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.     

Ramalin‐Mediated Apoptosis Is Enhanced by Autophagy Inhibition in Human Breast Cancer Cells

Breast cancer, the most commonly diagnosed cancer in women worldwide, is treated in various ways. Ramalin is a chemical compound derived from the Antarctic lichen Ramalina terebrata and is known to exhibit antioxidant and antiinflammatory activities. However, its effect on breast cancer cells remains unknown. We examined the ability of ramalin to induce apoptosis and its mechanisms in MCF‐7 and MDA‐MB‐231 human breast cancer cell lines. Ramalin inhibited cell growth and induced apoptosis in both cell lines in a concentration‐dependent manner. By upregulating Bax and downregulating Bcl‐2, ramalin caused cytochrome c and apoptosis‐inducing factor to be released from the mitochondria into the cytosol, thus activating the mitochondrial apoptotic pathway. In addition, activated caspase‐8 and caspase‐9 were detected in both types of cells exposed to ramalin, whereas ramalin activated caspase‐3 only in the MDA‐MB‐231 cells. Ramalin treatment also increased the levels of LC3‐II and p62. Moreover, the inhibition of autophagy by 3‐methyladenine or Atg5 siRNA significantly enhanced ramalin‐induced apoptosis, which was accompanied by a decrease in Bcl‐2 levels and an increase in Bax levels. Therefore, autophagy appears to be activated as a protective mechanism against apoptosis in cancer cells exposed to ramalin. These findings suggest that ramalin is a potential anticancer agent for the treatment of patients with non‐invasive or invasive breast cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

Fangchinoline Induces G1 Arrest in Breast Cancer Cells Through Cell‐Cycle Regulation

Fangchinoline, an alkaloid derived from the dry roots of Stephaniae tetrandrine S. Moore (Menispermaceae), has been shown to possess cytotoxic, anti‐inflammatory, and antioxidant properties. In this study, we used Fangchinoline to inhibit breast cancer cell proliferation and to investigate its underlying molecular mechanisms. Human breast cancer cell lines, MCF‐7 and MDA‐MB‐231, were both used in this study. We found that Fangchinoline significantly decreased cell proliferation in a dose‐dependent manner and induced G1‐phase arrest in both cell lines. In addition, upon analysis of expression of cell cycle‐related proteins, we found that Fangchinoline reduced expression of cyclin D1, cyclin D3, and cyclin E, and increased expression of the cyclin‐dependent kinase (CDK) inhibitors, p21/WAF1, and p27/KIP1. Moreover, Fangchinoline also inhibited the kinase activities of CDK2, CDK4, and CDK6. These results suggest that Fangchinoline can inhibit human breast cancer cell proliferation and thus may have potential applications in cancer therapy. Copyright © 2013 John Wiley & Sons, Ltd.     

F1012‐2 inhibits the growth of triple negative breast cancer through induction of cell cycle arrest,apoptosis, and autophagy

Sesquiterpene lactones (SLs) are plant‐derived constituents that have been proved to have potential antitumour activity. However, the intracellular molecular targets of SLs and the underlying molecular mechanisms have not been well elucidated. Here, we report that F1012‐2, a novel SL active fraction, isolated from Eupatorium lindleyanum DC., can significantly inhibit the growth of triple‐negative breast cancer (TNBC) cells (MDA‐MB‐231 and MDA‐MB‐468) but has no obvious inhibitory effect on the growth of human mammary epithelial cells (MCF‐10A). The related mechanisms on cell growth inhibition of F1012‐2 were demonstrated by inducing apoptosis in a caspase‐dependent manner through the intrinsic pathway and extrinsic pathway. F1012‐2 could also activate autophagy in TNBC cells. Simultaneously, we found that F1012‐2‐induced apoptosis was enhanced by inhibition of autophagy. Furthermore, F1012‐2 could induce cell cycle arrest at G2/M phase with decreasing expression of cyclin B1, cdc2, and upregulating p21, p‐cdc2. Also, F1012‐2 activated Akt and p38 signalling pathways. In vivo, F1012‐2 exhibited a potential antitumour effect in MDA‐MB‐231 xenografts without apparent toxicity. Taken together, our results identified that F1012‐2 inhibited cell growth via multiple signalling pathways in vitro and in vivo. These data suggest that F1012‐2 may be a potential natural active fraction for the treatment of TNBC.     

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.     

