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1.  Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways 
PLoS ONE  2013;8(10):e77270.
Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.
doi:10.1371/journal.pone.0077270
PMCID: PMC3790876  PMID: 24124611
2.  A Dietary Anthocyanidin Delphinidin Induces Apoptosis of Human Prostate Cancer PC3 Cells In vitro and In vivo: Involvement of Nuclear Factor-κB Signaling 
Cancer research  2008;68(20):8564-8572.
Delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables, possesses antioxidant, anti-inflammatory, and antiangiogenic properties. In this study, we provide evidence that it could be developed as a novel agent against human prostate cancer (PCa). We observed that delphinidin treatment to human PCa LNCaP, C4-2, 22Rν1, and PC3 cells resulted in a dose-dependent inhibition of cell growth without having any substantial effect on normal human prostate epithelial cells. We selected PC3 cells as a test model system because of their highly aggressive proliferative nature. Delphinidin treatment of cells resulted in a dose-dependent induction of apoptosis and arrest of cells in G2-M phase. This induction of apoptosis seems to be mediated via activation of caspases because N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluromethylketone significantly reduced apoptosis induced by delphinidin. We also observed that delphinidin treatment of cells resulted in a dose-dependent decrease in (a) phosphorylation of IκB kinase γ (NEMO), (b) phosphorylation of nuclear factor-κB (NF-κB) inhibitory protein IκBα, (c) phosphorylation of NF-κB/p65 at Ser536 and NF-κB/p50 at Ser529, (d) NF-κB/p65 nuclear translocation, and (e) NF-κB DNA binding activity. Delphinidin administration (2 mg, i.p. thrice weekly) to athymic nude mice implanted with PC3 cells resulted in a significant inhibition of tumor growth. Analysis of tumors from delphinidin-treated mice showed significant decrease in the expression of NF-κB/p65, Bcl2, Ki67, and PCNA. Taken together, our data suggest that delphinidin could be developed as an agent against human PCa.
doi:10.1158/0008-5472.CAN-08-2232
PMCID: PMC3149885  PMID: 18922932
3.  Delphinidin inhibits cell proliferation and invasion via modulation of Met receptor phosphorylation 
The HGF/Met signaling pathway is deregulated in majority of cancers and is associated with poor prognosis in breast cancer. Delphinidin, present in pigmented fruits and vegetables possesses potent anti-oxidant, anti-inflammatory and anti-angiogenic properties. Here, we assessed the anti-proliferative and anti-invasive effects of delphinidin on HGF-mediated responses in the immortalized MCF-10A breast cell line. Treatment of cells with delphinidin prior to exposure to exogenous HGF resulted in the inhibition of HGF-mediated (i) tyrosyl-phosphorylation and increased expression of Met receptor, (ii) phosphorylation of downstream regulators such as FAK and Src and (iii) induction of adaptor proteins including paxillin, Gab-1 and GRB-2. In addition, delphinidin treatment resulted in significant inhibition of HGF-activated (i) Ras-ERK MAPKs and (ii) PI3K/AKT/mTOR/p70S6K pathways. Delphinidin was found to repress HGF-activated NFκB transcription with a decrease in (i) phosphorylation of IKKα/β and IκBα, and (ii) activation and nuclear translocation of NFκB/p65. Inhibition of HGF-mediated membrane translocation of PKCα as well as decreased phosphorylation of STAT3 was further observed in delphinidin treated cells. Finally, decreased cell viability of Met receptor expressing breast cancer cells treated with delphinidin argues for a potential role of the agent in the prevention of HGF-mediated activation of various signaling pathways implicated in breast cancer.
doi:10.1016/j.taap.2008.03.023
PMCID: PMC2989819  PMID: 18499206
Delphinidin; Hepatocyte growth factor; Breast cancer
4.  Delphinidin, a dietary anthocyanidin in pigmented fruits and vegetables 
Cell cycle (Georgetown, Tex.)  2008;7(21):3320-3326.
In a recent publication, we have shown that delphinidin, an anthocyanidin induces apoptosis and cell cycle arrest in highly metastatic human prostate cancer (PCa) PC3 cells. Extending these studies, we provide additional evidence that delphinidin induces apoptosis and cell cycle arrest in androgen refractory human PCa 22Rν1 cells and that these effects are concomitant with inhibition of NFκB. We observed that delphinidin treatment to 22Rν1 cells resulted in a dose-dependent (i) G2/M phase cell cycle arrest, (ii) induction of apoptosis (iii) and inhibition of NFκB signaling. The induction of apoptosis by delphinidin was mediated via activation of caspases since a general caspase inhibitor Z-VAD-FMK significantly reversed this effect. Delphinidin treatment to cells resulted in a dose-dependent decrease in (i) phosphorylation of IKKγ (NEMO), (ii) phosphorylation of NFκB inhibitory protein IκBα, (iii) phosphorylation of NFκB/p65 at Ser536 and NFκB/p50 at Ser529, (iv) NFκB/p65 nuclear translocation, and (v) NFκB DNA binding activity. Taken together, our data show that delphinidin induces apoptosis of both androgen independent and androgen refractory human PCa cells via activation of caspases and in addition, this effect might be due to inhibition of NFκB signaling. We suggest that delphinidin could be developed as a novel agent against PCa.
