit Adhesion and EMT of Highly Metastatic Breast Cancer Cells MDA‐MB‐231

Recent evidence suggests that polyphenolic compounds from plants have anti‐invasion and anti‐metastasis capabilities. The Korean annual weed, Artemisia annua L., has been used as a folk medicine for treatment of various diseases. Here, we isolated and characterized polyphenols from Korean A. annua L (pKAL). We investigated anti‐metastatic effects of pKAL on the highly metastatic MDA‐MB‐231 breast cancer cells especially focusing on cancer cell adhesion to the endothelial cell and epithelial‐mesenchymal transition (EMT). Firstly, pKAL inhibited cell viability of MDA‐MB‐231 cells in a dose‐dependent manner, but not that of human umbilical vein endothelial cells (ECs). Polyphenols from Korean A. annua L inhibited the adhesion of MDA‐MB‐231 cells to ECs through reducing vascular cell adhesion molecule‐1 expression of MDA‐MB‐231 and ECs, but not intracellular adhesion molecule‐1 at the concentrations where pKAL did not influence the cell viability of either MDA‐MB‐231 cells nor EC. Further, pKAL inhibited tumor necrosis factor‐activated MDA‐MB‐231 breast cancer cell invasion through inhibition of matrix metalloproteinase‐2 and matrix metalloproteinase‐9 and EMT. Moreover, pKAL inhibited phosphorylation of Akt, but not that of protein kinase C. These results suggest that pKAL may serve as a therapeutic agent against cancer metastasis at least in part by inhibiting the cancer cell adhesion to ECs through suppression of vascular cell adhesion molecule‐1 and invasion through suppression of EMT. Copyright © 2016 John Wiley & Sons, Ltd.     

Zerumbone Suppresses IL‐1β‐induced Cell Migration and Invasion by Inhibiting IL‐8 and MMP‐3 Expression in Human Triple‐negative Breast Cancer Cells

Inflammation is a key regulatory process in cancer development. Prolonged exposure of breast tumor cells to inflammatory cytokines leads to epithelial‐mesenchymal transition, which is the principal mechanism involved in metastasis and tumor invasion. Interleukin (IL)‐1β is a major inflammatory cytokine in a variety of tumors. To date, the regulatory mechanism of IL‐1β‐induced cell migration and invasion has not been fully elucidated. Here, we investigated the effect of zerumbone (ZER) on IL‐1β‐induced cell migration and invasion in breast cancer cells. The levels of IL‐8 and matrix metalloproteinase (MMP)‐3 mRNA were analyzed by real‐time polymerase chain reaction. The levels of secreted IL‐8 and MMP‐3 protein were analyzed by enzyme‐linked immunosorbent assay and western blot analysis, respectively. Cell invasion and migration was detected by Boyden chamber assay. The levels of IL‐8 and MMP‐3 expression were significantly increased by IL‐1β treatment in Hs578T and MDA‐MB231 cells. On the other hand, IL‐1β‐induced IL‐8 and MMP‐3 expression was decreased by ZER. Finally, IL‐1β‐induced cell migration and invasion were decreased by ZER in Hs578T and MDA‐MB231 cells. ZER suppresses IL‐1β‐induced cell migration and invasion by inhibiting IL‐8 expression and MMP‐3 expression in TNBC cells. ZER could be a promising therapeutic drug for treatment of triple‐negative breast cancer patients. Copyright © 2014 John Wiley & Sons, Ltd.     

In Vitro and In Silico Evaluation of NF‐κB Targeted Costunolide Action on Estrogen Receptor‐Negative Breast Cancer Cells—A Comparison with Normal Breast Cells

Costunolide, a sesquiterpene lactone is a plant‐derived secondary metabolite found to be present in most of the pharmacologically active herbs, being the cause for their medicinal values. The present study aims to evaluate the cytotoxic effect of costunolide isolated from Costus speciosus rhizome extract on MDA‐MB‐231 cells and explore its targeted action in comparison with its action on the normal breast cells (MCF 10A). The effect of costunolide on cell viability of the cells was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide viability assay. The targeted action of the compound was analyzed comparing the effectiveness of the compound to alter the protein expression levels of NF‐κB subunits in the normal and the cancer cells using western blotting analysis. In silico studies were performed to predict the targeted interaction of costunolide with the NF‐κB subunit proteins. Costunolide inhibited the cell viability of MDA‐MB‐231 cells in a dose‐dependent manner leaving no significant change in the viability of the normal breast cells. The over expressed NF‐κB subunits – p65, 52 and 100 in the cancer cells were found to be downregulated when treated with costunolide at an effective dose of 20 and 40 μM costunolide. In silico results provided stable interactions between costunolide and the target proteins, supporting the in vitro results in addition. Thus, costunolide derived from C. speciosus plant source elevates a fresh conviction for its use in breast cancer therapy for its cytotoxic efficacy and non‐toxic nature. Copyright © 2014 John Wiley & Sons, Ltd.     

