Estrogen Receptor Negative Human Breast Research Articles

Strong Protection by 4-Hydroxyestrone against Erastin-Induced Ferroptotic Cell Death in Estrogen Receptor-Negative Human Breast Cancer Cells: Evidence for Protein Disulfide Isomerase as a Mechanistic Target for Protection.

Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E1), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E1 can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E1 forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI's catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E1 as well as cystamine (a known PDI inhibitor) against chemically induced ferroptosis in human breast cancer cells. Importantly, inhibition of PDI by 4-OH-E1 and cystamine or PDI knockdown by siRNAs each markedly reduces iNOS activity and NO accumulation, which has recently been demonstrated to play an important role in erastin-induced ferroptosis. In conclusion, this study demonstrates that 4-OH-E1 is a novel inhibitor of PDI and can strongly inhibit ferroptosis in human breast cancer cells in an estrogen receptor-independent manner. The mechanistic understanding gained from the present study may also aid in understanding the estrogen receptor-independent cytoprotective actions of endogenous estrogen metabolites in many noncancer cell types.

Sevoflurane modulates breast cancer cell survival via modulation of intracellular calcium homeostasis

BackgroundSome retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis.MethodsEstrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects.ResultsSevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell.ConclusionSevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.

Granulocyte-colony stimulating factor-associated aortitis in a woman with advanced breast cancer: a case report and review of the literature

BackgroundGranulocyte-colony stimulating factor (G-CSF) is increasingly been used to prevent febrile neutropenia (FN) associated with the administration of chemotherapy for various cancers. The most common adverse effects of G-CSF are bone pain and injection-site reactions and aortitis has rarely been reported. We report herein a rare case of G-CSF associated with aortitis in a woman with advanced breast cancer.Case presentationA 72-year-old woman with estrogen receptor-negative human epidermal growth factor 2-positive breast cancer with distant metastases in the lung was admitted. Her treatment was initiated with docetaxel in combination with trastuzumab and pertuzumab followed by the supportive use of a long-acting G-CSF, pegfilgrastim. After administration of pegfilgrastim on day 5, the patient had an intermittent fever (body temperature up to 39.6 °C) on day 9 which continued irrespective of taking levofloxacin. She visited our outpatient clinic on day 13 with no objective symptoms other than fever. Laboratory tests revealed a high neutrophil count (15,000/μl) and a high C-reactive protein (CRP) level (46.35 mg/dl) without any other abnormalities. There was no response upon administration of antimicrobial agents. An 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) revealed thickening of the wall of the descending thoracic aorta and left pleural effusion. Therefore, thoracic aortitis induced by pegfilgrastim was suspected. On day 19, the fever resolved spontaneously followed by a gradual reduction in the neutrophil count and CRP level. In the follow-up CT, the aortic wall thickness and pleural effusion had disappeared.ConclusionsG-CSF may cause aortitis due to stimulation of the production of inflammatory cytokines. In case of high continuous fever after administration of pegfilgrastim, aortitis should be suspected unless there are other infectious findings.

BMI is an independent prognostic factor for late outcome in patients diagnosed with early breast cancer: A landmark survival analysis

BMI is an independent prognostic factor for late outcome in patients diagnosed with early breast cancer: A landmark survival analysis

The effects of naloxone on human breast cancer progression: in vitro and in vivo studies on MDA.MB231 cells.

BackgroundNaloxone is viewed as a specific competitive opioid antagonist acting at the level of opioid receptors (μ, δ, and κ) with blended agonist-adversary or agonist action. The role of naloxone in tumor cell growth has been poorly studied in human cancer cell lines.Materials and methodsIn the present study, we report findings from in vitro and in vivo experiments performed to evaluate the effects of naloxone on human breast cancer cell growth and progression. In vitro assays were conducted on estrogen receptor-negative human breast carcinoma cells, MDA.MB231, treated with naloxone at different concentrations (10–100 μM). In vivo experiments were performed on a mouse model of human triple-negative breast cancer generated by using MDA.MB231 injected subcutaneously in mice. Naloxone was daily intraperitoneally injected in mice at 0.357 mg/kg for 2 weeks and at 0.714 mg/kg for the next 2 weeks. Microvessels formation was detected by fluorescein isothiocyanate-dextran (100 μL) injected into the tail vein of mice and confirmed by immunohistochemistry with CD31 on mice tumor sections.ResultsIn vitro tests showed that the cell proliferation of MDA.MB231 was inhibited by naloxone in a dose-dependent manner, whereas the cell death was increased. In vivo studies demonstrated that tumors of mice treated with naloxone were significantly smaller than those observed in the control groups, as long as naloxone was administered. Finally, naloxone was not able to impair the microvessel formation in tumors of treated mice.ConclusionOur data showed, for the first time, that naloxone reduced breast cancer progression without affecting angiogenesis.