PMCID: PMC2989799  PMID: 18948740
delphinidin; apoptosis; NFκB; prostate cancer
5.  Influence of Berry-Polyphenols on Receptor Signaling and Cell-Death Pathways: Implications for Breast Cancer Prevention 
Breast cancer is the most commonly diagnosed cancer among women worldwide. Many women have become more aware of the benefits of increasing fruit consumption, as part of a healthy lifestyle, for the prevention of cancer. The mechanisms by which fruits, including berries, prevent breast cancer can be partially explained by exploring their interactions with pathways known influence cell-proliferation and evasion of cell-death. Two receptor pathways- estrogen receptor (ER) and tyrosine kinase receptors, especially the epidermal growth factor receptor (EGFR) family- are drivers of cell-proliferation and play a significant role in the development of both primary and recurrent breast cancer. There is strong evidence to show that several phytochemicals present in berries such as cyanidin, delphinidin, quercetin, kaempferol, ellagic acid, resveratrol and pterostilbene, interact with and alter the effects of these pathways. Further, they also induce cell death (apoptosis and autophagy) via their influence on kinase signaling. In this review, we summarize in vitro data regarding the interaction of berry polyphenols with the specific receptors and the mechanisms by which they induce cell death. Further, we also present in vivo data of primary breast cancer prevention by individual compounds and whole berries. Finally, we present a possible role for berries and berry compounds in the prevention of breast cancer and our perspective on the areas that require further research.
doi:10.1021/jf204084f
PMCID: PMC3383353  PMID: 22300613
Berries; Berry Polyphenols; Breast Cancer; Ellagic Acid; Cyanidin; Delphinidin; Quercetin; Kaempherol; Resveratrol; Estrogen Receptor; Epidermal Growth Factor Receptor; Kinase Signaling; Apoptosis; Autophagy; ACI rats
6.  Effect of Berry Extracts and Bioactive Compounds on Fulvestrant (ICI 182,780) Sensitive and Resistant Cell Lines 
Fulvestrant (ICI 182,780; ICI) is approved for the treatment of advanced metastatic breast cancer that is unresponsive to other endocrine therapies. Berries are frequently consumed for their antioxidant, anti-inflammatory, and anticancer potential. In this study, we tested the efficacy of two berry extracts (Jamun-EJAE and red raspberry-RRE) and their bioactive compounds (Delphinidin-Del and Ellagic acid-EA) to inhibit cell proliferation with or without a sublethal dose of ICI in various breast cancer cell lines. ICI-sensitive (LCC1, ZR75-1, and BT474) and -resistant (LCC9, ZR75-1R) cells were subjected to treatment with berry extracts alone (0.1–100 μg/mL) or with a sub-lethal dose of ICI ( 1). EA, in doses tested, did not have any significant effects on any of the cell lines. Finally, we found that the extracts were more effective at lower, physiologically relevant concentrations than at higher experimental doses.
doi:10.1155/2012/147828
PMCID: PMC3549366  PMID: 23346406
7.  Modulation of the BRCA1 protein and induction of apoptosis in triple negative breast cancer cell lines by the polyphenolic compound curcumin 
In the current study, we sought to examine the effects of curcumin in a specific type of breast cancer called triple negative breast cancer. These cancers lack expression of the estrogen and progesterone receptors and do not over-express HER2. Current treatment for triple negative breast cancers is limited to cytotoxic chemotherapy, and upon relapse, there are not any therapies currently available. We demonstrate here that the bioactive food compound curcumin induces DNA damage in triple negative breast cancer cells in association with phosphorylation, increased expression, and cytoplasmic retention of the BRCA1 protein. In addition, curcumin promotes apoptosis and prevents anchorage-independent growth and migration of triple negative breast cancer cells. Apoptosis and BRCA1 modulation were not observed in non-transformed mammary epithelial cells, suggesting curcumin may have limited non-specific toxicity. This study suggests that curcumin and potentially curcumin analogues should be tested further in the context of triple negative breast cancer. These results are novel, having never been previously reported, and suggest that curcumin could provide a novel, non-toxic therapy, which could lead to improved survival for patients with triple negative breast cancer. Curcumin should be studied further in this subset of breast cancer patients, for whom treatment options are severely limited.
PMCID: PMC2756684  PMID: 19809577
mammary carcinoma; triple negative; curcumin; DNA damage; BRCA1
8.  Modulation of the BRCA1 Protein and Induction of Apoptosis in Triple Negative Breast Cancer Cell Lines by the Polyphenolic Compound Curcumin 
In the current study, we sought to examine the effects of curcumin in a specific type of breast cancer called triple negative breast cancer. These cancers lack expression of the estrogen and progesterone receptors and do not over-express HER2. Current treatment for triple negative breast cancers is limited to cytotoxic chemotherapy, and upon relapse, there are not any therapies currently available. We demonstrate here that the bioactive food compound curcumin induces DNA damage in triple negative breast cancer cells in association with phosphorylation, increased expression, and cytoplasmic retention of the BRCA1 protein. In addition, curcumin promotes apoptosis and prevents anchorage-independent growth and migration of triple negative breast cancer cells. Apoptosis and BRCA1 modulation were not observed in non-transformed mammary epithelial cells, suggesting curcumin may have limited non-specific toxicity. This study suggests that curcumin and potentially curcumin analogues should be tested further in the context of triple negative breast cancer. These results are novel, having never been previously reported, and suggest that curcumin could provide a novel, non-toxic therapy, which could lead to improved survival for patients with triple negative breast cancer. Curcumin should be studied further in this subset of breast cancer patients, for whom treatment options are severely limited.
