Receptor-negative Human Breast Cancer Cell 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.

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.

INFLUENCE OF EXOGENOUS LACTOFERRIN ON THE OXIDANT/ ANTIOXIDANT BALANCE AND MOLECULAR PROFILE OF HORMONE RECEPTOR-POSITIvE AND -NEGATIvE HUMAN BREAST CANCER CELLS IN VITRO

To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of "free" iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231and MDA-MB-468cells. These changes resulted in increased expression of proapoptotic Bax protein (p<0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4-1.7-fold, p< 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14-24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G2/M phase, especially in receptor-negative cell lines. The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, "free" iron and NO generation rate, resalting in the blocking of cell cycle at G2/M-phase and apoptosis of malignant cells.

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.

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.

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.

Genistein inhibits osteolytic bone metastasis and enhances bone mineral in nude mice

In this study, the effective activity of genistein on osteolytic bone metastasis and bone mineral was investigated. Female BALB/c-nu/nu mice were injected with estrogen receptor-negative human breast cancer cells, MDA-MB-231, into left cardiac ventricle to form osteolytic bone metastases, and administered genistein subcutaneously after radiologically small but defined osteolytic metastases had been observed (protocol 1), simultaneously with cancer cells inoculation (protocol 2) and prophylactically 7 days before inoculation of cancer cells (protocol 3). In all protocols, genistein (10 mg/kg/day) markedly reduced the number and volume of osteolytic bone metastases assessed by radiography and the number of osteoclasts. Furthermore, histomorphometrical analysis revealed that genistein markedly increased trabecular area (Tb.Ar%), trabecular thickness (Tb.Th) and trabecular number (Tb.N), and decreased trabecular separation (Tb.Sp). These results thus demonstrate that genistein could inhibit osteolytic bone metastases, suppress bone resorption, increase bone mass and improve bone microstructure in bone metastases of breast cancer.

Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac)

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin αvβ3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

Cadmium regulation of apoptotic and stress response genes in tumoral and immortalized epithelial cells of the human breast

Cadmium (Cd) is a widely-disseminated metal which can be imported and accumulated in living cells thereby drastically interfering with their biological mechanisms. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Concerning breast cancer, very limited studies have been produced so far on the role played by Cd on estrogen receptor-negative human breast cancer cells, that are expected to be insensitive to the already-proven metallo-estrogenic effect exerted by Cd on the estrogen receptor-positive cell counterparts. Here, we have examined the effects of long-term (96 h) exposure of estrogen receptor-negative MDA-MB231 malignant adenocarcinoma cells to CdCl 2 at 5 μM concentration, corresponding to the IC 50 for this time of incubation, by evaluating the expression levels of genes coding for stress response factors (e.g. heat shock proteins and metallothioneins), and for apoptosis-related factors and enzymes. In parallel, we tested the gene expression pattern of immortalized HB2 breast epithelial cells, taken as non-tumoral counterpart, after the same exposure to the metal which instead did not exert any change in their cell number with respect to controls. Our cumulative results indicate that, whilst HB2 cells appear to activate defense mechanisms against metal stress principally via metallothionein massive up-regulation and appearance of the spliced form of XBP-1 message, MDA-MB231 cells seem to couple the onset of a protective reaction (e.g. up-regulation of hsp27 and metallothioneins) to the switching-on of new intracellular pathways directing cells to a kind of death which shares several aspects with the apoptotic program, such as down-regulation of Bcl-2 and over-expression of Dap kinase and several caspases.

