Receptor Negative Breast Cancer Cells Research Articles

Low doses of bisphenol A stimulate the proliferation of breast cancer cells via ERK1/2/ERRγ signals

The effects and mechanisms of bisphenol A (BPA) on the development of breast cancer are still not well illustrated. The present study revealed that nanomolar BPA significantly promoted the proliferation of both estrogen receptor (ER) positive (MCF-7) and negative (SkBr3) breast cancer cells, which was confirmed by up regulation of proliferating cell nuclear antigen (PCNA) and Bcl-2. Neither ERα nor G-protein-coupled estrogen receptor (GPER) mediated this effect of BPA because their inhibitors had no effect on the BPA induced cell proliferation. However, silencing of estrogen related receptor gamma (ERRγ) by its specific siRNA significantly abolished BPA induced proliferation of breast cancer cells, while si-ERRα had no similar effect. Moreover, nanomolar BPA up regulated the mRNA and protein levels of ERRγ and triggered its nuclear translocation via a time dependent manner. Further studies revealed that 10−8M BPA obviously increased the phosphorylation of ERK1/2, while had no similar effect on the phosphorylation of JNK and p38 MAPK. Further, PD 98059, the inhibitor of ERK1/2, significantly abolished the BPA induced up regulation of ERRγ and proliferation of breast cancer cells. Collectively, our results revealed that nanomolar BPA can trigger the proliferation of breast cancer cells via ERK1/2/ERRγ signals. Given that nanomolar BPA has been widely detected in human tissues, the clinical relevance of BPA and breast cancer progression should be further investigated.

When science fails a scientist.

In August 2012, a phone call from Scotland told me that my sister had been diagnosed with advanced breast cancer. Six weeks later she was dead. In between was a blur of travel, family tensions, and—one bright spot—making my sister laugh one more time. But my sharpest memory of those weeks is the helplessness of sitting in a hospital office learning that estrogen receptor-negative breast cancer cells in my sister's body had metastasized to her bones, lungs, and brain. We could make her comfortable, the doctor said. That was it. ![Figure][1] ILLUSTRATION: ROBERT NEUBECKER > “My decision has been to grab life—now.” When I learned about my sister's cancer, I was in the midst of a career transition—the second in my professional life. Back in my 30s, I had decided to leave the bench because I felt I could have a greater impact on the human condition as a medical research foundation administrator than I could as a decent, but not great, neuroscientist. For 6 years, I was the chief scientific officer of a foundation that researched drug treatments for benign but deforming tumors that could become malignant. I was the one sitting at the other end of the table, facing people who were relying on science to change, and possibly save, the life of their loved one. “We are going to get there,” I would say. “We will find drugs that will work.” I truly believed it. I got the news about my sister after my husband and I had taken the radical step of moving from a city-centered life to the rural one we had fallen in love with. I was working from our new home, consulting for cancer foundations and walking my dog in the woods at lunchtime. For the first time, my career had slowed down. I was quietly torn between fully embracing this change and returning to the career fast track, but I couldn't quite figure out what it was that I missed. Was it the prestige, the salary, or leading the charge to find cancer treatments? Traveling to our annual international conference 6 years straight (always at the time of my June wedding anniversary)? Strategizing with 30 researchers and clinicians about curing brain tumors (which caused me to miss a celebration dear to my in-laws)? After the phone call from Scotland, I emailed my sister's medical reports to many of the doctors I'd worked with while at the foundation, in the hope they could help. But their responses, though sensitive and thoughtful, could only try to prepare me for my big sister's death at age 56. First, the helplessness grew. Then the anger: Why was hospice the only option she had? Weren't there off-label drugs or clinical trials? Shouldn't I know this? Had my sister been in denial? How could she not know she was riddled with cancer? She'd feared breast exams, so she didn't get them. We'd argued about that, and about her smoking. Eventually, my anger gave way to resignation: Her cancer had simply eluded the options science had to offer. We knew my mother's large family had a history of breast and ovarian cancer, and one cousin had a BRCA1 mutation. But it wasn't until after my sister's death that I investigated and mapped the family cancer tree. When my gynecologist saw it, she said wow. We'll never know if my sister had a BRCA1 mutation, but the summer after she died I learned that I did. The decision to have prophylactic surgery was easy. Science may have helped me protect my health, but I no longer assume that we'll have translated enough science into better drugs for cancer if I ever need them. Meanwhile, this difficult couple of years has helped me make peace with my decision to abandon the high-profile career ladder. Just a year before she died, my sister had retired and she was looking forward to freedom. So my decision has been to grab life—now. At 50, I finally have a work-life balance. I found a job in mental health administration where I can make an intellectual contribution, and which also allows me to go home and live life. I have given enough to the “cures” cause. It is a long road, and it is time for others to lead the way. [1]: pending:yes

