Migration Of MCF-7 Research Articles

Beta-Adrenoreceptor antagonists reduce cancer cell proliferation, invasion, and migration

Context: Propranolol, atenolol, and ICI118,551 are non-selective β-adrenergic receptor (AR), β1-AR, and β2-AR antagonists, respectively.Objective: We investigated the efficacy of propranolol, atenolol, and ICI118,551 on proliferation, migration, and invasion of non-stimulated breast (MCF7), colon (HT-29), and hepatocellular (HepG2) cancer cells.Materials and methods: β-AR expression profiling of cells was performed by real time PCR. Cell proliferation was determined by MTT. Boyden chamber and scratch assays were performed to evaluate invasion and migration.Results and discussion: All cell lines expressed β-ARs. ICI118,551 was the most cytotoxic, whereas atenolol was the least effective β-AR antagonist for 24, 48, and 72 h. Cell invasion was inhibited by ICI118,551 (45, 46, and 50% for MCF7, HT29, and HepG2, respectively) and propranolol (72, 65, and 90% for MCF7, HT29, and HepG2, respectively). Propranolol, atenolol, and ICI118,551 reduced migration of MCF7, HT-29, and HepG2 cells to varying extents depending on the application concentration and duration. Propranolol and atenolol reduced migration of MCF7 and HT-29 in a concentration-dependent manner, whereas migration of these cells decreased after 48 and 72 h of ICI118,551 applications.Conclusion: Beta2-AR antagonist seemed to be the most cytotoxic β-blocker on non-stimulated cancer cells. Propranolol and ICI118,551 were more effective than atenolol in inhibiting invasion and migration of non-stimulated MCF7 and HT-29 cells; ICI118,551 being the most potent. Concordantly, β2-selective blockage seemed to be more effective for non-stimulated cells. Effect of the selective β-AR antagonists showed variation depending on the concentration, incubation time, and histological origin of cells.

Xenon decreases cell migration and secretion of a pro-angiogenesis factor in breast adenocarcinoma cells: comparison with sevoflurane

While volatile agents have been implicated in metastasis-enhancing effects on cancer cells, the effects of xenon are unknown. We investigated xenon- and sevoflurane-mediated effects on migration and expression of angiogenesis biomarkers in human breast adenocarcinoma cells. MDA-MB-231 and MCF-7 cells were exposed to xenon 70% with O2 25%, CO2 5%; control gas containing O2 25%, CO2 5%, N2 70%; or sevoflurane 2.5 vol% administered in O2 60%, N2 37%, or control gas. Cell viability was determined by the MTT assay. Migration at 24 h was determined using the Oris™ Cell Migration Assay. Secretion of angiogenesis factors was measured using a membrane-based immunoassay array. Xenon reduced MDA-MB-231 migration to 59 (13%) after 1-h exposure (P=0.02), 64 (10%) after 3 h (P=0.01), and 71 (9%) after 5 h (P=0.04) compared with control gas, without affecting viability. Similarly, MCF-7 migration was significantly reduced at all timepoints [to 58 (12%) at 1 h, 65 (12%) at 3 h, and 65% (12%) at 5 h]. Sevoflurane did not affect migration when delivered in control gas. Glycine, an N-methyl-d-aspartate receptor co-agonist, antagonized the effects of xenon on migration. Expression of the pro-angiogenesis factor regulated on activation, normal T cell expressed and secreted (RANTES) was reduced in conditioned medium from xenon-exposed MDA-MB-231 cells compared with cells exposed to either control gas or sevoflurane [mean dot density 2.0 (0.2) compared with 3.0 (0.1) and 3.1 (0.3), respectively (P=0.02)]. Xenon, but not sevoflurane, inhibited migration in both oestrogen receptor positive and negative breast adenocarcinoma cells. Furthermore, xenon decreased release of the pro-angiogenic factor RANTES from MDA-MB-231 cells.