PMCID: PMC2756684  PMID: 19809577
mammary carcinoma; triple negative; curcumin; DNA damage; BRCA1
9.  Delphinidin, an Anthocyanidin in Pigmented Fruits and Vegetables, Induces Apoptosis and Cell Cycle Arrest in Human Colon Cancer HCT116 Cells 
Molecular carcinogenesis  2009;48(3):260-270.
Because of unsatisfactory treatment options for colon cancer, there is a need to develop novel preventive approaches for this malignancy. One such strategy is through chemoprevention by the use of non-toxic dietary substances and botanical products. Delphinidin, an anthocyanidin in pigmented fruits and vegetables, possesses strong antioxidant and anti-inflammatory properties. In the present study, we investigated the antiproliferative and proapoptotic properties of delphinidin in human colon cancer HCT116 cells. We found that treatment of cells with delphinidin (30–240 μM; 48 h) resulted in (i) decrease in cell viability (ii) induction of apoptosis, (iii) cleavage of PARP, (iv) activation of caspases-3, -8, and -9, (v) increase in Bax with a concomitant decrease in Bcl-2 protein, and (vi) G2/M phase cell cycle arrest. NF-κB provides a mechanistic link between inflammation and cancer, and is a major factor controlling the ability of both pre-neoplastic and malignant cells to resist apoptosis-based tumor surveillance mechanisms. We therefore, determined the effect of delphinidin on NF-κB signaling pathway. The immunoblot, ELISA and EMSA analysis demonstrated that the treatment of HCT116 cells with delphinidin resulted in the inhibition of (i) IKKα, (ii) phosphorylation and degradation of IκBα, (iii) phosphorylation of NF-κB/p65 at Ser536, (iv) nuclear translocation of NF-κB/p65, (v) NF-κB/p65 DNA binding activity, and (vi) transcriptional activation of NF-κB. Our results suggest that delphinidin treatment of HCT116 cells suppressed NF-κB pathway, resulting in G2/M phase arrest and apoptosis. We suggest that delphinidin could have potential in inhibiting colon cancer growth.
doi:10.1002/mc.20477
PMCID: PMC2946888  PMID: 18729103
apoptosis; cell cycle arrest; delphinidin; NF-κB
10.  A novel antitumor activity of deguelin targeting the insulin-like growth factor (IGF) receptor pathway via up-regulation of IGF-binding protein-3 expression in breast cancer 
Cancer letters  2013;332(1):102-109.
In this study, we investigated the antitumor effects of deguelin in several human breast cancer cells in vitro and in vivo. Deguelin inhibited cell viability and the anchorage-dependent and anchorage-independent colony formation of triple-negative (MDA-MB-231 and MDA-MB-468) and triple-positive (MCF-7) breast cancer cells, and it significantly reduced the growth of MCF-7 cell xenograft tumors. The induction of apoptosis, inhibition of insulin-like growth factor-1 receptor (IGF-1R) signaling activation, and up-regulation of IGF-binding protein-3 (IGFBP-3) expression may be associated with deguelin-mediated antitumor effects. Our findings suggest a potential therapeutic use for deguelin in patients with triple-negative breast cancer and for those with breast cancers who are sensitive to endocrine- and HER2-targeted therapies.
doi:10.1016/j.canlet.2013.01.022
PMCID: PMC3638119  PMID: 23348700
Deguelin; Breast cancer; Apoptosis; IGFR signaling; IGFBP-3
11.  Delphinidin-3-Glucoside Protects against Oxidized Low-Density Lipoprotein-Induced Mitochondrial Dysfunction in Vascular Endothelial Cells via the Sodium-Dependent Glucose Transporter SGLT1 
PLoS ONE  2013;8(7):e68617.
Delphinidin-3-glucoside (Dp) is a member of a family of bioactive compounds known as anthocyanins that occur naturally in pigmented plants and are known to ameliorate oxidative stress. Previous studies have showed that Dp decreased oxidative stress in vascular endothelial cells, however, the underlying mechanisms remain largely unknown. In the present study, we showed that pretreatment with Dp significantly suppressed oxidized low-density lipoprotein (oxLDL)-induced cell proliferation inhibition and apoptosis in primary human umbilical vein endothelial cells (HUVECs). Also, Dp pretreatment attenuated oxLDL-induced mitochondrial dysfunction via decreased reactive oxygen species (ROS) and superoxide anion generation, thereby repressing mitochondrial membrane potential and closing mitochondrial permeability transition pore. Furthermore, in vitro and in vivo data showed that Dp was transported into endothelial cells in a temperature, concentration, and time-dependent manner via the sodium-dependent glucose transporter (SGLT1). Suppression of SGLT1 by its substrate glucose, its inhibitor phlorizin or SGLT1 siRNA blocked Dp transportation. Repression of SGLT1 significantly inhibited Dp function of ameliorating mitochondrial dysfunction induced by pro-apoptotic factors (Apoptosis-inducing factor, Cytochrome c, Caspase-3 and Bax/Bcl-2 ratio). Taken together, our data indicate that Dp protects VECs via the SGLT1-ROS-mitochodria pathway. This new insight may help to elucidate the molecular mechanisms underlying the vascular protection afforded by Dp, and anthocyanins in general, in the context of prevention of endothelial dysfunction and atherosclerosis.