Abstract 4463: Riluzole effectively modulates cell cycle and cell death in a molecularly diverse set of breast cancer cell lines

Abstract Metabotropic glutamate receptor 1 (GRM1) has an established oncogenic role in melanoma. Growing evidence supports a similar role in breast cancer. We have previously shown that GRM1 is not only expressed in human breast cancers, but that levels of expression correlate with clinical outcomes with tamoxifen treatment, and that GRM1 knockdown reduces breast cancer cell viability and growth. Others have shown that GRM1 modulating drugs also reduce viability and growth of estrogen receptor (ER) negative breast cancer cell lines. Riluzole, a postulated inhibitor of glutamate release, has been evaluated in pre-clinical studies in melanoma. Early clinical trials have shown promising results for this disease. However, the mechanistic effects of riluzole have not been well-defined. Therefore, we evaluated the functional activity of riluzole in breast cancer. A molecularly diverse set of ER+ and ER- breast cancer cell lines with variable GRM1 expression were evaluated. Cell lines were treated with either riluzole or BAY 36-7620 (non-competitive GRM1 antagonist) or their combination. Treatment with either drug reduces cell number and viability, inhibits cell proliferation, and alters expression of cell cycle and apoptotic pathway genes as determined by gene microarray analysis. Drug treatment produces cell line-dependent effects on markers for proliferation, cell cycle, DNA damage, and apoptosis. Riluzole induces G2/M arrest in all cell lines tested. More specifically, riluzole induces mitotic arrest as demonstrated by changes in histone H3 phosphorylation at serine 10 and cyclin B level. Treatment with BAY 36-7620 induces a modest G2/M arrest and increase in the sub G1 population in a subset of cell lines. However, BAY 36-7620 does not induce mitotic arrest. The combination of riluzole and BAY 36-7620 results in increased cell death and decreased proliferation in all cell lines tested. Our data suggest that either riluzole or BAY 36-7620 induce breast cancer cell death. However, riluzole may modulate the activity of intracellular targets independent of GRM1. As riluzole is an FDA-approved drug, it could be quickly moved into a clinical trial and may provide a novel therapeutic approach in the treatment of breast cancer. Citation Format: Sonia C. Dolfi, Daniel J. Medina, Shridar Ganesan, Alexei Vazquez, Kim M. Hirshfield. Riluzole effectively modulates cell cycle and cell death in a molecularly diverse set of breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4463. doi:10.1158/1538-7445.AM2015-4463

Androgen receptor- and PIAS1-regulated gene programs in molecular apocrine breast cancer cells

We have analyzed androgen receptor (AR) chromatin binding sites (ARBs) and androgen-regulated transcriptome in estrogen receptor negative molecular apocrine breast cancer cells. These analyses revealed that 42% of ARBs and 39% androgen-regulated transcripts in MDA-MB453 cells have counterparts in VCaP prostate cancer cells. Pathway analyses showed a similar enrichment of molecular and cellular functions among AR targets in both breast and prostate cancer cells, with cellular growth and proliferation being among the most enriched functions. Silencing of the coregulator SUMO ligase PIAS1 in MDA-MB453 cells influenced AR function in a target-selective fashion. An anti-apoptotic effect of the silencing suggests involvement of the PIAS1 in the regulation of cell death and survival pathways. In sum, apocrine breast cancer and prostate cancer cells share a core AR cistrome and target gene signature linked to cancer cell growth, and PIAS1 plays a similar coregulatory role for AR in both cancer cell types.

Nitric Oxide-Releasing Aspirin Suppresses NF-κB Signaling in Estrogen Receptor Negative Breast Cancer Cells in Vitro and in Vivo.