MiR-886-5p Inhibition Inhibits Growth and Induces Apoptosis of MCF7 Cells

To explore the molecular mechanisms of miR-886-5p in breast cancer., we examined roles in inhibiting growth and migration of MCF-7 cells. MiR-886-5p mimics and inhibitors were used to express or inhibit MiR-886-5p, respectively, and MTT and clone formation assays were used to determine the survival and proliferation. Hoechst 33342/ PI double staining was applied to detect apoptosis. The expression of caspase-3, caspase-8, caspase-9, MT1-MMP, VEGF-C and VEGF-D was detected by Western blotting, and the levels of MMP2 and MMP9 secreted from MCF-7 cells were assessed by ELISA. MCF-7 cell migration was determined by wound healing and Transwell assays. We found that the growth of MCF-7 cells was inhibited upon decreasing miR-886-5p levels. Inhibiting miR-866-5p also significantly induced apoptosis and decreased the migratory capacity of these cells. The expression of VEGF-C, VEGF-D, MT1-MMP, MMP2, and MMP9 was also found to be decreased as compared to controls. Our data show that downregulation of miR-886-5p expression in MCF-7 cells could significantly inhibit cell growth and migration. This might imply that inhibiting miR-886-5p could be a therapeutic strategy in breast cancer.

High Glucose Level Promotes Migration Behavior of Breast Cancer Cells through Zinc and Its Transporters

BackgroundThe diabetes patients have been associated with an increased risk of mortality by breast cancer and there are difference between the breast cancer patients with diabetes, and their nondiabetic counterparts in the regimen choice and effects of breast cancer treatment. However, the pathophysiological relationships of diabetes and breast cancer have not yet been elucidated in detail. In this study, we investigate the breast cancer cell line, MCF-7 motility, which linked to invasion and metastasis, in high glucose level corresponding to hyperglycemia and the role of Zn and its transporter.Methodology/Principal findingsWe demonstrated the significant motility of MCF-7 cultured in hyperglycemic level (25 mM glucose) in comparison to normal physiological glucose level (5.5 mM glucose). The other hand, the osmotic control medium, 5.5 mM glucose with 19.5 mM mannitol or fructose had no effect on migratory, suggesting that high glucose level promotes the migration of MCF-7. Moreover, the activity of intracellular Zn2+ uptake significantly increased in high glucose-treated cells in comparison to 5.5 mM glucose, and the mRNA expression of zinc transporters, ZIP6 and ZIP10, was upregulated in 25 mM glucose-treated cells. The deficiency of ZIP6 or ZIP10 and intracellular Zn2+ significantly inhibited the high migration activity in 25 mM glucose medium, indicating that Zn2+ transported via ZIP6 and ZIP10 play an essential role in the promotion of cell motility by high glucose stimulation.Conclusion/SignificanceZinc and its transporters, ZIP6 and ZIP10, are required for the motility stimulated with high glucose level. These findings provide the first evidence proposing the novel strategies for the diagnosis and therapy of breast cancer with hyperglycemia.

Evaluating the Potential Bioactivity of a Novel Compound ER1626

BackgroundER1626, a novel compound, is a derivate of indeno-isoquinoline ketone. This study was designed to evaluate the biological activity and potential anti-tumor mechanism of ER1626.MethodMTT assay, scratch assay and flow cytometry were used to determine cell proliferation, cell migration and cell cycle distribution as well as cell apoptosis on human breast cancer MCF-7 cells and endometrial cancer Ishikawa cells. We also explored the antiangiogenic effect of ER1626 on HUVEC cells and chicken embryos. The expression of estrogen receptor protein was investigated with western-blot analysis.ResultsER1626 down-regulated the expression of estrogen receptor α protein and up-regulated β protein in MCF-7 and Ishikawa cells. The value of IC50 of ER1626 on MCF-7 and Ishikawa cells were respectively 8.52 and 3.08 µmol/L. Meanwhile, ER1626 decreased VEGF secretion of MCF-7 and Ishikawa cells, disturbed the formation of VEGF-stimulated tubular structure in HUVEC cells, and inhibited the angiogenesis on the chicken chorioallantoic membrane. Scratch assay revealed that ER1626 suppressed the migration of MCF-7, Ishikawa and HUVEC cells. In addition to induction tumor cell apoptosis, ER1626 arrested cell cycle in G1/G0 phase in MCF-7 cells and G2/M phase in Ishikawa cells.ConclusionIn conclusion, our results demonstrated that ER1626 has favorable bioactivities to be a potential candidate against breast cancer and angiogenesis.