doi:10.1371/journal.pone.0068617
PMCID: PMC3715513  PMID: 23874689
12.  Delphinidin, a dietary antioxidant, induces human epidermal keratinocyte differentiation but not apoptosis: studies in submerged and three-dimensional epidermal equivalent models 
Experimental dermatology  2013;22(5):10.1111/exd.12140.
Delphinidin (Del), [3,5,7,3′-,4′-,5′-hexahydroxyflavylium], an anthocyanidin and a potent antioxidant abundantly found in pigmented fruits and vegetables exhibits proapoptotic effects in many cancer cells. Here, we determined the effect of Del on growth, apoptosis and differentiation of normal human epidermal keratinocytes (NHEKs) in vitro in submerged cultures and examined its effects in a three-dimensional (3D) epidermal equivalent (EE) model that permits complete differentiation reminiscent of in vivo skin. Treatment of NHEKs with Del (10–40 μm; 24–48 h) significantly enhanced keratinocyte differentiation. In Del-treated cells, there was marked increase in human involucrin (hINV) promoter activity with simultaneous increase in the mRNA and protein expressions of involucrin and other epidermal differentiation markers including procaspase-14 and transglutaminase-1 (TGM1), but without any effect on TGM2. Del treatment of NHEKs was associated with minimal decrease in cell viability, which was not associated with apoptosis as evident by lack of modulation of caspases, apoptosis-related proteins including Bcl-2 family of proteins and poly(ADP-ribose) polymerase cleavage. To establish the in vivo relevance of our observations in submerged cultures, we then validated these effects in a 3D EE model, where Del was found to significantly enhance cornification and increase the protein expression of cornification markers including caspase-14 and keratin 1. For the first time, we show that Del induces epidermal differentiation using an experimental system that closely mimics in vivo human skin. These observations suggest that Del could be a useful agent for dermatoses associated with epidermal barrier defects including aberrant keratinization, hyperproliferation or inflammation observed in skin diseases like psoriasis and ichthyoses.
doi:10.1111/exd.12140
PMCID: PMC3777415  PMID: 23614741
caspase-14; delphinidin; epidermis; involucrin; keratinocyte differentiation; transglutaminase
13.  Significance of p27kip1 as potential biomarker for intracellular oxidative status 
Nutrition Research and Practice  2010;4(5):351-355.
Our previous proteomic study demonstrated that oxidative stress and antioxidant delphinidin regulated the cellular level of p27kip1 (referred to as p27) as well as some heat shock proteins in human colon cancer HT 29 cells. Current study was conducted to validate and confirm the regulation of these proteins using both in vitro and in vivo systems. The level of p27 was decreased by hydrogen peroxide in a dose-dependent manner in human colon carcinoma HCT 116 (p53-positive) cells while it was increased upon exposure to hydrogen peroxide in HT 29 (p53-negative) cells. However, high concentration of hydrogen peroxide (100 µM) downregulated p27 in both cell lines, but delphindin, one of antioxidative anthocyanins, enhanced the level of p27 suppressed by 100 µM hydrogen peroxide. ICR mice were injected with varying concentrations of hydrogen peroxide, delphinidin and both. Western blot analysis for the mouse large intestinal tissue showed that the expression of p27 was upregulated by 25 mg/kg BW hydrogen peroxide. To investigate the association of p27 regulation with hypoxia-inducible factor 1-beta (HIF-1β), the level of p27 was analyzed in wild-type mouse hepatoma hepa1c1c7 and Aryl Hydrocarbon Nuclear Translocator (arnt, HIF-1β)-defective mutant BPRc1 cells in the absence and presence of hydrogen peroxide and delphinidin. While the level of p27 was responsive to hydrogen peroxide and delphinidin, it remained unchanged in BPRc1, suggesting that the regulation of p27 requires functional HIF-1β. We also found that hydrogen peroxide and delphinidin affected PI3K/Akt/mTOR signaling pathway which is one of upstream regulators of HIFs. In conclusion, hydrogen peroxide and antioxidant delphinidin seem to regulate intracellular level of p27 through regulating HIF-1 level which is, in turn, governed by its upstream regulators comprising of PI3K/Akt/mTOR signaling pathway. The results should also encourage further study for the potential of p27 as a biomarker for intracellular oxidative or antioxidant status.
doi:10.4162/nrp.2010.4.5.351
PMCID: PMC2981716  PMID: 21103079
p27kip1; biomarker; antioxidant; oxidative status; delphinidin
14.  Delphinidin, One of the Major Anthocyanidins, Prevents Bone Loss through the Inhibition of Excessive Osteoclastogenesis in Osteoporosis Model Mice 
PLoS ONE  2014;9(5):e97177.