Estrogen Receptor-Positive and Estrogen Receptor-Negative Human Breast Cancer Cells: Regulation of Expression of Cancer-Related Genes by Estradiol and Tamoxifen

Breast cancer is one of the most prevalent forms of carcinomas among women world-wide. The oncogenes, tumor suppressor genes and antioxidant enzyme genes are cancer-related genes which play an important role in the initiation, metastasis and malignancy of many cancers, including breast cancer. Estrogen which is a key steroid hormone in the regulation and differentiation of the normal breast also appears to be involved in the carcinogenesis of this tissue. Antiestrogen therapy, such as the use of tamoxifen, targets the estrogen receptors (ER) through which estrogen exerts both its normal biological function as well as the molecular processes leading to cancer formation. The objectives of the present study were to characterize and quantify the expression of genes related to human breast carcinogenesis, using ER- positive (ZR-75-1) and ER-negative (MDA-MB-231) cells as in vitro cellular models of breast cancer. We have examined the expression of a number of genes which have an important clinical significance in human breast cancer, when expressed at levels below or above the normal range. Among these are the receptor oncogenes (EGFR, c-erbB2), other oncogenes (pS2, hMAM, MUC1 and CK-19), the tumor suppressor gene, p53, as well as four of the major antioxidant enzyme genes. These are superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), Se-glutathione peroxidase (GPx1) and catalase (CAT), all of which have been well characterized in human cells, and whose activities have been localized to different cellular compartments, such as the cytoplasm, mitochondria and peroxisomes. Gene expressions were determined using real-time PCR, in control untreated cells as well as when these two cell types were exposed to 17β-estradiol (E), or tamoxifen (TAM) or a combination of E and TAM. Our results show that basal level expressions of receptor oncogenes (EGFR and c-erbB2) as well as two of the four antioxidant enzyme genes (SOD2 and GPx1) were several folds lower in ER-positive breast cancer cells (ZR-75-1) as compared to ER-negative cells (MDAMB- 231). However, treatment with E and TAM, either individually or in combination, produced much greater effects on the ER-positive cells than on the ER-negative cells, with the result that these genes were greatly overexpressed in the ER-positive cells as compared to the ER-negative cells. Of these, the receptor oncogene, c-erbB2, and the antioxidant enzyme gene, SOD2, were the most affected, resulting in striking upregulations ranging over several hundred folds. This, and other observations, would suggest that the carcinogenic effects of estradiol and the anticarcinogenic effects of tamoxifen are not only mediated by the presence of estrogen receptors, but may also be regulated, either directly or indirectly, through other mechanisms, including the mitochondrial antioxidant system. Keywords: Breast cancer, estrogen and antiestrogens, tamoxifen, oncogenes, tumor suppressor genes, antioxidant enzyme genes, oxidative stress and antioxidants, quantitative real-time PCR

Aberrant Expression of Fra-1 in Estrogen Receptor-negative Breast Cancers and Suppression of their Propagation In Vivo by Ascochlorin, an Antibiotic that Inhibits Cellular Activator Protein-1 Activity

Estrogen receptor-negative breast cancers generally are highly malignant, resistant to chemotherapy and poorly prognostic. Here we demonstrate that estrogen receptor-negative human breast cancer cell lines highly express Fra-1, c-Fos and c-Jun, components of the transcription factor, activator protein-1 (AP-1). Retrospective observation of breast cancer tissues obtained by core needle biopsy before surgery from stages II and III patients demonstrates that Fra-1 expression is high in estrogen receptor-negative human breast cancers, and negatively correlated to paclitaxel sensitivity. Ascochlorin, which suppresses cellular AP-1 activity, selectively kills estrogen receptor-negative human and mouse breast cancer cell lines, and prolongs the survival time of mice implanted with an estrogen receptor-negative mammary carcinoma. These results suggest that chemotherapy targeting AP-1 activity is a potent strategy for estrogen receptor-negative human breast cancers.

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor‐positive and ‐negative human breast cancer cells via the intrinsic pathway

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.