Estrogen receptor negative (ER(−)) breast cancer is aggressive, responds poorly to current treatments and has a poor prognosis. The NF-κB signaling pathway is implicated in ER(−) tumorigenesis. Aspirin (ASA) is chemopreventive against ER(+) but not for ER(−) breast cancers. Nitric oxide-releasing aspirin (NO-ASA) is a safer ASA where ASA is linked to an NO-releasing moiety through a spacer. In vitro, we investigated anti-proliferation effects of NO-ASA (para- and meta-isomers) against ER(−) breast cancer cells MDA-MB-231 and SK-BR-23, effects on NF-κB signaling, and reactive oxygen species by standard techniques. In vivo, effects of NO-ASA were evaluated in a mouse xenograft model using MDA-MB-231 cells. p-NO-ASA inhibited the growth of MDA-MB-231 and SK-BR-3 cells at 24 h, the respective IC50s were 13 ± 2 and 17 ± 2 μM; ASA had an IC50 of >3000 μM in both cell lines. The IC50s for m-NO-ASA in MDA-MB-231 and SK-BR-3 were 173 ± 15 and 185 ± 12 μM, respectively, therefore, implying p-NO-ASA as a stronger inhibitor of growth p-NO-ASA reduced cell growth by inhibiting proliferation, inducing apoptosis and causing G0/G1 cell cycle block. Activation of NF-κB was inhibited by both isomers as demonstrated by decreases in NF-κB-DNA binding and luciferase activity at 24 h, However, m-NO-ASA produced transient effects at 3 h such as increased NF-κB-DNA-binding, increased levels of nuclear p50, even though both isomers inhibited IκB degradation. Increase in nuclear p50 by m-NO-ASA was associated with translocation of p50 in to the nucleus as observed by immunoflouresence at 3 h. NO-ASA induced reactive oxygen species (ROS) as evidenced by overall increases in both H2DCFDA (2′,7′-dichlorodihydrofluorescein) and DHE (dihydroethidium)-derived fluorescence. Inhibition of ROS by N-acetyl-cysteine reversed the m-NO-ASA-mediated translocation of p50 in to the nucleus. In xenografts, p-NO-ASA inhibited tumor growth by inhibiting proliferation (PCNA and tumor volume), inducing apoptosis (TUNEL positive cells) and reducing NF-κB expression. Both isomers inhibit cancer cells, inhibit NF-κB pathway and induce ROS, and have potential as anticancer compounds.

The chemomodulatory effects of resveratrol and didox on herceptin cytotoxicity in breast cancer cell lines.

Herceptin is considered an essential treatment option for double negative breast cancer. Resveratrol and didox are known chemopreventive agents with potential anticancer properties. The aim of the current study is to investigate the influence of resveratrol and didox on the cytotoxicity profile of herceptin in HER-2 receptor positive and HER-2 receptor negative breast cancer cell lines (T47D and MCF-7 cell lines, respectively). The IC50’s of herceptin in T47D and MCF-7 were 0.133 ± 0.005 ng/ml and 23.3795 ± 1.99 ng/ml respectively. Equitoxic combination of herceptin with resveratrol or didox in T47D significantly reduced the IC50 to 0.052 ± 0.001 and 0.0365 ± 0.001 ng/ml, respectively and similar results were obtained in MCF-7. The gene expression of BCL-xl was markedly decreased in T47D cells following treatment with herceptin/resveratrol compared to herceptin alone. Immunocytochemical staining of HER-2 receptor in T47D cells showed a significant reduction after treatment with herceptin/resveratrol combination compared to herceptin alone. On the contrary, herceptin/didox combination had no significant effect on HER-2 receptor expression. Cell cycle analysis showed an arrest at G2/M phase for both cell lines following all treatments. In conclusion, herceptin/resveratrol and herceptin/didox combinations improved the cytotoxic profile of herceptin in both T47D and MCF-7 breast cancer cell lines.

Synthesis, characterization and biological evaluation of transition metal complexes derived from N, S bidentate ligands.