Anthocyanins, one of the flavonoid subtypes, are a large family of water-soluble phytopigments and have a wide range of health-promoting benefits. Recently, an anthocyanin-rich compound from blueberries was reported to possess protective property against bone loss in ovariectomized (OVX) animal models. However, the active ingredients in the anthocyanin compound have not been identified. Here we show that delphinidin, one of the major anthocyanidins in berries, is a potent active ingredient in anti-osteoporotic bone resorption through the suppression of osteoclast formation. In vitro examinations revealed that delphinidin treatment markedly inhibited the differentiation of RAW264.7 cells into osteoclasts compared with other anthocyanidins, cyanidin and peonidin. Oral administration of delphinidin significantly prevented bone loss in both RANKL-induced osteoporosis model mice and OVX model mice. We further provide evidence that delphinidin suppressed the activity of NF-κB, c-fos, and Nfatc1, master transcriptional factors for osteoclastogenesis. These results strongly suggest that delphinidin is the most potent inhibitor of osteoclast differentiation and will be an effective agent for preventing bone loss in postmenopausal osteoporosis.
doi:10.1371/journal.pone.0097177
PMCID: PMC4019566  PMID: 24824988
15.  Long Chain Fatty Acyl-CoA Synthetase 4 Is a Biomarker for and Mediator of Hormone Resistance in Human Breast Cancer 
PLoS ONE  2013;8(10):e77060.
The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.
doi:10.1371/journal.pone.0077060
PMCID: PMC3796543  PMID: 24155918
16.  Plant Food Delphinidin-3-Glucoside Significantly Inhibits Platelet Activation and Thrombosis: Novel Protective Roles against Cardiovascular Diseases 
PLoS ONE  2012;7(5):e37323.
Delphinidin-3-glucoside (Dp-3-g) is one of the predominant bioactive compounds of anthocyanins in many plant foods. Although several anthocyanin compounds have been reported to be protective against cardiovascular diseases (CVDs), the direct effect of anthocyanins on platelets, the key players in atherothrombosis, has not been studied. The roles of Dp-3-g in platelet function are completely unknown. The present study investigated the effects of Dp-3-g on platelet activation and several thrombosis models in vitro and in vivo. We found that Dp-3-g significantly inhibited human and murine platelet aggregation in both platelet-rich plasma and purified platelets. It also markedly reduced thrombus growth in human and murine blood in perfusion chambers at both low and high shear rates. Using intravital microscopy, we observed that Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for vessel occlusion. Dp-3-g also significantly inhibited thrombus growth in a carotid artery thrombosis model. To elucidate the mechanisms, we examined platelet activation markers via flow cytometry and found that Dp-3-g significantly inhibited the expression of P-selectin, CD63, CD40L, which reflect platelet α- and δ-granule release, and cytosol protein secretion, respectively. We further demonstrated that Dp-3-g downregulated the expression of active integrin αIIbβ3 on platelets, and attenuated fibrinogen binding to platelets following agonist treatment, without interfering with the direct interaction between fibrinogen and integrin αIIbβ3. We found that Dp-3-g reduced phosphorylation of adenosine monophosphate-activated protein kinase, which may contribute to the observed inhibitory effects on platelet activation. Thus, Dp-3-g significantly inhibits platelet activation and attenuates thrombus growth at both arterial and venous shear stresses, which likely contributes to its protective roles against thrombosis and CVDs.
doi:10.1371/journal.pone.0037323
PMCID: PMC3356278  PMID: 22624015
17.  Delphinidin inhibits IL-1β-induced activation of NF-κB by modulating the phosphorylation of IRAK-1Ser376 in human articular chondrocytes 
Rheumatology (Oxford, England)  2013;52(6):998-1008.
Objective. In OA, there is enhanced expression of pro-inflammatory cytokines such as IL-1β in the affected joint. Delphinidin, an anthocyanidin found in pigmented fruits and vegetables, has been shown to possess anti-inflammatory and antioxidant properties. In the present study we determined whether delphinidin would inhibit the IL-1β-induced activation of NF-κB in human chondrocytes and determined the mechanism of its action.
Methods. PGE2 levels and activation of NF-κB p65 in human OA chondrocytes were determined by ELISA-based assays. Protein expression of cyclo-oxygenase-2 (COX-2) and phosphorylation of kinases was determined by western immunoblotting. Expression level of mRNAs was determined by TaqMan assays.
Results. Delphinidin inhibited IL-1β-induced expression of COX-2 and production of PGE2 in human chondrocytes. Delphinidin also inhibited IL-1β-mediated phosphorylation of IL-1 receptor-associated kinase-1Ser376, phosphorylation of IKKα/β, expression of IKKβ, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65. Phosphorylation of TGF-β-activated kinase 1 was not observed but NF-κB-inducing kinase (NIK) was phosphorylated and phosphorylation of NIK was blocked by delphinidin in IL-1β-treated human chondrocytes.