CRH suppressed TGFβ1-induced Epithelial–Mesenchymal Transition via induction of E-cadherin in breast cancer cells

Since its discovery in biopsies from breast cancer patients, the effect of corticotropin-releasing hormone (CRH) on carcinoma progression is still unclear. Transforming growth factorβ1 (TGFβ1) promotes Epithelial–Mesenchymal Transition (EMT) and induces Snail1 and Twist1 expressions. Loss of epithelial cadherin (E-cadherin) mainly repressed by Snail1 and Twist1, has been considered as hallmark of Epithelial–Mesenchymal Transition (EMT). Two breast cancer cell lines, MCF-7 and MDA-MB-231 were used to investigate the effect of CRH on TGFβ1-induced EMT by transwell chamber. And HEK293 cells were transiently transfected with CRHR1 or CRHR2 to explore the definite effects of CRH receptor. We reported that CRH inhibited migration of human breast cancer cells through downregulation of Snail1 and Twist1, and subsequent upregulation of E-cadherin. CRH inhibited TGFβ1-mediated migration of MCF-7 via both CRHR1 and CRHR2 while this inhibition in MDA-MB-231 was mainly via CRHR2. Ectopic re-expression of CRHR1 or CRHR2 respectively in HEK293 cells increased E-cadherin expression after CRH stimulation. Furthermore, CRH repressed expression of mesenchymal marker, N-cadherin and induced expression of Occludin, inhibiting EMT in MCF-7 & MDA-MB-231. Our results suggest that CRH may function as a tumor suppressor, at least partly by regulating TGFβ1-mediated EMT. These results may contribute to uncovering the effect of CRH in breast tumorigenesis and progression.

MiR-26b is down-regulated in carcinoma-associated fibroblasts from ER-positive breast cancers leading to enhanced cell migration and invasion

Carcinoma-associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor-positive cancers, and explore the influences of one of these, miR-26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR-26b was the most highly deregulated microRNA. Using qPCR, miR-26b was confirmed as down-regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR-26b expression changed breast fibroblast behaviour. Reduced miR-26b expression caused fibroblast migration and invasion to increase by up to three-fold in scratch-closure and trans-well assays. Furthermore, in co-culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR-26b expression enhanced both MCF7 migration in trans-well assays and MCF7 invasion from three-dimensional spheroids by up to five-fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR-26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR-26b. In addition, three novel miR-26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR-26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor-positive cancers, and we have identified key genes and molecular pathways that act downstream of miR-26b in CAFs. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Observations on the effects of Suppressor of Cytokine Signaling 7 (SOCS7) knockdown in breast cancer cells: their in vitro response to Insulin Like Growth Factor I (IGF-I)

Suppressor of cytokine signaling 7 (SOCS7) is a member of the SOCS family and is known to interact with phospholipase Cγ-1 (PLCγ-1), one of the insulin-like growth factor-I (IGF-I) receptor downstream molecules. In this study, we sought to observe the effect of knocking down SOCS7 gene on breast cancer cells in vitro growth and migration and to elucidate whether this involves IGF-I-PLCγ1 route using the PLCγ-1 blocker U73122. Suitable breast cancer cells (MCF7 and MDA-MB-231) were transfected with anti-SOCS7 ribozymal transgene, to create sub-lines with SOCS7 knockdown verified by RT-PCR. The growth and migration of the cells were evaluated in the presence or absence of IGF-I and PLCγ-1 inhibitor using growth assay, scratch-wound and electrical cell impedance sensing (ECIS) migration assays. IGF-I treatment produced more pronounced influence on MCF7 growth and migration and on MDA-MB-231 migration when SOCS7 gene was knocked down in both lines (p < 0.05). The absence of IGF-I-induced growth response in MDA-MB-231 could be due to the intrinsic characteristics of these cells. PLCγ-1 pharmacological inhibition during their in vitro migration seemed to only occur when SOCS7 gene was knocked down. To the best of our knowledge, this is the first report of the SOCS7 regulatory role in IGF-I induced in vitro functions in ER-positive and ER-negative breast cancer cells. IGF-I treatment and SOCS7 loss have synergistically resulted in increased growth and migration of MCF7 and in increased migration of MDA-MB-231 cells. The migratory effects could be due to a precise anti-PLCγ-1 role.