Profiling of gene expression regulated by 17β-estradiol and tamoxifen in estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines.

One area of great importance in breast cancer (BC) research is the study of gene expression regulated by both estrogenic and antiestrogenic agents. Although many studies have been performed in this area, most of them have only addressed the effects of 17β-estradiol (E2) and tamoxifen (TAM) on MCF7 cells. This study aimed to determine the effect of low doses of E2 and TAM on the expression levels of 84 key genes, which are commonly involved in breast carcinogenesis, in four BC cell lines differentially expressing estrogen receptor (ER) α and HER2 (MCF7, T47D, BT474, and SKBR3). The results allowed us to determine the expression patterns modulated by E2 and TAM in ERα+ and ERα− cell lines, as well as to identify differences in expression patterns. Although the MCF7 cell line is the most frequently used model to determine gene expression profiles in response to E2 and TAM, the changes in gene expression patterns identified in ERα+ and ERα− cell lines could reflect distinctive properties of these cells. Our results could provide important markers to be validated in BC patient samples, and subsequently used for predicting the outcome in ERα+ and ERα− tumors after TAM or hormonal therapy. Considering that BC is a molecularly heterogeneous disease, it is important to understand how well, and which cell lines, best model that diversity.

Activation of Akt characterizes estrogen receptor positive human breast cancers which respond to anthracyclines.

Anthracyclines are key components of human breast cancer chemotherapy. Here, we explored the role of Akt signaling in anthracycline resistance.The antitumor activity of doxorubicin and Akt inhibitor A-443654 alone or combined was examined in estrogen receptor (ER) positive and negative human breast cancer cell lines. Further, we examined mRNA changes induced by anthracyclines in locally advanced breast cancers biopsied before and after treatment in two clinical trials.Doxorubicin increased Akt phosphorylation in ER positive MCF7 and T47D cell lines, with no effect in ER negative MDA-MB231 breast cancer cells. A-443654 was significantly more cytotoxic in doxorubicin-resistant compared to doxorubicin-naïve MCF7. This difference was not observed in MDA-MB231. Among 24 patients, AKT1 gene expression increased 24 hrs after the initial epirubicin exposure in ER positive tumors responding to therapy (n=6), as compared to ER positive non-responders (n=7) or ER negative tumors (n=11). In contrast, AKT1 mRNA changes after 16 weeks of doxorubicin were unrelated to clinical response and ER status (n=30).In conclusion, rapid Akt activation was observed in ER positive breast cancers which responded to anthracyclines. Increased cytotoxicity of A-443654 in doxorubicin-resistant MCF7 cells indicates a possible role for Akt inhibitors in ER positive breast cancers where chemoresistance evolves.

NSG Mice Provide a Better Spontaneous Model of Breast Cancer Metastasis than Athymic (Nude) Mice.

Metastasis is the most common cause of mortality in breast cancer patients worldwide. To identify improved mouse models for breast cancer growth and spontaneous metastasis, we examined growth and metastasis of both estrogen receptor positive (T47D) and negative (MDA-MB-231, SUM1315, and CN34BrM) human breast cancer cells in nude and NSG mice. Both primary tumor growth and spontaneous metastases were increased in NSG mice compared to nude mice. In addition, a pattern of metastasis similar to that observed in human breast cancer patients (metastases to the lungs, liver, bones, brain, and lymph nodes) was found in NSG mice. Furthermore, there was an increase in the metastatic burden in NSG compared to nude mice that were injected with MDA-MB-231 breast cancer cells in an intracardiac experimental metastasis model. This data demonstrates that NSG mice provide a better model for studying human breast cancer metastasis compared to the current nude mouse model.

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.

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.

Tamoxifen induces apoptosis through cancerous inhibitor of protein phosphatase 2A-dependent phospho-Akt inactivation in estrogen receptor-negative human breast cancer cells.

