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1.  Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells 
Molecular Cancer  2011;10:97.
In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery.
AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis.
AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.
PMCID: PMC3199901  PMID: 21827643
Adeno-Associated Virus Type 2; AAV2; Breast cancer; Pro-apoptotic therapeutics; Apoptosis; Cell cycle; Rep proteins; c-Myc
2.  In vitro cytotoxicity of Strobilanthes crispus ethanol extract on hormone dependent human breast adenocarcinoma MCF-7 cell 
Strobilanthes crispus has been traditionally used as antidiabetic, anticancer, diuretic, antilytic and laxative agent. However, cytotoxicity and antiproliferative effect of S. crispus is still unclear.
Strobilanthes cripus was able to reduce cell viability and proliferation in MTT and BrdU assays. Both cell cycle progression and Tunel assay suggested that IC50 of S. crispus ethanol extract induced sub-G1 cell cycle phase, and DNA fragmentation. On the other hand, translocation of mitochondria cytochrome c release, induction of caspase 3/7 and p53 while suppress XIAP on treated MCF-7 cell were also observed in this study.
Our findings suggest that S. crispus ethanol extract induced apoptosis and DNA fragmentation on hormone dependent breast cancer cell line MCF-7 via mitochondria dependent p53 apoptosis pathway.
PMCID: PMC3377542  PMID: 22471785
Strobilanthes crispus; MCF-7; Apoptosis; p53
3.  An Aqueous Extract of Fagonia cretica Induces DNA Damage, Cell Cycle Arrest and Apoptosis in Breast Cancer Cells via FOXO3a and p53 Expression 
PLoS ONE  2012;7(6):e40152.
Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer.
Methodology/Principal Findings
Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as γ-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines.
Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression.
PMCID: PMC3384610  PMID: 22761954
4.  Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat 
Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.
TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.
Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.
This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
PMCID: PMC3446342  PMID: 22613095
Panobinostat; LBH589; triple-negative breast cancer; xenograft; histone deacetylase inhibitor; E-cadherin; CDH1; epithelial-to-mesenchymal transition
5.  Crude ethyl acetate extract of marine microalga, Chaetoceros calcitrans, induces Apoptosis in MDA-MB-231 breast cancer cells 
Pharmacognosy Magazine  2014;10(37):1-8.
Marine brown diatom Chaetoceros calcitrans and green microalga Nannochloropsis oculata are beneficial materials for various applications in the food, nutraceutical, pharmaceutical and cosmeceutical industries.
This study investigated cytotoxicity of different crude solvent extracts from C. calcitrans and N. oculata against various cancer cell lines.
Materials and Methods:
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out to screen the cytotoxic effects of hexane (Hex), dichloromethane (DCM), ethyl acetate, and methanol extract from C. calcitrans and N. oculata toward various cancer cell lines. Flow cytometry cell cycle was used to determine the cell cycle arrest while the mode of cell death was investigated through acridine orange/propidium iodide (AOPI) staining, Annexin V-Fluorescein Isothiocyanate (FITC) and Terminal deoxynucleotidyl transferase-mediated d-UTP Nick End Labeling (TUNEL) assays. Expression profile of apoptotic and proliferative-related genes was then determined using the multiplex gene expression profiler (GeXP).
Crude ethyl acetate (CEA) extract of C. calcitrans inhibited growth of MDA-MB-231 cells, with IC50 of 60 μg/mL after 72 h of treatment. Further studies were conducted to determine the mode of cell death at various concentrations of this extract: 30, 60 and 120 μg/mL. The mode of cell death was mainly apoptosis as shown through apoptosis determination test. The expression data from GeXP showed that caspase-4 was upregulated while B-cell leukemia/lymphoma 2(Bcl-2) was down regulated. Thus, caspase-4 induction endoplasmic reticulum death pathway is believed to be one of the mechanisms underlying the induction of apoptosis while Bcl-2 induced S and G2/M cell cycle phase arrest in MDA-MB-231 cells.
CEA extract of C. calcitrans showed the highest cytotoxicity on MDA-MB-231 via apoptosis.
PMCID: PMC3969653  PMID: 24696543
Apoptosis; Chaetoceros calcitrans; crude ethyl acetate extract; gene expression profiler; MDA-MB-231
6.  Antitumor Activity of Antimicrobial Peptides Containing CisoDGRC in CD13 Negative Breast Cancer Cells 
PLoS ONE  2013;8(1):e53491.
isoAsp-Gly-Arg (isoDGR) is a derivative of the Asn-Gly-Arg (NGR) motif, which is used as a targeted delivery tool to aminopeptidase N (CD13) positive cells. Recent studies have shown that cyclic isoDGR (CisoDGRC) has a more efficient affinity with αvβ3, a type of integrin that overexpresses in tumor cells. Antimicrobial peptides (AMPs) are an efficient antitumor peptide that specifically kills tumor cells. In the present study, we designed antimicrobial peptides containing the CisoDGRC motif (CDAK) and assessed its antitumor activity for CD13−/αvβ3+ breast cancer cells (MCF-7 and MDA-MB-231) in vitro and in vivo.
In vitro: We assessed the cytotoxicity of CDAK for MCF-7 and MDA-MB-231 breast cancer cells, the human umbilical vein endothelial cell (HUVEC), and human foreskin fibroblasts (HFF). We performed an apoptosis assay using Annexin-V/PI, DNA ladder, mitochondrial membrane potential, and Caspase-3 and Bcl-2. The effect on cell cycles and affinity with cell were tested using flow cytometry and fluorescent microscopy and the effect on invasion was analyzed using an invasion assay. CDAK was injected intravenously into tumor-bearing athymic nude mice in vivo experiment.
CDAK showed cytotoxic activity in MCF-7 and MDA-MB-231 cells, whereas HUVEC and HFF were less sensitive to the peptides. CDAK induced apoptosis, reduced mitochondrial membrane potential, promoted Caspase-3, and inhibited Bcl-2 expression in the two breast cancer cell lines. In addition, CDAK inhibited proliferation of cancer cell through S phase arrest, and own selective affinity with MCF-7 and MDA-MB-231cells, inhibited the invasion of MDA-MB-231 cells. In vivo, CDAK significant inhibited the progression of the tumor and the generation of neovascularization.
Antimicrobial peptides containing the CisoDGRC (CDAK) motif could efficiently exhibit the antitumor activity for CD13−/αvβ3+ breast cancer cells.
