Migration Of MCF-7 Cells Research Articles

Tricresyl phosphate isomers exert estrogenic effects via G protein-coupled estrogen receptor-mediated pathways

Tricresyl phosphates (TCPs), as representative aromatic organophosphate flame retardants (OPFRs), have received much attention due to their potential neurotoxicity and endocrine-disrupting effects. However, the role of estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) in their estrogen disrupting effects remains poorly understood. Therefore, in this study, three TCP isomers, tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) and tri-p-cresyl phosphate (TpCP), were examined for their activities on ERα by using two-hybrid yeast assay, and action on GPER by using Boyden chamber assay, cAMP production assay, calcium mobilization assay and molecular docking analysis. The results showed that three TCP isomers were found to act as ERα antagonists. Conversely, they had agonistic activity on GPER to promote GPER-mediated cell migration of MCF7 cells and SKBR3 cells. Both ToCP and TpCP activated GPER-mediated cAMP production and calcium mobilization, whereas TmCP had different mode of action, it only triggered GPER-mediated calcium mobilization, as evidenced by using the specific GPER inhibitor (G15) and GPER overexpressing experiments. Molecular docking further revealed that the way of interaction of TmCP and TpCP with GPER was different from that of ToCP with GPER, and higher activity of ToCP in activating GPER-mediated pathways might be associated with the alkyl substitution at the ortho position of the aromatic ring. Our results, for the first time, found a new target, GPER, for TCPs exerting their estrogen-disrupting effects, and demonstrated complex estrogen-disrupting effects of three TCP isomers involved their opposite activities toward ERα and GPER.

Natural killer cell-derived exosome-entrapped paclitaxel can enhance its anti-tumor effect.

To study the effectiveness of natural killer cell-derived exosome (NK-Exos)-entrapped paclitaxel (PTX-NK-Exos) in enhancing its anti-tumor effect. The NK-Exos were isolated through ultra-high-speed centrifugation, and the PTX-NK-Exos system was constructed via electroporation. The morphology, particle size, Zeta potential and entrapment rate of PTX-NK-Exos were evaluated using transmission electron microscope (TEM), dynamic light scattering (DLS), Western blotting and high-performance liquid chromatography (HPLC), respectively. The uptake of Exos in human breast cancer MCF-7 cells was observed under a laser confocal microscope. Moreover, the effect of PTX-NK-Exos on MCF-7 cell viability was determined through methyl thiazolyl tetrazolium (MTT) assay, flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) staining. The effects of PTX-NK-Exos on messenger ribonucleic acid (mRNA) and protein expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and Caspase-3 in MCF-7 cells were detected using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The NK-Exos were successfully isolated via ultra-high-speed centrifugation, and they had uniform particle size and high expression of markers for Exos. MCF-7 cells could take up Exos. The PTX-NK-Exos drug delivery system was successfully prepared using electroporation. In PTX group and NK-Exos group, the proliferation of MCF-7 cells declined, the nuclear apoptosis was evident and the apoptosis rate of MCF-7 cells rose compared with those in Control group. In PTX group and PTX-NK-Exos group, the migration of MCF-7 cells declined compared with that in Control group. According to the results of qRT-PCR and Western blotting, PTX-NK-Exos exerted an anti-tumor effect through inducing the up-regulation of Bax and Caspase-3 in the apoptotic signaling pathway in tumor cells. Exos isolated through ultra-high-speed centrifugation can be used to prepare the PTX-NK-Exos drug delivery system through electroporation. Drug-loaded Exos can effectively inhibit proliferation and induce apoptosis of tumor cells, thereby exerting an anti-tumor effect.

Effects of obatoclax combined with gemcitabine on breast cancer cells under hypoxia condition

