Estrogen Receptor Positive MCF-7 Research Articles

Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

BackgroundTriple-negative breast cancer (TNBC) is one of the most difficult subtypes of breast cancer to treat due to its aggressive, metastatic behavior, and a lack of a targeted therapy. Trivalent arsenic derivatives (arsenite, AsIII) with remarkable clinical efficacy in acute promyelocytic leukemia has been demonstrated to exhibit inhibitory effect against breast cancer cells. To provide novel insight into the development of new therapeutic strategies, antitumor activity of AsIII and tetrandrine (Tetra), a Chinese plant-derived alkaloid, against the TNBC cell line MDA-MB-231 in vitro and in vivo was investigated.MethodsCytotoxicity was evaluated using cell viability, lactate dehydrogenase leakage and cell cycle assay. Alterations of genes related to cell proliferation and death were analyzed using western blotting. In vivo antitumor activity of AsIII alone or in combination with Tetra was studied using MDA-MB-231 xenografts in nude mice.ResultsSynergistic cytotoxic effects of two drugs were observed in the cells. In vivo study also showed that co-administration of AsIII and Tetra significantly reduced tumor volume and weight, directly supporting its in vitro antitumor activity. No deaths and reduction of body-weight were observed after a long-term co-administration, indicating its good tolerability. S-phase arrest associated with the upregulation of FOXO3a, p27 along with decreased Cyclin D1 expression was observed in the cells treated with the combined regimen. A substantial upregulated p21 expression and downregulated phospho-FOXO3a and Cyclin D1 expression was observed in the tumor tissues of mice co-administered with AsIII and Tetra. Autophagy induction was observed in the combination treatment in vitro and in vivo. The addition of wortmannin, a potent autophagy inhibitor, significantly rescued MDA-MB-231 cells from their cytotoxicity of AsIII and Tetra.ConclusionsS-phase arrest, autophagic and necrotic cell death contribute to the cytocidal effects of the combined regimen of AsIII and Tetra. Considering our previous study showing synergistic cytotoxic effects of the combined regimen in estrogen receptor-positive breast cancer cell line MCF-7, these results suggest that development of the combination regimen of AsIII plus Tetra may offer many benefits to patients with different types of breast cancer.

PCP4/PEP19 upregulates aromatase gene expression via CYP19A1 promoter I.1 in human breast cancer SK-BR-3 cells.

The Purkinje cell protein 4/peptide 19 (PCP4/PEP19) is a novel breast cancer cell expressing peptide, originally found in the neural cells as an anti-apoptotic factor, could inhibit cell apoptosis and enhance cell migration and invasion in human breast cancer cell lines. The expression of PCP4/PEP19 is induced by estrogens in estrogen receptor-positive (ER+) MCF-7 cells but also highly expressed in ER- SK-BR-3 cells. In this study, we investigated the effects of PCP4/PEP19 on aromatase gene expression in MCF-7 and SK-BR-3 human breast cancer cells. In SK-BR-3 cells but not in MCF-7 cells, PCP4/PEP19 knockdown by siRNA silencing decreased the aromatase expression in gene transcriptional level. When PCP4/PEP19 was overexpressed by CMV promoter-driven PCP4/PEP19 expressing plasmid transfection, aromatase gene transcription increased in SK-BR-3 cells. This aromatase gene transcription is mainly mediated through promoter region PI.1, which is usually active in the placental tissue but not in the breast cancer tissue. These results indicate a new function of PCP4/PEP19 that would enhance aromatase gene upregulation to supply estrogens in heterogeneous cancer microenvironment.

