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.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

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.     

Upregulation of activin A gene by butyrate in human colon cancer cell lines

Activin A has been reported to play a role in the progression of colorectal cancer. Because dietary fiber protects against colorectal cancer, we hypothesized that butyrate, a fermentation product of dietary fiber, may affect the expression of activin A in colon cancer cells. Semiquantitative RT-PCR demonstrated that the activin A gene was upregulated by sodium butyrate in the human colon cancer cell lines HT-29 and Caco-2 in a concentration- and time-dependent manner. However, the activin A gene did not respond to sodium butyrate in the human normal colonic cell line FHC, rat normal intestinal epithelial cell (IEC) line IEC-6, and the explant of rat colon. Flow cytometry and agarose gel electrophoresis of genomic DNA revealed that cell cycle arrest and apoptosis were induced by sodium butyrate but not exogenous activin A in HT-29 cells, indicating that activin A could not act as an autocrine factor in colon cancer cells. By assuming that activin A promotes colorectal cancer spread as a paracrine factor, our findings suggest that butyrate could act as a tumor promoter in some circumstances.     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

Effect of recombinant human TNF on human ovarian cancer cell lines

Ovarian cancer is the second most common gynecologic malignancy. Standard therapeutic approaches to this disease, surgery followed by chemotherapy, have produced response rates of up to 80%. However, the five-year survival rate remains around 30%. Recently, Tumor Necrosis Factor (TNF) has received attention as either an alternative or an associated agent for chemotherapy of ovarian cancer. TNF is known to have direct cytotoxic and cytostatic effects on a variety of transformed cell lines "in vitro". Furthermore, TNF is known to enhance significantly the "in vitro" effects of a class of chemotherapeutic agents, specifically those targeted at DNA topoisomerase II. In this work we have investigated TNF-induced cytotoxicity in four established human epithelial ovarian cancer cell lines: A-2774; SV-626; SKOV-3 and Pa-1. TNF mediated cytotoxic activity was observed in a range of concentrations between 1 U/ml and 10-3 U/ml. A-2774 and SV-626 were the two most sensitive lines, especially when exposed to high concentrations of TNF.     

Specific binding of benzodiazepines to human breast cancer cell lines

Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.     

正丁酸钠对人胃腺癌细胞染色体数目和DNA含量的影响

After treatment with n-sodium butyrate for 7 days, the inhibition of growth rate of human gastric adenocarcinoma cell (MGc 80-3) in culture reaches 50.7%. About 90% of the cancer cells treated with the drug undergo obvious differentiation, and their ultrastructure is also changed. Moreover, the cancer cells which have hyperdiploid chromosomes increase from 78% to 96%; on the contrary, the percentage of hypertriploid cells decreases from 6% to 2%, while that of hypertetraploid cells diminishes from 14% to 2%. By using the combination of 3H-TdR autoradiography and Feulgen cytophotometry to measure the cellular DNA content of unlabelled cells (G1), it is shown that the DNA amount in the experimental group is lower than that in the control group. Furthermore, the DNA content of undifferentiated cells in the unlabelled cells (G1) of the experimental group is hyperhexaploid in amount (D1 = 3.76 and 3.56), and about 90% undifferentiated cells have a DNA value of over 6 C. On the other hand, the differentiated cells in the unlabelled cells on the above same slide have near-tetraploid DNA values (D1 = 2.03 and 1.99), and a DNA content below 4 C is found in about 60% differentiated cells. The difference in DNA amount between these two categories of cells is statistically significant. The results mentioned above suggest that although the amount of genetic material in the morphologically differentiated cells has markedly decreased, these cells still do not represent normal diploid cells.     

c-MET protects breast cancer cells from apoptosis induced by sodium butyrate

Sodium butyrate (NaBu) is regarded as a potential reagent for cancer therapy. In this study, a specific breast cancer cell population that is resistant NaBu treatment was identified. These cells possess cancer stem cell characters, such as the capability of sphere formation in vitro and high tumor incident rate (85%) in mouse model. Forty percent of the NaBu resistant cells express the cancer stem cells marker, the CD133, whereas only 10% intact cells present the CD133 antigen. Furthermore, the endogenous expressing c-MET contributes to the survival of cancer stem cell population from the treatment of NaBu. The CD133+ group also presents a higher level of c-MET. A combination treatment of MET siRNA and NaBu efficiently prohibited the breast cancer progression, and the incident rate of the tumor decrease to 18%. This study may help to develop a new and alternative strategy for breast cancer therapy.     