Objective: To explore the effects of obatoclax(OBX) combined with gemcitabine(GEM) on breast cancer cells MCF-7 and BT-20 cell activity, migration, invasion and apoptosis under hypoxia condition.Methods: Breast cancer cells MCF-7 and BT-20 were divided into normal group, hypoxia group, GEM group, OBX+GEM group. Normal group: Cells were cultured at 37℃, 5% CO2 for 24 h and 48 h; Hypoxia group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2 for 24 h and 48 h; GEM group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2, adding 10 μmol/L GEM for 24 h and 48 h; OBX + GEM group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2, adding 10 μmol/L GEM and 50 nmol/L OBX for 24 h and 48 h. Western blot method was used to detect the expressions of HIF-1α in MCF-7 and BT-20 cells under normal oxygen and hypoxia condition. CCK-8 method was used to detect cancer cell activity, each group was provided with 15 compound holes. Scratch experiment was used to detect cells migration ability, each group was provided with 6 compound holes. Western blot method was used to detect the expressions of vimentin, E-Cadherin and p53 protein in cells of each group. Results: Under hypoxia condition, the expression of HIF-1α in MCF-7 and BT-20 cells was much higher than that under normal oxygen(P＜0.05). Compared with hypoxia group, GEM could reduce MCF-7 and BT-20 cells migration ability(P＜0.01)and cell activity(P＜0.05), while decrease the expression of vimentin protein(P＜0.01)and promote the expressions of E-Cadherin (P＜0.01)and p53 protein(P＜0.01) in tumor cells under hypoxia condition. In OBX combined with GEM group, the cell activity and the migration ability of MCF-7 and BT-20 were reduced significantly(P＜0.01). The expression of vimentin in cells was further reduced(P＜0.01). The expressions of E-Cadherin(P＜0.01)and p53(P＜0.01) protein were increased significantly compared with GEM group. Conclusion: Under hypoxia condition, OBX combined with a low-dose of GEM can significantly inhibit the growth, migration and invasion of breast cancer cells, and enhance the pro-apoptotic effect of GEM, but the specific mechanism needs further study.

DNASE1L2, as a Carcinogenic Marker, Affects the Phenotype of Breast Cancer Cells Via Regulating Epithelial-Mesenchymal Transition Process.

Purpose: The authors explore the role of DNASE1L2 in breast cancer (BC) and its affect on the cell phenotype. Methods: Breast invasive ductal carcinoma RNA-Seq data set was downloaded from The Cancer Genome Atlas database for analyzing DNASE1L2 levels. Overall survival curve was plotted by Kaplan-Meier methods. The correlations between DNASE1L2 expression and clinical characteristics were analyzed by chi-square tests. Cox regression models were implemented for analyzing the potential prognosticators of BC. Small interference RNA-DNASE1L2 and pcDNA3.1-DNASE1L2 were transfected into BC cells to silence and overexpress DNASE1L2, respectively. Relative mRNA and protein levels were determined by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. Cell counting Kit-8, clone formation, and Transwell assays were employed to measure the proliferative, invasive, and migratory abilities. Results: Bioinformatics analysis showed that the levels of DNASE1L2 were found to be elevated in BC tissues, which was further proved by qRT-PCR tests. Besides, high expression of DNASE1L2 was dramatically led to a poor overall survival. Furthermore, DNASE1L2 expression was remarkably associated with age and pathologic-stage. Silencing DNASE1L2 showed an inhibitory effect on the proliferation, invasion, and migration of MCF7 cells, whereas overexpression of DNASE1L2 in BT549 cells presented the opposite results. Mechanistically, downregulation of DNASE1L2 could significantly enhance the levels of E-cadherin, as well as suppress the levels of Vimentin, N-cadherin and Snail, whereas upregulation of DNASE1L2 showed the reverse outcomes. Conclusion: This study for the first time demonstrated that DNASE1L2 was upregulated in BC cells, and acted as an oncogene to affect the phenotype of BC cells by modulating the epithelial-mesenchymal transition process, which suggested that DNASE1L2 might be considered as a useful biomarker for BC therapeutics.

Design, Synthesis, and Biological Evaluation of Proteolysis Targeting Chimeras (PROTACs) for the Dual Degradation of IGF-1R and Src.

A focused PROTAC library was developed to degrade both IGF-1R and Src proteins, which are associated with various cancers. PROTACs with IGF-1R and Src degradation potentials were synthesized by tethering different inhibitor warhead units and the E3 ligase (CRBN) recruiting-pomalidomide with various linkers. The designed PROTACs 12a–b inhibited the proliferation and migration of MCF7 and A549 cancer cells with low micromolar potency (1–5 μM) in various cellular assays.