Abstract 2862: Inhibition of DNA repair pathways in breast cancer is a potential mechanism of action of IT-139

Abstract IT-139 is a novel small-molecule drug that demonstrated a manageable safety profile at the maximum tolerated dose and modest antitumor activity as a monotherapy in a phase I clinical trial. A high affinity for albumin and unique chemistry allow increased uptake and activation of IT-139 within tumor cells, respectively. To date, IT-139 has been reported to inhibit the endoplasmic reticulum stress-sensing protein GRP78. However, different cancer cells show disparate responses to IT-139 and the precise mechanism of action in high-response versus low-response cancer cells remains unclear. In vitro studies have indicated that IT-139 induces cytostatic rather than cytotoxic effects as a monotherapy. In this study, to understand IT-139 mediated signaling mechanism in breast cancer cells, we used estrogen receptor-positive (ER+) MCF7 breast cancer cells to obtain gene-metabolite integrated models following IT-139 treatment (10 or 100 μM for 72 h). Our data showed that IT-139 significantly reduced expression of genes involved in the DNA repair pathway, including BRCA1 and RAD51, following treatment with IT-139 in MCF7 cells. Validation of our findings in other breast cancer cell lines including MDA-MB-175 (ER+), MDA-MB-231 and MDA-MB-468 (both ER-) cells also showed increased phosphorylation of H2AX (Ser139) following IT-139 treatment, suggesting lack of proper DNA surveillance and subsequent accumulation of DNA double-stranded breaks (DSB). Consequently, we investigated the effects of combination treatment approaches with IT-139 and other clinical anticancer therapies that interfere with nucleotide synthesis or DNA damage repair pathways, particularly in ER- breast cancers. Our data showed additive effects of IT-139 in combination with capecitabine (5-FU prodrug) or olaparib (PARP inhibitor) and a synergistic effect with carboplatin in ER- breast cancer cell lines. Further analysis of IT-139 mediated signal transduction in breast cancer cells will reveal whether it can be combined with aforementioned chemotherapies to circumvent resistance. Citation Format: Isabel Conde, Yassi Fallah, Diane Demas, Suzanne Bakewell, Filipa Lynce, Ayesha N. Shajahan-Haq. Inhibition of DNA repair pathways in breast cancer is a potential mechanism of action of IT-139 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2862.

Abstract 374: Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis

Abstract Metabolic reprogramming is a hallmark of tumor cells, owing to the increased energetic and biosynthetic demands associated with maintaining high rates of proliferation. Therapeutic strategies targeting unique, critical metabolic pathways of tumor cells are under investigation and in clinical trials. Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that provide population-based measurements and limited kinetic information. We developed a genetically encoded ATP sensor to enable direct, automated analysis of cellular ATP levels using IncuCyte® S3. In this study, our live cell imaging approach was utilized to evaluate the effect of cancer therapeutics on ATP levels in tumor cell lines, focusing primarily on compounds that target metabolic vulnerabilities. Cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated. ATP levels were monitored and analyzed using an IncuCyte® S3 equipped with a specialized filter set and data acquisition module. Cellular ATP levels were measured over the course of hours to days following compound treatment. Reductions in ATP levels could be observed within an hour of administration, highlighting the sensitivity and temporal resolution of the IncuCyte® S3 ATP sensor assay. Transient reductions in ATP followed by recovery, which would have been missed by typical endpoint assays, were noted using our live cell analysis approach. Sustained decreases in ATP were associated with enhanced antiproliferative efficacy compared to conditions under which recovery of ATP levels were observed. For example, triple-negative breast cancer (TNBC) cell lines have been shown to be more dependent on activity of glutaminase 1 (GLS1), which catalyzes the first step in utilization of glutamine to fuel mitochondrial metabolism, than their receptor-positive counterparts. Using our live cell analysis approach, we observed a rapid drop in ATP upon glutamine deprivation or inhibition of GLS1 by CB-839 in the TNBC cell line MDA-MB-231. ATP levels remained below that of vehicle-treated cells for the duration of the three-day time course. In contrast, the estrogen receptor-positive cell line MCF-7 displayed a more modest decrease in ATP and full recovery within 48 hours. Quantification of phase confluence confirmed that CB-839 had a stronger antiproliferative effect in MDA-MB-231 cells compared to MCF-7 cells. Visualization of morphology in tandem with automated ATP sensor analysis provide additional insight into the heterogeneity of cellular responses to compound addition. Overall, these data highlight the ability of IncuCyte® S3 to provide direct, kinetic measurement of ATP by live-cell analysis. Citation Format: Cicely L. Schramm, Grigory S. Filonov, Michael L. Bowe, Yong X. Chen, Laura A. Skerlos, Dyke P. McEwen, Daniel M. Appledorn. Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 374.

