Thioltransferase overexpression increases resistance of MCF-7 cells to adriamycin

Thioltransferase, a small redox protein with thiol-disulfide oxidoreductase and dehydroascorbate reductase activities, has been reported to be expressed at higher levels in Adriamycin-resistant MCF-7 human breast tumor cells (MCF-7 ADR(R)) when compared with Adriamycin sensitive MCF-7 WT (MCF-7 WT) cells. The present study examined the effects of stably transfecting MCF-7 WT cells with the cDNA for human thioltransferase and the effects of subsequent Adriamycin cytotoxicity in the MCF-7 WT transfected cells. All transfected cell lines overexpressing thioltransferase activity were more resistant to Adriamycin than untransfected MCF-7 WT cells, supporting the hypothesis that increases in thioltransferase expression are related to Adriamycin resistance. This resistance was independent of the ability of thioltransferase to catalyze reduction of dehydroascorbic acid to ascorbic acid, as the addition of an ascorbate generating derivative, L-ascorbic acid-2-phosphate, to the media did not additionally increase Adriamycin resistance.     

Visfatin stimulates proliferation of MCF-7 human breast cancer cells

Obesity, a condition characterized by increased fat content and altered secretion of adipokines, is a risk factor for postmenopausal breast cancer. Visfatin has recently been established as a novel adipokine that is highly enriched in visceral fat. Here we report that visfatin regulated proliferation of MCF-7 human breast cancer cells. Exogenous administration of recombinant visfatin increased cell proliferation and DNA synthesis rate in MCF-7 cells. Furthermore, visfatin activated G1-S phase cell cycle progression by upregulation of cyclin D1 and cdk2 expression. Visfatin also increased the expression of matrix metalloproteinases 2, matrix metalloproteinases 9, and vascular endothelial growth factor genes, suggesting that it may function in metastasis and angiogenesis of breast cancer. Taken together, these findings suggest that visfatin plays an important role in breast cancer progression.     

Cripto-1 overexpression leads to enhanced invasiveness and resistance to anoikis in human MCF-7 breast cancer cells

Cripto-1 (CR-1) is an epidermal growth factor (EGF)-CFC protein that has been shown to signal through nodal/Alk-4, PI3K/Akt, and/or ras/raf/MEK/MAPK pathways in mammalian cells, and that is frequently expressed in human primary breast carcinomas. In the present study, the human estrogen receptor positive, MCF-7 breast cancer cell line, that expresses low levels of endogenous CR-1, was transfected with a CR-1 expression vector. MCF-7 CR-1 cells expressed high levels of a 25 kDa recombinant CR-1 protein that was not detected in MCF-7 cells transfected with a control vector (MCF-7 neo). Overexpression of CR-1 did not induce an estrogen independent phenotype in MCF-7 cells. In fact, MCF-7 CR-1 cells showed a response to exogenous estrogens that was similar to MCF-7 neo cells, and failed to grow in immunosuppressed mice in absence of estrogen stimulation. However, MCF-7 CR-1 cells showed a rate of proliferation in serum free conditions, and an ability to form colonies in soft-agar that were higher as compared with MCF-7 neo cells. More importantly, overexpression of CR-1 enhanced the resistance to anoikis and the invasion ability of MCF-7 cells. MCF-7 CR-1 cells showed levels of activation of both Akt and Smad-2 that were significantly higher as compared with MCF-7 neo. These findings suggest that CR-1 overexpression might be associated with the progression towards a more aggressive phenotype in breast carcinoma, through the activation of both Akt and Smad-2 signalling pathways.     