Apolipoprotein-mediated regulation of lipid metabolism induces distinctive effects in different types of breast cancer cells

BackgroundThe highest incidence of breast cancer is in the Western world. Several aspects of the Western lifestyle are known risk factors for breast cancer. In particular, previous studies have shown that cholesterol levels can play an important role in the regulation of tumor progression.MethodsIn the present study, we modulated cholesterol metabolism in the human breast cancer cell lines MCF-7 and MDA-MB-231 using a genetic approach. Apolipoprotein A-I (apoA-I) and apolipoprotein E (apoE) were expressed in these cell lines to modulate cholesterol metabolism. The effects of these apolipoproteins on cancer cell properties were examined.ResultsOur results show that both apolipoproteins can regulate cholesterol metabolism and can control the epithelial-to-mesenchymal transition process. However, these effects were different depending on the cell type. We show that expressing apoA-I or apoE stimulates proliferation, migration, and tumor growth of MCF-7 cells. However, apoA-I or apoE reduces proliferation and migration of MDA-MB-231 cells.ConclusionsThese data suggest that modulating sterol metabolism may be most effective at limiting tumor progression in models of triple-negative cancers.

Corticotropin Releasing Factor Receptors in breast cancer: Expression and activity in hormone-dependent growth in vitro

Corticotropin Releasing Factor (CRF) neuropeptides coordinate the stress response via two distinct membrane receptors (CRF-Rs). We have previously shown expression of both CRF-Rs in human breast cancer tissues. In the present study, we examined in vitro using the MCF-7 cell line model, the regulation of CRF-Rs expression and their signaling in hormone-dependent breast cancer growth. Our findings show that similarly to breast cancer biopsies, the predominant receptor type expressed in the cell line is CRF-R2α. The transcription of CRF-R1 and CRF-R2 is up and down-regulated respectively by exposure to estradiol (E2); however this effect seems not to be exerted at the level of promoter gene methylation, although in human breast cancer specimens, CRF-R1 methylation was found to be positively associated with the presence of steroid hormone receptors. Finally, we showed that specific activation of CRF-R2 increased the migration of MCF-7 cells and potentiated an estrogen-inducing effect. Our data support an involvement of CRF-R signaling in breast cancer pathophysiology via a regulatory steroid-hormone interplay.

Extracts of edible, medicinal Thai plants inhibit the human breast cancer cells

Purpose: To evaluate the effects of ten edible, medicinal Thai plant extracts on MCF-7 cell viability and cell migration, as well as their mechanism(s) of action.
 Methods: Ethanolic plant extracts of ten edible, medicinal plants were tested for their cytotoxicity against MCF-7 cells using sulforhodamine B (SRB). To investigate the cytotoxic mechanism(s) of action of these extracts, the study was examined gene expression and protein expression by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Cell migration was studied by wound healing assay.
 Results: Four of the ten test extracts were potently cytotoxic, Careya sphaerica (CS), Azadirachta indica (AI), Piper nigrum (PN) and Oroxylum indicum (OI) with half maximal inhibitory concentrations (IC50) less than 100 μg/mL. All four extracts stimulated ROS overgeneration, increased caspase 3 activity and decreased growth-related gene expression including cdk2, cdk4, cdk6, cyclin D1 and cyclin E. Furthermore, the extracts significantly enhanced cyclin-dependent kinase inhibitor (CDKI) p21 levels and activated cancer cell death. The four extracts, CS, AI, PN and OI, also significantly reduced cancer cell migration, with PN being the most potent.
 Conclusion: Extract of the edible plants CS, AI, PN and OI have in vitro anticancer activity and are promising starting points for the development of breast cancer drugs.
 Keywords: Careya sphaerica (CS), Azadirachta indica (AI), Piper nigrum (PN), Oroxylum indicum (OI), Breast cancer, Cell death

Breast cancer-derived exosomes regulate cell invasion and metastasis in breast cancer via miR-146a to activate cancer associated fibroblasts in tumor microenvironment

ObjectiveTo explore the effects of breast cancer (BC)-derived exosomes on invasion and migration of BC cells. MethodsExosomes (Exo-MA, Exo-M7, Exo-M1) were extracted from normal breast epithelial cells (MCF-10A), BC cells (MCF-7/MDA-MB-231) and BC cells with miR-146a overexpression or knockdown using multi-step differential centrifugation. Morphologies and sizes of exosomes were observed by transmission electron microscope (TEM) and particle size analysis respectively. BC mouse models were injected with DIR labeled Exo-MA, Exo-M7 or Exo-M1. The epithelial-mesenchymal transition (EMT) in BC cells was determined by PCR and Western blot. PKH67 labeled Exo-MA, Exo-M7 and Exo-M1 were incubated with NFs or MCF-7 to measure the activation of CAFs. Cell invasion and migration abilities were determined by scratch test and Transwell assay. ResultsExo-MA, Exo-M7, Exo-M1 were successfully extracted with positive expressions of Alix, CD63 and TSG101. Contents of Ki67, N-cadherin, Vimentin and Snail-1 were increased but E-cadherin was decreased, compared to Exo-MA group. Exo-M7 or Exo-M1 could increase BC cell proliferation and enhance EMT in nude mouse. Exo-M7 and Exo-M1 could accelerate the transformation of NFs into CAFs and promote the recruitment of CAFs in MCF-7. Transfection of miR-146a could promote the transformation of NFs into CAFs and promote cell invasion and migration of MCF-7 cells. As a target gene of miR-146a, TXNIP could inhibit the activation of CAFs. miR-146a overexpression or TXNIP silence enhance the activation of Wnt signal pathway. ConclusionBC-derived exosomes promote the activation of CAFs through miR-146a/TXNIP axis to activate Wnt pathway, which in turn enhances invasion and metastasis of BC cells.