Cytokines in osteoblast-conditioned medium promote the migration of breast cancer cells

Bone is one of the most common metastatic sites for breast cancer. In this study, we observed a promoting effect of osteoblast-conditioned medium (OCM) on the migration of MCF-7, a noninvasive cell line of breast cancer cells. Cytokine antibody array was used to compare the cytokines of OCM with the conditioned medium of non-differentiated osteoblast cells, which consequently revealed factors related to migration, such as IL8, IL6, CSF2 (G-CSF), CSF3 (GM-CSF), and TNFRSF11B (osteoprotegerin). The expression of genes related to migration was also estimated with a PCR array, which showed that 9 genes were upregulated and 26 genes downregulated. Moreover, activated p38, ERK, and AKT pathways were found in the OCM treatment group. This finding indicated the migration ability of breast cancer cells, which move toward the bone depending on the presence of specific cytokines in its surrounding microenvironment.

Estrogen receptor β inhibits estradiol-induced proliferation and migration of MCF-7 cells through regulation of mitofusin 2

In the present study, we investigated whether estrogen receptor (ER) β affected the proliferation and migration of the human breast cancer cell line MCF-7 through regulation of mitofusin 2 (mfn2). A previous study reported that mfn2 may be regulated by ER through a non-classical pathway; in this pathway, the ER modulates the activities of other transcription factors by stabilizing their binding to DNA and/or recruiting coactivators to the complex. However, the previous study, unlike the study presented here, did not directly explore the interactions between ER and mfn2. Here, RT-PCR and western blot analysis were used to test the expression of mfn2 in MCF-7 cells after exposure to different doses of estradiol (E2). The ability of cells to proliferate and migrate was determined by MTT assay and a monolayer-wounding protocol, respectively. Finally, changes in MCF-7 cell biology after transfection with ERβ or mfn2 expression vectors were investigated, and the role of ERβ in mfn2 expression was also explored. Our results showed that E2 attenuated mfn2 expression in a dose-dependent manner, concomitant with the activation of proliferation and migration of MCF-7 cells. The mfn2 expression vector effectively suppressed E2-induced upregulation of PCNA and migration in MCF-7 cells. ERβ inhibited the E2-induced mfn2 downregulation that accompanied the inhibition of proliferation and migration in MCF-7 cells. Briefly, ERβ may inhibit E2-induced proliferation and migration of MCF-7 cells through regulation of mfn2.

Ani-survivin DNAzymes Inhibit Cell Proliferation and Migration in Breast Cancer Cell Line MCF-7

Survivin, a new member of the inhibitor of apoptosis protein (IAP) family, both inhibits apoptosis and regulates the cell cycle. It is overexpressed in breast tumor tissues. In this study, we designed two survivin specific DNAzymes (DRz1 and DRz2) targeting survivin mRNA. The results showed that DRz1 could decrease the expression of survivin by nearly 60%. Furthermore, DRz1 significantly inhibited cell proliferation, induced apoptosis and inhibited migration in MCF-7 cells. In addition, down-regulation of survivin expression was associated with increased caspase-3 and -9 activities in MCF-7 cells after 24 h transfection. In our experiments, the efficacy of DRz1 to influence survivin levels and associated effects were better than DRz2. Survivin-DRz1 might have anti-tumorigenic activity and may potentially provide the basis for a novel therapeutic intervention in breast cancer treatment.

Abstract P1-04-03: Knocking down Suppressor of Cytokine Signaling 7 in breast cancer: The role in Insulin-like Growth Factor - I/Phospholipase Cγ-1 signaling