PMCID: PMC3543424  PMID: 23326440
7.  Profiling of phenolic compounds and their antioxidant and anticancer activities in pandan (Pandanus amaryllifolius Roxb.) extracts from different locations of Malaysia 
Phytochemicals and antioxidants from plant sources are of increasing interest to consumers because of their roles in the maintenance of human health. Most of the secondary metabolites of herbs are used in a number of pharmaceutical products.
Secondary metabolites composition and content of five flavonoids and three phenolic acids were evaluated and determined in Pandanus amaryllifolius extracts from three different locations of Malaysia by RP-HPLC; Total phenolic and total flavonoid content were determined using Folin-Ciocalteau and aluminum chloride colorimetric assay; The antioxidant activity of the extracts was determined by the ferric reducing antioxidant potential (FRAP) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay was employed to screen anticancer activity of extracts against MCF-7 cancer cell line.
Highest value of total flavonoids (TF) and total phenolics (TP) was observed in pandan extract from Bachok locattion (1.87 mg/g DW and 6.72 mg/g DW) followed by Klang (1.32 mg/g DW; 5.07 mg/g DW) and Pontian (1.12 mg/g DW; 4.88 mg/g DW). Rutin just detected from Bachok location with value of 0.082 mg/g DW. High content of epicatechin (0.035 mg/g DW) and naringin (0.325 mg/g DW) were observed from Bachok location while, highest content of catechin (0.613 mg/g DW) and kaempferol (0.278 mg/g DW) was observed in pandan extract from Klang location. The extract of pandan from Bachok exhibited highest value of gallic acid (0.423 mg/g DW) and cinnamic acid (0.084 mg/g DW). Ferrulic acid just detected from pandan extract of Bachok location with concentration of 0.281mg/g DW. Between studied locations Bachok exhibited highest value of DPPH (64.27%) and FRAP (517.2 μm of Fe (II)/g) activity followed by Klang (52.16%; 448.6 μm of Fe (II)/g) and Pontian (50.10%; 314.8 μm of Fe (II)/g). The preliminary screening showed pandan extracts from 3 locations possessed anticancer promoting activity against MCF-7 cell line, with 78.3%, 70.5% and 67.4% inhibition rate, respectively. Maximum MCF-7cell line inhibition was observed in pandan extract from Bachok location.
The samples collected from the North (Bachok) exhibited the highest TP, TF antioxidant and anticancer activity while those from the Southern portion (Pontian) appeared to have the lowest content of TP, TF and antioxidant activity.
PMCID: PMC4220543  PMID: 24289290
Pandanus amaryllifolius; Flavonoids; HPLC; Antioxidant activity; DPPH; FRAP; Anticancer activity; MCF-7
8.  Evaluation of cytotoxic and chemotherapeutic properties of boldine in breast cancer using in vitro and in vivo models 
To date, plants have been the major source of anticancer drugs. Boldine is a natural alkaloid commonly found in the leaves and bark of Peumus boldus. In this study, we found that boldine potently inhibited the viability of the human invasive breast cancer cell lines, MDA-MB-231 (48-hour IC50 46.5±3.1 μg/mL) and MDA-MB-468 (48-hour IC50 50.8±2.7 μg/mL). Boldine had a cytotoxic effect and induced apoptosis in breast cancer cells as indicated by a higher amount of lactate dehydrogenase released, membrane permeability, and DNA fragmentation. In addition, we demonstrated that boldine induced cell cycle arrest at G2/M phase. The anticancer mechanism is associated with disruption of the mitochondrial membrane potential and release of cytochrome c in MDA-MB-231. Boldine selectively induced activation of caspase-9 and caspase-3/7, but not caspase-8. We also found that boldine could inhibit nuclear factor kappa B activation, a key molecule in tumor progression and metastasis. In addition, protein array and Western blotting analysis showed that treatment with boldine resulted in downregulation of Bcl-2 and heat shock protein 70 and upregulation of Bax in the MDA-MB-231 cell line. An acute toxicity study in rats revealed that boldine at a dose of 100 mg/kg body weight was well tolerated. Moreover, intraperitoneal injection of boldine (50 or 100 mg/kg) significantly reduced tumor size in an animal model of breast cancer. Our results suggest that boldine is a potentially useful agent for the treatment of breast cancer.
PMCID: PMC4057328  PMID: 24944509
boldine; breast cancer; caspase cascade; Bcl-2/Bax; heat shock protein 70; nuclear factor kappa B
9.  Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells 
Breast Cancer Research  2005;7(4):R422-R435.
Inhibitors of cyclo-oxygenase (COX)-2 are being extensively studied as anticancer agents. In the present study we evaluated the mechanisms by which a highly selective COX-2 inhibitor, celecoxib, affects tumor growth of two differentially invasive human breast cancer cell lines.
MDA-MB-231 (highly invasive) and MDA-MB-468 (moderately invasive) cell lines were treated with varying concentrations of celecoxib in vitro, and the effects of this agent on cell growth and angiogenesis were monitored by evaluating cell proliferation, apoptosis, cell cycle arrest, and vasculogenic mimicry. The in vitro results of MDA-MB-231 cell line were further confirmed in vivo in a mouse xenograft model.
The highly invasive MDA-MB-231 cells express higher levels of COX-2 than do the less invasive MDA-MB-468 cells. Celecoxib treatment inhibited COX-2 activity, indicated by prostaglandin E2 secretion, and caused significant growth arrest in both breast cancer cell lines. In the highly invasive MDA-MB-231 cells, the mechanism of celecoxib-induced growth arrest was by induction of apoptosis, associated with reduced activation of protein kinase B/Akt, and subsequent activation of caspases 3 and 7. In the less invasive MDA-MB-468 cells, growth arrest was a consequence of cell cycle arrest at the G0/G1 checkpoint. Celecoxib-induced growth inhibition was reversed by addition of exogenous prostaglandin E2 in MDA-MB-468 cells but not in MDA-MB-231 cells. Furthermore, MDA-MB-468 cells formed significantly fewer extracellular matrix associated microvascular channels in vitro than did the high COX-2 expressing MDA-MB-231 cells. Celecoxib treatment not only inhibited cell growth and vascular channel formation but also reduced vascular endothelial growth factor levels. The in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of celecoxib significantly reduced tumor growth of MDA-MB-231 cells, which was associated with reduced vascularization and increased necrosis in the tumor mass.
The disparate molecular mechanisms of celecoxib-induced growth inhibition in human breast cancer cells depends upon the level of COX-2 expression and the invasive potential of the cell lines examined. Data suggest a role for COX-2 not only in the growth of cancer cells but also in activating the angiogenic pathway through regulating levels of vascular endothelial growth factor.
