Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): Potential role of cyclin D1

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G1 phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 μM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G1 phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G1 phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G1 phase of the cell cycle.

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, through down-regulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and tumor development of HER-2/neu-overexpressing ovarian cancer cells. Here, we established stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. The expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle delay in the G1 phase independently of the HER-2/neu down-regulation. In an orthotopic breast cancer model, we showed that expression of PEA3 inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment in another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, which suggests that PEA3 possessed a strong growth inhibitory effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence that the PEA3 gene could function as an antitumor and gene therapy agent for human breast cancers. ^

Retinoic acid induces sodium/iodide symporter gene expression and radioiodide uptake in the MCF-7 breast cancer cell line

The sodium/iodide symporter (NIS) stimulates iodide uptake in normal lactating breast, but is not known to be active in nonlactating breast or breast cancer. We studied NIS gene regulation and iodide uptake in MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line. All-trans retinoic acid (tRA) treatment stimulated iodide uptake in a time- and dose-dependent fashion up to ≈9.4-fold above baseline. Stimulation with selective retinoid compounds indicated that the induction of iodide uptake was mediated by retinoic acid receptor. Treatment with tRA markedly stimulated NIS mRNA and immunoreactive protein (≈68 kDa). tRA stimulated NIS gene transcription ≈4-fold, as shown by nuclear run-on assay. No induction of iodide uptake was observed with RA treatment of an ER-negative human breast cancer cell line, MDA-MB 231, or a normal human breast cell line, MCF-12A. The iodide efflux rate of tRA-treated MCF-7 cells was slow (t1/2 = 24 min), compared with that in FRTL-5 thyroid cells (t1/2 = 3.9 min), favoring iodide retention in MCF-7 cells. An in vitro clonogenic assay demonstrated selective cytotoxicity with 131I after tRA stimulation of MCF-7 cells. tRA up-regulates NIS gene expression and iodide uptake in an ER-positive breast cancer cell line. Stimulation of radioiodide uptake after systemic retinoid treatment may be useful for diagnosis and treatment of some differentiated breast cancers.

Rôle de GPR40 dans la survie et la prolifération cellulaires induites par l’oléate dans les cellules de cancer du sein MDA-MB-231 et de cancer de la prostate DU145

La relation entre l’obésité et le cancer, bien qu’établie par des études épidémiologiques, est peu connue. Pourtant, environ 25 % des cancers pourraient y être attribuables. Parmi les cancers reliés à l’obésité, les cancers du côlon, du sein chez les femmes ménopausées et de la prostate sont les plus fréquents. Des études sur modèles animaux ont suggéré une association positive entre une diète riche en gras et le développement du cancer mammaire et de la prostate. Nous avons étudié les mécanismes moléculaires par lesquels les acides gras influencent le devenir de lignées de cellules cancéreuses du sein et de la prostate. Ces travaux ont montré que les acides gras insaturés, dont l’oléate, induisent la prolifération cellulaire tandis que les acides gras saturés, dont le palmitate, diminuent la prolifération. Un traitement à l’oléate stimule la formation de gouttelettes lipidiques dans le cytoplasme des cellules de cancer du sein MDA-MB-231 et de la prostate DU145 alors qu’un traitement au palmitate entraîne l’apoptose. Le mécanisme d’action de l’oléate sur la prolifération a été étudié de façon plus approfondie. L’utilisation d’inhibiteurs pharmacologiques nous a permis de déterminer que l’effet prolifératif de l’oléate implique la voie PI3K/Akt, la voie ERK1/2 et l’activation d’un ou de plusieurs récepteur(s) couplé(s) aux protéines G (GPCR). L’oléate induit la phosphorylation rapide des protéines Akt et ERK1/2 dans les cellules de cancer du sein MDA-MB-231 et de la prostate DU145. Au cours des dernières années, deux GPCRs ont été identifiés comme étant activables par des acides gras à moyennes et à longues chaînes, GPR40 et GPR120. GPR40 étant exprimé dans plusieurs lignées cellulaires de cancer du sein et de la prostate contrairement à l’expression de GPR120 qui était inexistante dans la plupart des lignées, nous avons étudié l’implication de GPR40 dans l’effet prolifératif de l’oléate. Ces deux récepteurs n’étant pas exprimés dans les cellules épithéliales mammaires humaines en culture primaire, ces cellules ne répondent pas aux effets de l’oléate sur la prolifération et l’activation des voies de signalisation. L’activation des voies Akt et ERK1/2 par l’oléate dans les cellules MDA-MB-231 et DU145 est potentialisée par la surexpression du récepteur GPR40 et inhibée par l’utilisation d’un siRNA dirigé contre ce récepteur. Cependant, la prolifération induite par l’oléate ne semble pas affectée par la présence d’un siRNA dirigé contre GPR40. L’oléate étant un acide gras, il est capable d’entrer librement dans les cellules et une partie de ses effets sur la prolifération pourrait être attribuée à sa métabolisation. Un agoniste de GPR40, le GW9508, est en mesure d’activer GPR40 sans toutefois entrer dans les cellules ni activer le métabolisme de l’oléate. Le GW9508 stimule la phosphorylation des protéines Akt et ERK1/2 dans les cellules du cancer du sein MDA-MB-231 et de la prostate DU145, mais il n’est pas en mesure d’induire la prolifération cellulaire comme le fait l’oléate. Ces résultats nous permettent de mieux comprendre le mécanisme d’action de l’oléate sur les cellules de cancer du sein et de la prostate. L’oléate induit la signalisation de GPR40 qui est impliquée dans l’activation rapide des voies de signalisation Akt et ERK1/2. De son côté, l’effet prolifératif induit par l’oléate s’effectue par un mécanisme GPR40-indépendant, possiblement lié au métabolisme de l’oléate.