A Polyphenol‐rich Pomegranate Fruit Extract Suppresses NF‐κB and IL‐6 Expression by Blocking the Activation of IKKβ and NIK in Primary Human Chondrocytes

Pomegranate fruit extract (PE) rich in polyphenols has been shown to exert chondroprotective effects, but the mechanism is not established. Here, we used an in vitro model of inflammation in osteoarthritis (OA) to investigate the potential of PE to suppress interleukin 1 beta (IL‐1β)‐stimulated expression of inflammatory cytokine IL‐6, generation of reactive oxygen species (ROS) levels, and investigated the mechanism of NF‐κB inhibition by analyzing the activation of the kinases upstream of IκBα in primary human chondrocytes. Total and phosphorylated forms of kinases and expression of IL‐6 were determined at protein and mRNA levels by western immunoblotting and Taqman assay, respectively. Dihydrorhodamine 123 staining estimated ROS generation. Pomegranate fruit extract inhibited the mRNA and protein expression of IL‐6, generation of ROS, and inhibited the IL‐1β‐mediated phosphorylation of inhibitor of nuclear factor kappa‐B kinase subunit beta (IKKβ), expression of IKKβ mRNA, degradation of IκBα, and activation and nuclear translocation of NF‐κB/p65 in human chondrocytes. Importantly, phosphorylation of NF‐κB‐inducing kinase was blocked by PE in IL‐1β‐treated human OA chondrocytes. Taken together, these data suggest that PE exerts the chondroprotective effect(s) by suppressing the production of IL‐6 and ROS levels. Inhibition of NF‐κB activation by PE was blocked via modulation of activation of upstream kinases in human OA chondrocytes. Copyright © 2017 John Wiley & Sons, Ltd.     

A New Isoflavone Glycitein 7‐O‐beta‐d‐Glucoside 4'‐O‐methylate,isolated from Cordyceps militaris grown on Germinated Soybeans Extract,Inhibits EGF‐induced Mucus Hypersecretion in the Human Lung Mucoepidermoid Cells

A new isoflavone glycitein 7‐O‐beta‐d ‐glucoside 4''‐O‐methylate (CGLM) has been isolated recently from Cordyceps militaris grown on germinated soybean extract and has antioxidant activity. In the present study, CGLM was investigated for its suppression of airway mucous hyper‐secretion in epidermal growth factor (EGF)‐treated human lung mucoepidermoid cells. NCI‐H292 cells were treated with CGLM for 1 h, followed by EGF treatment for 24 h. The decrease in cyclooxygenase‐2 (COX‐2) production was correlated with reduced levels of protein and mRNA of inducible matrix metalloproteinase 9 (MMP‐9) and also MUC5AC gene expression. CGLM directly inhibited down‐regulated NF‐κB activity, and significantly inhibited the phosphorylation of p38 and ERK1/2 (p42/p44) in NCI‐H292 cells. These results suggest that CGLM protects NCI‐H292 cells from EGF‐induced damage by down‐regulation of COX‐2, MMP‐9 and MUC5AC gene expression, mediated via blocking the NF‐kappa‐B and p38/ERK MAPK pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Regulation of adhesion molecules expression in TNF‐α‐stimulated brain microvascular endothelial cells by tanshinone IIA: involvement of NF‐κB and ROS generation

Pro‐inflammatory cytokine‐mediated expression of cell surface adhesion molecules plays a key role in endothelial cell injury, leading to vascular inflammation and the development of many cerebrovascular diseases. Thus, antiinflammatory agents targeting these adhesion molecules may represent potential drugs for the prevention and treatment of cerebrovascular diseases. The present study explored the effects of tanshinone IIA (Tan IIA), an active ingredient present in the Salvia miltiorrhiza root, on the expression of cellular adhesion molecules in TNF‐α‐stimulated brain microvascular endothelial cells (BMVECs). Treatment with Tan IIA was found to suppress the expression of vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1), resulting in inhibition of TNF‐α‐induced adhesion of neutrophils to BMVECs in a dose‐dependent manner. In addition, Tan IIA significantly inhibited TNF‐α‐induced production of reactive oxygen species (ROS), which was accompanied by decreased malondialdehyde (MDA) levels. Treatment with Tan IIA also inhibited nuclear factor‐kappa B (NF‐κB) activation. Together, these results suggest that Tan IIA regulates TNF‐α‐induced expression of VCAM‐1 and ICAM‐1 through inhibition of NF‐κB activation and ROS generation in BMVECs. Copyright © 2010 John Wiley & Sons, Ltd.     