PMCID: PMC1175053  PMID: 15987447
10.  A novel naproxen derivative capable of displaying anti-cancer and anti-migratory properties against human breast cancer cells 
BMC Cancer  2014;14(1):567.
Increasingly, the role of chronic inflammation and its mediators in tumor generation and progression is gaining importance in the field of cancer research. In this context, candidature of non steroidal anti-inflammatory drugs (NSAIDs) as potential anti-tumor therapeutic agent is being evaluated globally. In the present study we have evaluated the anti-cancer effect of a series of newly synthesized naproxen derivatives on human breast cancer cell lines.
MCF-7 (poorly invasive) and MDA-MB-231 (highly invasive) cells were treated with different concentrations of naproxen sodium and its derivatives in vitro, and the underlying mechanism of action was monitored by employing studies related to induction of apoptosis, activation of caspases, cell-cycle progression, synthesis of PGE2 and cellular migration.
After a preliminary screening using MCF-7 and MDA-MB-231 cells, it was evident that naproxen derivative 4 has a better killing property compared to its parent compound naproxen sodium (NS). On further investigation, it was apparent that the observed growth inhibitory activity on MDA-MB-231 cells after treatment with 4, was not due to cell cycle arrest but due to an early induction of apoptosis and subsequent induction of caspases 3 and 9. Derivative 4 could also inhibit COX activity in MDA-MB-231 cells as evidenced by reduction in prostaglandin E2 secretion. Moreover, 4 was capable of delaying the overall migration rate of MDA-MB-231 cells in vitro.
In this study we report that a naproxen-derivative (4) has powerful anti-inflammatory and anti-tumor properties as it induces appreciable amount of apoptosis in breast cancer cell line, and can also delay migration of cancer cells (MDA-MB-231) which would in turn delay cancer cell invasion and formation of secondary tumours in primary breast cancer patients. Thus, we propose that 4 is worthy of further investigation due to its potential as a therapeutic agent in anti-tumor treatment regimen.
PMCID: PMC4133615  PMID: 25098498
MCF-7; MDA-MB-231; MTT assay; Apoptosis; Inflammation; Metastasis
11.  Fenofibrate induces apoptosis of triple-negative breast cancer cells via activation of NF-κB pathway 
BMC Cancer  2014;14:96.
There are a lot of unmet needs in patients with triple-negative breast cancer (TNBC). Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPAR-α) agonist, has been used for decades to treat hypertriglyceridaemia and mixed dyslipidaemia. Recent studies show that it might have anti-tumor effects, however, the mechanism remains unclear. Here, we assessed the ability of fenofibrate to induce apoptosis of TNBC in vitro and in vivo and explored involved mechanisms.
MTT method was used to evaluate the anti-proliferation effect of fenofibrate, and invert microscope to observe the apoptotic morphological changes. The percentage of apoptotic cells and distribution ratios of cell cycle were determined by flow cytometric analysis. The related protein levels were measured by Western blot method. The changes of genes and pathways were detected by gene expression profiling. The tumor growth in vivo was assessed by MDA-MB-231 xenograft mouse model. Terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) assay was employed to estimate the percentage of apoptotic cells in vivo. In order to evaluate the safety of fenofibrate, blood sampled from rat eyes was detected.
We found that fenofibrate had anti-proliferation effects on breast cancer cell lines, of which the first five most sensitive ones were all TNBC cell lines. Its induction of apoptosis was independent on PPAR-α status with the highest apoptosis percentage of 41.8 ± 8.8%, and it occurred in a time- and dose-dependent manner accompanied by up-regulation of Bad, down-regulation of Bcl-xl, Survivin and activation of caspase-3. Interestingly, activation of NF-κB pathway played an important role in the induction of apoptosis by fenofibtate and the effect could be almost totally blocked by a NF-κB specific inhibitor, pyrrolidine dithiocarbamate (PDTC). In addition, fenofibrate led to cell cycle arrest at G0/G1 phase accompanied by down-regulation of Cyclin D1, Cdk4 and up-regulation of p21, p27/Kip1. In vivo, fenofibrate slowed down tumor growth and induced apoptosis with a good safety profile in the MDA-MB-231 xengograft mouse model.
It is concluded that fenofibrate induces apoptosis of TNBC via activation of NF-κB pathway in a PPAR-α independent way, and may serve as a novel therapeutic drug for TNBC therapy.
PMCID: PMC4015735  PMID: 24529079
Triple-negative breast cancer; Fenofibrate; Anti-proliferation; Apoptosis; NF-κB; Cell cycle arrest
12.  Flavokawain A Induces Apoptosis in MCF-7 and MDA-MB231 and Inhibits the Metastatic Process In Vitro 
PLoS ONE  2014;9(10):e105244.
The kava-kava plant (Piper methsyticum) is traditionally known as the pacific elixir by the pacific islanders for its role in a wide range of biological activities. The extract of the roots of this plant contains a variety of interesting molecules including Flavokawain A and this molecule is known to have anti-cancer properties. Breast cancer is still one of the leading diagnosed cancers in women today. The metastatic process is also very pertinent in the progression of tumorigenesis.
MCF-7 and MDA-MB231 cells were treated with several concentrations of FKA. The apoptotic analysis was done through the MTT assay, BrdU assay, Annexin V analysis, cell cycle analysis, JC-1 mitochondrial dye, AO/PI dual staining, caspase 8/9 fluorometric assay, quantitative real time PCR and western blot. For the metastatic assays, the in vitro scratch assay, trans-well migration/invasion assay, HUVEC tube formation assay, ex vivo rat aortic ring assay, quantitative real time PCR and western blot were employed.
We have investigated the effects of FKA on the apoptotic and metastatic process in two breast cancer cell lines. FKA induces apoptosis in both MCF-7 and MDA-MB231 in a dose dependent manner through the intrinsic mitochondrial pathway. Additionally, FKA selectively induces a G2/M arrest in the cell cycle machinery of MDA-MB231 and G1 arrest in MCF-7. This suggests that FKA's anti-cancer activity is dependent on the p53 status. Moreover, FKA also halted the migration and invasion process in MDA-MB231. The similar effects can be seen in the inhibition of the angiogenesis process as well.
FKA managed to induce apoptosis and inhibit the metastatic process in two breast cancer cell lines, in vitro. Overall, FKA may serve as a promising candidate in the search of a new anti-cancer drug especially in halting the metastatic process but further in vivo evidence is needed.