IntroductionTamoxifen, a selective estrogen receptor (ER) modulator, may affect cancer cell survival through mechanisms other than ER antagonism. In the present study, we tested the efficacy of tamoxifen in a panel of ER-negative breast cancer cell lines and examined the drug mechanism.MethodsIn total, five ER-negative breast cancer cell lines (HCC-1937, MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3) were used for in vitro studies. Cellular apoptosis was examined by flow cytometry and Western blot analysis. Signal transduction pathways in cells were assessed by Western blot analysis. The in vivo efficacy of tamoxifen was tested in xenograft nude mice.ResultsTamoxifen induced significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3 cells, but not in HCC-1937 cells. Tamoxifen-induced apoptosis was associated with inhibition of cancerous inhibitor of protein phosphatase 2A (CIP2A) and phospho-Akt (p-Akt) in a dose-dependent manner. Ectopic expression of either CIP2A or Akt protected MDA-MB-231 cells from tamoxifen-induced apoptosis. In addition, tamoxifen increased protein phosphatase 2A (PP2A) activity, and tamoxifen-induced apoptosis was attenuated by the PP2A antagonist okadaic acid in the sensitive cell lines, but not in resistant HCC-1937 cells. Moreover, silencing CIP2A by small interfering RNA sensitized HCC-1937 cells to tamoxifen-induced apoptosis. Furthermore, tamoxifen regulated CIP2A protein expression by downregulating CIP2A mRNA. Importantly, tamoxifen inhibited the in vivo growth of MDA-MB-468 xenograft tumors in association with CIP2A downregulation, whereas tamoxifen had no significant effect on CIP2A expression and anti-tumor growth in HCC-1937 tumors.ConclusionsInhibition of CIP2A determines the effects of tamoxifen-induced apoptosis in ER-negative breast cancer cells. Our data suggest a novel “off-target“ mechanism of tamoxifen and suggest that CIP2A/PP2A/p-Akt signaling may be a feasible anti-cancer pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0431-9) contains supplementary material, which is available to authorized users.

Gene expression profiles of NO- and HNO-donor treated breast cancer cells: insights into tumor response and resistance pathways

Nitric oxide (NO) synthase 2 (NOS2), a major inflammatory protein, modulates disease progression via NO in a number of pathologies, including cancer. The role of NOS2-derived NO is not only flux-dependent, which is higher in mouse vs human cells, but also varies based on spatial and temporal distribution both within tumor cells and in the tumor microenvironment. NO donors have been utilized to mimic NO flux conditions and to investigate the effects of varied NO concentrations. As a wide range of effects mediated by NO and other nitrogen oxides such as nitroxyl (HNO) have been elucidated, multiple NO- and HNO-releasing compounds have been developed as potential therapeutics, including as tumor modulators. One of the challenges is to determine differences in biomarker expression from extracellular vs intracellular generation of NO or HNO. Taking advantage of new NO and HNO releasing agents, we have characterized the gene expression profile of estrogen receptor-negative human breast cancer (MDA-MB-231) cells following exposure to aspirin, the NO donor DEA/NO, the HNO donor IPA/NO andtheir intracellularly-activated prodrug conjugates DEA/NO-aspirin and IPA/NO-aspirin. Comparison of the gene expression profiles demonstrated that several genes were uniquely expressed with respect to NO or HNO, such as miR-21, HSP70, cystathionine γ-lyase and IL24. These findings provide insight into targets and pathways that could be therapeutically exploited by the redox related species NO and HNO.

Dinuclear berenil-platinum (II) complexes as modulators of apoptosis in human MCF-7 and MDA-MB231 breast cancer cells.