MART-10, a 1α,25(OH)2D3 Analog, Potently Represses Metastasis of ER+ Breast Cancer Cells with VEGF-A Overexpression.

Breast cancer ranks second in the list of cancer-related deaths for women. Even under multidisciplinary treatment, 25-50% of patients with breast cancer still ultimately develop metastasis, leading to poor prognosis. In addition to inducing angiogenesis, vascular endothelial growth factor-A (VEGF-A) is believed to directly increase cancer cell metastatic potential and overexpression of VEGF-A is associated with higher invasiveness of breast cancer. 1α,25(OH)2D3, the active form of vitamin D, and its analogs have been widely applied as anticancer agents in the past. Western blot, migration and invasion assays, enzyme-linked immunosorbent assay, and immunofluorescent stain were applied in this study. VEGF-A increased cell migration and invasion in estrogen receptor-positive (ER+) breast cancer MCF-7 cells. VEGF-A induced an autocrine loop in MCF-7 cells as VEGF-A treatment increased both VEGF-A expression and secretion. The expression of VEGF receptor type 2 (VEGFR2) and neuropilin 1 was also up-regulated by VEGF-A in MCF-7 cells. In addition, F-actin synthesis and LIM domain kinase 1 (LIMK-1) phosphorylation were increased by VEGF-A. VEGF-A also increased β-catenin expression and nuclear translocation of both β-catenin and nuclear factor-ĸB (NF-ĸB), indicating increased β-catenin and NF-ĸB activity. 1α,25(OH)2D3 and MART-10, an analog of 1α,25(OH)2D3, effectively repressed VEGF-A-induced MCF-7 cell migration and invasion and other VEGF-A-induced effects on MCF-7 cells, with MART-10 being more potent than 1α,25(OH)2D3 Conclusion: MART-10 can be deemed as a promising agent for prevention and treatment of metastasis of ER+ breast cancer with VEGF-A overexpression.

Regulation of tamoxifen resistance in estrogen receptor positive MCF-7 breast cancer cells by microRNA-10b targeting histone deacetylase 4

Objective To investigate the effect of microRNA-10b (miR-10b) targeting histone deacetylase 4 (HDAC4) on the resistance of estrogen receptor positive (ER+ ) MCF-7 breast cancer cells to tamoxifen. Methods ER+ and tamoxifen resistant MCF-7 cell line MCF-7-TR was established, and miR-10b expression level in MCF-7 cells and MCF-7-TR cells was detected by reverse transcription real-time quantitative polymerase chain reaction (Real-time PCR). Methyl thiazol tetrazolium (MTT) and trypan blue were used to detect the proliferation and activity of MCF-7 cells and MCF-7-TR cells transfected with pre-miR-10b, miR-10b inhibitor and siHDAC4 at different tamoxifen concentrations (0, 5, 10, 15, 20, 30 μmol/L), respectively. The expression of HDAC4 protein was detected by Western blotting. Results Taken expression level and invasive ability of miR-10b in MCF-7 breast cancer cells as the standard, the miR-10b expression level and invasion ability of MCF-7-TR cell breast cancer cells were 7.2±1.1 and 5.3±1.3 respectively. With the increase of the concentration of tamoxifen, decreased the rate of cell proliferation in MCF-7 cells, which is most obvious, followed by MCF-7-TR+ miR-10b inhibitor + siHDAC4 and MCF-7+ pre-miR-10b+ HDAC4 cells, and the proliferation of MCF-7-TR+ siHDAC4 cells and MCF-7-TR cells decreased the rate of the lowest. The migration ability of MCF-7 cells and MCF-7-TR+ miR-10b cells of the inhibitor decreased most, MCF-7+ pre-miR-10b+ and tamoxifen (10 μmol/L) MCF-7-TR+ cells and tamoxifen (10 μmol/L) had no significant difference in the ability of cell migration (t=1.116, P=0.272), but was significantly higher than that of MCF-7 cells and MCF-7-TR+ miR-10b cells (t=0.124, 10.769, 14.569, 11.577, P=0.000). The expression level of HDAC4 protein in MCF-7+ pre-miR-10b cells was lower than that in MCF-7 cells, MCF-7-TR cells, HDAC4 protein expression level, MCF-7-TR+ miR-10b inhibitor cells, MCF-7+ siHDAC4 cells HDAC4 protein expression level was lower than MCF-7 cells. Conclusion The miR-10b-HDAC4 signaling pathway may be a molecular mechanism of tamoxifen resistance in breast cancer. Key words: Breast cancer; Estrogen receptor positive; Tamoxifen resistance; MicroRNA-10b; Histone deacetylase 4

Stat3/Oct-4/c-Myc signal circuit for regulating stemness-mediated doxorubicin resistance of triple-negative breast cancer cells and inhibitory effects of WP1066.