PMCID: PMC4186755  PMID: 25286005
13.  Critical Role of p53 Upregulated Modulator of Apoptosis in Benzyl Isothiocyanate-Induced Apoptotic Cell Death 
PLoS ONE  2012;7(2):e32267.
Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.
PMCID: PMC3281133  PMID: 22359675
14.  ABT-737 reverses the acquired radioresistance of breast cancer cells by targeting Bcl-2 and Bcl-xL 
Acquired radioresistance of cancer cells remains a fundamental barrier to attaining the maximal efficacy of radiotherapy for the treatment of breast cancer. Anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, play an important role in the radioresistance of cancer cells. In the present study, we aimed to determine if ABT-737, a BH3-only mimic, could reverse the acquired radioresistance of the breast cancer cell line MDA-MB-231R by targeting Bcl-2 and Bcl-xL.
The radiosensitivity of MDA-MB-231 and MDA-MB-231R cells was compared using colony formation assays. Reverse-transcription PCR and western blot were performed to detect the expression of Bcl-2 and Bcl-xL in the cancer cell lines. Annexin V flow cytometric analysis and caspase-3 colorimetric assay were used to evaluate apoptosis of the cancer cells. Cell viability was measured using the Cell Counting Kit-8. The animals used in this study were 4 to 6-week-old athymic female BALB/c nu/nu mice.
The MDA-MB-231R cells were more radioresistant than the MDA-MB-231 cells, and Bcl-2 and Bcl-xL were overexpressed in the MDA-MB-231R cells. While ABT-737 was able to restore the radiosensitivity of the MDA-MB-231R cells in vitro and in vivo experiment, it was not able to enhance the radiosensitivity of the MDA-MB-231 cells. In addition, ABT-737 increased radiation-induced apoptosis in the MDA-MB-231R cells. Bcl-2 and Bcl-xL were down regulated in the MDA-MB-231R cells following treatment with ABT-737.
Targeting of the anti-apoptotic proteins Bcl-2 and Bcl-xL with ABT-737 may reverse the acquired radioresistance of MDA-MB-231R cells in vitro and in vivo. These findings suggest an attractive strategy for overcoming the acquired radioresistance of breast cancer cells.
PMCID: PMC3541995  PMID: 23259599
ABT-737; Breast cancer; Acquired radioresistance; Radiation; Bcl-2; Bcl-xL
15.  The Anti-Cancer Activities of Vernonia amygdalina Extract in Human Breast Cancer Cell Lines Are Mediated through Caspase-Dependent and p53-Independent Pathways 
PLoS ONE  2013;8(10):e78021.
Breast cancer is currently the leading cause of cancer-related deaths among women globally. Notably, medicinal plant extracts may be a potential source for treatments of breast cancer. Vernonia amygdalina (VA) is a woody shrub reported to have not only diverse therapeutic effects but also anti-cancer properties. However, current research about the mechanisms of the anti-cancer potential of VA has been limited. This study aimed to investigate the mechanisms of action of VA that underlie its anti-cancer effects in human breast cancer cell lines (MCF-7 and MDA-MB-231 cells). Results from MTT assay revealed that VA inhibits the proliferation of MCF-7 and MDA-MB-231, in a time- and dose-dependent manner. The underlying mechanism of this growth inhibition involved the stimulation of cell-type specific G1/S phase cell cycle arrest in only MCF-7 cells, and not in MDA-MB-231 cells. While the growth arrest was associated with increased levels of p53 and p21, and a concomitant decrease in the levels of cyclin D1 and cyclin E, it was shown that VA causes cell cycle arrest through a p53-independent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Furthermore, this study revealed that VA induces apoptosis in the two cell lines, as indicated by the increase in Annexin V-positive cells and sub-G1 population, and that this VA-induced apoptosis occurred through both extrinsic and intrinsic apoptotic pathways. The apoptosis in MCF-7 cells was also likely to be caspase-dependent and not p53 transcriptional-dependent. Given that approximately 70% of diagnosed breast cancers express ER-α, a crucial finding was that VA inhibits the expression of ER-α and its downstream player, Akt, highlighting the potential clinical significance of VA. Moreover, VA exhibits synergism when combined with doxorubicin, suggesting that it can complement current chemotherapy. Overall, this study demonstrates the potential applications of VA as an anti-cancer drug for breast cancer treatment.
PMCID: PMC3812032  PMID: 24205071
16.  Chemotherapy cytotoxicity of human MCF-7 and MDA-MB 231 breast cancer cells is altered by osteoblast-derived growth factors. 
Molecular Medicine  1999;5(2):86-97.
One-third of women with breast cancer will develop bone metastases and eventually die from disease progression at these sites. Therefore, we analyzed the ability of human MG-63 osteoblast-like cells (MG-63 cells), MG-63 conditioned media (MG-63 CM), insulin-like growth factor I (IGF-I), and transforming growth factor beta 1 (TGF-beta1) to alter the effects of adriamycin on cell cycle and apoptosis of estrogen receptor negative (ER-) MDA-MB-231 and positive (ER+) MCF-7 breast cancer cells, using cell count, trypan blue exclusion, flow cytometry, detection of DNA fragmentation by simple agarose gel, and the terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method for apoptosis (TUNEL assay). Adriamycin arrested MCF-7 and MDA-MB-231 cells at G2/M phase in the cell cycle and inhibited cell growth. In addition, adriamycin arrested the MCF-7 cells at G1/G0 phase and induced apoptosis of MDA-MB-231 cells. Exogenous IGF-I partially neutralized the adriamycin cytotoxicity/cytostasis of cancer cells. MG-63 CM and TGF-beta1 partially neutralized the adriamycin cytotoxicity of MDA-MB-231 cells but enhanced adriamycin blockade of MCF-7 cells at G1/G0 phase. MG-63 osteoblast-like cells inhibited growth of MCF-7 cells while promoting growth and rescued MDA-MB-231 cells from adriamycin apoptosis in a collagen co-culture system. These data suggest that osteoblast-derived growth factors can alter the chemotherapy response of breast cancer cells. Conceivably, host tissue (bone)-tumor cell interactions can modify the clinical response to chemotherapy in patients with advanced breast cancer.
PMCID: PMC2230409  PMID: 10203574
17.  Suberoylanilide hydroxamic acid as a potential therapeutic agent for human breast cancer treatment. 
Molecular Medicine  2000;6(10):849-866.