Synergism between the Anticancer Actions of 2-Methoxyestradiol and Microtubule-Disrupting Agents in Human Breast Cancer

2-Methoxyestradiol (2-MeO-E(2)), a well-known nonpolar endogenous metabolite of 17beta-estradiol, has strong antiproliferative, apoptotic, and antiangiogenic actions in vitro and in vivo at pharmacologic concentrations. We determined in the present study whether 2-MeO-E(2) can enhance the anticancer actions of paclitaxel or vinorelbine (two commonly used microtubule-disrupting agents) in several human breast cancer cell lines, including the estrogen receptor-positive MCF-7 and T-47D cells and the receptor-negative MDA-MB-435s and MDA-MB-231 cells. 2-MeO-E(2) in combination with paclitaxel or vinorelbine exhibited a synergistic anticancer effect in these human breast cancer cells in vitro, and this synergistic effect was more pronounced when each of the drugs was used at relatively low concentrations. Additional experiments using female athymic BALB/c nu/nu mice showed that p.o. administration of 2-MeO-E(2) at 30 mg/kg body weight, once a week for 6 weeks, markedly enhanced the activity of paclitaxel or vinorelbine against the growth of the estrogen receptor-negative MDA-MB-231 human breast cancer xenografts in these animals. By contrast, combination of 2-MeO-E(2) with 5-fluorouracil only had a partial additive effect against the growth of these cell lines in culture, and no synergistic effect was observed. Interestingly, when doxorubicin was used in combination with 2-MeO-E(2), the antiproliferative effect of 2-MeO-E(2) was somewhat antagonized by doxorubicin when it was present at high concentrations. Our results showed that 2-MeO-E(2) at nontoxic or subtoxic doses selectively enhanced the effects of certain microtubule-disrupting agents (such as paclitaxel and vinorelbine) against the growth of the receptor-negative human breast cancer cells in culture and also in athymic nude mice.

Function and prognostic value of tumor suppressor gene LKB1 in human breast carcinoma

To study the function and prognostic value of the recently identified tumor suppressor gene LKB1 in human breast cancer. Estrogen receptor-positive human breast cancer cells of MCF-7 line and estrogen receptor-negative human breast cancer cells of the lines MDA-MB-435 and MDA-MB-231 were cultured and tested for the LKB1 expression. Plasmid pcDNA4 TOPO HisMAX/LKB1 expressing LKB1 was constructed and transfected into the MDA-MB-435 cells. PCR and Western blotting were used to detect the expressions of LKB1 mRNA and protein. Flow cytometry was used to determine the cell cycle. PCR and Western blotting were used to detect the expressions of LKB1 mRNA and protein in 121 human breast cancer samples. The association with relapse free survival (RFS) and overall survival (OS) was assessed by univariate analysis (log-rank test and Kaplan-Meier method). Human breast cells of MCF-7 line expressed LKB1 and the cells of MDA-MB-435 and MDA-MB-231 lines did not. Flow cytometry showed that 40.9% of the MDA-MB-435 transfected with pcDNA4 TOPO HisMAX/LKB1 was at the G(1) stage, a proportion significantly higher than that of the control (18.3%). The expressions of cyclin D1 and cyclin E were significantly down-regulated and the expression of p21(WAF1/CIP1) was significantly up-regualed. LKB1 was lowly expressed in 43 out of the 121 cases of breast cancer (35.4%) and highly expressed in 78 cases (64.6%). Low expression of LKB1 was correlated with pathological grade (P = 0.013), cancer size (P = 0.001), status of estrogen receptor (P = 0.048), and status of lymph nodes metastasis (P = 0.003). Univariate analysis showed that low expression of LKB1 was significantly correlated with shorter RFS (P = 0.048) and low OS (P = 0.003). Reintroducing LKB1 into breast cell of the lines lacking LKB1 expression restores LKB1 activity and induces growth suppression by G(1) cell cycle block. The LKB1 mediated G(1) cell cycle arrest is caused by up-regulation of the expression of p21(WAF1/CIP1) in MDA-MB-435 breast cancer cells.