Diallyl Trisulfide Inhibits Leptin-induced Oncogenic Signaling in Human Breast Cancer Cells but Fails to Prevent Chemically-induced Luminal-type Cancer in Rats.

Previous studies have demonstrated inhibitory effect of garlic component diallyl trisulfide (DATS) on growth of breast cancer cells in vitro and in vivo. This study investigated the effect of DATS on oncogenic signaling regulated by leptin, which plays an important role in breast carcinogenesis. Leptin-induced phosphorylation and nuclear translocation of STAT3 was inhibited significantly in the presence of DATS in MCF-7 (a luminal-type human breast cancer cell line) and MDA-MB-231 (a basal-like human breast cancer cell line). Leptin-stimulated cell proliferation, clonogenic cell survival, and migration and/or invasion ability in MCF-7 and/or MDA-MB-231 cells were also suppressed by DATS treatment. DATS exposure resulted in inhibition of leptin-stimulated expression of protein and/or mRNA levels of Bcl-2, Bcl-xL, Cyclin D1, vascular endothelial growth factor, and matrix metalloproteinase-2. Western blotting revealed a decrease in protein levels of phosphorylated STAT3 in breast cancer xenografts from DATS-treated mice when compared to controls in vivo. However, the incidence of N-methyl-N-nitrosourea-induced luminal-type breast cancer development in rats was not affected by oral administration of 5 mg/kg or 25 mg/kg DATS. The present study reveals that oncogenic signaling induced by leptin is inhibited in the presence of DATS but higher doses of this phytochemical may be required to achieve chemopreventive activity.

Abstract P6-04-18: The regulatory role of activating transcription factor-2 (ATF2) in modulating tamoxifen resistance in estrogen-receptor positive breast cancer

Abstract Introduction: Activating transcription factor-2 (ATF2), a member of the ATF and CREB group of bZIP transcription factors regulates gene transcription by forming homodimers or heterodimers with other AP-1 family members. We have previously shown that ATF2 is important for modulating the effect of tamoxifen, whereas Thr71-ATF2 phosphorylation predicts for improved outcome of ER-positive breast cancers receiving tamoxifen. In this study we further explore the role of ATF2 in modulating breast cancer endocrine therapy resistance. Methods: ATF2 silencing: The MCF7, TamR, LCC2, LCC9 breast cancer cell lines were maintained in their appropriate growth media. Cells were transfected using RNAiMAX (ThermoFisher Scientific) with two different siRNAs and a Silencer® negative control. Gene expression was assessed by quantitative real-time PCR (RT-qPCR) (TaqMan assays, ThermoFisher Scientific) and the protein levels were analysed by western blotting. Cell growth, invasion, migration: Sulforhodamine B assay was used for cell growth determination following manufactures’ protocol. Soft-agar colony formation was performed by seeding and incubating the cells in low-melting agarose plates. The resulting colonies were 5% MTT-stained and counted in a Gel Count™ (Oxford Optronix). Invasion/migration was performed using the transwell matrigel-coated (BD Biosciences) plates and the scratch wound healing assay. All the assays were performed in triplicates in three independent experiments. Microarray profiling: Human HT12_V4 microarray (Illumina) was performed to identify genes that were differentially regulated by ATF-2 silencing in TamR cells compared to MCF-7. Data analyses including log2 transformation and quantile normalisation were performed on R Bioconductor. Differentially-expressed genes were selected using FDR correction for multiple testing with p-value cut-off <0.05. Results: ATF-2 silencing significantly inhibited the proliferation (p<0.005), colony formation (p<0.005) and migratory ability (p<0.01) of TamR, LCC2 and LCC9 cells but had a no effect on the growth and migration of MCF-7 cells. Moreover, differential gene expression analysis in MCF7 versus TamR cells revealed a shift from the ER-enriched transcription factor network towards an ATF2-enriched and TGF2β signalling pathway (p=0.025) in TamR cells suggesting alternative hormone-independent growth mechanisms. We then considered differentially-expressed genes specific to the TamR cells (not differentially-expressed in MCF7) and found a reintroduction of ER and its signalling pathway upon ATF2 knockdown along with a reduction in MYC transcriptional activity (EnrichR). RT-qPCR and western blot confirmed the increased expression of ER and its targets TFF1, GREB1, NCOA3, in TamR cells after ATF2 silencing in both the gene and protein levels. Conclusions: In conclusion, for the first time, ATF-2 was identified as a novel transcription factor that can modulate endocrine resistance by regulating ER expression and activity. This data suggests that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity. Citation Format: Athina Giannoudis, Mohammed I Malki, Hisham Mohammed, Bharath Rudraraju, Suraj Menon, Simak Ali, Jason S Carroll, Carlo Palmieri. The regulatory role of activating transcription factor-2 (ATF2) in modulating tamoxifen resistance in estrogen-receptor positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-18.