Apoptotic effect of lambertianic acid through AMPK/FOXM1 signaling in MDA‐MB231 breast cancer cells

Though lambertianic acid (LA) was known to exert antitumor effect in liver and prostate cancers, its underlying anticancer mechanism is never reported in breast cancers so far. Thus, in this study, apoptotic mechanism of LA was elucidated in MDA‐MB‐231 breast cancer cells. Here, LA increased cytotoxicity in MCF‐7 and MDA‐MB‐231 cells; enhanced sub‐G1 population, G2/M arrest, and cleaved poly(ADP‐ribose) polymerase; activated phosphorylation of AMP‐activated protein kinase (AMPK)/acetyl‐CoA carboxylase pathway; and also suppressed phosphorylation of AKT and the expression of forkhead box M1 (FOXM1), X‐linked inhibitor of apoptosis protein, B‐cell lymphoma 2, and CyclinB1 in MDA‐MB‐231 cells. Furthermore, AMPK inhibitor compound C reversed the effect of LA on FOXM1, Cyclin B1, and cleaved poly(ADP‐ribose) polymerase in MDA‐MB‐231 cells. Notably, immunoprecipitation revealed that LA disturbed the direct binding of AKT and FOXM1 in MDA‐MB‐231 cells. Overall, these findings suggest that LA‐induced apoptosis is mediated via activation of AMPK and inhibition of AKT/FOXM1 signaling pathway.     

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.     

Rutin,a Quercetin Glycoside,Restores Chemosensitivity in Human Breast Cancer Cells

Several studies have documented the ability of flavonoids to sensitize cancer cells to chemotherapeutics and reverse multidrug resistance by inhibition of efflux pumps (adenosine triphosphate‐binding cassette transporters), apoptosis activation, and cell cycle arrest. In this study, the flavonoid rutin (quercetin 3‐O‐β‐d ‐rutinoside) was investigated as chemosensitizer towards two different human epithelial breast cancer cell lines: (i) MB‐MDA‐231, selected as representative for triple‐negative breast cancer and (ii) MCF‐7 used as a well‐characterized model of HER2‐negative breast cancer. To assess the cytocompatibility of rutin against non‐cancer cells, primary human mammary fibroblasts were used as control and non‐target cells. In MDA‐MB‐231 cells, 20 μM rutin enhanced cytotoxicity related to cyclophosphamide and methotrexate. Rutin significantly (p < 0.05) increased the anticancer activity of both chemotherapeutics, at 24–48–72 h, and decreased the activity of the adenosine triphosphate‐binding cassette transporters, namely, P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP). Flow cytometry analysis showed 20 μM and 50 μM rutin arrested cell cycle at G2/M and G0/G1 phases, respectively, significantly promoting cell apoptosis. Rutin, via non‐selective inhibition of P‐gp and BCRP pumps, efficiently reverses multidrug resistance and restores chemosensitivity to cyclophosphamide and cyclophosphamide of human chemoresistant, triple‐negative breast cancer cells, successfully arresting cell cycle progression. Copyright © 2017 John Wiley & Sons, Ltd.     

Growth inhibition and cell cycle arrest in the G0/G1 by schizandrin,a dibenzocyclooctadiene lignan isolated from Schisandra chinensis,on T47D human breast cancer cells

Schizandrin is one of the main dibenzocyclooctadiene lignans present in the fruit of Schisandra chinensis (Schisandraceae). Biological activities including hepatoprotective, antiviral and neuroprotective effects of schizandrin and other dibenzocyclooctadiene lignans have been reported previously. However, the antiproliferative effect of schizandrin against human cancer cells has been poorly determined to date. This study examined the antiproliferative effect of schizandrin in human breast cancer cells. Schizandrin exhibited growth inhibitory activities in cultured human breast cancer cells, and the effect was the more profound in estrogen receptor (ER)‐positive T47D cells than in ER‐negative MDA‐MB‐231 cells. When treated with the compound in T47D cells, schizandrin induced the accumulation of a cell population in the G0/G1 phase, which was further demonstrated by the induction of CDK inhibitors p21 and p27 and the inhibition of the expression of cell cycle checkpoint proteins including cyclin D1, cyclin A, CDK2 and CDK4. These results suggest that schizandrin inhibits cell proliferation through the induction of cell cycle arrest with modulating cell cycle‐related proteins in human breast cancer cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Antitumour effects of phyllanthus emblica L.: Induction of cancer cell apoptosis and Inhibition of in vivo tumour promotion and in vitro invasion of human cancer cells

Phyllanthus emblica Linn. (PE) is a medicinal fruit used in many Asian traditional medicine systems for the treatment of various diseases including cancer. The present study tested the potential anticancer effects of aqueous extract of PE in four ways: (1) against cancer cell lines, (2) in vitro apoptosis, (3) mouse skin tumourigenesis and (4) in vitro invasiveness. The PE extract at 50–100 µg/mL significantly inhibited cell growth of six human cancer cell lines, A549 (lung), HepG2 (liver), HeLa (cervical), MDA‐MB‐231 (breast), SK‐OV3 (ovarian) and SW620 (colorectal). However, the extract was not toxic against MRC5 (normal lung fibroblast). Apoptosis in HeLa cells was also observed as PE extract caused DNA fragmentation and increased activity of caspase‐3/7 and caspase‐8, but not caspase‐9, and up‐regulation of the Fas protein indicating a death receptor‐mediated mechanism of apoptosis. Treatment of PE extract on mouse skin resulted in over 50% reduction of tumour numbers and volumes in animals treated with DMBA/TPA. Lastly, 25 and 50 µg/mL of PE extract inhibited invasiveness of MDA‐MB‐231 cells in the in vitro Matrigel invasion assay. These results suggest P. emblica exhibits anticancer activity against selected cancer cells, and warrants further study as a possible chemopreventive and antiinvasive agent. Copyright © 2010 John Wiley & Sons, Ltd.     