Two bidentate NS ligands were synthesized by the condensation reaction of S-2-methylbenzyldithiocarbazate (S2MBDTC) with 2-methoxybenzaldehyde (2MB) and 3-methoxybenzaldehyde (3MB). The ligands were reacted separately with acetates of Cu(II), Ni(II) and Zn(II) yielding 1:2 (metal:ligand) complexes. The metal complexes formed were expected to have a general formula of [M(NS)2] where M = Cu2+, Ni2+, and Zn2+. These compounds were characterized by elemental analysis, molar conductivity, magnetic susceptibility and various spectroscopic techniques. The magnetic susceptibility measurements and spectral results supported the predicted coordination geometry in which the Schiff bases behaved as bidentate NS donor ligands coordinating via the azomethine nitrogen and thiolate sulfur. The molecular structures of the isomeric S2M2MBH (1) and S2M3MBH (2) were established by X-ray crystallography to have very similar l-shaped structures. The Schiff bases and their metal complexes were evaluated for their biological activities against estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cell lines. Only the Cu(II) complexes showed marked cytotoxicity against the cancer cell lines. Both Schiff bases and other metal complexes were found to be inactive. In concordance with the cytotoxicity studies, the DNA binding studies indicated that Cu(II) complexes have a strong DNA binding affinity.

Interleukin-8 upregulates integrin β3 expression and promotes estrogen receptor-negative breast cancer cell invasion by activating the PI3K/Akt/NF-κB pathway

Interleukin-8 (IL-8) possesses tumorigenic and proangiogenic properties and is overexpressed in many human cancers. The integrin family regulates a diverse array of cellular functions crucial to the initiation, progression and metastasis of solid tumors. However, the mechanisms of action of IL-8 and integrin in estrogen receptor-negative breast cancer are largely unknown. In this study, IL-8 and integrin β3 expression in human breast cancer cells and tissues was examined by real-time PCR, Western blot and immunochemistry analysis. Integrin β3 expression, invasive ability and the activation of PI3K/Akt and NF-κB pathways in IL-8 knockdown breast cancer cells were evaluated. In addition, reporter assay and ChIP were performed to assess integrin β3 promoter activity in IL-8 knockdown cells. We observed a positive correlation between integrin β3 and IL-8 expression, which was inversely correlated with ER status in breast cancer cell lines and tissues. IL-8 siRNA decreased the invasion and integrin β3 expression in human breast cancer cells. Moreover, IL-8 siRNA attenuated the phosphorylation of PI3K and Akt and inhibited NF-κB activity and binding on integrin β3 promoter. IL-8 siRNA diminished NF-κB nuclear translocation via blocking IκB phosphorylation in the cytoplasm. In conclusion, IL-8 activates the PI3K/Akt pathway, which in turn activates NF-κB, resulting in the upregulation of integrin β3 expression and increased invasion of estrogen receptor-negative breast cancer cells. IL-8/PI3K/Akt/NF-κB/integrin β3 axis may be exploited for therapeutic intervention to breast cancer metastasis.

Cyclopeptide RA-V inhibits cell adhesion and invasion in both estrogen receptor positive and negative breast cancer cells via PI3K/AKT and NF-κB signaling pathways

Cyclopeptide RA-V has potent anti-tumor and anti-angiogenic activities, but its potential anti-metastatic activity is unknown. Cancer cells acquire invasive ability to degrade and adhere to extracellular matrix (ECM), allowing them to migrate to adjacent tissues and ultimately metastasize. Hence, the present study aimed to investigate the effects of RA-V on cell adhesion, migration, invasion and matrix degradation, and its underlying mechanism in two human breast cancer cell lines MCF-7 (ER-positive) and MDA-MB-231 (ER-negative). Our results demonstrated that RA-V (12.5nM) can significantly inhibit breast cancer cell adhesion and migration via interfering cofilin signaling and chemokine receptors involved in cell migration. RA-V reduced the expressions of vascular intracellular adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), focal adhesion kinase (FAK) and integrins. The activities and expressions of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) and urokinase-type of plasminogen activator (uPA) were also inhibited by RA-V. Furthermore, RA-V inhibits the expressions of EGFR, PI3K/AKT and NF-κB signaling molecules, and reduces the binding of β-estradiol to ER via affecting binding ability of ER in MCF-7 cells. RA-V inhibits breast cancer cell migration, adhesion and ECM degradation in vitro, implying that RA-V is a potential anti-metastatic agent in breast cancer, and likely acts via PI3K/AKT and NF-κB signaling pathways in both ER-positive and ER-negative breast cancer cells.