Caspase-independent apoptosis,in human MCF-7c3 breast cancer cells,following photodynamic therapy,with a novel water-soluble phthalocyanine

A new water-soluble phthalocyanine derivative, 2,3,9,10,16,17,23,24-octakis(3-aminopropyloxy) phthalocyaninato zinc II (PoII) was studied as a photosensitizer for photodynamic therapy (PDT) in MCF-7c3 cells. We report here that PoII and red light induces apoptosis. However, the precise mechanism appears to differ from that induced by PDT with other known phthalocyanines. The present study provides evidence that in the case of PoII, caspases do not participate in the apoptotic response. PoII-PDT-treated cells exhibited chromatin condensation and phosphatidylserine (PS) externalization. In the absence of light activation, PoII had no detectable cytotoxic effect. An early event upon PoII-PDT was photodamage to lysosomes, suggesting that they are the primary sites of action. Moreover, the treatment induces Bid activation, mitochondrial swelling and translocation of apoptosis-inducing factor (AIF) to the nucleus. An atypical proteolysis of poly(ADP-ribose) polymerase (PARP) indicative of calpain-like activation was observed. These data support the notion that an alternative mechanism of caspase-independent apoptosis was found in PoII-photosensitized cells.     

Estrogen-stimulated glucuronidation of dihydrotestosterone in MCF-7 human breast cancer cells

The non-aromatizable androgen dihydrotestosterone (DHT) has been shown to exert a potent inhibitory effect on the proliferation of some human breast cancer cell lines. DHT, however, has little or no significant inhibition on MCF-7 cell proliferation in either the presence or absence of estradiol (E₂). Since the metabolism of DHT into non-active compounds may be responsible for the observed lack of androgenic effect in this cell line, we have investigated the metabolic fate of labeled DHT in MCF-7 cells. A time course incubation was performed with 1 nM [³H]DHT and analysis of the various metabolites formed revealed a time-dependent increase in glucuronidated steroids which was stimulated more than 4-fold by 0.1 nM E₂. The major glucuronidated steroid was androstane-3, 17β-diol in both control and E₂-stimulated cells, comprising 22 ± 1.2% and 30 ± 0.6% of the total radioactivity in the medium, respectively. Other steroid glucuronides observed included DHT, androstane-3β, 17β-diol, and androsterone, all of which were elevated in the E₂-treated cells relative to control values. The present data show that E₂ exerts a stimulatory effect on the glucuronidation of androgens and their metabolites in the estrogen-dependent breast cancer celll line MCF-7. Since glucuronidation is an effective means of cellular elimination of active steroids, such a pathway may be considered as a possible site of regulation of breast cancer cell growth by hormones.     

Androgens inhibit estrogen action in MCF-7 human breast cancer cells

We have observed that the estrogen-dependent augmentation of cytoplasmic progesterone receptor (PR_c) content in MCF-7 human breast cancer cells is completely inhibited in the presence of testosterone (T) or dihydrotestosterone (DHT)¹. This effect is neither a result of altered PR_c affinity for test ligand nor a result of the direct interaction of either androgen with PR_c. Furthermore, the antiestrogenic activity of DHT is blocked in the presence of the antiandrogen, cyproterone, indicating that it is mediated through the androgen receptor. Further investigations of this cell line may provide important insights into the effects of sex steroids upon the hormonal regulation of a variety of healthy and malignant human tissues.     

Estradiol inhibits glucocorticoid receptor expression and induces glucocorticoid resistance in MCF-7 human breast cancer cells

Our study has shown that treatment of MCF-7 human breast cancer cells with 17-beta estradiol (E(2)) produced significant decreases in glucocorticoid receptor (GR) concentrations and GR mRNA levels. E(2) pre-treatment of MCF-7 cells stably transfected with the GR responsive pMTV-CAT reporter (MCF-7-MTV cells), caused significant attenuation of dexamethasone (DEX)-induced chloramphenicol acetyl transferase (CAT). In MCF-7 cells transiently transfected with [(GRE)(3)-Luc] reporter plasmid, E(2) pre-treatment significantly suppressed DEX-induced luciferase, which was abolished by the estrogen receptor antagonist ICI 182,780. We examined the effect of chronic E(2) treatment as well as E(2) withdrawal on GR function and abundance. MCF-7-MTV cells were treated with vehicle (control) or E(2) for up to 16 days. A third group received E(2) for 5 days followed by E(2) withdrawal from day 6 to 16. Chronic E(2) treatment almost totally abrogated DEX-induced CAT and reduced GR to very low levels. Interestingly, in the group subjected to E(2) withdrawal, neither the DEX response nor GR abundance recovered and reached control values suggesting that the estrogen mediated suppression is long lasting and could not be easily reversed. The E(2) induced resistance to glucocorticoid action may be of potential clinical significance in a number of settings including breast cancer, neuroendocrine response to stress and osteoporosis and could possibly contribute to the differences in glucocorticoid responsiveness among patients.     