Cytotoxic effects of butanolic extract from Pfaffia paniculata (Brazilian Ginseng) on cultured human breast cancer cell line MCF-7

Roots of Pfaffia paniculata have been well documented for multifarious therapeutic values and have also been used for cancer therapy in folk medicine. This study has been performed in a human breast tumor cell line, the MCF-7 cells. These are the most commonly used model of estrogen-positive breast cancer, and it has been originally established in 1973 at the Michigan Cancer Foundation from a pleural effusion taken from a woman with metastatic breast cancer. Butanolic extract of the roots of P. paniculata showed cytotoxic effect MCF-7 cell line. as determined with crystal violet assay, cellular death with acridine orange/ethidium bromide staining, and cell proliferation with immunocytochemistry of bromodeoxyuridine (BrdU). Subcellular alterations were evaluated by electron microscopy. Cells treated With butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. The results show that this butanolic extract indeed presents cytotoxic substances, and its fractions merit further investigations. (C) 2008 Elsevier GmbH. All rights reserved.

Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line.

The expression of a hybrid gene formed by the promoter region of the Xenopus laevis vitellogenin gene B1 and the CAT coding region is regulated by estrogen when the gene is transfected into hormone-responsive MCF-7 cells. Furthermore, the 5' flanking region of the gene B1 alone can confer inducibility to heterologous promoters, although to a varying extent depending on the promoter used. Deletion mapping of he vitellogenin hormone-responsive sequences revealed that a 13 bp element 5'-AGTCACTGTGACC-3' at position -334 is essential for estrogen inducibility. We have shown previously that this 13 bp element is present upstream of several liver-specific estrogen-inducible genes.

Synergistic antitumor effect of TRAIL and adriamycin on the human breast cancer cell line MCF-7

The aim of the present study was to determine the effect of the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and adriamycin (ADM) on the human breast cancer cell line MCF-7 and to identify potential mechanisms of apoptosis. Cell viability was analyzed by the MTT assay and the synergistic effect was assessed by the Webb coefficient. Apoptosis was quantified using the annexin V-FITC and propidium iodide staining flow cytometry. The mRNA expression of TRAIL receptors was measured by RT-PCR. Changes in the quantities of Bax and caspase-9 proteins were determined by Western blot. MCF-7 cells were relatively resistant to TRAIL (IC50 >10 µg/mL), while MCF-7 cells were sensitive to ADM (IC50 <10 µg/mL). A subtoxic concentration of ADM (0.5 µg/mL) combined with 0.1, 1, or 10 µg/mL TRAIL had a synergistic cytotoxic effect on MCF-7 cells, which was more marked with the combination of TRAIL (0.1 µg/mL) and ADM (0.5 µg/mL). In addition, the combined treatment with TRAIL and ADM significantly increased cell apoptosis from 9.8% (TRAIL) or 17% (ADM) to 38.7%, resulting in a synergistic apoptotic effect, which is proposed to be mediated by up-regulation of DR4 and DR5 mRNA expression and increased expression of Bax and caspase-9 proteins. These results suggest that the combination of TRAIL and ADM might be a promising therapy for breast cancer.

Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro

It has been postulated that the R- and S-equol enantiomers have different biological properties given their different binding affinities for the estrogen receptor. S-(-)equol is produced via the bacterial conversion of the soy isoflavone daidzein in the gut. We have compared the biological effects of purified S-equol to that of racemic (R and S) equol on breast and prostate cancer cells of varying receptor status in vitro. Both racemic and S-equol inhibited the growth of the breast cancer cell line MDA-MB-231 (> or = 10 microM) and the prostate cancer cell lines LNCaP (> or = 5 microM) and LAPC-4 (> or = 2.5 microM). The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30 microM) was unable to prevent DNA damage in MCF-7 or MCF-10A breast cells following exposure to 2-hydroxy-4-nonenal, menadione, or benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. In contrast, racemic equol (10, 30 microM) prevented DNA damage in MCF-10A cells following exposure to 2-hydroxy-4-nonenal or menadione. These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol.

Diallyl disulfide-induced apoptosis in a breast-cancer cell line (MCF-7) may be caused by inhibition of histone de-acetylation

The health benefits of garlic have been proven by epidemiological and experimental studies. Diallyl disulphide (DADS), the major organosulfur compound found in garlic oil, is known to lower the incidence of breast cancer both in vitro and in vivo. The studies reported here demonstrate that DADS induces apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G0 population and substantially halts DNA synthesis. DADS also induces phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane and activates caspase-3. Further studies revealed that DADS modulates the cellular levels of Bax, Bcl-2, Bcl-xL and Bcl-w in a dose-dependent manner, suggesting the involvement of Bcl-2 family proteins in DADS induced apoptosis. Histone deacetylation inhibitors (HDACi) are known to suppress cancer growth and induce apoptosis in cancer cells. Here it is shown that DADS has HDACi properties in MCF-7 cells as it lowers the removal of an acetyl group from an acetylated substrate and induces histone-4 (H4) hyper-acetylation. The data thus indicate that the HDACi properties of DADS may be responsible for the induction of apoptosis in breast cancer cells.

Prolactin upregulates sphingosine kinase-1 expression and activity in the human breast cancer cell line MCF7 and triggers enhanced proliferation and migration

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

ECHOGENIC LIPOSOMES FOR NITRIC OXIDE DELIVERY AND BREAST CANCER TREATMENT

Liposomes, also known as nontoxic, biodegradable, and non-immunogenic therapeutic delivery vehicles, have been proposed as a carrier for drugs and antitumor agents in cancer chemotherapy. Echogenic liposomes (ELIP) have the potential to entrap air or bioactive gas to enhance acoustic reflectivity in ultrasound and are used as a contrast agent. The innovative part of this study is based on a novel concept to encapsulate nitric oxide (NO) gas into ELIP, deliver it to breast cancer cells, and control its release via direct ultrasound exposure. Studies on the effect of NO in tumor biology have shown that a high levels of NO (> 300 nM) leads to cytostasis or apoptosis by decreasing the translation of several cell cycle proteins and stimulating cancer cell death by activating the p53 pathway. The central hypothesis is that NO gas can be packaged and delivered through a delivery methodology to breast cancer cells to facilitate tumor regression with minimal systemic toxicity. The primary goal of this thesis is to develop an echogenic liposomal solution that has the ability to encapsulate NO, to release NO locally upon ultrasound exposure, and to induce breast cancer cell death. NO-containing echogenic liposomes (NO-ELIP) were prepared by the freezing-under-pressure method previously developed in our laboratory. It was necessary to evaluate stability of NO-ELIP and release of NO from NO-ELIP by measuring echogenicity using intravascular ultrasound images. Breast cancer cell lines, MDA-MB-231 and MDA-MB-468, were selected to investigate the cytotoxic effects of NO liberated from NO-ELIP and their response to NO concentration. Ultrasound-triggered NO release from NO-ELIP using ultrasound activation was studied. It was demonstrated that NO-ELIP remained stable for 5 hours in bovine serum albumin. Delivery of NO using NO-ELIP induced cytotoxicity and programmed cell death of MDA-MB-231 and MDA-MB-468 after 5 hours of incubation. Enhancement of the NO-ELIP effect for therapeutic application was observed with ultrasound activation. This work demonstrates that NO-ELIP can incorporate and deliver NO to breast cancer cells providing increased NO stability and ultrasound-controlled NO release. Improved therapeutic effect with the use of NO-ELIP is expected to be found for breast cancer treatment.

Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy.

Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN-con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers.

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading. (C) 2003 Elsevier B.V. All rights reserved.