English

The identification of medicinal plants and derived natural products for development as anti-cancer agents is of long standing interest. We have investigated the anti-proliferative properties of Lupinus angustifolius on breast cancer and, in particular, whether the extracts of roots and shoots of L. angustifolius can be considered as candidates for primary and secondary cancer prevention. Ethanolic extracts of roots and shoots of L. angustifolius were analysed for their substance classes by pyrolysis-field ionization mass spectrometry. Various concentrations of these extracts (0.1, 1, 10, 25, 50, 100 and 200 µg/ml, incubation period: 24 h) were studied with respect to their effects on cell proliferation and cytotoxicity against the breast cancer cell lines MCF-7 (ER-α+, ER-β+) and BT20 (ER-α-, ER-β-). L. angustifolius ethanolic root and shoot extracts inhibited cell proliferation in the MCF-7 and BT20 cells; root extract: strongest inhibition MCF-7 (200 μg/ml 71.03 ± 12.62%); BT20 (200 μg/ml 99.72 ± 6.48%), IC50-values: MCF-7 (52.3 ± 9.39 μg/ml); BT20 (66.86 ± 7.02 μg/ml); shoot extract: strongest inhibition MCF-7 (200 μg/ml 64.7 ± 10.07 %); BT20 (200 μg/ml 86.32 ± 9.19%), IC50-values: MCF-7 (18.06 ±4.49 μg/ml); BT20 (70.27 ± 0.76 μg/ml). Thus, extracts of L. angustifolius roots and shoots have anti-tumour activity against receptor-positive and receptor-negative breast cancer cell lines.   Key words: Anti-tumour activity, Lupinus angustifolius, breast cancer, MCF-7, BT20

Cdx2 polymorphism affects the activities of vitamin D receptor in human breast cancer cell lines and human breast carcinomas.

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression.

Differential effect of phosphorylation-defective survivin on radiation response in estrogen receptor-positive and -negative breast cancer.

Survivin is a key member of the inhibitor of apoptosis protein family, and is considered a promising therapeutic target due to its universal overexpression in cancers. Survivin is implicated in cellular radiation response through its role in apoptosis, cell division, and DNA damage response. In the present study, analysis of publically available data sets showed that survivin gene expression increased with breast cancer stage (p < 0.00001) and was significantly higher in estrogen receptor-negative cancers as compared to estrogen receptor-positive cancers (p = 9e-46). However, survivin was prognostic in estrogen receptor-positive tumors (p = 0.03) but not in estrogen receptor-negative tumors (p = 0.28). We assessed the effect of a survivin dominant-negative mutant on colony-formation (2D) and mammosphere-formation (3D) efficiency, and radiation response in the estrogen receptor-positive MCF7 and estrogen receptor-negative SUM149 breast cancer cell lines. The colony-formation efficiency was significantly lower in the dominant-negative survivin-transduced cells versus control MCF7 cells (0.42 vs. 0.58, p < 0.01), but it was significantly higher in dominant-negative population versus control-transduced SUM149 cells (0.29 vs. 0.20, p < 0.01). A similar, non-significant, trend in mammosphere-formation efficiency was observed. We compared the radiosensitivity of cells stably expressing dominant-negative survivin with their controls in both cell lines under 2D and 3D culture conditions following exposure to increasing doses of radiation. We found that the dominant-negative populations were radioprotective in MCF7 cells but radiosensitive in SUM149 cells compared to the control-transduced population; further, Taxol was synergistic with the survivin mutant in SUM149 but not MCF7. Our data suggests that survivin modulation influences radiation response differently in estrogen receptor-positive and estrogen receptor-negative breast cancer subtypes, warranting further investigation.

Equol, an Isoflavone Metabolite, Regulates Cancer Cell Viability and Protein Synthesis Initiation via c-Myc and eIF4G

Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via up-regulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER-) metastatic breast cancer cells, equol induced elevated levels of eIF4G, which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNAs translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of c-Myc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant down-regulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer-promoting effects of equol via up-regulation of eIF4GI and selective initiation of translation on mRNAs that utilize non-canonical initiation, including certain oncogenes.

Sulforaphane inhibited tumor necrosis factor-α induced migration and invasion in estrogen receptor negative human breast cancer cells

Sulforaphane significantly (p<0.05) inhibited tumor necrosis factor (TNF)-α induced cellular migration and invasion in MCF10DCIS.com human breast cancer cells, compared with controls. mRNA and protein expressions of MMPs, including MMP-2, MMP-9, and MMP-13, and the enzymatic activities of MMP-2 and MMP-9 were suppressed by sulforaphane treatments at 1, 5, and 10 μM concentration in MCF10DCIS.com cells. Sulforaphane should be considered as a potent agent for retardation of mammary tumorigenesis.