BACKGROUND: Suberoylanilide hydroxamic acid (SAHA) is a prototype of the newly developed, second-generation, hybrid polar compounds. It is a novel histone deacetylase inhibitor with high potency for inducing cell differentiation of cultured murine erythroleukemia cells. Studies with SAHA have primarily been performed with hematopoietic tumor cells. Here we extent these studies with SAHA to human breast cancer cell lines in an attempt to find better therapeutic agents for breast cancer treatment. MATERIALS AND METHODS: Human breast cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-435, as well as normal cells, including the normal breast epithelial cell line MCF-10A, and fibroblasts, were treated with SAHA. Cells assayed for cell survival by using trypan blue exclusion assay, colony formation assay, and cell cycle and apoptosis analysis. The effects of SAHA on cell cycle and apoptosis regulatory proteins were examined by Western blots analysis. The identification of additional target genes was carried out by differential display (DD) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: SAHA inhibited clonogenic growth of MCF7, MDA-MB-231, and MDA-MB-435 breast cancer cells. These cells were more sensitive to SAHA-mediated cytotoxic effects than normal breast epithelial cells and fibroblasts. The cytotoxic effects of SAHA on breast cancer cells were manifested by G1 and G2/M cell cycle arrest and eventual apoptosis. The pan-caspase inhibitor, Z-VAD.fmk, blocked SAHA-induced cell death, DNA laddering, and cleavage of poly(ADP-ribose) polymerase, indicating the involvement of caspases in SAHA-mediated apoptosis. In addition, SAHA modulated cell cycle and apoptosis regulatory proteins. For example, cyclin-dependent kinase (CDK) inhibitors p21WAF1/Cip1 and p27Kip1 were induced, and retinoblastoma protein pRb was hypophosphorylated. Moreover, SAHA induced several genes associated with differentiation and/ or growth inhibition. These genes encode gelsolin, isopentenyl-diphosphate delta isomerase (IDI1), and 1,25-dihydroxyvitamin D-3 up-regulated protein 1 (VDUP1), the last two of which were identified by DD. Induction of these genes may contribute to SAHA-mediated pro-differentiating and antiproliferative effects. CONCLUSIONS: SAHA induced growth inhibition, cell cycle arrest, and eventual apoptosis in human breast cancer cells, possibly by modulating cell cycle and apoptosis regulatory proteins, such as CDK inhibitors p21 and p27, pRb, and other differentiation and/or growth inhibition-associated genes, including gelsolin, IDI1 and VDUP1. This, together with the low toxicity in normal cells, suggests that SAHA might have therapeutic potential for the treatment of human breast cancers.
PMCID: PMC1949918  PMID: 11126200
18.  CXCL12-CXCR4 axis promotes the natural selection of breast cancer cell metastasis 
Tumour Biology  2014;35(8):7765-7773.
CXCR4 and its ligand CXCL12 can promote the proliferation, survival, and invasion of cancer cells. They have been shown to play an important role in regulating metastasis of breast cancer to specific organs. High CXCR4 expression was also correlated to poor clinical outcome. Previous study also showed that tumor cells express a high level of CXCR4 and that tumor metastasis target tissues (lung, liver, and bone) express high levels of the ligand CXCL12, allowing tumor cells to directionally migrate to target organs via a CXCL12-CXCR4 chemotactic gradient. However, the exact mechanisms of how CXCR4 and CXCL12 enhance metastasis and/or tumor growth and their full implications on breast cancer progression are unknown. Yet it is likely that chemokine receptor signaling may provide more than just a migrational advantage by also helping the metastasized cells establish and survive in secondary environments. In this study, we investigated CXCR4 and CXCL12 expression in breast cancer and analyzed its association with clinicopathological factors by immunohistochemistry first. Then, we detected the mRNA and protein expression of CXCR4 and CXCL12 in breast cancer cell lines by Western blot and RT-PCR. The MDA-MB-231 has CXCR4 expression and very weak CXCL12 expression. So, we constructed the functional CXCL12 expression in MDA-MB-231 using a gene transfection technique. Further experiments were conducted to evaluate the effect of CXCL12 transfection on the biological behaviors of MDA-MB-231. The cell proliferation of MDA-MB-231–CXCL12 was accessed by MTT assay; the apoptosis was analyzed by an AnnexinV-FITC/propidium iodide double staining of flow cytometry method; and the cell invasive ability was examined by Matrigel invasion assay. Immunohistochemical analysis showed the co-expression of CXCR4 and CXCL12 correlated with lymph node metastasis and TNM stage (p < 0.01). It suggested that the chemokine CXCL12 and its sole ligand CXCR4 play important role in the malignance of breast cancer. To gain a deeper insight into it, we picked CXCR4-expressing cells MDA-MB-231 to be transfected with CXCL12 stably. The decreased cellular proliferation, increased apoptosis, and invasive ability were found in MDA-MB-231 with successful CXCL12 transfection (p < 0.05). Our findings underlined the CXCL12-CXCR4 axis correlated tightly with breast cancer metastasis. CXCL12-CXCR4 axis can increase the invasion and apoptosis of MDA-MB-231 simultaneously. These data strongly support the hypothesis that CXCL12-CXCR4 axis promotes the natural selection of breast cancer cell metastasis. Our findings could have significant implications in terms of breast cancer aggressiveness and the effectiveness of targeting the receptors and downstream signaling pathways for the treatment of breast cancer.
PMCID: PMC4158177  PMID: 24810923
CXCR4; CXCL12; Breast cancer
19.  Mitotic Arrest and Apoptosis in Breast Cancer Cells Induced by Origanum majorana Extract: Upregulation of TNF-α and Downregulation of Survivin and Mutant p53 
PLoS ONE  2013;8(2):e56649.
In the present study, we investigated the effect of Origanum majorana ethanolic extract on the survival of the highly proliferative and invasive triple-negative p53 mutant breast cancer cell line MDA-MB-231.
We found that O. majorana extract (OME) was able to inhibit the viability of the MDA-MB-231 cells in a time- and concentration-dependent manner. The effect of OME on cellular viability was further confirmed by the inhibition of colony growth. We showed, depending on the concentration used, that OME elicited different effects on the MDA-MB 231 cells. Concentrations of 150 and 300 µg/mL induced an accumulation of apoptotic–resistant population of cells arrested in mitotis and overexpressing the cyclin-dependent kinase inhibitor, p21 and the inhibitor of apoptosis, survivin. On the other hand, higher concentrations of OME (450 and 600 µg/mL) triggered a massive apoptosis through the extrinsic pathway, including the activation of tumor necrosis factor-α (TNF-α), caspase 8, caspase 3, and cleavage of PARP, downregulation of survivin as well as depletion of the mutant p53 in MDA-MB-231 cells. Furthermore, OME induced an upregulation of γ-H2AX, a marker of double strand DNA breaks and an overall histone H3 and H4 hyperacetylation.