The metabolism of alkylating agents is accompanied by the generation of reactive oxygen species. The aim of this study was to treat estrogen receptor-positive and estrogen receptor-negative human breast cancer cells, MCF-7 and MDA-MB-231, respectively, with cisplatin and five different berenil-platinum (II) complexes, and then to investigate the oxidative modifications of DNA, lipid and protein, and to compare them with the profile of various pro- and antiapoptotic proteins. Changes in the levels of 8-hydroxy-2'-deoxyguanosine, 4-hydroxynonenal, carbonyl groups, dityrosine, active caspases 3, 8 and 9, as well as the expression of Bcl-2, Bax, cytochrome c, and p53 were subsequently examined. Activities of superoxide dismutase, catalase and glutathione, vitamin C levels were also investigated. Cellular reactions to cisplatin and the berenil-platinum (II) derivatives were more pronounced in MCF-7 cells as compared with the MDA-MB231 cells. Furthermore, the berenil-platinum (II) derivatives were found to be more effective than cisplatin. All of the complexes reduced the activity of superoxide dismutase and catalase, and also lowered the levels of non-enzymatic antioxidants. Increased level of lipid, protein as well as DNA damage markers was also observed after berenil-platinum (II) derivatives treatment. Similarly, the increase in the levels of the proapoptotic factors, were detected in MCF-7 and MDA-MB231 cells. Incubation of examined cells with the berenil-platinum (II) complexes also led to the increase in the levels of active caspases 3, 8 and 9. In conclusion, the results of the present study demonstrated that berenil-platinum (II) complexes more efficiently mediate cellular oxidative modifications and proapoptotic metabolism, particularly in MCF-7 cells, compared to cisplatin. Pt2(isopropylamine)4berenil2 and Pt2(piperidine)4(berenil)2 notably affected the cellular metabolism of estrogen-positive breast cancer cells. Thus, these complexes may be valuable for the design of additional anticancer drugs.

Chemopreventive activity of Cnidii Rhizoma for breast cancer

Chemopreventive potential for human breast cancer was assessed in vitro with Cnidii Rhizoma extract. Cnidii Rhizoma inhibited cell proliferation in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) human breast carcinoma cell lines. Cytochrome P450 (CYP) 1A1-mediated ethoxyresorufin O-deethylase (EROD) activity was inhibited by Cnidii Rhizoma in a concentration-dependent manner. In addition, Cnidii Rhizoma extract caused inhibition of microsomal aromatase (estrogen synthase) activity. Ornithine decarboxylase (ODC) activity was reduced to 40.3% of the control after 6 h treatment with Cnidii Rhizoma (5 mg/mL) in MCF-7 breast cancer cells. Cnidii Rhizoma extract markedly reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated matrix metalloproteinase (MMP)-9 activity. These results suggest that Cnidii Rhizoma could be of therapeutic value in preventing human breast cancer.

Antiproliferative Activity of a Dietary Supplement on Estrogen Receptor Positive and Negative Human Breast Adenocarcinoma Cell Lines

A commercial food supplement called Citozym (CIZ), was tested for antiproliferative activity against estrogen receptor (ER)-positive MCF-7 and (ER)-negative BT-20 human breast cancer cells, performing two independent experiments using SRB assay. A CIZ concentration of 100mg/mL, showed the most potent antiproliferative activity for cells in a potential range for further investigation on estrogen-insensitive breast cancer therapy

Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.

Obesity increases the incidence of distant metastases in oestrogen receptor-negative human epidermal growth factor receptor 2-positive breast cancer patients

BackgroundObesity is a major negative determinant of breast cancer outcome. However, there are contrasting data on the differential impact of obesity on specific breast cancer subtypes. In particular, very little is known on human epidermal growth factor receptor 2-positive (HER2+) tumours. Patients and methodsWe assessed the prognostic role of increased body mass index (BMI) on a consecutive series of non-metastatic HER2+ patients treated at our institution before the introduction of adjuvant Trastuzumab. We separately analysed oestrogen receptor-positive (ER+) and -negative (ER−) HER2+ cases. ResultsIn ER−/HER2+ tumours we observed a significantly worse overall survival (Hazard ratio (HR) 1.79, p-value 0.041) and cumulative incidence of distant metastases (HR 2.03, p-value 0.019) in obese (BMI>30) versus normal/underweight (BMI<25) patients. Local relapses appeared to be non-significantly reduced in obese patients, masking the overall effect on disease-free survival. Outcome in ER+ tumours, instead, was not significantly different between BMI groups. ConclusionsObesity significantly correlates with worse overall survival and cumulative incidence of distant metastases in ER−/HER2 positive breast cancer. Differences in the biology of breast tumours may determine individual susceptibility to obesity. The biology of the underlying tumour should be taken into account in the design of dietary intervention trials in breast cancer.