Inhibition of cell proliferation and migration by Oroxylum indicum extracts on breast cancer cells via Rac1 modulation

In this study, we investigated how Oroxylum indicum leaf and fruit extracts affect the viability and migration of MCF-7 breast cancer cells and the mechanisms of action responsible for these effects. MCF-7 cells treated with the extracts were examined using the sulforhodamine B, colony formation and caspase 3 activity assays, and by Western blotting. O. indicum extracts were found to inhibit MCF-7 cell growth in a concentration- and time-dependent manner, with 48 h IC50 values of 57.02 ± 2.85 μg/mL and 131.3 ± 19.2 μg/mL for leaf and fruit extracts, respectively. Further, the O. indicum leaf extract caused a reduction in MCF-7 cell viability, induction of MCF-7 cell apoptosis and ROS formation, and an increase in caspase 3 activity. Also, the two extracts inhibited MCF-7 cell migration and reduced both MMP 9 and ICAMP1 gene expression and MMP9 protein expression. Additionally, O. indicum extracts greatly reduced expression of the cell cycle regulatory protein Rac1 in the mevalonate pathway. In summary, O. indicum leaf and fruit extracts reduce breast cancer cell growth, cell viability and cell migration. O. indicum constituents could, therefore, be useful for augmenting the activity of chemotherapeutic drugs employed to treat breast cancer.

BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

BackgroundBRD7 is a tumor suppressor known to inhibit cell proliferation and cell cycle progression and initiate apoptosis in breast cancer. However, the function and underlying molecular events of BRD7 in tumor invasion and metastasis in breast cancer are not fully understood.MethodsBRD7 expression was assessed in two stable cell lines MDA231 and MCF7 with BRD7 overexpression and one stable cell line MDA231 with BRD7 interference using qRT-PCR and western blotting. CCK8 assay was used to examine the proliferation ability of MDA231 and MCF7 cells. Scratch wound healing assay was used to evaluate cell migration in MDA231 and MCF7 cells. Both Matrigel and three-dimensional invasion assays were performed to investigate the cell invasion ability after BRD7 overexpression or silencing or YB1 restoration in MDA231 and MCF7 cells. The potential interacting proteins of BRD7 were screened using co-immunoprecipitation combined with mass spectrometry and verified by co-immunoprecipitation in HEK293T cells. Additionally, we confirmed the specific binding region between BRD7 and YB1 in HEK293T cells by constructing a series of deletion mutants of BRD7 and YB1 respectively. Finally, xenograft and metastatic mouse models using MDA231 cells were established to confirm the effect of BRD7 on tumor growth and metastasis.ResultsHere, the results of a series of assays in vitro indicated that BRD7 has the ability to inhibit the mobility, migration and invasion of breast cancer cells. In addition, YB1 was identified as a novel interacting protein of BRD7, and BRD7 was found to associate with the C-terminus of YB1 via its N-terminus. BRD7 decreases the expression of YB1 through negatively regulating YB1 phosphorylation at Ser102, thereby promoting its proteasomal degradation. Furthermore, gene set enrichment analysis revealed that epithelial-mesenchymal transition (EMT) is the common change occurring with altered expression of either BRD7 or YB1 and that BRD7 represses mesenchymal genes and activates epithelial genes. Moreover, restoring the expression of YB1 antagonized the inhibitory effect of BRD7 on tumorigenicity, EMT, invasiveness and metastasis through a series of in vitro and in vivo experiments. Additionally, BRD7 expression was negatively correlated with the level of YB1 in breast cancer patients. The combination of low BRD7 and high YB1 expression was significantly associated with poor prognosis, distant metastasis and advanced TNM stage.ConclusionsCollectively, these findings uncover that BRD7 blocks tumor growth, migration and metastasis by negatively regulating YB1-induced EMT, providing new insights into the mechanism by which BRD7 contributes to the progression and metastasis of breast cancer.