Our findings provide strong evidence that O. majorana may be a promising chemopreventive and therapeutic candidate against cancer especially for highly invasive triple negative p53 mutant breast cancer; thus validating its complementary and alternative medicinal use.
PMCID: PMC3579842  PMID: 23451065
20.  Autophagic Cell Death, Polyploidy and Senescence Induced in Breast Tumor Cells by the Substituted Pyrrole JG-03-14, a Novel Microtubule Poison 
Biochemical pharmacology  2007;74(7):981-991.
JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB 231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on β-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time dependent cell death in the MCF-7 and MDA-MB 231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB 231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (< 10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but < 20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer.
PMCID: PMC2000298  PMID: 17692290
21.  Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner 
Molecular Biology Reports  2013;40(8):4995-5004.
The aim of the study was to analyze the consequence of silencing genes coding for the key subunits of the telomerase complex, i.e. TERT, TERC and TP1 in human breast cancer MCF7 and MDA-MB-231cells. The transfection was performed using Lipofectamine2000 and pooled siRNAs. The cytotoxic and/or antiproliferative effect of siRNA was measured by the SRB assay, the cell cycle was analysed by flow cytometry and DNA fragmentation by TUNEL analysis. Telomerase activity was assessed by TRAP, followed by PAGE and ELISA assays. Telomerase downregulation was also assessed using qPCR in order to estimate the changes in the expression profile of genes engaged in apoptosis. It was revealed that treatment of breast cancer cells with different siRNAs (100 nM) resulted in a cell type and time-dependent effects. The downregulation of telomerase subunits was followed by reduction of telomerase activity down to almost 60 % compared to control cells. However, a significant effect was only observed when the TERT subunit was downregulated. Its silencing resulted in a significant (p < 0.05) increase of apoptosis (over 10 % in MCF7 and about 5 % in MDA-MB-231 cells, corresponding to the Annexin V assay) and DNA fragmentation (almost 30 % in MCF7 and over 25 % in MDA-MB-231 cells). Interestingly, also several proapoptotic genes were induced after the downregulation of the key telomerase subunit, including Bax, Bik or caspase-1 and caspase-14, as well as NGFR and TNFSF10 which were upregulated twice and more.
PMCID: PMC3723976  PMID: 23677713
Adenocarcinoma therapy; Breast cancer; Telomerase; Telomere
22.  Cellular reprogramming by the conjoint action of ERα, FOXA1, and GATA3 to a ligand-inducible growth state 
Estrogen receptor α (ERα), FOXA1, and GATA3 form a functional enhanceosome in MCF-7 breast carcinoma cell that is significantly associated with active transcriptional features such as enhanced p300 co-activator and RNA Pol II recruitment as well as chromatin opening.The enhanceosome exerts significant impact and optimal transcriptional control in the regulation of E2-responsive genes.The presence of FOXA1 and GATA3 is indispensable in restoring the ERα growth-response machinery in the ERα-negative cells and recapitulating the appropriate expression cassette.
Estrogen receptor α (ERα) is a ligand-inducible hormone nuclear receptor that has important physiology and pathology roles in reproduction, cancer, and cardiovascular biology. The regulation of ERα involves its binding to the DNA recognition sequence also known as estrogen-response elements (EREs) and recruits a variety of co-activators, corepressors, and chromatin remodeling enzymes to initiate transcription machinery. In our previous (Lin et al, 2007) and recent (Joseph et al, 2010) studies, we have identified high confidence ERα binding sites in MCF-7 human mammary carcinoma cells. With known motif scanning and de novo motif detection, we identified that FOXA1 and GATA3 motifs were commonly enriched around ERα binding sites. Moreover, numerous microarray studies have documented the co-expression of ERα, FOXA1, and GATA3 in primary breast tumors (Badve et al, 2007; Wilson and Giguere, 2008). This evidence suggests that these three transcription factors (TFs) may cluster on DNA binding sites and contribute to the breast cancer phenotype. However, there is little understanding as to the nature of their coordinated interaction at the genome level or the biological consequences of their detailed interaction.
We mapped the genome-wide binding profiles of ERα, FOXA1, and GATA3 using the massive parallel chromatin immunoprecipitation-sequencing (ChIP-seq) approach. We observed that ERα, FOXA1, and GATA3 colocalized in a coordinated manner where ∼30% of all ERα binding sites were overlapped with FOXA1 and GATA3 bindings upon estrogen (E2) stimulation. Moreover, we found that the ERα+FOXA1+GATA3 conjoint sites were associated with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Such results indicate that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. And such enhanceosome binding sites appear to regulate the genes driving core ERα function.
To further validate that ERα+FOXA1+GATA3 co-binding represents an optimal configuration for E2-mediated transcriptional activation, we have performed luciferase reporter assays on GREB1 locus that actively engages ERα enhanceosome sites in gene regulation (Figure 5C). The presence of ERα induced the GREB1 luciferase activity to ∼246% (as compared with the control construct). The individual presence of FOXA1 and GATA3 or combination of both only produced subtle changes to the GREB1 luciferase activity. The combination of ERα+FOXA1 and ERα+GATA3 has increased the luciferase activity to ∼330%. Interestingly, the assemblage of ERα+FOXA1+GATA3 provided the optimal ER responsiveness to 370%. This suggests that ERα provides the fundamental gene regulatory module but that FOXA1 and GATA3 incrementally improve ERα-regulated transcriptional induction.
It is known that ERα is a ligand-activated TF that mediates the proliferative effects of E2 in breast cancer cells. Garcia et al (1992) showed inhibited growth in MDA-MB-231 cells with forced expression of ERα upon E2 treatment. The rationale for these different outcomes has remained elusive. We posited that these higher order regulatory mechanisms of ERα function such as the formation and composition of enhanceosomes may explain the establishment of transcriptional regulatory cassettes favoring either growth enhancement or growth repression.
