Hs578Bst Cells Research Articles

K-D:rib dampens Hs 578T cancer cell chemoinvasion and proliferation

Cancer cells, even in the presence of available oxygen, have a glycolysis enhancement. The “aerobic glycolysis” is known as the Warburg effect and it is considered one of the fundamental hallmarks of metabolic alteration during malignant transformation. A feature of many tumors is also a change into ions equilibrium, with particular reference to K+ intracellular concentration. Another hallmark in cancer is the reprogrammed chemotaxis pathways in favour of tumor cell dissemination. The doubling population time of 5 mM K:D-rib treated Hs 578T (HTB-126 ® ATCC) cell line is reduce by 30% respect to the control. During the chemotactic invasion assay, the relative number of motile and invasive cells, counted inside the FBS-AGAR spot, shows a decrease with the maintenance of the treatment reaching the 25% after nine days. Hs 578Bst (HTB-125 ® ATCC) non-tumor cell line treated for nineteen days with 5 mM K:D-rib was split twice as well as the control. No morphological change was visible in the treated respect to untreated cells. We demonstrate that the synergic action of potassium bicarbonate and D-ribose has effect on Hs 578T cancer cell line proliferation reducing the cell cycle time. At 5 mM concentration, K:D-rib is able to modify the tumorigenic potential of human breast cancer cell line Hs 578T, interfering in vitro with the capability of Hs 578 T cell line to migrate under chemotactic stimuli. Despite this, K:D-rib solution, does not exhibit any appreciable toxicity as confirmed by the proliferation assay accomplished on Hs 578Bst cell line.

Recombinant human erythropoietin alters gene expression and stimulates proliferation of MCF-7 breast cancer cells

BackgroundFunctional erythropoietin (EPO) signaling is not specific only to erythroid lineages and has been confirmed in several solid tumors, including breast. Three different isoforms of erythropoietin receptor (EPOR) have been reported, the soluble (EPOR-S) and truncated (EPOR-T) forms acting antagonistically to the functional EPOR. In this study, we investigated the effect of human recombinant erythropoietin (rHuEPO) on cell proliferation, early gene response and the expression of EPOR isoforms in the MCF-7 breast cancer cell line.Materials and methodsThe MCF-7 cells were cultured with or without rHuEPO for 72 h or 10 weeks and assessed for their growth characteristics, expression of early response genes and different EPOR isoforms. The expression profile of EPOR and EPOR-T was determined in a range of breast cancer cell lines and compared with their invasive properties.ResultsMCF-7 cell proliferation after rHuEPO treatment was dependent on the time of treatment and the concentration used. High rHuEPO concentrations (40 U/ml) stimulated cell proliferation independently of a preceding long-term exposure of MCF-7 cells to rHuEPO, while lower concentrations increased MCF-7 proliferation only after 10 weeks of treatment. Gene expression analysis showed activation of EGR1 and FOS, confirming the functionality of EPOR. rHuEPO treatment also slightly increased the expression of the functional EPOR isoform, which, however, persisted throughout the 10 weeks of treatment. The expression levels of EPOR-T were not influenced. There were no correlations between EPOR expression and the invasiveness of MCF-7, MDA-MB-231, Hs578T, Hs578Bst, SKBR3, T-47D and MCF-10A cell lines.ConclusionsrHuEPO modulates MCF-7 cell proliferation in time- and concentration-dependent manner. We confirmed EGR1, FOS and EPOR as transcription targets of the EPO-EPOR signaling loop, but could not correlate the expression of different EPOR isoforms with the invasiveness of breast cancer cell lines.

Correlation between erythropoietin receptor(s) and estrogen and progesterone receptor expression in different breast cancer cell lines

Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and Gprotein-coupled estrogen receptor1 [GPER; also known as Gprotein-coupled receptor30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor2 receptor, β, low‑affinity (CSF2RB) and ephrin type-B receptor4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan Gprotein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these tworeceptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways or to evade their inhibition. Therefore, breast cancer classification upon ESR, PGR and human epidermal growth factor receptor2 (HER2) may not be sufficient for the selection of suitable treatment protocol. The expression of EPOR, GPER and EPHB4 may be considered as additional classification factors.