MicroRNA-671-3p suppresses proliferation and invasion of breast cancer cells by targeting DEPTOR

To investigate the effects of miR-671-3p on the proliferation and invasion of breast cancer cells and explore the possible mechanism. We examined the expressions of miR-671-3p in human normal epithelial cells (MCF-10A) and breast cancer cell lines (MCF-7, MDA-MB-231, and SK-BR3) using RT-PCR. The effects of transfection with a miR-671-3p mimic or inhibitor on the proliferation, migration and invasion of MCF-7 cells were evaluated using CCK-8 assay and Transwell chamber assay. The target gene of miR-671-3p was predicated with Targetscan and validated by a dual luciferase reporter system and Western blotting. The expression of miR-671-3p was significantly lower in breast cancer cells than in normal breast epithelial cells. Compared with negative control group, MCF-7 cells with miR-671-3p overexpression exhibited significantly reduced proliferation and invasion, whereas inhibition of miR-671-3p obviously promoted the cell proliferation and invasion. Luciferase reporter assay demonstrated that DEPTOR was the target gene of miR-671-3p, and miR-671-3p overexpression caused significant down-regulation of the protein expression of DEPTOR. MiR-671-3p suppresses the proliferation and invasion of breast cancer cell line MCF-7 by directly targeting DEPTOR protein.

Trichostatin A reverses epithelial-mesenchymal transition and attenuates invasion and migration in MCF-7 breast cancer cells.

Breast cancer remains one of the leading causes of mortality in women, and epithelial-mesenchymal transition (EMT) serves an indispensable role in the invasion and migration of breast cancer cells. As a representative of classical histone deacetylase inhibitors (HDACIs), trichostatin A (TSA) has been demonstrated to reverse EMT in certain types of non-tumor cells and tumor cells. In the present study, the invasive and migratory abilities of MCF-7 cells were examined following treatment with TSA. TSA-induced changes in the expression of an epithelial biomarker epithelial cadherin (E-cadherin), a mesenchymal biomarker (vimentin), and a transcription factor [zinc finger protein SNAI2 (SLUG)] were also investigated. Transwell invasion and migration assays, and wound healing assays, revealed that the invasive and migratory abilities of MCF-7 cells were suppressed significantly upon treatment with TSA. Treatment with TSA led to an increased expression level of E-cadherin, and decreased expression of vimentin and, in MCF-7 cells. The overexpression of SLUG decreased the expression level of E-cadherin, but increased vimentin expression, and upon treatment with TSA, these effects were reversed. Additionally, SLUG knockdown also led to upregulation of E-cadherin expression, downregulation of vimentin expression, and suppression of the invasion and migration of MCF-7 cells. Taken together, these results suggest that TSA is able to reverse EMT via suppressing SLUG and attenuate the invasion and migration of MCF-7 cells in vitro, thereby providing a potential avenue for chemotherapeutic intervention in the treatment of breast cancer.

Effect of Ganoderma applanatum polysaccharides on MAPK/ERK pathway affecting autophagy in breast cancer MCF-7 cells