Abstract BACKGROUND: Suppressor of Cytokine Signaling 7 (SOCS7) is a member of the SOCS family and is known to interact with Phospholipase Cγ-1 (PLCγ-1), one of the Insulin like Growth Factor - I (IGF-I) receptor downstream molecules. In the present study, we sought to examine the effect of knocking down SOCS7 gene on breast cancer cells growth and migration and to elucidate whether this has involved IGF-I — PLCγ-1 signaling using the PLCγ-1 blocker U73122. METHODS: Suitable breast cancer cells (MCF7 and MDA-MB-231) were transfected with anti-SOCS7 ribozymal transgene, to create sublines with SOCS7 knockdown that was verified by RT-PCR. The growth and migration of the cells were evaluated in the presence or absence of IGF-I and PLCγ-1 inhibitor using in vitro growth assay and Electrical Cell Impedance Sensing (ECIS) migration assay. RESULTS: MCF7ΔSOCS7 and MDA-MB-231ΔSOCS7 (SOCS7 knockdown) were constructed. Both sublines showed a higher rate of growth compared to control cells with and without IGF-I stimulation. U73122 treatment did not appear to change this growth outcome. Using ECIS migration assay, it was shown that knocking down SOCS7 had a significant positive effect on the migration of MCF7 and MDA-MB-231 cells, and that both IGF-I treatment and SOCS7 knockdown had a synergistic positive influence on their migration (p &amp;lt; 0.05). We further demonstrated that the impact of U73122 on the IGF-I migratory effect was dependent upon SOCS7 knockdown as it has significantly blocked the stimulatory effect of IGF-I on MCF7ΔSOCS7 and MDA-MB-231ΔSOCS7 migration but not that of the control cells. While SOCS7 has acted to control the IGF-I effect, it appeared to cancel the inhibitory function of U73122, indicating a specific anti - PLCγ-1 role for SOCS7 in IGF-I induced breast cancer cellular migration. CONCLUSION: SOCS7 loss resulted in increased growth and migration of breast cancer cells and this had a synergistic effect on their response to IGF-I. This role could be related to its interaction with PLCγ-1 during the cellular migration but not the growth. It may be possible that SOCS7 acts through different mechanism to control the cellular growth. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-03.

Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells

Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines. Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post hoc tests were performed. Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs. We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.

Hypoxia can induce EMT and promote invasion of breast cancer cells

Objective To study the effect of hypoxia on EMT molecule E-cadherin, Vimentin and invasion of human breast cancer MCF-7 cells, reveal the mechanism of breast cancer invasion and metastasis and provide experimental and theoretical basis. Methods Western blot was used to observe the change of HIF-1α, E-cadherin and Vimeutin during hypoxia on MCF-7. MTT was used to study the effects of viability.Transwell chamber was used to detect the ability of invasion and metastasis. Results The expression of E-cadherin was significantly lower (0.09±0.02)(t=30.98,P=0.0007) and the expression of Vimentin was significantly higher (0.69±0.04) (t=915,P=0.0000) with the extension of hypoxia.The capability of adhesion (81.23±0.74) (t=82.05,P=0.000),invasion(120±6) (t=22.78,P=0.0009) and migration(190±6) (t=23.49,P=0.000)was significantly increased after 72 h hypoxia(P＜0.05).Conclusion Hypoxia can downregulate E-cadherin and upregulate Vimentin and enhance the adhesion,invasion and migration of MCF-7. Key words: Breast neoplasms; Anoxia; Epithelium; Stromalcells; Neoplasm invasiveness; Cel migration

Breast cancer associated fibroblasts promote MCF-7 invasion in vitro by secretion of HGF

This study was aimed to explore the influence of breast cancer associated fibroblasts (CAFs) in migration and invasion of breast cancer cell line MCF-7, and investigate whether hepatocyte growth factor (HGF) is involved in this process. Primary breast CAFs and their corresponding normal breast fibroblasts (NFs) were obtained by collagenase digestion. On the basis of the co-culture, the migration and invasion capacity of MCF-7 cells was compared between CAFs and NFs by Transwell. The difference in the HGF expression between them was detected by ELISA. The secretion of HGF was knocked down by using RNA interference technology in CAFs. Then the changes of migration and invasion capacity of MCF-7 cells were investigated by Transwell. Eventually, we isolated high-purity CAFs and NFs, and the CAFs had a stronger ability in promoting MCF-7 migration and invasion than the NFs. ELISA results demonstrated that CAFs secreted higher HGF, and the capacity of MCF-7 migration and invasion was declined after knocking down the secretion of HGF in CAFs by RNA interference. It is suggested that CAFs can promote MCF-7 migration and invasion through HGF in vitro.