The combination of rapamycin and resveratrol blocks autophagy and induces apoptosis in breast cancer cells.

Hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) is a frequent event in breast cancer and current efforts are aimed at targeting the mTORC1 signaling pathway in combination with other targeted therapies. However, patients often develop drug resistance in part due to activation of the oncogenic Akt signaling and upregulation of autophagy, which protects cancer cells from apoptosis. In the present study we investigated the effects of combination therapy of rapamycin (an allosteric mTORC1 inhibitor) together with resveratrol (a phytoestrogen that inhibits autophagy). Our results show that combination of these drugs maintains inhibition of mTORC1 signaling, while preventing upregulation of Akt activation and autophagy, causing apoptosis. Additionally, this combination was effective in estrogen receptor positive and negative breast cancer cells, underscoring its versatility.

Selectivity of Pinus sylvestris extract and essential oil to estrogen-insensitive breast cancer cells Pinus sylvestris against cancer cells.

Background:So far, the anticancer action of pine tree extracts has mainly been shown for the species distributed widely around the Asian countries.Objective:Therefore, this study was performed to examine the potential cytotoxicity of Scots pine (Pinus sylvestris L.) native also to the European region and growing widely in Estonia.Materials and Methods:The cytotoxic activity of methanol extract and essential oil of Scots pine needles was determined by sulforhodamine B assay in different human cancer cell lines.Results:This needle extract was found to suppress the viability of several human cancer cell lines showing some selectivity to estrogen receptor negative breast cancer cells, MDA-MB-231(half maximal inhibitory concentration [IC50] 35 μg/ml) in comparison with estrogen receptor-positive breast cancer cells, MCF-7 (IC50 86 μg/ml). It is the strongest cytotoxic effect at all measured, thus far for the needles and leaves extracts derived from various pine species, and is also the first study comparing the anticancer effects of pine tree extracts on molecularly different human breast cancer cells. The essential oil showed the stronger cytotoxic effect to both negative and positive breast cancer cell lines (both IC50 29 μg/ml) than pine extract (IC50 42 and 80 μg/ml, respectively).Conclusion:The data from this report indicate that Scots pine needles extract and essential oil exhibits some potential as chemopreventive or chemotherapeutic agent for mammary tumors unresponsive to endocrine treatment.

The palladacycle, AJ-5, exhibits anti-tumour and anti-cancer stem cell activity in breast cancer cells

Breast cancer is the most common malignancy amongst women worldwide but despite enormous efforts to address this problem, there is still limited success with most of the current therapeutic strategies. The current study describes the anti-cancer activity of a binuclear palladacycle complex (AJ-5) in oestrogen receptor positive (MCF7) and oestrogen receptor negative (MDA-MB-231) breast cancer cells as well as human breast cancer stem cells. AJ-5 is shown to induce DNA double strand breaks leading to intrinsic and extrinsic apoptosis and autophagy cell death pathways which are mediated by the p38 MAP kinase. This study provides evidence that AJ-5 is potentially an effective compound in the treatment of breast cancer.

The activation of G protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative breast cancer cells in vitro and in vivo.

There is an urgent clinical need for safe and effective treatment agents and therapy targets for estrogen receptor negative (ER−) breast cancer. G protein-coupled receptor 30 (GPR30), which mediates non-genomic signaling of estrogen to regulate cell growth, is highly expressed in ER− breast cancer cells. We here showed that activation of GPR30 by the receptor-specific agonist G-1 inhibited the growth of ER− breast cancer cells in vitro. Treatment of ER− breast cancer cells with G-1 resulted in G2/M-phase arrest, downregulation of G2-checkpoint regulator cyclin B, and induction of mitochondrial-related apoptosis. The G-1 treatment increased expression of p53 and its phosphorylation levels at Serine 15, promoted its nuclear translocation, and inhibited its ubiquitylation, which mediated the growth arrest effects on cell proliferation. Further, the G-1 induced sustained activation and nuclear translocation of ERK1/2, which was mediated by GPR30/epidermal growth factor receptor (EGFR) signals, also mediated its inhibition effects of G-1. With extensive use of siRNA-knockdown experiments and inhibitors, we found that upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was also involved in G-1-induced cell growth arrest. In vivo experiments showed that G-1 treatment significantly suppressed the growth of SkBr3 xenograft tumors and increased the survival rate, associated with proliferation suppression and upregulation of p53, p21 while downregulation of cyclin B. The discovery of multiple signal pathways mediated the suppression effects of G-1 makes it a promising candidate drug and lays the foundation for future development of GPR30-based therapies for ER− breast cancer treatment.