Ethanolic extracts of California mugwort (Artemisia douglasiana Besser) are cytotoxic against normal and cancerous human cells

California mugwort (Artemisia douglasiana Besser) is used by many tribes throughout California to treat a variety of conditions, including colds, allergies, and pain. California mugwort is also utilized as women's medicine. Its use is on the rise outside of Native communities, often without the guidance of a traditional healer or experienced herbalist. Because it has been shown to have antiproliferative activity against plant and animal cells, we investigated whether California mugwort extracts have an effect on normal human cells as well as estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast cancer cells. Ethanolic and aqueous extracts of A. douglasiana leaves were tested for cytotoxicity against unstimulated normal human peripheral blood mononuclear cells (hPBMC), as well as against an ER+ human breast cancer cell line (BT-474) and an ER- human breast cancer cell line (MDA-MB-231). An ethanolic leaf extract killed hPBMC, BT-474, and MDA-MB-231 cells with IC50 values of 23.6 ± 0.3, 27 ± 5, and 37 ± 4 μg/ml, respectively. An aqueous extract killed hPBMC with an IC50 value of 60 ± 10 μg/ml, but had no effect on the two cancer cell lines at concentrations up to 100 μg/ml. The results of this study indicate that the cytotoxicity of California mugwort extends to normal human cells, as well as cancerous cells. Therefore, until further is known about the safety of this medicine, caution should be taken when consuming extracts of California mugwort, whether as a tincture or as a tea.

Effects of steroidal aromatase inhibitors on sensitive and resistant breast cancer cells: Aromatase inhibition and autophagy

Several therapeutic approaches are used in estrogen receptor positive (ER+) breast cancers, being one of them the use of aromatase inhibitors (AIs). Although AIs demonstrate higher efficacy than tamoxifen, they can also exhibit de novo or acquired resistance after prolonged treatment. Recently, we have described the synthesis and biochemical evaluation of four steroidal AIs, 3β-hydroxyandrost-4-en-17-one (1), androst-4-en-17-one (12), 4α,5α-epoxyandrostan-17-one (13a) and 5α-androst-2-en-17-one (16), obtained from modifications in the A-ring of the aromatase substrate, androstenedione. In this study, it was investigated the biological effects of these AIs in different breast cancer cell lines, an ER+ aromatase-overexpressing human breast cancer cell line (MCF-7aro cells), an estrogen-receptor negative (ER−) human breast cancer cell line (SK-BR-3 cells), and a late stage of acquired resistance cell line (LTEDaro cells). The effects of an autophagic inhibitor (3-methyladenine) plus AIs 1, 12, 13a or exemestane in LTEDaro cells were also studied to understand the involvement of autophagy in AI acquired resistance. Our results showed that these steroids inhibit aromatase of MCF-7aro cells and decrease cell viability in a dose- and time-dependent manner. The new AI 1 is the most potent inhibitor, although the AI 12 demonstrates to be the most effective in decreasing cell viability. Besides, and in advantage over exemestane, AIs 12 and 13a also reduced LTEDaro cells viability. The use of the autophagic inhibitor allowed AIs to diminish viability of LTEDaro cells, presenting a similar behavior to the sensitive cells. Thus, inhibition of autophagy may sensitize hormone-resistant cancer cells to anti-estrogen therapies.

Apoptotic effect of tolfenamic acid on MDA-MB-231 breast cancer cells and xenograft tumors

Recent studies have indicated that non-steroidal anti-inflammatory drug (NSAID), particularly tolfenamic acid, can inhibit proliferation and induce apoptosis invarious cancer cells. Breast cancer represents one-third of all cancers diagnosed in women and is the second leading cause of cancer death in Western European and North American women. In the present study, we investigated the apoptotic effect of tolfenamic acid in MDA-MB-231 estrogen receptor-negative human breast carcinoma cells and in a xenograft tumor model. Treatment of cells with tolfenamic acid significantly decreased cell viability in a concentration-dependent manner. Notably, tolfenamic acid increased apoptosis-related proteins, such as p53 and p21, within 48 h. Furthermore, in vivo experiments showed that tolfenamic acid treatment resulted in a significant reduction in tumor volume over 5 weeks. Immunohistochemistry results showed that apoptosis-related protein induction by tolfenamic acid was significantly higher in the 50 mg/kg-treated group compared to the control group. Together, these results indicate that tolfenamic acid induces apoptosis in MDA-MB-231 breast cancer cells and tumor xenograft model and it may be a potential chemotherapeutic agent against breast cancer.