Expression of estrogen and progesterone receptors and estrogen metabolizing enzymes in different breast cancer cell lines

Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer. Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms DNA-damaging species. In the present study, we aimed to provide a better understanding of estrogen metabolism and actions in breast cancer, and to characterize model breast cancer cell lines. We determined the expression profiles of the genes for the estrogen and progesterone receptors, and for 18 estrogen-metabolizing enzymes in eight cell lines: MCF-7, MCF-10A, T47D, SKBR3, MDA-MB-231, MDA-MB-361, Hs-578T and Hs-578Bst cells. Similar gene expression profiles of these receptors and enzymes for the formation of estradiol via the aromatase and sulfatase pathways were observed in the MCF-7 and T47D metastatic cell lines. The MDA-MB-361 cells expressed ESR1, ESR2 and PGR as well, but differed in expression of the estrogen-metabolizing enzymes. In the MDA-MB-231 and SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the lack of ESR1 and the low levels of ESR2 expression suggested that the estrogens can only act via non-ER mediated pathways. In the non-tumorigenic MCF-10A cell line, the key enzymes of the aromatase pathway were not expressed, and the sulfatase pathway also had a marginal role. The comparison between gene expression profiles of the non-tumorigenic Hs-578Bst cells and the cancerous Hs-578T cells revealed that they can both form estrogens via the sulfatase pathway, while the aromatase pathway is less important in the Hs-578Bst cells. The Hs-578T cells showed low levels of ESR1, ESR2 and PGR expression, while only ESR1 and ESR2 expression was detected in the Hs-578Bst cells. Our data show that the cell lines examined provide the full range of model systems and should further be compared with the expression profiles of breast cancer specimens.

Common Polymorphism in the Phosphatase PHLPP2 Results in Reduced Regulation of Akt and Protein Kinase C

PHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt and protein kinase C (PKC) activity by specifically dephosphorylating these kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1). Here we identify a polymorphism that results in an amino acid change from a Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces phosphatase activity toward Akt both in vitro and in cells, in turn resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in breast cancer cells revealed the Ser-1016 variant is less functional toward both Akt and PKC. In pair-matched high grade breast cancer samples we observed retention of only the Ser allele from heterozygous patients (identical results were observed in a pair-matched normal and tumor cell line). Thus, we have identified a functional polymorphism that impairs the activity of PHLPP2 and correlates with elevated Akt phosphorylation and increased PKC levels.

Zinc retention differs between primary and transformed cells in response to zinc deprivation

Previous studies in our laboratory have demonstrated that reducing the availability of zinc with the extracellular metal chelator DTPA (diethylenetriaminepentaacetate) enhances, rather than inhibits, the thyroid hormone induction of growth hormone mRNA in GH3 rat anterior pituitary tumor cells. To understand the actions of the chelator on cellular zinc status, we observed the effects of DTPA on 65Zn uptake and retention. DTPA reduced the uptake of 65Zn by GH3 cells from the medium, but when GH3 cells were prelabeled with 65Zn, it resulted in greater retention of the isotope. In primary hepatocytes, DTPA both reduced the uptake of 65Zn from the medium and increased efflux from prelabeled cells. To investigate this difference, we studied the effects of DTPA on radioactive zinc flux in the H4IIE (rat hepatoma), MCF-7 (human breast cancer) and Hs578Bst (nontransformed human mammary) cell lines and in rat primary anterior pituitary cells. DTPA reduced the uptake of 65Zn in all cell lines examined. DTPA increased the retention of 65Zn in prelabeled H4IIE, MCF-7 and Hs578Bst cells but reduced it in primary pituitary cells. Time course experiments showed that 65Zn efflux is shut down rapidly by DTPA in transformed cells, whereas the chelator causes greater efflux from primary hepatocytes over the first 6 h. Experiments with 14C-labeled DTPA confirmed that this chelator does not cross cell membranes, showing that it operates entirely within the medium. Expression of ZnT-1, the efflux transporter, was not affected by DTPA in H4IIE cells. Thus, zinc deprivation enhanced zinc retention in established cell lines but increased efflux from primary cells, perhaps reflecting differing requirements for this mineral.

Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level

Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed non-invasive assay, we estimate the average NADH concentration in cancer cells (168 ± 49 μM) to be ∼1.8-fold higher than in breast normal cells (99 ± 37 μM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18 ± 0.08) and enzyme-bound (0.82 ± 0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25 ± 0.08; bound: 0.75 ± 0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.

Human prolactin receptor variants in breast cancer: low ratio of short forms to the long-form human prolactin receptor associated with mammary carcinoma.

Prolactin plays an essential role in the development of rodent mammary tumors and is a potent mitogen in human normal and cancerous breast tissues/cells. In this study, we have analyzed the expression of prolactin receptors, including the long receptor form (LF; stimulatory) and two novel short forms (SFs; S1a and S1b) derived from alternative splicing that are inhibitory of the activation induced by prolactin through the LF. Southern analysis of breast cancer profiling arrays revealed that 29 patients (group I) expressed an elevated LF, 10 patients (group II) showed decreased LF, and 8 patients (group III) had no change relative to the adjacent normal tissue. Their respective SF expression was increased in 21 patients of group I and generally decreased in groups II and III. However, the ratio of SF to LF was significantly decreased in 76% of the breast tumors and distributed evenly among the groups. Quantification of differential expression of prolactin receptor variants by real-time PCR in 15 pairs of human normal and tumor breast-matched tissues revealed a similar significant decrease in the ratio of SF to LF in the tumor tissue. Consistent lower ratio of SFs to LFs was confirmed in 8 of ten different breast cancer cell lines compared with normal mammary Hs578Bst and MCF10A cells. Because SFs act as dominant negative regulators of the stimulatory actions of the LF in vitro, their relatively reduced expression in cancer could cause gradations of unopposed prolactin-mediated LF stimulatory function and contribute to breast tumor development/progression.

Production of immunoreactive polymorphonuclear leucocyte elastase in human breast cancer cells: possible role of polymorphonuclear leucocyte elastase in the progression of human breast cancer.

Breast cancer cells are known to express various proteolytic enzymes, which make them invasive and favour their dissemination to distant sites. However, it is unclear whether breast cancer cells have the ability to produce polymorphonuclear leucocyte elastase (PMN-E). We measured immunoreactive (ir) PMN-E content in the conditioned medium of two breast cancer cell lines, MCF-7 and ZR-75-1, and two normal breast epithelial cell lines, HBL-100 and Hs 578Bst, using a highly specific and sensitive enzyme immunoassay. Furthermore, ir-PMN-E content was determined in tissue extracts from 62 human breast cancers. ir-PMN-E content in the culture medium of MCF-7 cells and ZR-75-1 cells increased as a function of time, regardless of the presence or absence of oestradiol. On the other hand, no detectable ir-PMN-E was secreted into the culture medium of HBL-100 and Hs 578Bst cells. ir-PMN-E was detectable in 59 of 62 tissue extracts prepared from human breast cancers, the concentration ranging from 0.12 to 19.17 micrograms per 100 mg of protein. When 62 breast cancer specimens were categorised into four groups in terms of clinical stage, ir-PMN-E content in breast cancer tissue was significantly higher in stage III (8.90 +/- 5.13 micrograms 100 mg-1 protein) and stage IV (12.19 +/- 5.44 micrograms 100 mg-1 protein) patients than in stage I (1.64 +/- 1.54 micrograms 100 mg-1 protein) and stage II (4.23 +/- 3.74 micrograms 100 mg-1 protein) patients. Breast cancer patients with high levels of ir-PMN-E showed significantly shorter disease-free survival and overall survival than those with low levels of ir-PMN-E at the cut-off point of 8.99 micrograms 100 mg-1 protein. In the multivariate analysis, ir-PMN-E content was found to be a significant prognostic factor for disease recurrence and death in human breast cancer.

Two syngeneic cell lines from human breast tissue: the aneuploid mammary epithelial (Hs578T) and the diploid myoepithelial (Hs578Bst) cell lines.

We characterized two human cell lines (Hs578T and Hs578Bst), which provide several unique features that should be useful in the study of breast disease. Hs578T, derived from a carcinosarcoma, is epithelial in origin. Hs578Bst, established from normal tissue peripheral to the tumor, is myoepithelial in origin. This is the first report of companion cell lines, one malignant and one normal, established from the same organ.