Effect of sodium butyrate on human breast cancer cell lines

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 m m . The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G₂M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.     

Sodium butyrate induces differentiation in breast cancer cell lines expressing the estrogen receptor

Addition of sodium butyrate (NaB) to 6 cultured human breast carcinoma cell lines results in a dose and time-dependent growth inhibition. Kinetic evidence, related to the growth of a minority cell population which decreases in size with time of exposure, is presented to indicate that the NaB effect is reversible. In those cell lines that express the estrogen receptor (ER), growth inhibition is accompanied by a more differentiated phenotype, which is characterized by increased accumulation of lipid and milk-fat globule membrane glycoproteins. The potential for differentiation is not blocked by tamoxifen, indicating that the relationship to ER expression is likely secondary to the association of ER expression with a particular stage of secretory cell differentiation that is susceptible to NaB induction. Of the 3 lines shown to respond in this way (MCF-7, ZR-75-1, and MDA-134), ZR-75-1 is an extreme example that may serve as a model for studies of gene expression during human mammary epithelial cell differentiation.     

Associated effects of sodium butyrate on histone acetylation and estrogen receptor in the human breast cancer cell line MCF-7

Sodium butyrate at a concentration of 5mM causes significant hyperacetylation of the core histones in the human breast cancer cell line MCF-7. Histone hyperacetylation was achieved in rapidly-growing cells at 40% confluency after 24 hours in 5mM sodium butyrate. More nearly confluent cells did not reach as high a level of histone hyperacetylation. Upon assaying the estrogen receptors, both cytosolic and KCl-extractable nuclear, we found that butyrate treatment had lowered the estrogen receptor levels in both compartments. To our knowledge this is the first report of an effect of sodium butyrate on estrogen receptor levels.     

Association of interferon-gamma induced growth inhibition and modulation of epidermal growth factor receptor gene expression in squamous cell carcinoma cell lines

Interferons (IFN) possess the ability to inhibit proliferation of certain transformed cell lines. Down modulation of the abnormal expression of certain oncogenes by IFN has been associated with phenotypic reversion of src, myc, or ras transformed cells. It has already been shown that some squamous cell carcinoma (SCCa) cell lines express elevated levels of the epidermal growth factor receptor (EGFR). Previously, in A431, an SCCa cell line, it was shown that IFN-gamma-induced growth inhibition was associated with both enhanced expression of EGFR and terminal differentiation. This study examines the effect of IFN-beta or IFN-gamma on five additional cervical SCCa cell lines. One cell line was shown to have amplification of the EGFR gene. An IFN-gamma induced antiproliferative response, observed in four of the five cell lines, was associated with increased expression of EGFR mRNA and induction of the IFN-inducible genes, HLA-A3 class I antigen and 2-5 oligoadenylate synthetase. These data suggest that the increased expression of the EGFR gene in a particular SCCa may predict response to IFN-gamma.     

Regulation of epidermal growth factor receptor gene expression

Synthesis and metabolism of the epidermal growth factor (EGF) receptor are extensively regulated to modulate cellular responses to ligand. To study regulation of EGF receptor gene expression, the 5' region of the gene was isolated from a human placental genomic library. A 5' proximal 1.1-kilobase fragment (-1100 to -19 relative to the ATG translation start site) and subfragments of this were subcloned in both forward and reverse orientations into the luciferase expression vector pSVOAL delta 5' and transfected into human cell lines. Luciferase activity was stimulated by treatment of transfected HeLa cells with EGF, 12-O-tetradecanoylphorbol 13-acetate (TPA), (Bu)2 cAMP, retinoic acid, and dexamethasone. Deletion analysis indicated full retention of activity after removal of the -1100 to -485 region (-485 to -19 fragment), but a 5-fold reduction in activity on removal of the -485 to -153 region (-153 to -19 fragment). Despite a reduction in basal activity, the proximal 134-basepair fragment retained responses to all inducers. Additivity was observed in response to maximal concentrations of TPA plus retinoic acid and of TPA plus (Bu)2 cAMP; the response to a combination of four inducers exceeded that to the RSV-LTR strong promoter. Differences in stimulated responses were observed in various recipients, with hepatoma HepG2 cells lacking responses to (Bu)2 cAMP and glioblastoma T98G cells lacking responses to EGF and TPA. These results indicate that a 134-basepair DNA fragment closely adjacent to the translation start site contains elements responsible for directing basal and stimulated expression of the EGF receptor gene.     

Expression and biosynthetic variation of the epidermal growth factor receptor in human hepatocellular carcinoma-derived cell lines.