In vitro anti-metastatic activity of enterolactone, a mammalian lignan derived from flax lignan, and down-regulation of matrix metalloproteinases in MCF-7 and MDA MB 231 cell lines

Actin cytoskeleton is involved in actin-based cell adhesion, cell motility, and matrix metalloproteinases(MMPs) MMP2, MMP9, MMP11 and MMP14 are responsible for cell invasion in breast cancer metastasis. The dietary intake of lignan from flax seed gets converted to enterolactone (EL) and enterodiol in the human system. Here we show that the enterolactone has a very significant anti-metastatic activity as demonstrated by its ability to inhibit adhesion and invasion and migration in MCF-7 and MDA MB231 cell lines. Migration inhibition assay, actin-based cell motility assay along with reverse transcriptase polymerase chain reaction (RT-PCR) for MMP2, MMP9, MMP11 and MMP14 genes were performed in MCF-7 and MDA MB 231 cell lines. Enterolactone seems to inhibit actin-based cell motility as evidenced by confocal imaging and photo documentation of cell migration assay. The results are supported by the observation that the enterolactone in vitro significantly down-regulates the metastasis-related metalloproteinases MMP2, MMP9 and MMP14 gene expressions. No significant alteration in the MMP11 gene expression was found. Therefore we suggest that the anti-metastatic activity of EL is attributed to its ability to inhibit cell adhesion, cell invasion and cell motility. EL affects normal filopodia and lamellipodia structures, polymerization of actin filaments at their leading edges and thereby inhibits actin-based cell adhesion and cell motility. The process involves multiple force-generating mechanisms of actin filaments i.e. protrusion, traction, deadhesion and tail-retraction. By down-regulating the metastasis-related MMP2, MMP9 and MMP14 gene expressions, EL may be responsible for cell invasion step of metastasis.

Comparison of Anti‐Invasive Activity of Parthenolide and 3‐Isopropyl‐2‐Methyl‐4‐Methyleneisoxazolidin‐5‐One (MZ‐6) – A New Compound with α‐Methylene‐γ‐Lactone Motif – on Two Breast Cancer Cell Lines

The biological activities of parthenolide, a sesquiterpene lactone isolated from feverfew, have been attributed to the presence of the α-methylene-γ-lactone skeleton. The lactone skeleton can react via the Michael type addition with sulfhydryl groups of enzymes and other functional proteins, interfering with key biological processes in the cell. In the present study, we describe an efficient method of preparation of 3-isopropyl-2-methyl-4-methyleneisoxazolidin-5-one (MZ-6), a synthetic compound with α-methylene-γ-lactone ring, as in parthenolide, additionally modified by introduction of a nitrogen atom. Furthermore, we investigated the cytotoxic activity and anti-metastatic potential of MZ-6 in comparison with parthenolide. Both compounds showed considerable cytotoxicity against breast cancer MCF-7 and MDA-MB-231 adenocarcinoma cells in vitro and were then evaluated for their anti-metastatic potential. The experimental results showed that MZ-6 and parthenolide suppressed, to a similar degree, migration of MCF-7, but not more aggressive MDA-MB-231 cells. In both cell lines, tested compounds down-regulated mRNA and protein levels of metalloproteinase-9 and urokinase plasminogen activator, the key proteases involved in the degradation of extracellular matrix and dissemination of cancer cells. The obtained results indicate that simple analogs of α-methylene-γ-lactones can be good substitutes for more complex structures isolated from plants.

Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivotumor growth

IntroductionMicroRNAs (miRNAs) are a class of small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression. A key oncomir in carcinogenesis is miR-21, which is consistently up-regulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and tissues, and searched for miR-21 targets.MethodsWe used in vitro and in vivo assays to explore the role of miR-21 in the malignant progression of human breast cancer, using miR-21 knockdown. Using LNA silencing combined to microarray technology and target prediction, we screened for potential targets of miR-21 and validated direct targets by using luciferase reporter assay and Western blot. Two candidate target genes (EIF4A2 and ANKRD46) were selected for analysis of correlation with clinicopathological characteristics and prognosis using immunohistochemistry on cancer tissue microrrays.ResultsAnti-miR-21 inhibited growth and migration of MCF-7 and MDA-MB-231 cells in vitro, and tumor growth in nude mice. Knockdown of miR-21 significantly increased the expression of ANKRD46 at both mRNA and protein levels. Luciferase assays using a reporter carrying a putative target site in the 3' untranslated region of ANKRD46 revealed that miR-21 directly targeted ANKRD46. miR-21 and EIF4A2 protein were inversely expressed in breast cancers (rs = -0.283, P = 0.005, Spearman's correlation analysis).ConclusionsKnockdown of miR-21 in MCF-7 and MDA-MB-231 cells inhibits in vitro and in vivo growth as well as in vitro migration. ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that inhibitory strategies against miR-21 using peptide nucleic acids (PNAs)-antimiR-21 may provide potential therapeutic applications in breast cancer treatment.