Evaluation of the antioxidative and genotoxic effects of sodium butyrate on breast cancer cells

Oncogenic stimulation shows a rise in reactive oxygen species (ROS), and ROS can eventually induce carcinogenesis by causing DNA damage. In this context, this study aims to evaluate some biochemical and genotoxic changes in the control of cell death caused by NaBu (Sodium butyrate). treatment in breast cancer cells. NaBu’s impact on cell proliferation was determined via WST-1 assay. The lipid peroxidation (MDA), reduced glutathione (GSH), Nitric Oxide (NO), hydrogen peroxide (H₂O₂), and superoxide dismutase (SOD) enzyme levels were determined biochemically. NaBu-induced genotoxic damage was estimated via single-cell gel electrophoresis (SCGE). NaBu reduced cell viability and potentially induced GSH, but decreased SOD enzyme activity and the level of MDA and NO decreased also H₂O₂ decreased at different times and NaBu concentrations. Higher NaBu concentrations amplified DNA damage in MCF-7 cells compared to the control group. NaBu shows anticancer and genotoxic effects, especially through antioxidant enzymes, one of the oxidative stress parameters in breast cancer. However, the anticancer and genotoxic effects of NaBu is changed in the oxidative stress parameters with time and treatment concentration of NaBu in MCF-7 cells. Furthermore, his oxidative stress-dependent effect changes need to be clarified by further evaluation with molecular and more biochemical parameters.     

Selective cytotoxicity of ascochlorin in ER-negative human breast cancer cell lines

While agents targeting estrogen receptors are most effective in adjuvant therapy for human breast cancers expressing estrogen receptors after surgery, breast cancers lacking estrogen receptor are clinically serious, because they are highly malignant and exhibit resistance to the usual anti-cancer drugs, including estrogen receptor-antagonists and DNA breaking agents. Here, we found that MX-1, a human breast cancer cell line lacking estrogen receptors, exhibited higher AP-1 activity and expressed higher levels of c-Jun, c-Fos, and Fra-1 when compared with conventional estrogen receptor-positive human breast cancer cell lines. The prenylphenol antibiotic ascochlorin suppressed the AP-1 activity of MX-1 cells, and selectively killed MX-1 cells, partly due to induction of apoptosis. Our results suggest that AP-1 is an effective clinical target molecule for the treatment of estrogen receptor-negative human breast cancer.     

Variability of the paracrine-induced osteoclastogenesis by human breast cancer cell lines

Breast cancer frequently metastasizes to the bone, often leading to the formation of osteolytic lesions. This work compares the paracrine-induced osteoclastogenesis mediated by four human breast cancer cell lines, the estrogen-receptor positive T47D and MCF-7 and the estrogen-negative SK-BR-3 and Hs-578T cell lines. Human osteoclast precursor cells were cultured in the presence of conditioned media from the breast cancer cell lines (10% and 20%), collected at different culture periods (48 h, 7 days, and 14 days). Cultures performed in the absence or the presence of M-CSF and RANKL served as negative and positive control, respectively. Results showed that the cell lines differentially expressed several osteoclastogenic genes. All cell lines exhibited a significant osteoclastogenic potential, evidenced by a high TRAP activity and number of osteoclastic cells, expression of several osteoclast-related genes, and, particularly, a high calcium phosphate resorption activity. Differences among the osteoclastogenic potential of the cell lines were noted. T47D and MCF-7 cell lines displayed the highest and the lowest osteoclastogenic response, respectively. Despite the variability observed, MEK and NF-κB signaling pathways, and, at a lesser extent, PGE2 production, seemed to have a central role on the observed osteoclastogenic response. In conclusion, the tested breast cancer cell lines exhibited a high osteoclastogenic potential, although with some variability on the cell response profile, a factor to be considered in the development of new therapeutic approaches for breast cancer-induced bone metastasis.     

Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines

Purified L-asparaginase of Tetrahymena pyriformis is a multi-subunit enzyme exhibiting protein kinase activity as well. The enzyme's L-asparaginase activity is affected by its phosphorylation state. Both native and dephosphorylated L-asparaginase show antiproliferative activity on three breast cancer cell lines (T47D, BT20 and MCF-7) and on Walker 256 cells. These cells do not possess measurable L-asparaginase or L-asparagine synthetase activity. When T47D cells are treated for different times with L-asparaginase and then placed in fresh medium, the growth of cells treated for 1, 3, or 6 hours is initiated and parallels control curve, while the growth of cells treated for 24 or 48 hours with L-asparaginase stays at the same inhibitory level (24 h treatment) or continues to drop (48 h treatment). Addition of D-asparagine, a competitive inhibitor of T. pyriformis L-asparaginase, counteracts the antiproliferative activity of L-asparaginase, indicating that L-asparaginase and not the kinase activity is responsible for that effect.     

Cellular localization of tissue factor in human breast cancer cell lines

Expression of tissue factor (TF), the cellular receptor of clotting factor VII/VIIa, is a feature of certain malignant tumours. The TF gene has been classified as an immediate early gene responsive to serum and cytokines. Thus, the regulation of TF gene expression seems to play a role in cell growth. Recently, we have shown that constitutive TF expression in MCF-7 breast cancer cells is modulated by such growth factors as EGF, TGFα, and IL-1. The present study deals with the immunocytochemically detectable cellular distribution of TF in human breast cancer cell lines MCF-7 and MaTu stimulated by EGF and TGFα. In MCF-7 cells growing logarithmically, stimulation led to a significant increase of TF mRNA after 2 h (in situ hybridization, Northern blot) and to maximum TF expression after 6 h (immunohistochemistry). When decorated by monoclonal antibodies, TF protein showed a pronounced localization at ruffled membrane areas, cell edges, and processes of spreading cells after 6 and 20 h. In more flattened cells TF was concentrated in peripheric lamellae and microspikes communicating with neighbouring cells. After epithelial colony pattern had established, TF was predominantly accumulated at the intercellular boundaries. The vary same distribution patterns as seen in MCF-7 cells were true for the stimulated MaTu cell line. The dynamics and cellular distribution patterns of stimulated TF expression support the hypothesis that TF could be of importance for morphogenic events associated with the growth and differentiation of breast cancer cells in culture.     

Antiproliferative effects of COX-2 inhibitor celecoxib on human breast cancer cell lines

The inducible COX-2 enzyme is over-expressed in human breast cancer and its over-expression generally correlates with angiogenesis, deregulation of apoptosis and worse prognosis. This observation may explain the beneficial effect of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors on breast cancer treatment. Here, we evaluated the antiproliferative activity of celecoxib, a selective COX-2 inhibitor, and its nitro-oxy derivative on human breast cancer cells characterized by low and high COX-2 expression, respectively. In ERα(+) MCF-7 cells celecoxib and its derivative induce a strong inhibition of cell growth, inhibition that is associated with the reduction of ERα expression and activation. These effects may be directly associated with ERK and Akt suppression and with PP2A and PTEN induction. In this cell line the drugs exert only weak effect on COX-2 level while they are able to reduce aromatase expression. On the contrary, in ERα(-) MDA-MB-231 cells, both drugs induce a marked inhibition of COX-2, inhibition that is associated with the reduction of aromatase expression and of cell proliferation. In both cell lines the effects of the drugs are associated with the suppression of cell invasion.     

Effect of bisphenols on telomerase expression and activity in breast cancer cell lines

Molecular Biology Reports - Bisphenol A (BPA), a monomer of polycarbonates and resins, was shown to induce the expression of telomerase enzyme which has been associated with breast cancer...