To test this hypothesis, we stably transfected the MDA-MB-231 cells with individual ERα, FOXA1, GATA3, or in combinations (Figure 6A). We observed inhibited growth in cells with enforced expression of ERα or FOXA1. There was unaltered growth in cells with expression of GATA3. Co-expression of ERα+FOXA1 or ERα+GATA3 exhibited inhibition of cell proliferation as compared with control cells. However, the co-expression of ERα together with FOXA1 and GATA3 resulted in marked induction of cell proliferation under E2 stimulation. We have recapitulated this cellular reprogramming in another ERα-negative breast cancer cell line, BT-549 and observed similar E2-responsive growth induction in the ERα+FOXA1+GATA3-expressing BT-549 cells. This suggests that only with the full activation of conjoint binding sites by the three TFs will the proliferative phenotype associated with ligand induced ERα be manifest.
To assess the nature of this transcriptional reprogramming, we asked the question if the reprogrammed MDA-MB-231 cells display any similarity in the expression profile of the ERα-positive breast cancer cell line, MCF-7 (Figure 6C). We combined the E2-regulated genes from these differently transfected MDA-MB-231 cells, and compared their expressions in these MDA-MB-231-transfected cells and MCF-7 cells. Strikingly, we found that the expression profiles of ERα+FOXA1+GATA3-expressing MDA-MB-231 cells display a good correlation (R=0.42) with the E2-induced expression profile of MCF-7. We did not observe such correlation between the expression profiles of MDA-MB-231 transfected with ERα only (R=−0.21). Furthermore, we observed that there is marginal induced expression of luminal marker genes and reduced expression of basal genes in the ERα+FOXA1+GATA3-expressing MDA-MB-231 as compared with the vector control cells. This suggests that the enhanceosome component is competent to partially reprogramme the basal cells to resemble the luminal cells.
Taken together, we have uncovered the genomics impact as well as the functional importance of an enhanceosome comprising ERα, FOXA1, and GATA3 in the estrogen responsiveness of ERα-positive breast cancer cells. This enhanceosome exerts significant combinatorial control of the transcriptional network regulating growth and proliferation of ERα-positive breast cancer cells. Most importantly, we show that the transfection of the enhanceosome component was necessary to reprogramme the ERα-negative cells to restore the estrogen-responsive growth and to transcriptionally induce a basal to luminal transition.
Despite the role of the estrogen receptor α (ERα) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERα into ERα-negative cells paradoxically has been growth inhibition. We mapped the binding profiles of ERα and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF-7 breast carcinoma cells, and observed that these three TFs form a functional enhanceosome that regulates the genes driving core ERα function and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERα-negative MDA-MB-231 and BT-549 cells to restore the estrogen-responsive growth resembling estrogen-treated ERα-positive MCF-7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERα, FOXA1, and GATA3 are necessary for the full repertoire of cancer-associated effects of the ERα.
PMCID: PMC3202798  PMID: 21878914
enhanceosome; estrogen receptor α; FOXA1; GATA3; synthetic phenotypes
23.  Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1 
PLoS Medicine  2007;4(6):e186.
While vascular endothelial growth factor (VEGF) expression in breast tumors has been correlated with a poor outcome in the pathogenesis of breast cancer, the expression, localization, and function of VEGF receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR or FLK1), as well as neuropilin 1 (NRP1), in breast cancer are controversial.
Methods and Findings
We investigated the expression and function of VEGF and VEGF receptors in breast cancer cells. We observed that VEGFR1 expression was abundant, VEGFR2 expression was low, and NRP1 expression was variable. MDA-MB-231 and MCF-7 breast cancer cells, transfected with antisense VEGF cDNA or with siVEGF (VEGF-targeted small interfering RNA), showed a significant reduction in VEGF expression and increased apoptosis as compared to the control cells. Additionally, specifically targeted knockdown of VEGFR1 expression by siRNA (siVEGFR1) significantly decreased the survival of breast cancer cells through down-regulation of protein kinase B (AKT) phosphorylation, while targeted knockdown of VEGFR2 or NRP1 expression had no effect on the survival of these cancer cells. Since a VEGFR1-specific ligand, placenta growth factor (PGF), did not, as expected, inhibit the breast cancer cell apoptosis induced by siVEGF, and since VEGFR1 antibody also had no effects on the survival of these cells, we examined VEGFR1 localization. VEGFR1 was predominantly expressed internally in MDA-MB-231 and MCF-7 breast cancer cells. Specifically, VEGFR1 was found to be colocalized with lamin A/C and was expressed mainly in the nuclear envelope in breast cancer cell lines and primary breast cancer tumors. Breast cancer cells treated with siVEGFR1 showed significantly decreased VEGFR1 expression levels and a lack of VEGFR1 expression in the nuclear envelope.
This study provides, to our knowledge for the first time, evidence of a unique survival system in breast cancer cells by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR1. These results may lead to an improved strategy for tumor therapy based on the inhibition of angiogenesis.
Shalom Avraham and colleagues' study provides evidence of a survival system in breast cancer cells by which VEGF acts as an internal autocrine survival factor through its binding to VEGFR1.
Editors' Summary
One woman in eight will develop breast cancer during her lifetime. Most of these women live for many years after their diagnosis and many are cured of their cancer. However, sometimes the cancer grows inexorably and spreads (metastasizes) around the body despite the efforts of oncologists. Characteristics of the tumor known as prognostic factors can indicate whether this spreading is likely to happen. Large tumors that have metastasized have a poorer prognosis than small tumors that are confined to the breast. The expression of specific proteins within the tumor also provides prognostic information. One protein whose expression is associated with a poor prognosis is vascular endothelial growth factor (VEGF). VEGF stimulates angiogenesis—the growth of new blood vessels. Small tumors get the nutrients needed for their growth from existing blood vessels but large tumors need to organize their own blood supply. They do this, in part, by secreting VEGF. This compound binds to proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels), which then send a signal into the cell instructing it to make new blood vessels. Angiogenesis inhibitors, including molecules that block the activity of VEGF receptors, are being developed for the treatment of cancer.
Why Was This Study Done?
Some breast cancer cell lines (cells isolated from breast cancers and grown in the laboratory) make VEGF and VEGF receptors (VEGFR1, VEGFR2, and neuropilin 1 [NRP1]). But, although some studies have reported an association between VEGFR1 expression in breast tumors and a poor prognosis, other studies have found no expression of VEGFR1 in breast tumors. Consequently, the role of VEGF receptors in breast cancer is unclear. In this study, the researchers analyzed the expression and function of VEGF and its receptors in breast cancer cells to investigate whether and how VEGF helps these cells to survive.
What Did the Researchers Do and Find?