Digitalis-Induced Signaling by Na+/K+-ATPase in Human Breast Cancer Cells

Because beneficial effects of digitalis treatment in breast cancer patients have been suggested by epidemiological studies, we explored the mechanism of the growth inhibitory effects of these drugs on the estrogen receptor-negative human breast cancer cell line MDA-MB-435 s. Ouabain concentrations (100 nM or lower) that caused less than 25% inhibition of the pumping function of Na+/K+-ATPase had no effect on cell viability but inhibited proliferation. At the same concentrations, ouabain 1) activated Src kinase and stimulated the interaction of Src and Na+/K+-ATPase with epidermal growth factor receptor (EGFR); 2) caused a transient and then a sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2); 3) increased the expression of p21Cip1 but decreased that of p53; and 4) activated c-Jun NH2-terminal kinase (JNK) but not p38 kinase. These data, in conjunction with our previous findings on the signaling role of Na+/K+-ATPase in other cells, suggest that ouabain-induced activation/transactivation of Src/EGFR by Na+/K+-ATPase leads to activation of ERK1/2, the resulting increase in the level of cell cycle inhibitor p21Cip1, and growth arrest. Cooperation of JNK with ERK1/2 in this process is also suggested. Digoxin and digitoxin concentrations close to or at the therapeutic plasma levels had effects on proliferation and ERK1/2 similar to those of ouabain, supporting the proposed potential value of digitalis drugs for the treatment of breast cancer.

Estradiol metabolites induce apoptosis in human breast cancer cells

Certain catechol estrogens, endogenous estradiol meta-bolites, have been shown to elicit antiproliferative and antiangiogenic properties in human breast cancer cells and vascular endothelial cells at high concentrations. In the present study we investigated whether this effect can be attributed to apoptotic or cytotoxic actions. The receptor-positive (MCF-7) and the receptor-negative (MDA-MB-231) human breast cancer cell line were used. The estradiol metabolites were tested in the concentrations of 5, 10 and 20µM for 24h. Apoptosis was measured by a cell death assay and cytotoxicity using a formazan assay. In both cell lines apoptotic but no cytotoxic effects were induced by 2-OHE1, 2-OHE2, 2-MeOE1 and 2-MeOE2. In contrast 4-OHE1 and 4-OHE2 were cytotoxic. 4-MeOE1 and 4-MeOE2 did not act either apoptotic or cytotoxic. Our results indicate that catechol estrogens may be suitable for breast cancer treatment when used in high dosages. This may be of special importance for therapy, since some of these metabolites have been shown to be tolerated much better than cytostatics, even in high dosages. In addition, some elicit only low estrogenic activity in terms of the reproductive system.

Effect of His-Gly-Lys Motif Derived from Domain 5 of High Molecular Weight Kininogen on Suppression of Cancer Metastasis Both in Vitro and in Vivo

We have demonstrated previously that kinin-free high molecular weight kininogen, its domain 5 (D5H, Gly402-Lys502), and peptides derived from D5H inhibited vitronectin-mediated migration and invasion of cancer cells in vitro (Kamiyama, F., Maeda, T., Yamane, T., Li, Y. H., Ogikubo, O., Otsuka, T., and Ohkubo, I. (2001) Biochem. Biophys. Res. Commun. 288, 975-980). In this study, we found that the amino acid sequence His-Gly-Lys (HGK) in D5H is the core motif for inhibition of adhesion and invasion of MDA-MB-231 cells in vitro. P-5m (484GHGKHKNK491, Gly484-Lys491), an octapeptide including the HGK motif derived from D5H, and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro. However, an octapeptide designated P-5m (K487R), in which Lys487 was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect. Recombinant GST-D5H expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5H (K487R) in which Lys487 was changed to Arg. Furthermore, P-5m (Gly484-Lys491) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect. These data strongly indicate that both the HGK motif and lysine residue (Lys487) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo. We tried to identify the HGK motif binding protein on the surface of cancer cells. A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5H by P-5 (His479-Lys493) peptide but not by P-1 (Gly402-Lys420) peptide originating from the N-terminal region of D5H.