Doxorubicin (Dox) is widely used in the treatment of triple-negative breast cancer cells (TNBCs), however resistance limits its effectiveness. Cancer stem cells (CSCs) are associated with Dox resistance in MCF-7 estrogen receptor positive breast cancer cells. Signal transducer and activator of transcription3 (Stat3) may functionally shift non-CSCs towards CSCs. However, whether Stat3 drives the formation of CSCs during the development of resistance in TNBC, and whether a Stat3 inhibitor reverses CSC-mediated Dox resistance, remains to be elucidated. In the present study, human MDA-MB-468 and murine4T1 mammary carcinoma cell lines with the typical characteristics of TNBCs, were compared with estrogen receptor-positive MCF-7 cells as a model system. The MTT assay was used to detect cytotoxicity of Dox. In addition, the expression levels of CSC-specific markers and transcriptional factors were measured by western blotting, immunofluorescence staining and flow cytometry. The mammosphere formation assay wasusedto detect stem cell activity. Under long-term continuous treatment with Dox at a low concentration, TNBC cultures not only exhibited a drug-resistant phenotype, but also showed CSC properties. These Dox-resistant TNBC cells showed activation of Stat3 and high expression levels of pluripotency transcription factors octamer-binding transcription factor-4 (Oct-4) and c-Myc, which was different from the high expression of superoxide dismutase2(Sox2) in Dox-resistant MCF-7 cells. WP1066 inhibited the phosphorylation of Stat3, and decreased the expression of Oct-4 and c-Myc, leading to a reduction in the CD44-positive cell population, and restoring the sensitivity of the cells to Dox. Taken together, a novel signal circuit of Stat3/Oct-4/c-Myc was identified for regulating stemness-mediated Dox resistance in TNBC. The Stat3 inhibitor WP1066 was able to overcome the resistance to Dox through decreasing the enrichment of CSCs, highlighting the therapeutic potential of WP1066 as a novel sensitizer of Dox-resistant TNBC.

Impairment of the Gnα11-controlled expression of claudin-1 and MMP-9 and collective migration of human breast cancer MCF-7 cells by DHEAS

Although dehydroepiandrosterone sulfate (DHEAS) constitutes the most abundant steroid in humans, in-depth investigations of its effects are rather scarce. We address here DHEAS effects on the estrogen receptor-positive metastatic human breast cancer cell line MCF-7. We focus on DHEAS-mediated signaling that might influence expression of claudin-1 and matrix metalloproteinase-9 (MMP-9), both known to be critical factors for migration and invasiveness of various cancers, including breast cancer cells.Physiological concentrations of DHEAS trigger persistent phosphorylation of Erk1/2 in MCF-7 cells. Exposure of these cells for 24 h to 1 μM DHEAS also leads to a significant reduction of claudin-1 expression that cannot be prevented by high concentrations of the steroid sulfatase inhibitor STX64, indicating that desulfation and further conversion of DHEAS to some other steroid hormone is not required for this action. In addition, exposure of MCF-7 cells to the same concentration of DHEAS completely abolishes MMP-9 expression and considerably impairs cell migratory behavior.Abrogation of Gnα11 expression by siRNA prevents the stimulatory effect of DHEAS on Erk1/2 phosphorylation, consistent with a G-protein-coupled receptor being involved in the DHEAS-induced signaling. Nevertheless, Gnα11 also has direct effects that do not depend on DHEAS; thus, when Gnα11 expression is suppressed, expression of claudin-1 and MMP-9 as well as cell migration are significantly reduced.This is the first report demonstrating direct involvement of DHEAS and Gnα11 in the regulation of claudin-1 and MMP-9 expression and migration of MCF-7 cells.