Antiestrogen action of 2-hydroxyestrone on MCF-7 human breast cancer cells

The estrogen responsive human breast cancer MCF-7 cell culture was examined for its response to 2-hydroxyestrone a principal metabolite of estradiol. Addition of 2-hydroxyestrone to the cell cultures in concentration of 10(-9) - 10(-6) M had no effect on cell growth and proliferation because of rapid O-methylation of the catechol estrogen by catechol O-methyltransferase which is highly active in these cells. In the presence of quinalizarin, a potent catechol O-methyltransferase inhibitor which reduces the O-methylation of the steroid, 10(-7) M and 10(-8) M 2-hydroxyestrone markedly suppresses the growth and proliferation of the cells. The tumor cell growth-inhibitory action of the catechol estrogen was neutralized by the presence of 10(-9) M estradiol. The catechol estrogen inhibition of cell growth is not observed in the estrogen receptor-negative human breast cancer cell lines MDA-MB-231 and MDA-MB-330 providing evidence that the inhibition is specific and is estrogen receptor-mediated. In contrast, the 16 alpha-hydroxylated metabolites of estradiol, estriol and 16 alpha-hydroxyestrone, are effective stimulators of MCF-7 cell proliferation with the latter exhibiting potency in excess of that expected from its estrogen receptor affinity. The present results represent the first observation of a specific receptor-mediated antiestrogenic action of 2-hydroxyestrone and suggest that the physiological regulation of the agonist activity of the primary estrogen may involve in situ generation of catechol estrogen.     

Silibinin induces protective superoxide generation in human breast cancer MCF-7 cells

Abstract

The pharmacological activity of polyphenolic silibinin from milk thistle (Silybum marianum) is primarily due to its antioxidant property. However, this study found that silibinin promoted sustained superoxide (O₂^·–) production that was specifically scavenged by exogenous superoxide dismutase (SOD) in MCF-7 cells, while the activity of endogenous SOD was not changed by silibinin. Previous work proved that silibinin induced MCF-7 cell apoptosis through mitochondrial pathway and this study further proved that O₂^·– generation induced by silibinin was also related to mitochondria. It was found that respiratory chain complexes I, II and III were all involved in silibinin-induced O₂^·– generation. Moreover, it was found that silibinin-induced O₂^·– had protective effect, as exogenous SOD markedly enhanced silibinin-induced apoptosis.     

Effects of differentiation-inducing agents on maturation of human MCF-7 breast cancer cells

The effects of the differentiation inducing agents (DIAS), sodium butyrate (NaBu), retinoic acid (RA), dimethylformamide (DMF), hexamethylene bisacetamide (HMBA), forskolin, and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the growth, morphology, and estrogen receptor (ER) content and epithelial membrane antigen (EMA) expression on a serumless human breast cancer cell line (MCF-7) were compared. All these agents reversibly caused a concentration-dependent growth inhibition in monolayers and markedly reduced colony-forming efficiency in soft agar. A twofold increase in doubling time was obtained with RA (1 microM), but cell replication ceased with NaBu (1 mM), forskolin (50 microM), DMF (1%), HMBA (5 mM), and TPA (8 nM). Total growth arrest induced by these last compounds was preceded by an accumulation of cells in G0/G1 phase observed at 24 h by flow cytometry and accompanied by a change in cell morphology as seen by light and electronic microscopy. An increase in cell volume and the presence of lipid droplets was noted in treated cells that were spread out, as compared with controls. The acquisition of a more mature phenotype was confirmed by an increased expression of EMA monitored by flow cytometry. A specific reduction in the number of ER without any constant dissociation (Kd) modification was also observed after treatment with the 5 DIAs. No modification of morphological or biochemical characteristics, including EMA expression and ER binding, were observed for RA (1 microM)-treated cells. All these results suggest that induction of a more differentiated phenotype is associated with a block in G1 cell cycle phase, resulting in total growth arrest. Apparently, RA (1 microM)-treated cells did not fulfill these criteria, since only a slight accumulation in G1 and a slowed growth rate were evaluated.     