β‐Bisabolene,a Sesquiterpene from the Essential Oil Extract of Opoponax (Commiphora guidottii), Exhibits Cytotoxicity in Breast Cancer Cell Lines

The essential oils from Commiphora species have for centuries been recognized to possess medicinal properties. Here, we performed gas chromatography‐mass spectrometry on the essential oil from opoponax (Commiphora guidotti) and identified bisabolene isomers as the main constituents of this essential oil. Opoponax essential oil, a chemical component; β‐bisabolene and an alcoholic analogue, α‐bisabolol, were tested for their ability to selectively kill breast cancer cells. Only β‐bisabolene, a sesquiterpene constituting 5% of the essential oil, exhibited selective cytotoxic activity for mouse cells (IC₅₀ in normal Eph4: >200 µg/ml, MG1361: 65.49 µg/ml, 4T1: 48.99 µg/ml) and human breast cancer cells (IC₅₀ in normal MCF‐10A: 114.3 µg/ml, MCF‐7: 66.91 µg/ml, MDA‐MB‐231: 98.39 µg/ml, SKBR3: 70.62 µg/ml and BT474: 74.3 µg/ml). This loss of viability was because of the induction of apoptosis as shown by Annexin V‐propidium iodide and caspase‐3/7 activity assay. β‐bisabolene was also effective in reducing the growth of transplanted 4T1 mammary tumours in vivo (37.5% reduction in volume by endpoint). In summary, we have identified an anti‐cancer agent from the essential oil of opoponax that exhibits specific cytotoxicity to both human and murine mammary tumour cells in vitro and in vivo, and this warrants further investigation into the use of β‐bisabolene in the treatment of breast cancers. Copyright © 2015 John Wiley & Sons, Ltd.     

Drug‐like dietary vanilloids induce anticancer activity through proliferation inhibition and regulation of bcl‐related apoptotic proteins

In this study, a series of 20 structurally similar vanilloids (Vn) were tested for their antiproliferative effects against 12 human cancer cells: human breast (MCF‐7 and MDA‐MB‐231), cervical (HeLa), ovarian (Caov‐3), lung (A549), liver (HepG2), colorectal (HT‐29 and HCT116), nasopharyngeal (CNE‐1 and HK‐1), and leukemic (K562 and CEM‐SS) cancer cells. Among all the tested vanilloids, Vn16 (6‐shogaol) exhibited the most potent cytotoxic effects against human colorectal cancer cells (HT‐29). The apoptotic induction effects exhibited by Vn16 on HT‐29 cells were confirmed using dual staining fluorescence microscopy and enzyme‐linked immunosorbent assay. The effects of Vn16 on regulation of 43 apoptotic‐related markers were determined in HT‐29. The results suggested that 8 apoptotic markers (caspase 8, BAD, BAX, second mitochondrial‐derived activator, caspase 3, survivin, bcl‐2, and cIAP‐2) were either upregulated or downregulated. These results further support the chemopreventive properties of foods that contain vanilloids.     

Norstictic Acid Inhibits Breast Cancer Cell Proliferation,Migration, Invasion,and In Vivo Invasive Growth Through Targeting C‐Met

Breast cancer is a major health problem affecting the female population worldwide. The triple‐negative breast cancers (TNBCs) are characterized by malignant phenotypes, worse patient outcomes, poorest prognosis, and highest mortality rates. The proto‐oncogenic receptor tyrosine kinase c‐Met is usually dysregulated in TNBCs, contributing to their oncogenesis, tumor progression, and aggressive cellular invasiveness that is strongly linked to tumor metastasis. Therefore, c‐Met is proposed as a promising candidate target for the control of TNBCs. Lichens‐derived metabolites are characterized by their structural diversity, complexity, and novelty. The chemical space of lichen‐derived metabolites has been extensively investigated, albeit their biological space is still not fully explored. The anticancer‐guided fractionation of Usnea strigosa (Ach.) lichen extract led to the identification of the depsidone‐derived norstictic acid as a novel bioactive hit against breast cancer cell lines. Norstictic acid significantly suppressed the TNBC MDA‐MB‐231 cell proliferation, migration, and invasion, with minimal toxicity to non‐tumorigenic MCF‐10A mammary epithelial cells. Molecular modeling, Z'‐LYTE biochemical kinase assay and Western blot analysis identified c‐Met as a potential macromolecular target. Norstictic acid treatment significantly suppressed MDA‐MB‐231/GFP tumor growth of a breast cancer xenograft model in athymic nude mice. Lichen‐derived natural products are promising resources to discover novel c‐Met inhibitors useful to control TNBCs. 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.     

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.