Fungal polysaccharides have become hotspots in the field of health foods due to their antitumor activity in recent years. In this experiment, antitumor effect of the medicinal fungus Ganoderma applanatum polysaccharide (GAP) was investigated in human breast cancer MCF-7 cells, as well as the molecular mechanism of its effect on autophagy. Results showed that GAP contains three polysaccharides with molecular weights of 6.36 × 105 Da, 4.25 × 105 Da, and 2.53 × 105 Da and which composed of rhamnose, glucose, arabinose, fucose in the molar ratio of 1:22:16.1:3.2. GAP inhibited the proliferation and migration of MCF-7 cells in a time-dose-dependent manner, the maximum inhibition rate reached 50.2% at 500 μg/mL in 48 h. Flow cytometry analysis showed that GAP could induce apoptosis, treatment of cells with GAP could result in up-regulation of gene and protein levels of autophagy-associated markers LC3 and Beclin-1; addition of autocrine late inhibitor CQ significantly raised the protein expression level of LC3II. The mitogen-activated protein kinases (MAPK) signaling pathway was not only related to the apoptotic pathway but also to the autophagy pathway; Western blot analysis showed that MAPK signaling pathway is involved in GAP-induced autophagy in MCF-7 cells. Detection of the relevant signaling pathway protein showed that the expression of p-ERK1/2 protein was down-regulated, however the expression of p-p38 and p-JNK protein was up-regulated. These results indicate that GAP could induce early autophagy in MCF-7 cells via the MAPK/ERK pathway. In conclusion, GAP showed strong antitumor activity by inducing apoptosis and autophagy through MAPK signaling pathway in MCF-7 cells, suggesting the molecular mechanism of fungal polysaccharide on its antitumor activity.

MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer.

Background: Breast cancer is one of the most frequent malignant tumors worldwide, with 1.67 million newly-diagnosed cases and 522,000 deaths each year. Therefore, seeking the novel biomarkers and therapeutic targets that contribute to postoperative recurrence and metastasis in patients with breast cancer is emerging and facilitates the development of innovative therapeutics.Methods: Retrieving the dataset of patients with hormone receptor (HR)-positive breast cancers from Gene Expression Omnibus (GEO) and collecting the data from the patients with HR-positive breast cancers enrolled in the First Affiliated Hospital of China Medical University are so as to identify the miRNAs associated with metastasis and distant metastasis-free survival (DMFS). Then MTT and Transwell migration assays were used to validate the effect of miRNAs on cell proliferation and migration of estrogen receptor-positive breast cancer T47D and MCF7 cells in vitro, respectively.Results: From GSE59829 dataset, the miRNA expression levels of miR-891a-5p, miR-383-5p and miR-1295a were significantly downregulated while the levels of miR-128-3p, miR-661 and miR-296-3p were significantly upregulated in breast cancers from patients with metastasis as compared to the matched non-metastatic group. Moreover, low expression levels of miR-891a-5p, miR-383-5p and miR-1295a or high expression levels of miR-128-3p, miR-661 and miR-296-3p were respectively associated with low DMFS in patients with breast cancer. Our clinical cohort study supported that the levels of miR-891a-5p, miR-383-5p and miR-1295a were significantly lower in breast cancers from the metastasis group when compared with non-metastatic group. However, there is no significant difference with regard to the levels of miR-128-3p, miR-661 and miR-296-3p in breast cancer between these two groups. Moreover, low expression levels of miR-891a-5p and miR-383-5p but not miR-1295a in breast cancer were significantly associated with low DMFS in patients, implying that the expression of miR-891a-5p and miR-383-5p were the potential prognosis markers for metastatic human breast cancers. Further investigation disclosed that miR-891a-5p but not miR-383-5p restrained both proliferation and migration of T47D and MCF7 cells. In silico analysis of miRNAs target gene through online computational algorithms revealed that A Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is the downstream target for miR-891a-5p. Further study confirmed that miR-891a-5p impeded ADAM10 expression by directly binding to its 3'UTR, leading to the inhibition of breast cancer cells proliferation and migration. Moreover, silencing ADAM10 inhibited T47D and MCF7 cells growth and migration.Conclusion: miR-891a-5p is the vital prognostic marker for HR-positive breast cancer. In addition, miR-891a-5p and miR-383-5p are the potential targets for HR-positive breast cancer therapeutics.

Novel Melatonin, Estrogen, and Progesterone Hormone Therapy Demonstrates Anti-Cancer Actions in MCF-7 and MDA-MB-231 Breast Cancer Cells.