Cytotoxicity of Tanshinone IIA combined with Taxol on drug-resist breast cancer cells MCF-7 through inhibition of Tau.

Drug resistance represents a major obstacle to improving the overall response and survival of cancer patients. Taxol is one of the most commonly used chemotherapy agents in breast cancer. As with many cancer therapeutic agents, resistance remains a significant problem when using Taxol to treat malignancies. In this study, estrogen receptor positive breast cancer cells MCF-7 were induced Taxol resistance. And Tanshinone IIA combined with Taxol was chosen to treat it. The drugs combination showed additive effect in most drug concentrations. Drug resistance cancer cells showed a higher microtubule associated protein (Tau) expression, which was considered as one of the reasons for Taxol resistance. Tanshinone IIA inhibited the expression of Tau in MCF-7 cells and resulted in higher sensibility of Taxol. Moreover, Tanshinone IIA also showed cytotoxicity to MCF-7, which might be related to its estrogenicity effect. In conclusion, the combination of Tanshinone IIA and Taxol showed higher cytotoxicity to Taxol resistant MCF-7 cells, which might be related to the inhibition of Tau.

Synthesis and application of 131I-fulvestrant as a targeted radiation drug for endocrine therapy in human breast cancer.

The aim of this study was to label fulvestrant (anendocrine therapy drug for breast cancer) with radioiodine and to evaluate the effect of 131I-fulvestrant on inhibiting the growth of human breast cancer and its influence on major organs in nude mice. Fulvestrant was labeled with radioiodine using a modified chloramine Tmethod, and its chemical properties were assessed using traditional methods. The binding affinity of 131I-fulvestrant was measured by radioligand binding assays, and its antiproliferative activity was determined by MTT assays. The ability of 131I-fulvestrant to kill MCF-7 and MDA-MB-231 cells was also detected by MTT assays. We established MCF-7 cell xenografts in nude mice and monitored tumor growth and critical organ function. When the labeling reactions were conducted for 5min at room temperature at pH7.5, the radiochemical yield of the 131I labeling to fulvestrant was 62.34±1.8%, the radiochemical purity was 98.6±3.4%, and the half maximal inhibitory concentration (IC50) at 48h was 35µCi. 131I-fulvestrant was stable, and its binding affinity to estrogen receptor-positive (ER+) MCF-7 cells was also retained. In addition, 131I-fulvestrant exhibited similar cytotoxicity in MCF-7 and MDA-MB-231 cells, although MCF-7 cells showed a slightly more pronounced response. 131I-fulvestrant continuously exerted a tumor suppressive effect on MCF-7 cells but not on MDA-MB-231 cells (P<0.05). Upon intravenous injection of 131I-fulvestrant into nude mice, the radioactivity distribution corresponded to ER expression patterns and was primarily confined to the tumor. 131I-fulvestrant exhibited a precise growth inhibition effect on MCF-7 breast cancer cells, and its effects on general conditions of nude mice and their major organs were manageable. Therefore, radioiodine labeling of fulvestrant was successful and could be used to develop novel drugs for breast cancer by superimposing the benefits of radiotherapy and endocrine therapy.

Tunicamycin-induced ER stress in breast cancer cells neither expresses GRP78 on the surface nor secretes it into the media.

GRP78 (an Mr 78 kDa calcium dependent glucose binding protein) is located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr. Paraffin sections from N-glycosylation inhibitor tunicamycin treated ER-/PR-/HER2+ (double negative) breast tumor in athymic nude mice exhibited reduced N-glycan but increased GRP78 expression. We have evaluated the effect of tunicamycin on cellular localization of GRP78 in metastatic human breast cancer cells MDA-MB-231 (ER-/PR-/HER2-). Tunicamycin inhibited cell proliferation in a time and dose-dependent manner. Nonmetastatic estrogen receptor positive (ER+) MCF-7 breast cancer cells were also equally effective. GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/mL) treated MCF-7 and MDA-MB-231 cells. GRP78 is an ER stress marker, so we have followed its intracellular localization using immunofluorescence microscopy after subjecting the cancer cells to various stress conditions. Unfixed cells stained with either FITC-conjugated Concanavalin A (Con A) or Texas-red conjugated wheat germ agglutinin (WGA) exhibited surface expression of N-glycans but not GRP78. GRP78 became detectable only after a brief exposure of cells to ice-cold methanol. Western blotting did not detect GRP78 in conditioned media of cancer cells whereas it did for MMP-1. The conclusion, GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. Our study therefore suggests strongly that anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast and other sold tumors.