High Expression of Transient Receptor Potential Channels in Human Breast Cancer Epithelial Cells and Tissues: Correlation with Pathological Parameters

Background: Transient Receptor Potential (TRP) channels are expressed in many solid tumors. However, their expression in breast cancer remains largely unknown. Here, we investigated the profile expression of 13 TRP channels in human breast ductal adenocarcinoma (hBDA) and performed a correlation between their overexpression and pathological parameters. Methods: The TRP channels expression was determined by RT-PCR in hBDA tissue, in human breast cancer epithelial (hBCE) primary culture and in MCF-7 cell line. The TRP protein level was evaluated by immunohistochemistry in hBDA tissue samples of 59 patients. Results: TRPC1, TRPC6, TRPM7, TRPM8, and TRPV6 channels were overexpressed in hBDA compared to the adjacent non-tumoral tissue. Most interestingly, TRPC1, TRPM7 and TRPM8 expression strongly correlated with proliferative parameters (SBR grade, Ki67 proliferation index, and tumor size), and TRPV6 was mainly overexpressed in the invasive breast cancer cells. Using laser capture microdissection, we found that TRPV6 expression was higher in invasive areas, compared to the corresponding non-invasive ones. Moreover, TRPV6 silencing inhibited MDA-MB-231 migration and invasion, and MCF-7 migration. Conclusion: TRP channels are aberrantly expressed in hBDA, hBCE primary cultures, and cell lines, and associated with pathological parameters. The high expression of TRP channels in tumors suggests the potential of these channels for diagnostic, prognosis and/or therapeutic approaches in human breast ductal adenocarcinoma.

Abstract 565: Effects of zoledronic acid on the interaction between mesenchymal stem cells and breast cancer cells

Abstract Zoledronic acid (ZA) is the mainstay of treatment of bone metastases associated with a wide variety of tumors, including breast cancer. It has been recently shown that ZA increases the progression free-survival of estrogen receptor (ER)-positive breast cancer patients by reducing both loco-regional and distant metastases, suggesting that this compound might act directly on breast cancer cells. However, ZA very rapidly concentrates in the bone following intravenous administration, and therefore this drug is unlikely to have a direct effect on cell populations outside the bone. Recent reports have shown that bone-marrow-derived mesenchymal stem cell (MSCs) are recruited to the stroma of developing tumors where they increase the metastatic potential of breast cancer cells by secreting cytokines, chemokines and growth factors that sustain breast cancer migration, invasion and metastasis. In particular, the chemokine CCL5 (RANTES) plays an important role in this phenomenon. Therefore, we investigated the effects of ZA on the ability of primary MSCs to secrete cytokines and growth factors that might be involved in breast cancer progression. We found that treatment with ZA produced marginal effects on the growth of human primary MSCs with an approximately 25% growth inhibition at 20 μM. In contrast, treatment with similar concentrations of ZA almost completely suppressed the ability of MSCs to secrete RANTES (&amp;gt;90% reduction). The effect of ZA on RANTES was quite specific, since marginal inhibition of the secretion of different angiogenic growth factors, such as VEGF, IL-8 and bFGF was observed. Treatment of MSCs with ZA also produced a 50% reduction in the levels of secretion of IL-6 that has been previously shown to act as a potent paracrine growth factor for human breast cancer cells. Both RANTES and IL-6 transcripts were significantly reduced in MSCs following exposure to ZA, suggesting a transcriptional regulation of these cytokines by ZA. Conditioned medium from ZA-treated MSCs showed a reduced ability to promote the migration of ER-positive MCF-7 breast cancer cells through a fibronectin-coated membrane as compared with conditioned medium from untreated cells. In addition, antibodies anti-RANTES and anti-IL-6, alone or in combination, produced an approximately 50% reduction of the migration of MCF-7 induced by conditioned medium from untreated MSCs. In co-culture assays, treatment with ZA reduced the ability of MSCs to sustain the growth of breast cancer cells. Finally, the migration of MSCs was reduced by approximately 45% following treatment with ZA. Taken together, these data suggest that ZA might exert its antitumor activity in the bone marrow microenvironment by inhibiting the migration of MSCs and by blocking the ability of MSCs to secrete factors involved in breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 565.