Nicotine increases cancer stem cell population in MCF-7 cells

Epidemiological studies have suggested that cigarette smoking is related to increased breast cancer risk. Nicotine is most likely related to the risk in cigarette smoking. However, the mechanisms by which nicotine promotes cancer development are not fully understood. It has recently been suggested that development of breast cancer are originated from cancer stem cells, which are a minor population of breast cancer. In the present study, we investigated the effects of nicotine on the population of cancer stem cells in MCF-7 human breast cancer cells, using flow cytometry with a cancer stem cell marker aldehyde dehydrogenase (ALDH). We found that nicotine increased ALDH-positive cell population in a dose-dependent manner. We further demonstrated that a PKC-Notch pathway is involved in the effect of nicotine. In addition, the effect of nicotine was blocked by treatment with the α7 subunit-selective antagonist of nicotinic acetylcholine receptors (nAChR) α-Bungarotoxin. These data suggest that nicotine increases the stem cell population via α7-nAChR and the PKC-Notch dependent pathway in MCF-7 cells. These findings reveal a relationship between nicotine and the cancer stem cells in human breast cancer.     

Melatonin increases p53 and p21WAF1 expression in MCF-7 human breast cancer cells in vitro.

The aim of the present work was to study whether melatonin, at physiological concentrations, exerts its antiproliferative effects on MCF-7 human breast cancer cells by inducing the expression of some of the proteins involved in the control of the cell cycle. MCF-7 cells were cultured for 48 h in DMEM media containing either melatonin (1 nM) or the diluent (0.001% ethanol). At this concentration, after 48 hours of incubation, melatonin reduced the number of viable cells in relation to controls. The decreased cell proliferation was coincident with a significant increase in the expression of p53 as well as p21WAF1 proteins. These results demonstrate that melatonin inhibits MCF-7 cell proliferation by inducing an arrest of cell cycle dependent on an increased expression of p21WAF1 protein, which is mediated by the p53 pathway.     

槟榔碱对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的：观察槟榔碱对人乳腺癌细胞（MCF-7）增殖和凋亡的影响,并探讨其机制。方法：采用四甲基偶氮唑盐（MTT）法检测不同浓度（0、10、30、50、100、300、500μmol/L）槟榔碱对MCF-7细胞增殖的影响,Hoechst 33342染色和流式细胞术检测细胞凋亡,Western blot法检测Bax,Bcl-2和P53蛋白表达。结果：低浓度（0、10、30、50μmol/L）槟榔碱不影响细胞的增殖和凋亡;而高浓度（100、300、500 μmol/L）槟榔碱呈浓度依赖性抑制MCF-7细胞增殖、诱导MCF-7细胞凋亡、提高P53和Bax蛋白表达、降低Bcl-2蛋白表达。结论：高浓度槟榔碱抑制MCF-7细胞增殖、诱导凋亡,其机制可能与提高P53和Bax蛋白表达,降低Bcl-2蛋白表达有关。     

Estrogenic effects of two derivatives of icariin on human breast cancer MCF-7 cells

The aims of the present study were to determine the estrogenic activities of icariin (ICA) and its derivatives and their structure–estrogenic activity relationship. Therefore, icaritin (ICT) and desmethylicaritin (DICT) were derived from ICA. The estrogenic activities of ICA, ICT and DICT were examined by cell proliferation and progestogen receptor mRNA expression of estrogen-receptor-positive MCF-7 cells. Current studies exhibited that ICT and DICT both markedly enhanced the proliferation of MCF-7 cells; as compared to estradiol (100%), their relative proliferative effects (RPE) were 90% and 94%, respectively. Cell proliferation induced by ICT and DICT was completely antagonized by ICI182,780. ICT and DICT increased progestogen receptor (PR) at mRNA levels at 48 h after treatment, although the effects were not as prominent as 17β-estradiol (E₂). These phenomena were not observed with ICA. Results demonstrate that ICT and DICT (nonconjugated forms) possess estrogen-like activity; however, ICA appears to have no estrogenicity in the MCF-7 cell line model in vitro.