A novel melatonin, estrogen, and progesterone hormone therapy was developed as a safe bio-identical alternative hormone therapy for menopausal women based on the Women’s Health Initiative findings that PremPro™ increased breast cancer risk and mortality of all types of breast cancer in postmenopausal women. For HER2 breast cancer, melatonin, estrogen, and progesterone delayed tumor onset and reduced tumor incidence in neu female mice. For other breast cancers, its actions are unknown. In this study, melatonin, estrogen, and progesterone hormone therapy were assessed in human ER+ (MCF-7) and triple negative breast cancer (MDA-MB-231) cells, and found to decrease proliferation and migration of both breast cancer lines. Inhibition of MEK1/2 and 5 using PD98059 and BIX02189, respectively, inhibited proliferation and migration in MDA-MB-231 cells and proliferation in MCF-7 cells; however, when combined with melatonin, estrogen, and progesterone, BIX02189 blocked melatonin, estrogen, and progesterone–mediated inhibition of migration in MCF-7 cells and induced Elf-5. For MDA-MB-231 cells, BIX02189 combined with melatonin, estrogen, and progesterone inhibited proliferation and increased pERK1/2 and β1-INTEGRIN; levels of pERK5 remained low/nearly absent in both breast cancer lines. These findings demonstrate novel anti-cancer actions of melatonin, estrogen, and progesterone in ER+ and triple negative breast cancer cells through intricate MEK1/2- and MEK5-associated signaling cascades that favor anti-proliferation and anti-migration.

Adipocyte-derived SFRP5 inhibits breast cancer cells migration and invasion through Wnt and epithelial-mesenchymal transition signaling pathways.

ObjectiveObesity is closely associated with metastasis in breast cancer patients. Secreted frizzled-related protein 5 (SFRP5), one of the novel adipokines with anti-inflammatory properties, is associated with obesity. This study aims to study the role of SFRP5 in the crosstalk between obesity and breast cancer metastasis and identify the underlying mechanism.Methods3T3-L1 pre-adipocytes were differentiated to mature adipocytes and a hypertrophic adipocyte model was induced with palmitic acid (PA). Cell motility was measured in MDA-MB-231 and MCF-7 breast cancer cells co-cultured with adipocytes conditioned medium (CM) with or without SFRP5 protein. Wnt and epithelial-mesenchymal transition (EMT) signal pathways were investigated by western blot. Circulating SFRP5 level in 218 breast cancer patients and the association with clinicopathologic characteristics of breast cancer were further determined. Online databases ENCORI and PREDICT Plus were used to exam the link between SFRP5 and prognosis.ResultsReduced SFRP5 level was detected in the hypertrophic adipocyte model. Recombinant SFRP5 protein inhibited MDA-MB-231 and MCF-7 cells invasion and migration induced by PA-treated adipocyte CM, and SFRP5 inhibition by specific antibody reversed the effect of SFRP5. Furthermore, SFRP5 significantly inhibited Wnt and downstream EMT in breast cancer cells. Low circulating SFRP5 level correlated with body mass index (BMI), lymph node (LN) metastasis, TNM stage and high Ki67 expression in breast cancer patients. Increased SFRP5 level was associated with favorable predicted survival. Kaplan-Meier curves showed high SFRP5 level in tumor tissue was associated with better outcome of breast cancer patients.ConclusionsOur findings demonstrated SFRP5 is a vital adipokine that mediates the crosslink between obesity and the metastatic potential of breast cancer. Promotion of SFRP5 expression in the adipose microenvironment may represent a novel approach for preventing breast cancer metastasis.

Methyl Linderone Suppresses TPA-Stimulated IL-8 and MMP-9 Expression Via the ERK/STAT3 Pathway in MCF-7 Breast Cancer Cells.

Methyl linderone (ML), a cyclo-pentenedione, was isolated from the fruit of Lindera erythrocarpa Makino (family Lauraceae). This plant has well-known anti-inflammatory effects; however, the anti-cancer effects of ML have not yet been reported. Thus, in the present study we investigated the effects of ML on the metastasis of human breast cancer cells. We used 12-O-tetradecanoyl phorbol-13-acetate (TPA)-stimulated MCF-7 cells as the cell model to study the effects of ML on invasion and migration. ML was found to reduce the invasion and migration rate of TPA-stimulated MCF-7 cells. Moreover, it inhibited two metastasis-related factors, matrix metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8), at the mRNA and protein expression levels, in TPA-treated MCF-7 cells. The mechanism by which ML exerted these effects was through the inhibition of translocation of activator protein-1 (AP-1) and signal transducer and activator of transcription-3 (STAT3), mediated via phosphorylation of extracellular signal-regulated kinase (ERK). Taken together, our findings indicated that ML attenuated the TPA-stimulated invasion and migration of MCF-7 cells by suppressing the phosphorylation of ERK and its downstream factors, AP-1 and STAT3. Therefore, ML is a potential agent for the treatment of breast cancer metastasis.