Astragaloside IV inhibits TGF‐β1‐induced epithelial‐mesenchymal transition through inhibition of the PI3K/Akt/NF‐κB pathway in gastric cancer cells

Astragaloside IV (AS‐IV) has been reported to possess anti‐metastasis activity in cancer cells. However, it is unknown whether AS‐IV could inhibit epithelial‐mesenchymal transition (EMT), a cellular de‐differentiation program that promotes metastasis, in cancer cells. The aim of this study was to study the effect and mechanism of AS‐IV on EMT in gastric cancer (GC) cells. The results showed that AS‐IV significantly inhibited cell viability, invasion, and migration of GC cells. The E‐cadherin to N‐cadherin switch and expression of Vimentin and metastasis‐related genes were induced by transforming growth factor β1 (TGF‐β1), whereas AS‐IV reversed the induction. In addition, AS‐IV inhibited TGF‐β1‐induced activation of PI3K/Akt/NF‐κB. Inhibition of the PI3K/Akt/NF‐κB pathway reversed TGF‐β1‐induced EMT. In conclusion, AS‐IV inhibited TGF‐β1‐induced EMT through inhibition of the PI3K/Akt/NF‐κB pathway in GC cells. AS‐IV might be an effective candidate for the treatment for GC.     

Trametenolic Acid B Reverses Multidrug Resistance in Breast Cancer Cells Through Regulating the Expression Level of P‐Glycoprotein

Trametenolic acid B (TAB) is the main active composition of Trametes lactinea (Berk.) Pat which possesses anti‐tumor activities. There was no report its antitumor effect through regulating P‐glycoprotein (P‐gp) so far, due to P‐gp over expression is one of the most important mechanisms contributing to the multiple drug resistance phenotype. The present aim was to investigate the effects of TAB on P‐gp in multidrug‐resistant cells; Paclitaxel‐resistant cell line MDA‐MB‐231/Taxol was established by stepwise exposure for 10 months. MDA‐MB‐231 cells and MDA‐MB‐231/Taxol cells were treated with TAB, and their growth was evaluated using MTT assays. Paclitaxel accumulation in the cells was analyzed by high performance liquid chromatogram (HPLC). The activity of P‐gp was detected by intracellular accumulation of rhodamine123 (Rho123), and the protein expression of P‐gp was evaluated using western blot. Results indicated that the IC₅₀ of MDA‐MB‐231/Taxol to paclitaxel (Taxol) was 33 times higher than that of nature MDA‐MB‐231. TAB increased the intracellular concentration of Taxol and inhibited the activity of P‐gp and suppressed the expression of P‐gp in MDA‐MB‐231/Taxol cells. Our present results showed that TAB could reverse Taxol resistance in MDA‐MB‐231/Taxol cells, mainly inhibiting the activity of P‐gp and down‐regulating the expression level of P‐gp, and then enhancing the accumulation of chemotherapy agents. © 2013 The Authors Phytotherapy Research Published by John Wiley & Sons Ltd.     

Anticancer activity and molecular mechanisms of α‐conidendrin,a polyphenolic compound present in Taxus yunnanensis,on human breast cancer cell lines

α‐Conidendrin is a polyphenolic compound found mainly in Taxus yunnanensis, as the source of chemotherapy drug paclitaxel, which has been used in traditional medicine for treatment of cancer. This study aimed to investigate the anticancer activity and molecular mechanisms of α‐conidendrin on breast cancer cell lines. The results of the present study show that α‐conidendrin possesses potent antiproliferative effects on breast cancer cell lines MCF‐7 and MDA‐MB‐231. α‐Conidendrin significantly induced apoptosis in breast cancer cells via reactive oxygen species generation, upregulation of p53 and Bax, downregulation of Bcl‐2, depolarization of mitochondrial membrane potential (MMP), release of cytochrome c from mitochondria, and activation of caspases‐3 and ‐9. α‐Conidendrin remarkably inhibited the proliferation of breast cancer cells through induction of cell cycle arrest by upregulating p53 and p21 and downregulating cyclin D1 and CDK4. Unlike breast cancer cells, the antiproliferative effect of α‐conidendrin on human foreskin fibroblast cells (normal cells) was very small. In normal cells, reactive oxygen species levels, loss of MMP, release of cytochrome c, mRNA expression of p53, p21, cyclin D1, CDK4, Bax, and Bcl‐2 as well as mRNA expression and activity of caspases‐3 and ‐9 were significantly less affected by α‐conidendrin compared with cancer cells. These results suggest that α‐conidendrin can be a promising agent for treatment of breast cancer with little or no toxicity against normal cells.     