Influence of morphine on TLR4/ NF-kB signaling pathway of MCF-7 cells

Morphine affects the risk of metastasis in cancer. The TLR4 gene promotes migration in adenocarcinoma cells. We investigated the effect of morphine on TLR4, MyD88 and NF-κ B-expression and migration. Migration of estrogen receptor-positive MCF7 breast cancer cells was studied after 24 and 48 hours incubation with morphine, with boyden chamber method. Morphine effect on TLR4, MyD88 and NF-κB mRNA expression was determined by quantitative Real-Time polymerase chain reaction. Migration was reduced at the doses of 0.5 and 5 µM (p < 0.05). However, TLR4, MyD88 and NF-κBmRNA expression was decreased at the doses of 0.5, 5 and 500 µM. Morphine at the dose of 50 µM increased the expression of mentioned genes. MCF-7 cell line after 48 hours incubation with the dose of 0.5 µM morphine decreased the migration and at the dose of 0.5 µM down-regulated the mRNA expression of TLR4, MyD88 and NF-κB, however, the higher doses increased the expression of TLR4, MyD88 and NF-κB. Morphine affects TLR4expression in breast cancer cell, which depends on time and concentration (Tab. 1, Fig. 5, Ref. 24).

Identification of d-seco modified steroid derivatives with affinity for estrogen receptor α and β isoforms using a non-transcriptional fluorescent cell assay in yeast

Synthesis and biological evaluation of steroidal derivatives with anticancer properties is an active area of drug discovery. Here we measured the relative affinities of d-seco modified steroidal derivatives for estrogen receptor α, estrogen receptor β or androgen receptor ligand binding domains using an optimized non-transcriptional fluorescent cell assay in yeast. Ligand binding domains of steroid receptors were expressed in-frame with yellow fluorescent protein in the yeast Saccharomyces cerevisiae. Addition of known steroid ligands to yeast expressing the appropriate cognate receptor results in increased fluorescence intensity, enabling estimation of receptor binding affinities in a dose-response and time-dependent manner. Relative binding affinities of d-seco modified steroidal derivatives 1–4 were then evaluated using this yeast system by live cell fluorimetry and fluorescence microscopy, coupled with in vitro cytotoxicity and in silico molecular docking studies. d-Seco estratriene derivative 2displayed strong affinity for both estrogen receptor α and β ligand binding domains and negligible affinity for the androgen receptor ligand binding domain. Compound 2 also showed moderate cytotoxicity against estrogen receptor positive MCF-7 breast adenocarcinoma cells. In addition to identification of new ligands for steroid receptors, this assay could also be used to filter out compounds with potential for off-target interactions with steroid receptors during the early stages of compound screening.

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

Equol Enhances Apoptosis-inducing Activity of Genistein by Increasing Bax/Bcl-xL Expression Ratio in MCF-7 Human Breast Cancer Cells

ABSTRACTAnticancer activities of soy isoflavones, such as genistein and equol, a bioactive metabolite of daidzein, have been extensively studied because of possible involvement in the prevention of breast cancer. However, their interactions still remain unclear. We investigated here whether cytotoxic activity of genistein was enhanced by equol, using estrogen receptor positive MCF-7, HER2-positive SK-BR-3, and triple-negative MDA-MB-468 cell lines. Although cytotoxicity of genistein did not significantly differ between three subtypes of breast cancer cells, cytotoxic activities of genistein were significantly enhanced in combination with 50 μM equol in MCF-7 cells, but not in SK-BR-3 and MDA-MB-468 cells. In fluorescence activated cell sorting (FACS) analyses, MCF-7 cells were arrested at the G2/M by genistein but at G1/S by equol. Combination treatment arrested cells at G2/M but abolished equol-induced G1 block, indicating an antagonistic activity of genistein against equol in cell-cycle progression. Although apoptosis was not so evident with genistein alone, the combination made a drastic induction of apoptosis, accompanied by the increase of Bax/Bcl-xL expression ratio, without affecting the activities of Akt and mTOR. Taken together, these data suggest that enhancement of genistein activity by equol would be mainly mediated by augmented induction of apoptosis rather than arrest or delay of the cell cycle.