Abstract 5114: The chemomodulatory effects of resveratrol and didox on herceptin cytotoxicity in breast cancer cell lines

Abstract Herceptin is considered an essential treatment option for double negative breast cancer. Resveratrol and didox are known chemopreventive agents with potential anticancer properties. The aim of the current study is to investigate the influence of resveratrol and didox on the cytotoxicity profile of herceptin in her-2 receptor positive and her-2 receptor negative breast cancer cell lines. The IC50's of herceptin in T47D and MCF-7 were 0.126±0.01 ng/ml and 26.71±1.85 ng/ml respectively. Equitoxic combination of herceptin with resveratrol or didox in T47D significantly reduced the IC50 to 0.05±0.01 and 0.029±0.006 ng/ml, respectively and similar results were obtained in MCF-7. The gene expression of Bcl-xl was markedly decreased in T47D cells following treatment with herceptin/resveratrol compared to herceptin alone. Immunocytochemical staining of her-2 receptor in T47D cells showed a significant reduction after treatment with herceptin/resveratrol combination compared to herceptin alone. On the other hand, combination of herceptin and didox had no significant effect on her-2 receptor expression. Cell cycle analysis showed an arrest at G2/M phase for both cell lines following treatment with herceptin alone and in either combinations regimen. In conclusion the combination of herceptin with resveratrol and didox improved the cytotoxic profile of herceptin in T47D (her-2 receptor positive cell line) and to a lesser extent in MCF-7 (her-2 receptor negative cell line). Citation Format: Ghada A. Abd Ellatif, Marianne Georges Tadros, Ashraf Bahey Eldin Abd El Naim, Amany Ibrahim Khalifa, Ahmad M. Alabd. The chemomodulatory effects of resveratrol and didox on herceptin cytotoxicity in breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5114. doi:10.1158/1538-7445.AM2014-5114

Abstract 620: Evidence for combination tamoxifen and betulinic acid to treat hormone responsive- and unresponsive breast cancer by attenuation of Pygopus expression

Abstract The presence of the estrogen and progesterone hormone receptors (ER and PR) in advanced breast cancer tumour nuclei is predictive for response to hormone disruptive therapy. However, the accuracy of ER and PR testing can be variable and although patients receive significant benefit from hormone therapy, they ultimately become refractory to treatment. The chromatin remodeling protein: human Pygopus2 (hPygo2), is important for oncogenic growth and cell cycle progression and may serve as a diagnostic or targetable biomarker for breast cancer. Here we demonstrate that ER-alpha (Erα)−Sp1 transcription factor (Sp1) complexes associate with chromatin at adjacent motifs within the hPygo2 promoter in both hormone receptor positive and negative breast cancer cells. Erα agonists induced, while antagonists inhibited hPygo2 mRNA and protein expression. Erα and Sp1 occupancy at the hPygo2 promoter required intact promoter binding sites and functional DNA binding domains. Estrogen (E2) increased, while Fulvestrant decreased, Erα binding to the hPygo2 promoter with no effect on Sp1. Furthermore, Sp1-directed siRNA reduced Sp1 occupancy at the hPygo2 promoter and decreased hPygo2 mRNA and protein expression in both Erα-expressing (+) and Erα-nonexpressing (-) cells. Betulinic acid, an Sp1 inhibitor, caused cell cycle arrest, reduced Sp1 binding to the hPygo2 promoter and reduced hPygo2 expression in Erα- cell lines. The activation of hPygo2 expression either cooperatively or independently by Erα and Sp1 suggests their potential diagnostic and therapeutic benefit for both hormone responsive and unresponsive breast cancer. Citation Format: Youlian Tzenov, Philip Andrews, Cathy Popadiuk, Kenneth R. Kao. Evidence for combination tamoxifen and betulinic acid to treat hormone responsive- and unresponsive breast cancer by attenuation of Pygopus expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 620. doi:10.1158/1538-7445.AM2014-620