1,2,3,6‐tetra‐O‐galloyl‐β‐D‐allopyranose gallotannin isolated,from Euphorbia jolkini,attenuates LPS‐induced nitric oxide production in macrophages

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation and macrophage‐mediated immunity. Macrophages express inducible NO synthase (iNOS) and produce NO after lipopolysaccharide (LPS) stimulation. Gallotannins are water‐soluble polyphenols with wide‐ranging biological activities. Various chemical structures of gallotannins occurring in medicinal and food plants that are used worldwide showed several remarkable biological and pharmacological activities. In the present study, we examined the inhibitory effects of gallotannin 1,2,3,6‐tetra‐O‐galloyl‐β‐D‐allopyranose (GT24) isolated from Euphorbia jolkini on the LPS‐induced NO production and underlying mechanisms of action. GT24 dose‐dependently decreased LPS‐induced NO production and iNOS expression in J774A.1 macrophages. In addition, GT24 inhibited LPS‐induced activation of nuclear factor (NF)‐κB as indicated by inhibition of degradation of I‐κBα, nuclear translocation of NF‐κB, and NF‐κB dependent gene reporter assay. Our results suggest that GT24 possesses an inhibitory effect on the LPS‐induced inflammatory reaction. Copyright © 2010 John Wiley & Sons, Ltd.     

Theacrine attenuates epithelial mesenchymal transition in human breast cancer MDA‐MB‐231 cells

Theacrine, a purine alkaloid structurally similar to caffeine, has recently become of interest as a potential therapeutic compound. Here, we investigated the antimetastatic potential of theacrine on human breast cancer MDA‐MB‐231 cells. We observed that theacrine can reverse epithelial‐to‐mesenchymal transition (EMT), which resulted in a decrease in the levels of mesenchymal markers (Fibronectin, Vimentin, N‐cadherin, Twist, and Snail) and an increase in the levels of epithelial markers (Occludin and E‐cadherin) in the cells. Additionally, theacrine attenuates TGF‐β‐induced EMT, cell adhesion, migration, and invasion in MDA‐MB‐231 cells. Overall, our results suggest that theacrine may inhibit the breast cancer cell metastasis by reversing the EMT process.     

Apoptosis Induction by 13‐Acetoxyrolandrolide through the Mitochondrial Intrinsic Pathway

The aim of this study was to evaluate the mechanisms of cytotoxicity of the sesquiterpene lactone 13‐acetoxyrolandrolide, a nuclear factor kappa B (NF‐κB) inhibitor that was previously isolated from Rolandra fruticosa. The effects associated with the inhibition of the NF‐κB pathway included dose‐dependent inhibition of the NF‐κB subunit p65 (RelA) and inhibition of upstream mediators IKKβ and oncogenic Kirsten rat sarcoma (K‐Ras). The inhibitory concentration of 13‐acetoxyrolandrolide on K‐Ras was 7.7 µm . The downstream effects of the inhibition of NF‐κB activation were also investigated in vitro. After 24 h of treatment with 13‐acetoxyrolandrolide, the mitochondrial transmembrane potential was depolarized in human colon cancer (HT‐29) cells. The mitochondrial oxidative phosphorylation was also negatively affected, and reduced levels of nicotinamine adenine dinucleotide phosphate (NAD(P)H) were detected after 2 h of 13‐acetoxyrolandrolide exposure. Furthermore, the expression of the pro‐apoptotic protein caspase‐3 increased in a concentration‐dependent manner. Cell flow cytometry showed that 13‐acetoxyrolandrolide induced cell cycle arrest at G₁, indicating that the treated cells had undergone caspase‐3‐mediated apoptosis, indicating negative effects on cancer cell proliferation. These results suggest that 13‐acetoxyrolandrolide inhibits NF‐κB and K‐Ras and promotes cell death mediated through the mitochondrial apoptotic pathway. Copyright © 2013 John Wiley & Sons, Ltd.     