The β-glucan from Lentinus edodes suppresses cell proliferation and promotes apoptosis in estrogen receptor positive breast cancers.

Breast cancer is now the most common cancer in worldwide women, and novel interventions are needed to overcome the resistance occurring in the estrogen-targeted endocrine therapy. Herein, we demonstrate that the β-glucan from Lentinus edodes (LNT) exhibited a profound inhibition ratio of ∼53% against estrogen receptor positive (ER+) MCF-7 tumor growth in nude mice similar to the positive control of cisplatin. Immunohistochemistry images showed that LNT evidently suppressed cell proliferation and promoted apoptosis in MCF-7 tumor tissues. The Western blotting analysis indicated that LNT up-regulated the tumor suppressor p53, phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2), cleaved-Caspase 3 and poly [ADP (ribose)] polymerase 1 (PARP 1) protein levels, and reduced the expression of mouse double minute 2 (MDM2), telomerase reverse transcriptase (TERT), nuclear factor-kappa B (NF-κB) p65, B-cell lymphoma-2 (Bcl-2), estrogen receptor α (ERα), etc. in tumor tissues. Moreover, LNT significantly suppressed phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt) and mammalian target of rapamycin (mTOR) protein levels. It was thus proposed that LNT inhibited MCF-7 tumor growth through suppressing cell proliferation and enhancing apoptosis possibly via multiple pathways such as PI3K/Akt/mTOR, NF-κB-, ERK-, ERα-, caspase- and p53-dependent pathways. Interestingly, the cell viability assay, siRNA transfection, Western blotting and flow cytometric analysis suggested that LNT targeted p53/ERα to only suppress cell proliferation via cell cycle arrest at G2/M phase without apoptosis in vitro. The big difference between in vivo and in vitro data suggested that the immune responses triggered by the polysaccharide should mainly contribute to the apoptotic effect in vivo. Overall, this work provides a novel strategy to treat ER+ breast cancers by using a naturally occurring β-glucan from mushrooms.

Targeted gadofullerene for sensitive magnetic resonance imaging and risk-stratification of breast cancer

Molecular imaging of cancer biomarkers is critical for non-invasive accurate cancer detection and risk-stratification in precision healthcare. A peptide-targeted tri-gadolinium nitride metallofullerene, ZD2-Gd3N@C80, is synthesised for sensitive molecular magnetic resonance imaging of extradomain-B fibronectin in aggressive tumours. ZD2-Gd3N@C80 has superior r1 and r2 relaxivities of 223.8 and 344.7 mM−1 s−1 (1.5 T), respectively. It generates prominent contrast enhancement in aggressive MDA-MB-231 triple negative breast cancer in mice at a low dose (1.7 µmol kg−1, 1 T), but not in oestrogen receptor-positive MCF-7 tumours. Strong tumour contrast enhancement is consistently observed in other triple negative breast cancer models, but not in low-risk slow-growing tumours. The dose of the contrast agent for effective molecular MRI is only slightly higher than that of ZD2-Cy5.5 (0.5 µmol kg−1) in fluorescence imaging. These results demonstrate that high-sensitivity molecular magnetic resonance imaging with ZD2-Gd3N@C80 may provide accurate detection and risk-stratification of high-risk tumours for precision healthcare of breast cancer.

Inhibitors of PI3K/ERK1/2/p38 MAPK Show Preferential Activity Against Endocrine-Resistant Breast Cancer Cells.