The researchers first examined the expression of VEGF receptors in several human breast cancer cell lines. All of them expressed VEGFR1, some expressed NRP1, but VEGFR2 expression was universally low. They then investigated the function of VEGF and its receptors in two human breast cancer cell lines (MDA-MB-231 and MCF-7). In both cell lines, blocking the expression of VEGF or of VEGFR1 (but not of the other two receptors) reduced cell survival by stimulating a specific process of cell death called apoptosis. Unexpectedly, adding VEGF to the cultures did not reverse the effect of blocking VEGF expression, a result that suggests that VEGF and VEGFR1 do not affect breast cancer cell survival by acting at the cell surface. Accordingly, when the researchers examined where VEGFR1 occurs in the cell, they found it on the membranes around the nucleus of the breast cancer cell lines and not on the cell surface; several primary breast tumors and normal breast tissue had the same localization pattern. Finally, the researchers showed that inhibitors of VEGF action that act at the cell surface did not affect the survival of the breast cancer cell lines.
What Do These Findings Mean?
These findings suggest that VEGF helps breast cancer cells to survive in a unique way: by binding to VEGFR1 inside the cell. In other words, whereas VEGF normally acts as a paracrine growth factor (it is released by one cell and affects another cell), in breast cancer cells it might act as an internal autocrine (intracrine) survival factor, a factor that affects the cells in which it is produced. These findings need confirming in more cell lines and in primary breast cancers but could have important implications for the treatment of breast cancer. Inhibitors of VEGF and VEGFR1 that act inside the cell (small molecule drugs) might block breast cancer growth more effectively than inhibitors that act at the cell surface (for example, proteins that bind to the receptor), because internally acting inhibitors might both kill the tumor directly and have antiangiogenic effects, whereas externally acting inhibitors could only have the second effect.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute information for patients and professionals on breast cancer (in English and Spanish) and on angiogenesis (in English and Spanish)
MedlinePlus Encyclopedia information for patients on breast cancer (in English and Spanish)
CancerQuest, information from Emory University on cancer biology and on angiogenesis and angiogenesis inhibitors (in several languages)
Wikipedia pages on VEGF (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC1885450  PMID: 17550303
24.  Boswellia sacra essential oil induces tumor cell-specific apoptosis and suppresses tumor aggressiveness in cultured human breast cancer cells 
Gum resins obtained from trees of the Burseraceae family (Boswellia sp.) are important ingredients in incense and perfumes. Extracts prepared from Boswellia sp. gum resins have been shown to possess anti-inflammatory and anti-neoplastic effects. Essential oil prepared by distillation of the gum resin traditionally used for aromatic therapy has also been shown to have tumor cell-specific anti-proliferative and pro-apoptotic activities. The objective of this study was to optimize conditions for preparing Boswellea sacra essential oil with the highest biological activity in inducing tumor cell-specific cytotoxicity and suppressing aggressive tumor phenotypes in human breast cancer cells.
Boswellia sacra essential oil was prepared from Omani Hougari grade resins through hydrodistillation at 78 or 100 oC for 12 hours. Chemical compositions were identified by gas chromatography-mass spectrometry; and total boswellic acids contents were quantified by high-performance liquid chromatography. Boswellia sacra essential oil-mediated cell viability and death were studied in established human breast cancer cell lines (T47D, MCF7, MDA-MB-231) and an immortalized normal human breast cell line (MCF10-2A). Apoptosis was assayed by genomic DNA fragmentation. Anti-invasive and anti-multicellular tumor properties were evaluated by cellular network and spheroid formation models, respectively. Western blot analysis was performed to study Boswellia sacra essential oil-regulated proteins involved in apoptosis, signaling pathways, and cell cycle regulation.
More abundant high molecular weight compounds, including boswellic acids, were present in Boswellia sacra essential oil prepared at 100 oC hydrodistillation. All three human breast cancer cell lines were sensitive to essential oil treatment with reduced cell viability and elevated cell death, whereas the immortalized normal human breast cell line was more resistant to essential oil treatment. Boswellia sacra essential oil hydrodistilled at 100 oC was more potent than the essential oil prepared at 78 oC in inducing cancer cell death, preventing the cellular network formation (MDA-MB-231) cells on Matrigel, causing the breakdown of multicellular tumor spheroids (T47D cells), and regulating molecules involved in apoptosis, signal transduction, and cell cycle progression.
Similar to our previous observations in human bladder cancer cells, Boswellia sacra essential oil induces breast cancer cell-specific cytotoxicity. Suppression of cellular network formation and disruption of spheroid development of breast cancer cells by Boswellia sacra essential oil suggest that the essential oil may be effective for advanced breast cancer. Consistently, the essential oil represses signaling pathways and cell cycle regulators that have been proposed as therapeutic targets for breast cancer. Future pre-clinical and clinical studies are urgently needed to evaluate the safety and efficacy of Boswellia sacra essential oil as a therapeutic agent for treating breast cancer.
PMCID: PMC3258268  PMID: 22171782
25.  The bisphosphonate zoledronic acid impairs membrane localisation and induces cytochrome c release in breast cancer cells 
British Journal of Cancer  2002;86(9):1479-1486.
Bisphosphonates are well established in the management of cancer-induced bone disease. Recent studies have indicated that these compounds have direct inhibitory effects on cultured human breast cancer cells. Nitrogen-containing bisphosphonates including zoledronic acid have been shown to induce apoptosis associated with PARP cleavage and DNA fragmentation. The aim of this study was to identify the signalling pathways involved. Forced expression of the anti-apoptotic protein bcl-2 attenuated bisphosphonate-induced loss of cell viability and induction of DNA fragmentation in MDA-MB-231 cells. Zoledronic acid-mediated apoptosis was associated with a time and dose-related release of mitochondrial cytochrome c into the cytosol in two cell lines. Rescue of cells by preincubation with a caspase-3 selective inhibitor and demonstration of pro-caspase-3 cleavage products by immunoblotting suggests that at least one of the caspases activated in response to zoledronic acid treatment is caspase-3. In both MDA-MB-231 and MCF-7 breast cancer cells, zoledronic acid impaired membrane localisation of Ras indicating reduced prenylation of this protein. These observations demonstrate that zoledronic acid-mediated apoptosis is associated with cytochrome c release and consequent caspase activation. This process may be initiated by inhibition of the enzymes in the mevalonate pathway leading to impaired prenylation of key intracellular proteins including Ras.
British Journal of Cancer (2002) 86, 1479–1486. DOI: 10.1038/sj/bjc/6600297
© 2002 Cancer Research UK
PMCID: PMC2375368  PMID: 11986784
bisphosphonates; breast cancer; apoptosis; caspases; Ras; cytochrome c

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