Conclusion. These data identify delphinidin as a novel inhibitor of IL-1β-induced production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expression and provide new insight into the mechanism of its action. Our results also identify inhibition of IRAK1Ser376 phosphorylation by delphinidin in IL-1β-induced activation of NF-κB in human chondrocytes. Given the important role played by IL-1β-induced NF-κB activation, COX-2 expression and PGE2 production in OA, our results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA.
doi:10.1093/rheumatology/kes363
PMCID: PMC3651611  PMID: 23392593
osteoarthritis; IL-1β; delphinidin; chondrocytes; NF-κB; COX-2; PGE2
18.  GREB1 Functions as a Growth Promoter and Is Modulated by IL6/STAT3 in Breast Cancer 
PLoS ONE  2012;7(10):e46410.
Background
Growth Regulation by Estrogen in Breast cancer (GREB1) was an estrogen receptor (ER) target gene, and GREB1 expression inversely correlated with HER2 status, possibly as a surrogate marker for ER status and a predictor for tamoxifen resistance in breast cancer patients. In the present study, we examine the function and regulation of GREB1 in breast cancer, with the goal to develop GREB1 as a biomarker in breast cancer with de novo and acquired tamoxifen resistance.
Methods
We overexpressed GREB1 using adenovirus containing the full length GREB1 cDNA (Ad-GREB1) in breast cancer cell lines. The soft agar assay was used as a measure of anchorage independent growth. The effects of GREB1 on cell proliferation in MCF-7 cells transduced with Ad-GREB1 were also measured by the me olic activity using AlamarBlue assay. We tested whether there was interaction between STAT3 and ER, which could repress GREB1 expression by immunoprecipitation assay. The effects of IL-6/JAK/STAT3 cascade activation on estrogen-induced GREB1 promoter activity were determined by luciferase assay and those on gene expression were measured by real time reverse transcription polymerase chain reaction (qRT-PCR).
Results
We found that the ability of breast cancer cells to grow in soft agar is enhanced following GREB1 transfection. In MCF-7 cells transduced with Ad-GREB1 or transfected with siRNA GREB1, the metabolic activity was increased or completely abolished, suggesting that GREB1 may function as a growth promoter in breast cancer. E2 treatment increased GREB1 promoter luciferase activity. IL-6 inhibited E2-induced GREB1 transcription activity and GREB1 mRNA expression. Constitutively expressing active STAT3 construct (STAT3-C) dramatically decreased GREB1 transcription.
Conclusions
These data indicate that overexpression of GREB1 promotes cell proliferation and increases the clonogenic ability in breast cancer cells. Moreover, Il6/STAT3 modulates estrogen-induced GREB1 transcriptional activity in breast cancer cells.
doi:10.1371/journal.pone.0046410
PMCID: PMC3463574  PMID: 23056300
19.  Delphinidin Attenuates Neoplastic Cell Transformation by Directly Inhibiting Raf/MEK/ERK Activities 
Recent studies suggest that anthocyanidins play a pivotal role in the chemopreventive effects of fruits and vegetables. However, the underlying molecular mechanisms and targets remain unknown. Neoplastic transformation of cells and inflammation are considered to be major events contributing to carcinogenesis. Here we report that delphinidin, a major dietary anthocyanidin, inhibits tumor-promoter-induced transformation and cyclooxygenase-2 (COX-2) expression in JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells by directly targeting Raf and MEK. Delphinidin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation and COX-2 expression at both the protein and transcriptional levels. The activation of AP-1 (activator protein-1) and NF-κB (nuclear factor-κB) induced by TPA was dose-dependently inhibited by delphinidin treatment. Kinase assays and Western blot data revealed that delphinidin strongly inhibited Raf1 and MEK1 kinase activities and subsequently attenuated TPA-induced phosphorylation of MEK, ERK, p90RSK, and MSK. Although delphinidin suppressed ERK and JNK activities, it was more effective at inhibiting Raf1 or MEK1 activities. Pull-down and competition assays revealed that delphinidin binds with Raf1 or MEK1 noncompetitively with ATP. Delphinidin also dose-dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor and H-Ras, both of which are known to be involved in the activation of Raf/MEK/ERK signaling. Together these findings suggested that the targeted inhibition of Raf1 and MEK activities and COX-2 expression by delphinidin contribute to the chemopreventive potential of fruits and vegetables.
doi:10.1158/1940-6207.CAPR-08-0071
PMCID: PMC2832759  PMID: 19139002
Delphinidin; Raf1; MEK1; cell transformation; chemoprevention
20.  CCN1 a Candidate Target for Zoledronic Acid Treatment in Breast Cancer 
Molecular cancer therapeutics  2011;10(5):10.1158/1535-7163.MCT-10-0836.