Current mainstream pharmacological options for the treatment of endocrine-resistant breast cancer have limitations in terms of their side effect profile and lack of discrimination between normal and cancer cells. In the current study, we assessed the responses of normal breast epithelial cells MCF10A, estrogen receptor-positive (ER+) MCF-7, and ER-silenced pII breast cancer cells to inhibitors (either individually or in combination) of downstream signaling molecules. The expression/activity of ERK1/2, p38 MAPK, and Akt was determined by Western blotting. Cell proliferation, motility, and invasion were determined using MTT, wound healing, and Matrigel assays, respectively. Morphological changes in response to variation in external pH were assessed by light microscopy. Our results demonstrated that the inhibitors of ERK1/2 (PD0325901), p38 MAPK (SB203580), and PI3K (LY294002) preferentially reduce breast cancer cell proliferation. In pII cells, they also reduced motility, invasion, and bleb formation induced by alkaline conditions. Combination treatment with lower concentrations of inhibitors was significantly more effective than single agents and was more effective against the cancer cell lines than the normal MCF10A. In contrast, the commonly used cytotoxic agent paclitaxel did not sufficiently discriminate between the MCF10A and the cancer cells. We concluded that combination therapy using ERK1/2 inhibitor and either p38 MAPK or PI3K inhibitor may provide a greater therapeutic benefit in treating breast cancer by specifically targeting cancer cells with lower doses of each drug than needed individually, potentially reducing unwanted side effects.

Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer activity against breast cancer cell lines

Forskolin C1-isoxazole derivatives (3,5-regioisomers) (11a–e, 14, 15a–h and 15, 16a–g) were synthesized regioselectively by adopting 1,3-dipolar cycloadditions. These derivatives were tested using estrogen receptor positive breast cancer cell lines MCF-7 and BT-474. Majority of the compounds exhibited activity against the p53-positive MCF-7 breast cancer cells but not against the p53-negative BT-474 breast cancer cells. Among forskolin derivatives, compounds 11a, 11c, 14a, 14f, 14g, 14h, 15b, 16g and 17b exhibited higher anti-cancer activity against MCF-7 cell line with an IC50≤1µM. The derivative 14f exhibited highest activity in both p53-positive (MCF-7) and p53-negative (BT-474) breast cancer cell lines with an IC50 of 0.5µM.

The IL-6/STAT3 Signaling Pathway Is an Early Target of Manuka Honey-Induced Suppression of Human Breast Cancer Cells.

There is renewed interest in the potential use of natural compounds in cancer therapy. Previously, we demonstrated the anti-tumor properties of manuka honey (MH) against several cancers. However, the underlying mechanism and molecular targets of this activity remain unknown. For this study, the early targets of MH and its modulatory effects on proliferation, invasiveness, and angiogenic potential were investigated using two human breast cancer cell lines, the triple-negative MDA-MB-231 cells and estrogen receptor-positive MCF-7 cells, and the non-neoplastic breast epithelial MCF-10A cell line. Exposure to MH at concentrations of 0.3–1.25% (w/v) induced a dose-dependent inhibition of the proliferation of MDA-MB-231 and MCF-7, but not MCF-10A, cells. This inhibition was independent of the sugar content of MH as a solution containing equivalent concentrations of its three major sugars failed to inhibit cell proliferation. At higher concentrations (>2.5%), MH was found to be generally deleterious to the growth of all three cell lines. MH induced apoptosis of MDA-MB-231 cells through activation of caspases 8, 9, 6, and 3/7 and this correlated with a loss of Bcl-2 and increased Bax protein expression in MH-treated cells. Incubation with MH induced a time-dependent translocation of cytochrome c from mitochondria to the cytosol and Bax translocation from the cytosol into the mitochondria. MH also induced apoptosis of MCF-7 cells via the activation of caspases 9 and 6. Low concentrations of MH (0.03–1.25% w/v) induced a rapid reduction in tyrosine-phosphorylated STAT3 (pY-STAT3) in MDA-MB-231 and MCF-7 cells. Maximum inhibition of pY-STAT3 was observed at 1 h with a loss of >80% and coincided with decreased interleukin-6 (IL-6) production. Moreover, MH inhibited the migration and invasion of MDA-MB-231 cells as well as the angiogenic capacity of human umbilical vein endothelial cells. Our findings identify multiple functional pathways affected by MH in human breast cancer and highlight the IL-6/STAT3 signaling pathway as one of the earliest potential targets in this process.