Nerolidol Protects Against LPS‐induced Acute Kidney Injury via Inhibiting TLR4/NF‐κB Signaling

Acute kidney injury (AKI) is a critical care syndrome, resulting in acute reduction of renal function and up to 22% mortality of hospitalized patients. Nerolidol is a major component in several essential oils that possesses various pharmacological properties. The present study aimed to investigate the potential effect of nerolidol on lipopolysaccharide (LPS)‐induced AKI. Nerolidol dose‐dependently reduced the pathological injuries of kidney induced by LPS in rats. Nerolidol significantly decreased the levels of blood urea nitrogen and creatinine in LPS‐treated rats in a dose‐dependent manner. In addition, nerolidol inhibited LPS‐induced decrease of cell viability in NRK‐52E rat proximal tubular cells, which effect was concentration dependent. Nerolidol notably inhibited the increase of TNFα and IL‐1β in LPS‐treated rats and the mRNA expression of TNFα and IL‐1β in LPS‐treated NRK‐52E cells. Nerolidol suppressed the increase of toll‐like receptor 4 (TLR4) expression, phosphorylation and nuclear translocation of p65 NF‐κB in kidneys of LPS‐treated rats and LPS‐treated NRK‐52E cells. Overexpression of TLR4 and p65 NF‐κB significantly suppressed nerolidol‐induced inhibition of TNFα and IL‐1β expression and increase of cell viability in LPS‐treated cells. In summary, we found that nerolidol played a critical anti‐inflammatory effects through inhibition of TLR4/NF‐κB signaling and protected against LPS‐induced AKI. Copyright © 2017 John Wiley & Sons, Ltd.     

Artichoke Leaf Extract Inhibits AKR1B1 and Reduces NF‐κB Activity in Human Leukemic Cells

The human intracellular enzyme AKR1B1 belongs to the aldo‐keto reductase superfamily. The AKR1B1‐catalyzed reduction of aldehydes is part of the intracellular inflammatory pathway leading to the activation of NF‐κB and the expression of pro‐inflammatory genes. The present study is aimed at determining the inhibition of AKR1B1 brought about by an extract of artichoke leaves (bracts), and the effects of this extract and three participating compounds on the expression of AKR1B1, COX‐2, and MMP‐2 proteins in THP‐1 cells. It seeks to identify the ability of the test substances to modulate the lipopolysaccharide (LPS)‐induced activation of NF‐κB in cells and the intracellular oxidant effect of test substances after incubation with LPS. Low concentrations of the extract inhibit the enzyme AKR1B1. After stimulation by LPS, the extract attenuated the activity of NF‐κB in THP‐1 cells, but no changes in the expression of AKR1B1 were recorded. The extract diminished the expression of the inflammation‐related enzymes COX‐2 and MMP‐2, probably by inhibiting the activity of NF‐κB. The extract significantly diminished the intracellular reactive oxygen species after a brief LPS incubation, which may also have reduced intracellular inflammation. The diminished activity of NF‐κB in the cells could be linked to the inhibition of the activity of AKR1B1. Copyright © 2017 John Wiley & Sons, Ltd.     

Cytotoxic Impact of Costunolide Isolated from Costus speciosus on Breast Cancer via Differential Regulation of Cell Cycle—An In‐vitro and In‐silico Approach

Costunolide, a sesquiterpene lactone, is a biologically active molecule found in most of the medicinally valuable plants. The present study aims to evaluate the anticancer property of costunolide isolated from Costus speciosus against breast cancer cell lines (MCF‐7 and MDA‐MB‐231). Costunolide effectively reduced the viability of both MCF‐7 and MDA‐MB‐231 cell lines at an IC₅₀ value of 40 μM. Flow cytometric analysis revealed costunolide mediated cell cycle arrest at G2/M phase in both the cell types. Western blotting results confirmed the alterations in the expression of cell cycle regulators (cyclin D1, D3, CDK‐4, CDK‐6, p18 INK4c, p21 CIP1/Waf‐1 and p27 KIP1) and apoptosis inducers (caspase‐3 and caspase‐9) upon costunolide treatment in comparison with their expressions in normal breast cell line (MCF‐10A). Costunolide mediated downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators were related to the induction of apoptosis in cancer cells. The above results were validated with in‐silico results that predicted stable interactions between costunolide and cancer targets. Thus costunolide effectively induced breast cancer cell apoptosis targeting cell cycle regulation, and the compound can be used as an effective herbal therapeutic molecule to treat breast cancer with further explorations. Copyright © 2015 John Wiley & Sons, Ltd.