CCN1, also known as CYR61, is a survival and pro-angiogenic factor overexpressed in about 30% of invasive breast carcinomas, and particularly in triple negative breast carcinomas (TNBC). CCN1 expression in breast cancer promotes tumorigenicity, metastasis, antihormone and chemo-resistance. TNBCs often develop bone metastasis, thus the vast majority of patients receive bisphosphonate treatment as a companion to chemotherapy. Zoledronic acid (ZOL), a bisphosphonate currently in use, inhibits bone resorption, prevents development of new osteolytic lesions induced by tumor metastasis, and has a direct anti-tumor activity in breast cancer cells and tumors. We have shown that ZOL inhibits anchorage independent growth as well as branching and morphogenesis in CCN1 overexpressing cells. However, the mechanism is not yet well understood. In this study, we investigate the effect of ZOL in breast cancer cells with high and with undetectable CCN1 expression levels. We demonstrate that CCN1 expressing cells are more sensitive to ZOL, that ZOL induces downregulation of the CCN1 promoter activity and CCN1 protein expression in a dose dependent manner, and that ZOL is associated with a decrease in phosphorylated Akt and translocation of FOXO3a, a negative regulator of CCN1 expression, to the nucleus. Deletion of the FOXO3a binding site in the CCN1 promoter prevents ZOL inhibition of the CCN1 promoter activity demonstrating that FOXO3a transcriptional activation is necessary for ZOL to induce CCN1 inhibition. This study provides evidence that ZOL targets the pro-angiogenic factor (CCN1) through FOXO3a, and reveals a new mechanism of ZOL action in breast cancer cells.
doi:10.1158/1535-7163.MCT-10-0836
PMCID: PMC3857108  PMID: 21393426
CYR61/CCN1; bisphosphonates; Zoledronic acid; growth; breast cancer
21.  Lapatinib–induced NF-kappaB activation sensitizes triple-negative breast cancer cells to proteasome inhibitors 
Breast Cancer Research : BCR  2013;15(6):R108.
Introduction
Triple-negative breast cancer (TNBC), a subtype of breast cancer with negative expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), is frequently diagnosed in younger women and has poor prognosis for disease-free and overall survival. Due to the lack of known oncogenic drivers for TNBC proliferation, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed.
Methods
Triple-negative breast cancer cell lines were treated with proteasome inhibitors in combination with lapatinib (a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor). Their in vitro and in vivo viability was examined by MTT assay, clonogenic analysis, and orthotopic xenograft mice model. Luciferase reporter gene, immunoblot, and RT-qPCR, immunoprecipitation assays were used to investigate the molecular mechanisms of action.
Results
Our data showed that nuclear factor (NF)-κB activation was elicited by lapatinib, independent of EGFR/HER2 inhibition, in TNBCs. Lapatinib-induced constitutive activation of NF-κB involved Src family kinase (SFK)-dependent p65 and IκBα phosphorylations, and rendered these cells more vulnerable to NF-κB inhibition by p65 small hairpin RNA. Lapatinib but not other EGFR inhibitors synergized the anti-tumor activity of proteasome inhibitors both in vitro and in vivo. Our results suggest that treatment of TNBCs with lapatinib may enhance their oncogene addiction to NF-κB, and thus augment the anti-tumor activity of proteasome inhibitors.
Conclusions
These findings suggest that combination therapy of a proteasome inhibitor with lapatinib may benefit TNBC patients.
doi:10.1186/bcr3575
PMCID: PMC3979035  PMID: 24216290
22.  Optical imaging of metabolism in HER2 overexpressing breast cancer cells 
Biomedical Optics Express  2011;3(1):75-85.
The optical redox ratio (fluorescence intensity of NADH divided by that of FAD), was acquired for a panel of breast cancer cell lines to investigate how overexpression of human epidermal growth factor receptor 2 (HER2) affects tumor cell metabolism, and how tumor metabolism may be altered in response to clinically used HER2-targeted therapies. Confocal fluorescence microscopy was used to acquire NADH and FAD auto-fluorescent images. The optical redox ratio was highest in cells overexpressing HER2 and lowest in triple negative breast cancer (TNBC) cells, which lack HER2, progesterone receptor, and estrogen receptor (ER). The redox ratio in ER-positive/HER2-negative cells was higher than what was seen in TNBC cells, but lower than that in HER2 overexpressing cells. Importantly, inhibition of HER2 using trastuzumab significantly reduced the redox ratio in HER2 overexpressing cells. Furthermore, the combinatorial inhibition of HER2 and ER decreased the redox ratio in ER+/HER2+ breast cancer cells to a greater extent than inhibition of either receptor alone. Interestingly, trastuzumab had little impact upon the redox ratio in a cell line selected for acquired resistance to trastuzumab. Taken together, these data indicate that the optical redox ratio measures changes in tumor metabolism that reflect the oncogenic effects of HER2 activity within the cell, as well as the response of the cell to therapeutic inhibition of HER2. Therefore, optical redox imaging holds the promise of measuring response and resistance to receptor-targeted breast cancer therapies in real time, which could potentially impact clinical decisions and improve patient outcome.
doi:10.1364/BOE.3.000075
PMCID: PMC3255344  PMID: 22254170
(170.1790) Confocal microscopy; (170.2520) Fluorescence microscopy; (170.1530) Cell analysis; (170.1610) Clinical applications
23.  Dual blockade of HER2 in HER2-overexpressing tumor cells does not completely eliminate HER3 function 
Purpose
Dual blockade of HER2 with trastuzumab with lapatinib or with pertuzumab is a superior treatment approach compared to single agent HER2 inhibitors. However, many HER2-overexpressing breast cancers still escape from this combinatorial approach. Inhibition of HER2 and downstream phosphatidylinositol-3 kinase (PI3K)/AKT causes a transcriptional and post-translational upregulation of HER3 which, in turn, counteracts the antitumor action of the HER2-directed therapies. We hypothesized that suppression of HER3 would synergize with dual blockade of HER2 in breast cancer cells sensitive and refractory to HER2 antagonists.