Expression of the epidermal growth factor (EGF) was analyzed in six human hepatocellular carcinoma-derived and one human hepatoblastoma-derived cell line, each of which retained the differentiated phenotype and functions of the parenchymal hepatocyte. The level of receptor expression of each hepatoma cell line was similar to that of the normal human fibroblast, approximately 10(5) molecules per cell. However, NPLC/PRF/5, a subline of the PLC/PRF/5 cell line obtained following reestablishment of a xenograft tumor in vitro, was found to express 4 x 10(6) high-affinity EGF receptor molecules per cell. Proliferation of the NPLC/PRF/5 cell line was inhibited in the presence of nanomolar quantities of ligand. Receptor overexpression was found to result from EGF receptor gene amplification without apparent rearrangement of the EGF receptor coding sequences. Although cell-specific variability in posttranslational processing of EGF receptor N-linked oligosaccharides in the hepatoma cell lines was found, no difference between the receptors in PLC/PRF/5 and NPLC/PRF/5 was observed and no aberrant receptor-related species were detected. EGF receptor gene amplification in the NPLC/PRF/5 cell line is probably a reflection of genome instability and selection of variants with augmented growth potential in limiting concentrations of EGF in vivo. When viewed in this light, EGF receptor overexpression could represent a manifestation of tumor progression in the EGF-responsive hepatocyte.     

Modulation of estrogen receptor and epidermal growth factor receptor mRNAs by phorbol ester in MCF 7 breast cancer cells

Previous studies have demonstrated an inverse relationship between estrogen receptor (ER) and epidermal growth factor receptor (EGF-R) gene expression in human breast cancer cells. This relationship was further investigated in MCF 7 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Exposure to 10 nM TPA resulted in a time-dependent increase in EGF-R mRNA, first apparent at 3 h and maximal between 9 and 24 h. There was a concomitant fall in ER mRNA with a maximum decline to 15-20% of control between 12 and 24 h. Although EGF-R mRNA levels declined between 24 and 72 h, both EGF-R mRNA and EGF-R binding remained above control levels and this was accompanied by a sustained depression of ER mRNA. These data support the view that ER and EGR-R gene expression is inversely regulated in human breast cancer and describe for the first time an inhibitory effect of a phorbol ester on steroid hormone receptor gene expression.     

Modeling the estrogen receptor to growth factor receptor signaling switch in human breast cancer cells

Breast cancer cells develop resistance to endocrine therapies by shifting between estrogen receptor (ER)-regulated and growth factor receptor (GFR)-regulated survival signaling pathways. To study this switch, we propose a mathematical model of crosstalk between these pathways. The model explains why MCF7 sub-clones transfected with HER2 or EGFR show three GFR-distribution patterns, and why the bimodal distribution pattern can be reversibly modulated by estrogen. The model illustrates how transient overexpression of ER activates GFR signaling and promotes estrogen-independent growth. Understanding this survival-signaling switch can help in the design of future therapies to overcome resistance in breast cancer.     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.     

Effect of sodium butyrate on gene expression in a rat myogenic cell line

Sodium butyrate, when added in millimolar concentration to a culture of myoblasts of the L6 cell line, inhibits reversibly cell proliferation and differentiation. In the present work, we have studied the effect of Na butyrate on the translational efficiency of the overall poly (A)+ RNA. The mRNA from treated cells was translated in vitro as efficiently as proliferating myoblasts mRNA, while a decrease of translation efficiency was observed with myotubes mRNA. In addition this RNA directs the synthesis of several new polypeptides. on the switch on of alpha actin and myosin heavy chains (MHC), muscle specific genes by the dot blot and Northern blot techniques using cloned probes. Na butyrate prevented the expression of MHC and allowed the switch on of alpha actin gene but at a lesser extent than in normal myotubes. In addition the drug prevented the translocation of alpha actin mRNA into the cytoplasm.     

Differential gene expression profiling of human epidermal growth factor receptor 2-overexpressing mammary tumor

Human epidermal growth factor receptor 2 (HER2) is highly expressed in approximately 30% of breast cancer patients, and substantial evidence supports the relationship between HER2 overexpression and poor overall survival. However, the biological function of HER2 signal transduction pathways is not entirely clear. To investigate gene activation within the pathways, we screened differentially expressed genes in HER2-positive mouse mammary tumor using two-directional suppression subtractive hybridization combined with reverse dot-blotting analysis. Forty genes and expressed sequence tags related to transduction, cell proliferation/growth/apoptosis and secreted/extracellular matrix proteins were differentially expressed in HER2-positive mammary tumor tissue. Among these, 19 were already reported to be differentially expressed in mammary tumor, 11 were first identified to be differentially expressed in mammary tumor in this study but were already reported in other tumors, and 10 correlated with other cancers. These genes can facilitate the understanding of the role of HER2 signaling in breast cancer.