Experimental Design
Inhibition of HER2/HER3 in HER2+ breast cancer cell lines was evaluated by western blot. We analyzed drug-induced apoptosis and 2- and 3-dimensional growth in vitro. Growth inhibition of PI3K was examined in vivo in xenografts treated with combinations of trastuzumab, lapatinib, and the HER3 neutralizing monoclonal antibody U3-1287.
Results
Treatment with U3-1287 blocked the upregulation of total and phosphorylated HER3 that followed treatment with lapatinib and trastuzumab and, in turn, enhanced the anti-tumor action of the combination against trastuzumab-sensitive and -resistant cells. Mice bearing HER2+ xenografts treated with lapatinib, trastuzumab, and U3-1287 exhibited fewer recurrences and better survival compared to mice treated with lapatinib and trastuzumab.
Conclusions
Dual blockade of HER2 with trastuzumab and lapatinib does not eliminate the compensatory upregulation of HER3. Therapeutic inhibitors of HER3 should be considered as part of multi-drug combinations aimed at completely and rapidly disabling the HER2 network in HER2-overexpressing breast cancers.
doi:10.1158/1078-0432.CCR-12-2024
PMCID: PMC3563762  PMID: 23224399
HER2; HER3; lapatinib; trastuzumab; breast cancer
24.  Anti-tumor effects of retinoids combined with trastuzumab or tamoxifen in breast cancer cells: induction of apoptosis by retinoid/trastuzumab combinations 
Introduction
HER2 and estrogen receptor (ER) are important in breast cancer and are therapeutic targets of trastuzumab (Herceptin) and tamoxifen, respectively. Retinoids inhibit breast cancer growth, and modulate signaling by HER2 and ER. We hypothesized that treatment with retinoids and simultaneous targeting of HER2 and/or ER may have enhanced anti-tumor effects.
Methods
The effects of retinoids combined with trastuzumab or tamoxifen were examined in two human breast cancer cell lines in culture, BT474 and SKBR3. Assays of proliferation, apoptosis, differentiation, cell cycle distribution, and receptor signaling were performed.
Results
In HER2-overexpressing/ER-positive BT474 cells, combining all-trans retinoic acid (atRA) with tamoxifen or trastuzumab synergistically inhibited cell growth, and altered cell differentiation and cell cycle. Only atRA/trastuzumab-containing combinations induced apoptosis. BT474 and HER2-overexpressing/ER-negative SKBR3 cells were treated with a panel of retinoids (atRA, 9-cis-retinoic acid, 13-cis-retinoic acid, or N-(4-hydroxyphenyl) retinamide (fenretinide) (4-HPR)) combined with trastuzumab. In BT474 cells, none of the single agents except 4-HPR induced apoptosis, but again combinations of each retinoid with trastuzumab did induce apoptosis. In contrast, the single retinoid agents did cause apoptosis in SKBR3 cells; this was only modestly enhanced by addition of trastuzumab. The retinoid drug combinations altered signaling by HER2 and ER. Retinoids were inactive in trastuzumab-resistant BT474 cells.
Conclusions
Combining retinoids with trastuzumab maximally inhibits cell growth and induces apoptosis in trastuzumab-sensitive cells. Treatment with such combinations may have benefit for breast cancer patients.
doi:10.1186/bcr2625
PMCID: PMC2949655  PMID: 20696059
25.  The TRAIL to targeted therapy of breast cancer 
Advances in cancer research  2009;103:43-73.
Breast cancers can be classified into those which express the estrogen (ER) and progesterone (PR) receptors, those with HER-2 amplification, and those without expression of ER, PR, or amplified HER-2 (referred to as triple-negative or basal-like breast cancer). Tumor Necrosis Factor Apoptosis Inducing Ligand (TRAIL) activates apoptosis upon binding to its receptors in many tumor types and the ligand and agonist antibodies are currently being studied in patients in clinical phase I and phase II trials. Cell line studies suggest that many breast cancer cell lines are very resistant to TRAIL-induced apoptosis. However, recent data suggest that a subset of triple-negative/basal-like breast cancer cells is sensitive to TRAIL as a single agent. In addition many studies have demonstrated that resistance to TRAIL-mediated apoptosis in breast cancer cells can be overcome by combinations of TRAIL with chemotherapy, radiation, and various targeted agents. This review will discuss the current understanding of the mechanisms which control TRAIL-mediated apoptosis in breast cancer cells. The preclinical data supporting the use of TRAIL ligands and agonistic antibodies alone and in combination in breast cancer will also be discussed.
doi:10.1016/S0065-230X(09)03003-6
PMCID: PMC3538140  PMID: 19854352

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